Springer Handbook of Enzymes, 2nd Edition, Supplement Volume S3 (Class 2 Transferases EC 2.7.11.1–2.7.11.16) [2nd ed.] 3540856986, 9783540856986

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Springer Handbook of Enzymes Supplement Volume S3

Dietmar Schomburg and Ida Schomburg (Eds.)

Springer Handbook of Enzymes Supplement Volume S3 Class 2 Transferases EC 2.7.11.1–2.7.11.16 coedited by Antje Chang

Second Edition

13

Professor Dietmar Schomburg e-mail: [email protected] Dr. Ida Schomburg e-mail: [email protected]

Technical University Braunschweig Bioinformatics & Systems Biology Langer Kamp 19b 38106 Braunschweig Germany

Dr. Antje Chang e-mail: [email protected]

Library of Congress Control Number: applied for ISBN 978-3-540-85698-6

2nd Edition Springer Berlin Heidelberg New York

The first edition was published as the “Enzyme Handbook, edited by D. and I. Schomburg”.

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springer.com # Springer-Verlag Berlin Heidelberg 2009 Printed in Germany The use of general descriptive names, registered names, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and free for general use. The publisher cannot assume any legal responsibility for given data, especially as far as directions for the use and the handling of chemicals and biological material are concerned. This information can be obtained from the instructions on safe laboratory practice and from the manufacturers of chemicals and laboratory equipment. Cover design: Erich Kirchner, Heidelberg Typesetting: medionet Publishing Services Ltd., Berlin Printed on acid-free paper

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Preface

Today, as the full information about the genome is becoming available for a rapidly increasing number of organisms and transcriptome and proteome analyses are beginning to provide us with a much wider image of protein regulation and function, it is obvious that there are limitations to our ability to access functional data for the gene products – the proteins and, in particular, for enzymes. Those data are inherently very difficult to collect, interpret and standardize as they are widely distributed among journals from different fields and are often subject to experimental conditions. Nevertheless a systematic collection is essential for our interpretation of genome information and more so for applications of this knowledge in the fields of medicine, agriculture, etc. Progress on enzyme immobilisation, enzyme production, enzyme inhibition, coenzyme regeneration and enzyme engineering has opened up fascinating new fields for the potential application of enzymes in a wide range of different areas. The development of the enzyme data information system BRENDAwas started in 1987 at the German National Research Centre for Biotechnology in Braunschweig (GBF), continued at the University of Cologne from 1996 to 2007, and then returned to Braunschweig, to the Technical University, Institute of Bioinformatics & Systems Biology. The present book “Springer Handbook of Enzymes” represents the printed version of this data bank. The information system has been developed into a full metabolic database. The enzymes in this Handbook are arranged according to the Enzyme Commission list of enzymes. Some 5,000 “different” enzymes are covered. Frequently enzymes with very different properties are included under the same EC-number. Although we intend to give a representative overview on the characteristics and variability of each enzyme, the Handbook is not a compendium. The reader will have to go to the primary literature for more detailed information. Naturally it is not possible to cover all the numerous literature references for each enzyme (for some enzymes up to 40,000) if the data representation is to be concise as is intended. It should be mentioned here that the data have been extracted from the literature and critically evaluated by qualified scientists. On the other hand, the original authors’ nomenclature for enzyme forms and subunits is retained. In order to keep the tables concise, redundant information is avoided as far as possible (e.g. if Km values are measured in the presence of an obvious cosubstrate, only the name of the cosubstrate is given in parentheses as a commentary without reference to its specific role). The authors are grateful to the following biologists and chemists for invaluable help in the compilation of data: Cornelia Munaretto and Dr. Antje Chang. Braunschweig Spring 2009

Dietmar Schomburg, Ida Schomburg

VII

List of Abbreviations

A Ac ADP Ala All Alt AMP Ara Arg Asn Asp ATP Bicine C cal CDP CDTA CMP CoA CTP Cys d dDFP DNA DPN DTNB DTT EC E. coli EDTA EGTA ER Et EXAFS FAD FMN Fru Fuc G Gal

adenine acetyl adenosine 5’-diphosphate alanine allose altrose adenosine 5’-monophosphate arabinose arginine asparagine aspartic acid adenosine 5’-triphosphate N,N’-bis(2-hydroxyethyl)glycine cytosine calorie cytidine 5’-diphosphate trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid cytidine 5’-monophosphate coenzyme A cytidine 5’-triphosphate cysteine deoxy(and l-) prefixes indicating configuration diisopropyl fluorophosphate deoxyribonucleic acid diphosphopyridinium nucleotide (now NAD+ ) 5,5’-dithiobis(2-nitrobenzoate) dithiothreitol (i.e. Cleland’s reagent) number of enzyme in Enzyme Commission’s system Escherichia coli ethylene diaminetetraacetate ethylene glycol bis(-aminoethyl ether) tetraacetate endoplasmic reticulum ethyl extended X-ray absorption fine structure flavin-adenine dinucleotide flavin mononucleotide (riboflavin 5’-monophosphate) fructose fucose guanine galactose

IX

List of Abbreviations

GDP Glc GlcN GlcNAc Gln Glu Gly GMP GSH GSSG GTP Gul h H4 HEPES His HPLC Hyl Hyp IAA IC 50 Ig Ile Ido IDP IMP ITP Km lLeu Lys Lyx M mM mMan MES Met min MOPS Mur MW NAD+ NADH NADP+ NADPH NAD(P)H

X

guanosine 5’-diphosphate glucose glucosamine N-acetylglucosamine glutamine glutamic acid glycine guanosine 5’-monophosphate glutathione oxidized glutathione guanosine 5’-triphosphate gulose hour tetrahydro 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid histidine high performance liquid chromatography hydroxylysine hydroxyproline iodoacetamide 50% inhibitory concentration immunoglobulin isoleucine idose inosine 5’-diphosphate inosine 5’-monophosphate inosine 5’-triphosphate Michaelis constant (and d-) prefixes indicating configuration leucine lysine lyxose mol/l millimol/l metamannose 2-(N-morpholino)ethane sulfonate methionine minute 3-(N-morpholino)propane sulfonate muramic acid molecular weight nicotinamide-adenine dinucleotide reduced NAD NAD phosphate reduced NADP indicates either NADH or NADPH

List of Abbreviations

NBS NDP NEM Neu NMN NMP NTP oOrn pPBS PCMB PEP pH Ph Phe PHMB PIXE PMSF p-NPP Pro Q10 Rha Rib RNA mRNA rRNA tRNA Sar SDS-PAGE Ser T tH Tal TDP TEA Thr TLCK Tm TMP TosTPN Tris Trp TTP Tyr U

N-bromosuccinimide nucleoside 5’-diphosphate N-ethylmaleimide neuraminic acid nicotinamide mononucleotide nucleoside 5’-monophosphate nucleoside 5’-triphosphate orthoornithine paraphosphate-buffered saline p-chloromercuribenzoate phosphoenolpyruvate -log10[H+ ] phenyl phenylalanine p-hydroxymercuribenzoate proton-induced X-ray emission phenylmethane-sulfonylfluoride p-nitrophenyl phosphate proline factor for the change in reaction rate for a 10 C temperature increase rhamnose ribose ribonucleic acid messenger RNA ribosomal RNA transfer RNA N-methylglycine (sarcosine) sodium dodecyl sulfate polyacrylamide gel electrophoresis serine thymine time for half-completion of reaction talose thymidine 5’-diphosphate triethanolamine threonine Na-p-tosyl-l-lysine chloromethyl ketone melting temperature thymidine 5’-monophosphate tosyl- (p-toluenesulfonyl-) triphosphopyridinium nucleotide (now NADP+ ) tris(hydroxymethyl)-aminomethane tryptophan thymidine 5’-triphosphate tyrosine uridine

XI

List of Abbreviations

U/mg UDP UMP UTP Val Xaa XAS Xyl

XII

mmol/(mg*min) uridine 5’-diphosphate uridine 5’-monophosphate uridine 5’-triphosphate valine symbol for an amino acid of unknown constitution in peptide formula X-ray absorption spectroscopy xylose

Index of Recommended Enzyme Names

EC-No.

Recommended Name

2.7.11.4 2.7.11.15 2.7.11.11 2.7.11.12 2.7.11.3 2.7.11.8 2.7.11.9 2.7.11.16 2.7.11.10 2.7.11.5 2.7.11.7 2.7.11.1 2.7.11.13 2.7.11.2 2.7.11.14 2.7.11.6

[3-methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase b-adrenergic-receptor kinase . . . . . . . . . . . . . . . . . . cAMP-dependent protein kinase . . . . . . . . . . . . . . . . . cGMP-dependent protein kinase . . . . . . . . . . . . . . . . . dephospho-[reductase kinase] kinase . . . . . . . . . . . . . . . Fas-activated serine/threonine kinase . . . . . . . . . . . . . . . Goodpasture-antigen-binding protein kinase. . . . . . . . . . . . G-protein-coupled receptor kinase . . . . . . . . . . . . . . . . IkB kinase. . . . . . . . . . . . . . . . . . . . . . . . . . . [Isocitrate dehydrogenase (NADP+ )] kinase . . . . . . . . . . . . myosin-heavy-chain kinase . . . . . . . . . . . . . . . . . . . non-specific serine/threonine protein kinase . . . . . . . . . . . . protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . [pyruvate dehydrogenase (acetyl-transferring)] kinase . . . . . . . rhodopsin kinase. . . . . . . . . . . . . . . . . . . . . . . . [tyrosine 3-monooxygenase] kinase . . . . . . . . . . . . . . .

Page 167 400 241 288 163 203 207 448 210 178 186 1 325 124 370 184

XIII

Description of Data Fields

All information except the nomenclature of the enzymes (which is based on the recommendations of the Nomenclature Committee of IUBMB (International Union of Biochemistry and Molecular Biology) and IUPAC (International Union of Pure and Applied Chemistry) is extracted from original literature (or reviews for very well characterized enzymes). The quality and reliability of the data depends on the method of determination, and for older literature on the techniques available at that time. This is especially true for the fields Molecular Weight and Subunits. The general structure of the fields is: Information – Organism – Commentary – Literature The information can be found in the form of numerical values (temperature, pH, Km etc.) or as text (cofactors, inhibitors etc.). Sometimes data are classified as Additional Information. Here you may find data that cannot be recalculated to the units required for a field or also general information being valid for all values. For example, for Inhibitors, Additional Information may contain a list of compounds that are not inhibitory. The detailed structure and contents of each field is described below. If one of these fields is missing for a particular enzyme, this means that for this field, no data are available.

1 Nomenclature EC number The number is as given by the IUBMB, classes of enzymes and subclasses defined according to the reaction catalyzed. Systematic name This is the name as given by the IUBMB/IUPAC Nomenclature Committee Recommended name This is the name as given by the IUBMB/IUPAC Nomenclature Committee Synonyms Synonyms which are found in other databases or in the literature, abbreviations, names of commercially available products. If identical names are frequently used for different enzymes, these will be mentioned here, cross references are given. If another EC number has been included in this entry, it is mentioned here.

XV

Description of Data Fields

CAS registry number The majority of enzymes have a single chemical abstract (CAS) number. Some have no number at all, some have two or more numbers. Sometimes two enzymes share a common number. When this occurs, it is mentioned in the commentary.

2 Source Organism For listing organisms their systematic name is preferred. If these are not mentioned in the literature, the names from the respective literature are used. For example if an enzyme from yeast is described without being specified further, yeast will be the entry. This field defines the code numbers for the organisms in which the enzyme with the respective EC number is found. These code numbers (form ) are displayed together with each entry in all fields of BRENDA where organism-specific information is given.

3 Reaction and Specificity Catalyzed reaction The reaction as defined by the IUBMB. The commentary gives information on the mechanism, the stereochemistry, or on thermodynamic data of the reaction. Reaction type According to the enzyme class a type can be attributed. These can be oxidation, reduction, elimination, addition, or a name (e.g. Knorr reaction) Natural substrates and products These are substrates and products which are metabolized in vivo. A natural substrate is only given if it is mentioned in the literature. The commentary gives information on the pathways for which this enzyme is important. If the enzyme is induced by a specific compound or growth conditions, this will be included in the commentary. In Additional information you will find comments on the metabolic role, sometimes only assumptions can be found in the references or the natural substrates are unknown. In the listings, each natural substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included only if the respective authors were able to demonstrate the formation of the specific product. If only the disappearance of the substrate was observed, the product is included without organisms of references. In cases with unclear product formation only a ? as a dummy is given. Substrates and products All natural or synthetic substrates are listed (not in stoichiometric quantities). The commentary gives information on the reversibility of the reaction,

XVI

Description of Data Fields

on isomers accepted as substrates and it compares the efficiency of substrates. If a specific substrate is accepted by only one of several isozymes, this will be stated here. The field Additional Information summarizes compounds that are not accepted as substrates or general comments which are valid for all substrates. In the listings, each substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included if the respective authors demonstrated the formation of the specific product. If only the disappearance of the substrate was observed, the product will be included without organisms or references. In cases with unclear product formation only a ? as a dummy is given. Inhibitors Compounds found to be inhibitory are listed. The commentary may explain experimental conditions, the concentration yielding a specific degree of inhibition or the inhibition constant. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. Cofactors, prosthetic groups This field contains cofactors which participate in the reaction but are not bound to the enzyme, and prosthetic groups being tightly bound. The commentary explains the function or, if known, the stereochemistry, or whether the cofactor can be replaced by a similar compound with higher or lower efficiency. Activating Compounds This field lists compounds with a positive effect on the activity. The enzyme may be inactive in the absence of certain compounds or may require activating molecules like sulfhydryl compounds, chelating agents, or lipids. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. Metals, ions This field lists all metals or ions that have activating effects. The commentary explains the role each of the cited metal has, being either bound e.g. as Fe-S centers or being required in solution. If an ion plays a dual role, activating at a certain concentration but inhibiting at a higher or lower concentration, this will be given in the commentary. Turnover number (min- 1) The kcat is given in the unit min-1 . The commentary lists the names of the substrates, sometimes with information on the reaction conditions or the type of reaction if the enzyme is capable of catalyzing different reactions with a single substrate. For cases where it is impossible to give the turnover number in the defined unit (e.g., substrates without a defined molecular weight, or an undefined amount of protein) this is summarized in Additional Information.

XVII

Description of Data Fields

Specific activity (U/mg) The unit is micromol/minute/milligram of protein. The commentary may contain information on specific assay conditions or if another than the natural substrate was used in the assay. Entries in Additional Information are included if the units of the activity are missing in the literature or are not calculable to the obligatory unit. Information on literature with a detailed description of the assay method may also be found. Km-Value (mM) The unit is mM. Each value is connected to a substrate name. The commentary gives, if available, information on specific reaction condition, isozymes or presence of activators. The references for values which cannot be expressed in mM (e.g. for macromolecular, not precisely defined substrates) are given in Additional Information. In this field we also cite literature with detailed kinetic analyses. Ki-Value (mM) The unit of the inhibition constant is mM. Each value is connected to an inhibitor name. The commentary gives, if available, the type of inhibition (e.g. competitive, non-competitive) and the reaction conditions (pH-value and the temperature). Values which cannot be expressed in the requested unit and references for detailed inhibition studies are summerized under Additional information. pH-Optimum The value is given to one decimal place. The commentary may contain information on specific assay conditions, such as temperature, presence of activators or if this optimum is valid for only one of several isozymes. If the enzyme has a second optimum, this will be mentioned here. pH-Range Mostly given as a range e.g. 4.0–7.0 with an added commentary explaining the activity in this range. Sometimes, not a range but a single value indicating the upper or lower limit of enzyme activity is given. In this case, the commentary is obligatory. Temperature optimum ( C) Sometimes, if no temperature optimum is found in the literature, the temperature of the assay is given instead. This is always mentioned in the commentary. Temperature range ( C) This is the range over which the enzyme is active. The commentary may give the percentage of activity at the outer limits. Also commentaries on specific assay conditions, additives etc.

XVIII

Description of Data Fields

4 Enzyme Structure Molecular weight This field gives the molecular weight of the holoenzyme. For monomeric enzymes it is identical to the value given for subunits. As the accuracy depends on the method of determination this is given in the commentary if provided in the literature. Some enzymes are only active as multienzyme complexes for which the names and/or EC numbers of all participating enzymes are given in the commentary. Subunits The tertiary structure of the active species is described. The enzyme can be active as a monomer a dimer, trimer and so on. The stoichiometry of subunit composition is given. Some enzymes can be active in more than one state of complexation with differing effectivities. The analytical method is included. Posttranslational modifications The main entries in this field may be proteolytic modification, or side-chain modification, or no modification. The commentary will give details of the modifications e.g.: – proteolytic modification (, propeptide Name) [1]; – side-chain modification (, N-glycosylated, 12% mannose) [2]; – no modification [3]

5 Isolation / Preparation / Mutation / Application Source / tissue For multicellular organisms, the tissue used for isolation of the enzyme or the tissue in which the enzyme is present is given. Cell-lines may also be a source of enzymes. Localization The subcellular localization is described. Typical entries are: cytoplasm, nucleus, extracellular, membrane. Purification The field consists of an organism and a reference. Only references with a detailed description of the purification procedure are cited. Renaturation Commentary on denaturant or renaturation procedure. Crystallization The literature is cited which describes the procedure of crystallization, or the X-ray structure.

XIX

Description of Data Fields

Cloning Lists of organisms and references, sometimes a commentary about expression or gene structure. Engineering The properties of modified proteins are described. Application Actual or possible applications in the fields of pharmacology, medicine, synthesis, analysis, agriculture, nutrition are described.

6 Stability pH-Stability This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. Temperature stability This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. Oxidation stability Stability in the presence of oxidizing agents, e.g. O2, H2 O2, especially important for enzymes which are only active under anaerobic conditions. Organic solvent stability The stability in the presence of organic solvents is described. General stability information This field summarizes general information on stability, e.g., increased stability of immobilized enzymes, stabilization by SH-reagents, detergents, glycerol or albumins etc. Storage stability Storage conditions and reported stability or loss of activity during storage.

References Authors, Title, Journal, Volume, Pages, Year.

XX

Non-specific serine/threonine protein kinase

2.7.11.1

1 Nomenclature EC number 2.7.11.1 Systematic name ATP:protein phosphotransferase (non-specific) Recommended name non-specific serine/threonine protein kinase Synonyms 14-3-3 [426] 3-phosphoinositide dependent protein kinase-1 [1, 26, 34] 3-phosphoinositide-dependent kinase 1 [413] 30 kDa protein kinase homolog [219, 220, 221, 222, 230] 5’-AMP-activated protein kinase, catalytic a-1 chain [185, 186] 5’-AMP-activated protein kinase, catalytic a-2 chain [187, 188, 197, 198] 90S6K [439] A-T, mutated A-T, mutated homolog A-kinase AKT1 [39, 439] AKT2 [75, 103] AKin10 [208] AMH type II receptor AMPK [198, 413] AP50 kinase ATP-protein transphosphorylase Akt protein kinase [37] Akt-3 [53] Akt3 [52] Anti-sigma B factor rsbT Apg1p [179] AsfK [436] Ataxia telangiectasia mutated Ataxia telangiectasia mutated homolog BUB1 protein kinases [355] breast-tumor-amplified kinase CD43-associated serine/threonine kinase [319] CDC25 suppressing protein kinase

1

Non-specific serine/threonine protein kinase

2.7.11.1

CDPK [466] CDPK-related protein kinase [182] CK-2 [251] CK1 [215, 413, 439, 465] CK1 g 1 [247] CK1 g 2 [247] CK1 g 3 [247] CK1a [443] CK1d [413] CK1e-1 [444] CK2 [267, 276, 413, 422, 429, 435, 439, 454, 456, 459, 465, 470, 471] CK2-a CKI CKI-a [231] CKI-b [231] CKII [264, 437] CKIe-2 [444] CKIe-3 [444] CLK1 [167] CLP-36 interacting kinase CPK1 [466] CSNK1D [240] CSNK1G1 [248] CSNK1G2 [243] calcium-dependent protein kinase C calcium/phospholipid-dependent protein kinase Cdc7Hs protein [370] cell division cycle 2-like Chk1 [127, 128, 413] Chk1 kinase [212, 213] DAP-kinase DCAMKL1 [132] DM kinase [89] DM-kinase [90] DMCK1 [241] DMPK [88] DNA damage response protein kinase DUN1 [169, 170, 171] DNA-PKcs DNPK1 EC 2.7.1.70 EDPK [369] Ern1p [391] G11 protein G2-specific protein kinase fin1 [114] GSK-3 [430, 431, 438] GSK-3b [430, 434, 465] 2

2.7.11.1

Non-specific serine/threonine protein kinase

GSK3 [414] GSK3b [409, 413, 427, 432, 433] Gin4p protein kinase [200] HIPK2 [320, 418] HRR25 [228] HSPK 21 HSPK 36 Hank’s type serine/threonine kinase [421] Hpr kinase HsCdc7 [371] HuCds1 [145] Hunk [142] ISPK-1 [85] IpL1protein kinase [121] Ire1p kinase [390] KIAA0369 [133, 134, 211] kinase interacting with stathmin Kiz-1 [16] Krct [367] LIM domain kinase 1 [12, 13, 14, 15, 16, 17, 18, 19, 20] LIMK [14] lymphocyte-oriented kinase M phase-specific cdc2 kinase MAP kinase activated protein kinase 2 [176, 177] MAP kinase-activated protein kinase 2 [6, 174, 176] MAPK-activated protein kinase 1a [413] MAPK-activated protein kinase 2 [413] MAPK-activated protein kinase-2 [174] MAPKAP kinase-2 [177] MAPKAP-K1a [413] MAPKAP-K2 [413] MAPKAPK-2 [174] MIL proto-oncogene serine/threonine-protein kinase [5] MPSK MT-PK [96] myristoylated and palmitoylated serine-threonine kinase NDR protein kinase [428] NDR1 [428] NPH1 [56] NTHK2 kinase [446] NY-REN-55 antigen ORF47 protein [437] P-CIP2 P460 PAK-3 [284] PAK4 [285] PASK [283] 3

Non-specific serine/threonine protein kinase

2.7.11.1

PDK1 [34, 413, 414, 420] PK1 [449] PK2 [449] PKA PKB [414] PKBa [413] PKD2 [440] PKG [413] PKL12 [368] PKN [423] PKN1 [458] PRP4 kinase PRP4 pre-mRNA processing factor 4 homolog PSK-H1 PSKH1 [116] Pak1 [301] Pak1 protein [294] Phototropin [56, 447] Pim-2h PkB kinase PkaG [436] PknB [412, 425] PknD [412] PknE [412] PknF [412] PknH [421] PknI [448] pre-mRNA protein kinase protein kinase B kinase protein kinase Krct protein kinase MST protein kinase PKL12 RAC-PKg [106] RAC-a serine/threonine kinase [37, 39, 46, 47, 48, 49, 50, 74, 81] RAC-b serine/threonine protein kinase [75, 76, 81, 103] RAC-g serine/threonine protein kinase [38, 51, 52, 53, 54, 106] RAC/Akt kinase [47] RKIN1 protein [194] RMIL serine/threonine-protein kinase [11, 21] RP1 protein RPS2 [237] RSK [426] RSK1 [426] RSK2 [457] RSK3 [102] Raf kinase 4

2.7.11.1

Non-specific serine/threonine protein kinase

Raf-1 Rio2 [451] Rsk-1 S6 kinase [107] S-receptor kinase S6K [417] S6K1 [413, 415] S6K2 [108, 415] S6KII a [59] SAST [445] SCD10.09 [436] SGK [92] SGK1 [439] SNF1-like protein kinase [114] SNF1-related protein kinase KIN10 [207, 208] SPAK [281] SPAK/STE20 [455] SPS1-related proline alanine-rich kinase [455] SRK SSTK [442] STE [439] STE20-like kinase MST1 STE20-like kinase MST2 STE20-like kinase MST3 STE20/SPS1-related proline-alanine rich protein kinase [281, 282, 283] STE20/SPS1-related, proline alanine-rich kinase [281] STK11 [203] Ser/Thr kinase [447] Ser/Thr protein kinase [412, 419, 453] serine/threonine kinase 15 serine/threonine kinase Ayk1 serine/threonine-protein kinase NRK2 serine/threonine-protein kinase NYD-SPK SpkA [462] Ste20-like kinase Ste20-related protein kinase [455] Ste20p-like protein kinase CaCla4p [279] StkP [453, 463] StpK [419] Switch protein/serine kinase T-antigen kinase TPK I [409] TSSK5 [411] t-tubulin kinase 1 [465] Titin, heart isoform N2-B [325, 326] Tssk4 [464] Twitchin kinase 5

Non-specific serine/threonine protein kinase

2.7.11.1

UNC51 [137] US3 kinase [468] Unc51.1 [137] WNK1 [416, 450] WNK1 kinase [410] WNK4 [416] WNK4 kinase [410] XEEK1 kinase [210] autophagy serine/threonine-protein kinase APG1 [179, 180] bIIPKC c-mil protein [5] cAMP-dependent protein kinase A calcium-dependent protein kinase [159, 466] calcium-dependent protein kinase 2 [175] calcium-dependent protein kinase SK5 [160] calcium-dependent protein kinase, isoform 1 [183] calcium-dependent protein kinase, isoform 11 [184] calcium-dependent protein kinase, isoform 2 [184] calcium-dependent protein kinase, isoform AK1 [195, 196] carbon catabolite derepressing protein kinase [149, 150, 178, 191, 194] casein kinase casein kinase 1 [439, 465] casein kinase 1 d [413] casein kinase 1 g1 [248] casein kinase 1a [443] casein kinase 1e-1 [444] casein kinase 1e-2 [444] casein kinase 1e-3 [444] casein kinase 2 [255, 422, 429, 435, 465] casein kinase I [215, 460] casein kinase I g 2 [243] casein kinase I homolog 1 [28, 223, 224, 225] casein kinase I homolog 2 [224, 225, 226] casein kinase I homolog 3 [232] casein kinase I homolog HRR25 [227, 228, 229] casein kinase I homolog cki1 [114, 233, 234] casein kinase I homolog cki3 [114, 216] casein kinase I homolog hhp1 [114, 235, 236] casein kinase I homolog hhp2 [114, 235, 236] casein kinase I, a isoform [6, 231, 238, 239, 242, 244] casein kinase I, b isoform [231] casein kinase I, d isoform [240, 245, 246] casein kinase I, d isoform like [214, 237] casein kinase I, e isoform [31, 249] casein kinase I, g 1 isoform [247, 248] 6

2.7.11.1

Non-specific serine/threonine protein kinase

casein kinase I, g 2 isoform [243, 247] casein kinase I, g 3 isoform [247, 250] casein kinase II [251, 253, 254, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 270, 272, 273, 274, 276, 437] casein kinase Ie [217] casein kinase-2 casein kinase-II [452] cell cycle protein kinase DBF2 [70, 71] cell cycle protein kinase hsk1 [114, 377] cell cycle protein kinase spo4 [114, 378] cell division control protein 15 [287, 288, 289] cell division control protein 7 [373, 374, 375, 376] cell division cycle 7-related protein kinase [370, 371, 372, 379] checkpoint kinase 1 [413] checkpoint serine/threonine-protein kinase bub1 [114, 362, 363, 364] cyclic AMP-dependent protein kinase cyclic AMP-dependent protein kinase A cyclic monophosphate-dependent protein kinase cyclic nucleotide-dependent protein kinase cyclin-dependent kinase cytidine 3’,5’-cyclic monophosphate-responsive protein kinase dSTPK61 death-associated protein kinase 1 [181] discs overgrown protein kinase [217, 218] ethylene receptor protein NTHK2 [446] eukaryotic-type serine/threonine protein kinase [453] glycogen synthase kinase 3 [414] glycogen synthase kinase 3 b [432, 433] glycogen synthase kinase 3b [409, 427] glycogen synthase kinase-3 [430, 431, 434, 438] glycogen synthase kinase-3ab [441] glycogen synthase kinase-3b [424, 465] hARK1 hBUBR1 [361] hPAK1 [300] hPDK1 hPSK histone kinase homeodomain-interacting protein kinase 2 [317, 318, 319, 320, 418] hormonally upregulated neu tumor-associated kinase [142, 143] hydroxyalkyl-protein kinase insulin-stimulated protein kinase [85] kinase, casein (phosphorylating) kinase, protamine (phosphorylating) 7

Non-specific serine/threonine protein kinase

2.7.11.1

kinase, protein (phosphorylating) kinase, protein, A (phosphorylating) kinase, protein, C (phosphorylating) kinase-related apoptosis-inducing protein kinase 1 [123] mBub1b [360] mPDK1 meiosis-specific serine/threonine-protein kinase MEK1 [114, 118, 119, 120] mitogen-activated S6 kinase mitosis inducer protein kinase cdr1 [114, 151, 152] mitosis inducer protein kinase cdr2 [114, 189, 190] mitotic checkpoint serine/threonine-protein kinase BUB1 [354, 355, 356, 357, 358, 359] mitotic checkpoint serine/threonine-protein kinase BUB1 b [356, 357, 359, 360, 361] mitotic control element nim1+ [152] myotonic dystrophy protein kinase [88, 89, 90, 91] myotonin-protein kinase [88, 89, 91, 96, 97, 98, 99, 100, 101] negative regulator of sexual conjugation and meiosis [114, 344] nitrogen permease reactivator protein [345] nonphototropic hypocotyl protein 1 [56, 57, 58, 271] nuclear Dbf2-related protein kinase [428] ovarian-specific serine/threonine-protein kinase Lok [6, 136] p46Eg265 p46XlEg22 p53-related protein kinase [7, 277] p54 S6 kinase 2 [108] p70 S6 kinase [65, 110] p70 ribosomal S6 kinase 1 [415] p70(S6k) [65] p82 kinase p90 ribosomal S6 kinase [426] pEg2 peripheral plasma membrane protein CASK [129, 130, 131, 139, 209] phosphoinositide-dependent kinase-1 [420] phosphorylase b kinase kinase piD261 [386] pp39-mos pp90rsk Ser/Thr kinase [102] protamine kinase protein casein kinase II [452] protein glutamyl kinase protein kinase (phosphorylating) protein kinase 2 [73] protein kinase B a [47] protein kinase B g [51, 54] 8

2.7.11.1

Non-specific serine/threonine protein kinase

protein kinase B/Akt [469] protein kinase Ba [413] protein kinase Bg [54] protein kinase C, mu type [104, 105] protein kinase CK2 [454, 456, 459, 461, 467, 470, 471] protein kinase Cbk1p [327] protein kinase Cm [104] protein kinase D [105] protein kinase D2 [440] protein kinase DBF20 [70] protein kinase DC1 [30] protein kinase DC2 [30] protein kinase Doa [312] protein kinase HIPK2 [317] protein kinase KIN1 [153, 155] protein kinase N1 [458] protein kinase PKX1 [87] protein kinase PVPK-1 [62] protein kinase Rim15p [322, 323] protein kinase Sgk [35, 36] protein kinase cds1 [114, 199] protein kinase cek1 [114, 324] protein kinase dsk1 [114, 311] protein kinase p58 protein phosphokinase protein serine kinase protein serine-threonine kinase protein serine/threonine kinase [459] protein-aspartyl kinase protein-cysteine kinase protein-serine kinase protein-serine/threonine kinase rDRAK1 [123] rac-PK [76] ratAurA ribosomal S6 kinase (Rsk-2) [82] ribosomal S6 kinase 2 [415, 457] ribosomal S6 protein kinase [417] ribosomal protein S6 kinase [65, 66, 67, 68, 72] ribosomal protein S6 kinase II ribosomal protein S6 kinase II a [59, 63] ribosomal protein S6 kinase II b [59] ribosomal protein S6 kinase a 1 [63, 86, 107] ribosomal protein S6 kinase a 2 [86, 102, 112] ribosomal protein S6 kinase a 3 [82, 83, 84, 85, 86] ribosomal protein S6 kinase a 6 [111] ribosomal protein S6 kinase b 2 [108, 109, 110, 113] 9

Non-specific serine/threonine protein kinase

2.7.11.1

serine kinase serine protein kinase serine(threonine) protein kinase serine-specific protein kinase serine/threonine kinase [425, 436, 455] serine/threonine kinase 17A [123, 144] serine/threonine kinase 17B [144] serine/threonine kinase PRP4 homolog [306] serine/threonine protein kinase [328, 329, 419, 442, 448, 458] serine/threonine protein kinase 16 [365, 366, 367, 368, 369] serine/threonine protein kinase 31 [389] serine/threonine protein kinase BUD32 [386, 387, 388] serine/threonine protein kinase afsK [336, 338, 339] serine/threonine protein kinase pkaA [336, 337] serine/threonine protein kinase pkaB [336, 337] serine/threonine-protein kinase 1 [380, 381, 382, 383, 384, 385] serine/threonine-protein kinase 11 [201, 202, 203, 204, 205, 210] serine/threonine-protein kinase ASK1 [2, 172] serine/threonine-protein kinase ASK2 [172, 173] serine/threonine-protein kinase AtPK1/AtPK6 [78, 79, 80, 271] serine/threonine-protein kinase AtPK19 [271] serine/threonine-protein kinase CBK1 [226, 327] serine/threonine-protein kinase CLA4 [278, 279, 292] serine/threonine-protein kinase CTR1 [22, 23] serine/threonine-protein kinase Chk1 [114, 126, 127, 128, 154, 161, 162, 163, 164, 212] serine/threonine-protein kinase Chk2 [31, 145, 146, 147, 148] serine/threonine-protein kinase DCAMKL1 [125, 132, 133, 134, 135, 211] serine/threonine-protein kinase GIN4 [200] serine/threonine-protein kinase H1 [116, 117] serine/threonine-protein kinase IPL1 [121] serine/threonine-protein kinase IRE1 precursor [28, 390, 391, 392] serine/threonine-protein kinase KIN3 [287, 288, 393, 394] serine/threonine-protein kinase KIN4 [192, 193] serine/threonine-protein kinase KSP1 [28, 350] serine/threonine-protein kinase Kist [277, 313, 314, 316] serine/threonine-protein kinase NEK2 [342] serine/threonine-protein kinase NEK3 [342] serine/threonine-protein kinase PAK 1 [284, 290, 291, 300, 301] serine/threonine-protein kinase PAK 2 [302, 303, 304] serine/threonine-protein kinase PAK 3 [280, 284, 290] serine/threonine-protein kinase PAK 4 [285] serine/threonine-protein kinase PAK 7 [7] 10

2.7.11.1

Non-specific serine/threonine protein kinase

serine/threonine-protein kinase PAK1 [168] serine/threonine-protein kinase PTK1/STK1 [348, 349] serine/threonine-protein kinase PTK2/STK2 [349, 351, 352] serine/threonine-protein kinase Pk61C [6, 33] serine/threonine-protein kinase RCK1 [165, 166] serine/threonine-protein kinase RCK2 [166, 167] serine/threonine-protein kinase RIM15 [323] serine/threonine-protein kinase SAT4 [346, 347] serine/threonine-protein kinase SCH9 [27, 28, 29] serine/threonine-protein kinase SKM1 [298, 299] serine/threonine-protein kinase SKS1 [227, 353] serine/threonine-protein kinase STE20 [28, 295, 296] serine/threonine-protein kinase STE20 homolog [305] serine/threonine-protein kinase Sgk [35, 36, 40, 41, 42, 43, 44, 45, 92, 93, 94, 95] serine/threonine-protein kinase ULK1 [137, 138, 140, 141] serine/threonine-protein kinase YPK1 [60, 61] serine/threonine-protein kinase YPK2/YKR2 [60, 64] serine/threonine-protein kinase ark1 [115] serine/threonine-protein kinase cot-1 [77] serine/threonine-protein kinase fused [6, 156, 157, 158] serine/threonine-protein kinase nrc-2 [55] serine/threonine-protein kinase orb6 [25] serine/threonine-protein kinase pak1/shk1 [114, 293, 294] serine/threonine-protein kinase pkn2 [334] serine/threonine-protein kinase pkn5 [335] serine/threonine-protein kinase pkn6 [335] serine/threonine-protein kinase pknA [332, 333] serine/threonine-protein kinase pknD [330, 331] serine/threonine-protein kinase prp4 [114, 315] serine/threonine-protein kinase sck1 [32] serine/threonine-protein kinase shk2 [114, 297] serine/threonine-protein kinase ssp1 [114, 124] serine/threonine-protein kinase transforming protein Rmil [8, 9, 10] serine/threonine-protein kinase transforming protein mil [3, 4] serine/threonine-protein kinase unc-51 [206] serine/threonine-specific protein kinase [449] spermatozoon associated protein kinase [69] sporulation-specific protein 1 [286] syntrophin-associated serine/threonine kinase [445] t-protein kinase I [409] testis-specific serine/threonine kinase [411] testis-specific serine/threonine protein kinase 5 variant a [464] testis-specific serine/threonine protein kinase 5 variant b [464] testis-specific serine/threonine protein kinase 5 variant d [464] testis-specific serine/threonine protein kinase 5 variant g [464] 11

Non-specific serine/threonine protein kinase

2.7.11.1

threonine-specific protein kinase type-2 casein kinase Additional information ( PKD2 belongs to the PKD family of serine/threonine kinases [440]; PKN belongs to the AGC subfamily of protein kinases [423]; see also EC 2.7.11.26 [409, 424, 427, 430, 432, 433, 438, 441]; see also EC 2.7.11.26 and EC 2.7.11.11 [431]; the enzyme belongs to the Rio family, Rio2 subfamily, of serine/threonine protein kinases [451]; the enzyme TSSK3 belongs to the family of testis-specific serine-threonine kinases [420]; TSSK5 is a member of the testis-specific serine/threonine kinase family [411]; cf. EC 2.7.11.26 [465]; the enzyme belongs to the CKI family of serine/threonine protein kinases [460]; the enzyme belongs to the p90RSK, RSK, family of proteins [457]) [409, 411, 420, 423, 424, 427, 430, 431, 432, 433, 438, 440, 441, 451, 457, 460, 465] CAS registry number 191808-15-8 (phosphoinositide dependent protein kinase 1) 37278-10-7 377752-08-4 (ribosomal protein S6 kinase 2) 389133-24-8 (ribosomal S6 kinase 3) 52660-18-1 (casein kinase, protein kinase CK2) 9026-43-1 (this CAS Reg. No. encompasses a great variety of protein kinases including the serine/threonine specific kinases) 90698-26-3 (ribosomal protein S6 kinase 1)

2 Source Organism Drosophila melanogaster (no sequence specified) [423, 460] Mus musculus (no sequence specified) [415, 420, 423, 441, 457] Homo sapiens (no sequence specified) [409, 413, 414, 418, 422, 423, 427, 428, 429, 430, 432, 433, 437, 438, 439, 440, 441, 443, 452, 454, 456, 458, 459, 460, 461, 465, 469, 470, 471] Rattus norvegicus (no sequence specified) [395, 397, 408, 410, 413, 416, 423, 426, 431, 434, 439, 445, 450, 467] Sus scrofa (no sequence specified) [399] Saccharomyces cerevisiae (no sequence specified) [396,439] Bos taurus (no sequence specified) [398,401,402,403,404,405,423,424] Oryctolagus cuniculus (no sequence specified) [400] Arabidopsis thaliana (no sequence specified) [447] Streptococcus pneumoniae (no sequence specified) [419, 453, 463] Xenopus laevis (no sequence specified) [423, 460] Caenorhabditis elegans (no sequence specified) [423] Mycobacterium tuberculosis (no sequence specified) [412, 421, 425, 448] Streptomyces coelicolor (no sequence specified) [436] Synechocystis sp. (no sequence specified) [462]

12

2.7.11.1



























Non-specific serine/threonine protein kinase

Archaeoglobus fulgidus (no sequence specified) [451] Salmo gairdneri (no sequence specified) [406, 407] herpes simplex virus type 1 (no sequence specified) [468] varicella-zoster virus (no sequence specified) [437] Avian retrovirus MH2 (UNIPROT accession number: P00531) [3, 4] Gallus gallus (UNIPROT accession number: P05625) [5] Avian retrovirus IC10 (UNIPROT accession number: P10533) [8, 9] avian rous-associated virus type 1 (UNIPROT accession number: P27966) [10] Coturnix coturnix japonica (UNIPROT accession number: P34908) [11] Homo sapiens (UNIPROT accession number: P53667) [12, 13, 14, 15] Mus musculus (UNIPROT accession number: P53668) [16, 17, 18, 19] Rattus norvegicus (UNIPROT accession number: P53669) [20] Gallus gallus (UNIPROT accession number: Q04982) [21] Arabidopsis thaliana (UNIPROT accession number: Q05609) [22, 23] Schizosaccharomyces pombe (UNIPROT accession number: O13310) [25] Rattus norvegicus (UNIPROT accession number: O55173) [26] Saccharomyces cerevisiae (UNIPROT accession number: P11792) [27, 28, 29] Drosophila melanogaster (UNIPROT accession number: P16911) [30] Schizosaccharomyces pombe (UNIPROT accession number: P50530) [32] Drosophila melanogaster (UNIPROT accession number: Q9W0V1) [6, 33] Mus musculus (UNIPROT accession number: Q9Z2A0) [34] Homo sapiens (UNIPROT accession number: O15530) [1] Schizosaccharomyces pombe (UNIPROT accession number: Q12701) [1] Mus musculus (UNIPROT accession number: Q9WVC6) [35] Homo sapiens (UNIPROT accession number: O00141) [36, 40, 41, 42, 43, 44, 45] Homo sapiens (UNIPROT accession number: P31749) [37, 39, 46, 47, 48, 49, 50] Homo sapiens (UNIPROT accession number: Q9Y243) [38, 51, 52, 53, 54] Neurospora crassa (UNIPROT accession number: O42626) [55] Arabidopsis thaliana (UNIPROT accession number: O48963) [56, 57, 58, 271] Xenopus laevis (UNIPROT accession number: P10665) [59] Xenopus laevis (UNIPROT accession number: P10666) [59] Saccharomyces cerevisiae (UNIPROT accession number: P12688) [60, 61] Phaseolus vulgaris (UNIPROT accession number: P15792) [62] Drosophila melanogaster (UNIPROT accession number: P16912) [30] Gallus gallus (UNIPROT accession number: P18652) [63] Mus musculus (UNIPROT accession number: P18653) [63] Saccharomyces cerevisiae (UNIPROT accession number: P18961) [60,64] Rattus norvegicus (UNIPROT accession number: P21425) [65,66,67,68]

13

Non-specific serine/threonine protein kinase



























14

2.7.11.1

Aplysia californica (UNIPROT accession number: P21901) [69] Saccharomyces cerevisiae (UNIPROT accession number: P22204) [70,71] Homo sapiens (UNIPROT accession number: P23443) [72] Dictyostelium discoideum (UNIPROT accession number: P28178) [73] Mus musculus (UNIPROT accession number: P31750) [74] Homo sapiens (UNIPROT accession number: P31751) [75,76] Saccharomyces cerevisiae (UNIPROT accession number: P32328) [70] Neurospora crassa (UNIPROT accession number: P38679) [77] Arabidopsis thaliana (UNIPROT accession number: P42818) [78, 79, 80, 271] Rattus norvegicus (UNIPROT accession number: P47196) [81] Rattus norvegicus (UNIPROT accession number: P47197) [81] Homo sapiens (UNIPROT accession number: P51812) [82,83,84,85,86] Homo sapiens (UNIPROT accession number: P51817) [87] Mus musculus (UNIPROT accession number: P54265) [88,89,90,91] Bos taurus (UNIPROT accession number: Q01314) [48,50] Rattus norvegicus (UNIPROT accession number: Q06226) [92,93,94,95] Homo sapiens (UNIPROT accession number: Q09013) [89, 91, 96, 97, 98, 99, 100, 101] Homo sapiens (UNIPROT accession number: Q15349) [86,102] Homo sapiens (UNIPROT accession number: Q15418) [86] Arabidopsis thaliana (UNIPROT accession number: Q39030) [271] Mus musculus (UNIPROT accession number: Q60823) [103] Mus musculus (UNIPROT accession number: Q62101) [104,105] Rattus norvegicus (UNIPROT accession number: Q63484) [106] Rattus norvegicus (UNIPROT accession number: Q63531) [107] Homo sapiens (UNIPROT accession number: Q9UBS0) [108,109,110] Homo sapiens (UNIPROT accession number: Q9UK32) [111] Mus musculus (UNIPROT accession number: Q9WUA6) [51,54] Mus musculus (UNIPROT accession number: Q9WUT3) [112] Mus musculus (UNIPROT accession number: Q9Z1M4) [113] Schizosaccharomyces pombe (UNIPROT accession number: Q10292) [114] Schizosaccharomyces pombe (UNIPROT accession number: O59790) [114, 115] Homo sapiens (UNIPROT accession number: P11801) [116,117] Saccharomyces cerevisiae (UNIPROT accession number: P24719) [118, 119, 120] Saccharomyces cerevisiae (UNIPROT accession number: P38991) [121] Plasmodium falciparum (UNIPROT accession number: Q27731) [122] Schizosaccharomyces pombe (UNIPROT accession number: P50526) [114, 124] Rattus norvegicus (UNIPROT accession number: O08875) [125] Oryctolagus cuniculus (UNIPROT accession number: Q9GM70) [123] Homo sapiens (UNIPROT accession number: O14757) [126,127,128] Homo sapiens (UNIPROT accession number: O14936) [129,130,131] Homo sapiens (UNIPROT accession number: O15075) [132,133,134,135]

2.7.11.1

























Non-specific serine/threonine protein kinase

Mus musculus (UNIPROT accession number: O35280) [127,128] Drosophila melanogaster (UNIPROT accession number: O61267) [6,136] Mus musculus (UNIPROT accession number: O70405) [137,138] Mus musculus (UNIPROT accession number: O70589) [139] Schizosaccharomyces pombe (UNIPROT accession number: O74536) [114] Homo sapiens (UNIPROT accession number: O75385) [140, 141] Mus musculus (UNIPROT accession number: O88866) [142, 143] Homo sapiens (UNIPROT accession number: O94768) [144] Homo sapiens (UNIPROT accession number: O96017) [31, 145, 146, 147, 148] Saccharomyces cerevisiae (UNIPROT accession number: P06782) [149, 150] Schizosaccharomyces pombe (UNIPROT accession number: P07334) [114, 151, 152] Saccharomyces cerevisiae (UNIPROT accession number: P13185) [153] Saccharomyces cerevisiae (UNIPROT accession number: P13186) [153] Schizosaccharomyces pombe (UNIPROT accession number: P22987) [155] Drosophila melanogaster (UNIPROT accession number: P23647) [6, 156, 157, 158] Daucus carota (UNIPROT accession number: P28582) [159] Glycine max (UNIPROT accession number: P28583) [160] Schizosaccharomyces pombe (UNIPROT accession number: P34208) [114, 161, 162, 163, 164] Saccharomyces cerevisiae (UNIPROT accession number: P38147) [154] Saccharomyces cerevisiae (UNIPROT accession number: P38622) [165, 166] Saccharomyces cerevisiae (UNIPROT accession number: P38623) [166, 167] Saccharomyces cerevisiae (UNIPROT accession number: P38990) [168] Saccharomyces cerevisiae (UNIPROT accession number: P39009) [169, 170, 171] Arabidopsis thaliana (UNIPROT accession number: P43291) [2, 172] Arabidopsis thaliana (UNIPROT accession number: P43292) [172, 173] Drosophila melanogaster (UNIPROT accession number: P49071) [6, 174] Zea mays (UNIPROT accession number: P49101) [175] Homo sapiens (UNIPROT accession number: P49137) [176, 177] Candida albicans (UNIPROT accession number: P52497) [178] Saccharomyces cerevisiae (UNIPROT accession number: P53104) [179, 180] Homo sapiens (UNIPROT accession number: P53355) [181] Daucus carota (UNIPROT accession number: P53681) [182] Oryza sativa (UNIPROT accession number: P53682) [183] Oryza sativa (UNIPROT accession number: P53683) [184] Oryza sativa (UNIPROT accession number: P53684) [184] Rattus norvegicus (UNIPROT accession number: P54645) [185, 186] 15

Non-specific serine/threonine protein kinase

2.7.11.1

Homo sapiens (UNIPROT accession number: P54646) [187, 188] Schizosaccharomyces pombe (UNIPROT accession number: P87050) [114, 189, 190] Candida glabrata (UNIPROT accession number: Q00372) [191] Saccharomyces cerevisiae (UNIPROT accession number: Q01919) [192, 193] Secale cereale (UNIPROT accession number: Q02723) [194] Arabidopsis thaliana (UNIPROT accession number: Q06850) [195, 196, 466] Rattus norvegicus (UNIPROT accession number: Q09137) [197, 198] Schizosaccharomyces pombe (UNIPROT accession number: Q09170) [114, 199] Saccharomyces cerevisiae (UNIPROT accession number: Q12263) [200] Homo sapiens (UNIPROT accession number: Q13131) [185] Homo sapiens (UNIPROT accession number: Q15831) [201, 202, 203, 204, 205] Caenorhabditis elegans (UNIPROT accession number: Q23023) [206] Arabidopsis thaliana (UNIPROT accession number: Q38997) [207, 208] Rattus norvegicus (UNIPROT accession number: Q62915) [209] Xenopus laevis (UNIPROT accession number: Q91604) [210] Mus musculus (UNIPROT accession number: Q9JLM8) [211] Homo sapiens (UNIPROT accession number: Q9UEE5) [144] Xenopus laevis (UNIPROT accession number: Q9YI18) [212, 213] Mus musculus (UNIPROT accession number: Q9Z265) [148] Plasmodium falciparum (UNIPROT accession number: O15726) [215] Schizosaccharomyces pombe (UNIPROT accession number: O74135) [114, 216] Drosophila melanogaster (UNIPROT accession number: O76324) [217, 218] Vaccinia virus (UNIPROT accession number: P16913) [219, 220, 221] Vaccinia virus (UNIPROT accession number: P20505) ( SGT1 [222]) [222] Saccharomyces cerevisiae (UNIPROT accession number: P23291) [28, 223, 224, 225] Saccharomyces cerevisiae (UNIPROT accession number: P23292) [28, 223, 224, 225, 226] Saccharomyces cerevisiae (UNIPROT accession number: P29295) [227, 228, 229] Variola virus (UNIPROT accession number: P33800) [230] Bos taurus (UNIPROT accession number: P35506) [231] Bos taurus (UNIPROT accession number: P35507) [231] Saccharomyces cerevisiae (UNIPROT accession number: P39962) [232] Schizosaccharomyces pombe (UNIPROT accession number: P40233) [114, 233, 234] Schizosaccharomyces pombe (UNIPROT accession number: P40234) [114, 233]

16

2.7.11.1

Non-specific serine/threonine protein kinase

Schizosaccharomyces pombe (UNIPROT accession number: P40235) [114, 235, 236] Schizosaccharomyces pombe (UNIPROT accession number: P40236) [114, 235, 236] Arabidopsis thaliana (UNIPROT accession number: P42158) [214, 237] Homo sapiens (UNIPROT accession number: P48729) [238, 239] Homo sapiens (UNIPROT accession number: P48730) [240] Homo sapiens (UNIPROT accession number: P49674) [31, 238] Drosophila melanogaster (UNIPROT accession number: P543670) [6, 241] Gallus gallus (UNIPROT accession number: P70065) [242] Homo sapiens (UNIPROT accession number: P78368) [243] Rattus norvegicus (UNIPROT accession number: P97633) [244] Rattus norvegicus (UNIPROT accession number: Q06486) [245, 246] Rattus norvegicus (UNIPROT accession number: Q62761) [247] Rattus norvegicus (UNIPROT accession number: Q62762) [247] Rattus norvegicus (UNIPROT accession number: Q62763) [247] Homo sapiens (UNIPROT accession number: Q9HCP0) [248] Mus musculus (UNIPROT accession number: Q9JMK2) [249] Homo sapiens (UNIPROT accession number: Q9Y6M4) [250] Drosophila melanogaster (UNIPROT accession number: P08181) [6, 268, 272] Rattus norvegicus (UNIPROT accession number: P19139) [251, 263] Saccharomyces cerevisiae (UNIPROT accession number: P15790) [252, 253, 268] Homo sapiens (UNIPROT accession number: P19138) [7, 254, 260, 261, 263, 266] Zea mays (UNIPROT accession number: P28523) [255] Oryctolagus cuniculus (UNIPROT accession number: P33674) [256] Caenorhabditis elegans (UNIPROT accession number: P19138) [257] Xenopus laevis (UNIPROT accession number: P28020) [258] Dictyostelium discoideum (UNIPROT accession number: Q02720) [259] Bos taurus (UNIPROT accession number: P20427) [260] Homo sapiens (UNIPROT accession number: P19784) [261] Gallus gallus (UNIPROT accession number: P21868) [262] Gallus gallus (UNIPROT accession number: P21869) [262] Arabidopsis thaliana (UNIPROT accession number: Q08466) [264, 271] Arabidopsis thaliana (UNIPROT accession number: Q08467) [195, 264] Theileria parva (UNIPROT accession number: P28547) [265] Mus musculus (UNIPROT accession number: O54833) [267, 276] Saccharomyces cerevisiae (UNIPROT accession number: P19454) [268, 269, 275] Schizosaccharomyces pombe (UNIPROT accession number: P40231) [114, 270, 274] Mus musculus (UNIPROT accession number: Q60737) [273] Candida albicans (UNIPROT accession number: O14427) [279] Homo sapiens (UNIPROT accession number: O75914) [280] 17

Non-specific serine/threonine protein kinase

2.7.11.1

Rattus norvegicus (UNIPROT accession number: O88506) [281, 282, 283] Mus musculus (UNIPROT accession number: O88643) [284] Homo sapiens (UNIPROT accession number: O96013) [285] Saccharomyces cerevisiae (UNIPROT accession number: P08458) [286] Saccharomyces cerevisiae (UNIPROT accession number: P27636) [287, 288, 289] Rattus norvegicus (UNIPROT accession number: P35465) [290, 291] Saccharomyces cerevisiae (UNIPROT accession number: P48562) [278, 292] Schizosaccharomyces pombe (UNIPROT accession number: P50527) [114, 293, 294] Saccharomyces cerevisiae (UNIPROT accession number: Q03497) [28, 295, 296] Schizosaccharomyces pombe (UNIPROT accession number: Q10 056) [114, 297] Saccharomyces cerevisiae (UNIPROT accession number: Q12469) [298, 299] Homo sapiens (UNIPROT accession number: Q13153) [300, 301] Homo sapiens (UNIPROT accession number: Q13177) [302, 303, 304] Mus musculus (UNIPROT accession number: Q61036) [284] Rattus norvegicus (UNIPROT accession number: Q62829) [290] Candida albicans (UNIPROT accession number: Q92212) [305] Homo sapiens (UNIPROT accession number: Q9P286) [7] Homo sapiens (UNIPROT accession number: Q9UEW8) [281] Mus musculus (UNIPROT accession number: Q9Z1W9) [281] Homo sapiens (UNIPROT accession number: Q13523) [306, 309, 310] Mus musculus (UNIPROT accession number: Q61136) [277, 308, 310] Homo sapiens (UNIPROT accession number: Q9UPE1) [307] Schizosaccharomyces pombe (UNIPROT accession number: P36616) [114, 311] Drosophila melanogaster (UNIPROT accession number: P49762) [312] Mus musculus (UNIPROT accession number: P97343) [277, 313, 314] Schizosaccharomyces pombe (UNIPROT accession number: Q07538) [114, 315] Rattus norvegicus (UNIPROT accession number: Q63285) [313, 316] Homo sapiens (UNIPROT accession number: Q9H2X6) [317, 318] Mus musculus (UNIPROT accession number: Q9QZR5) [317, 319, 320, 321] Rattus norvegicus (UNIPROT accession number: Q63802) [24] Saccharomyces cerevisiae (UNIPROT accession number: P43565) [322, 323] Streptomyces granaticolor (UNIPROT accession number: O54229) [340] Streptomyces granaticolor (UNIPROT accession number: O54228) [340] Homo sapiens (UNIPROT accession number: Q13188) [341, 342, 343] Homo sapiens (UNIPROT accession number: Q13043) [341] Homo sapiens (UNIPROT accession number: P51956) [342, 343] 18

2.7.11.1

Non-specific serine/threonine protein kinase

Homo sapiens (UNIPROT accession number: P51955) [342, 343] Schizosaccharomyces pombe (UNIPROT accession number: P08092) [114, 344] Saccharomyces cerevisiae (UNIPROT accession number: P22211) [345] Saccharomyces cerevisiae (UNIPROT accession number: P25333) [346, 347] Saccharomyces cerevisiae (UNIPROT accession number: P36002) [348, 349] Saccharomyces cerevisiae (UNIPROT accession number: P38691) [28, 350] Saccharomyces cerevisiae (UNIPROT accession number: P47116) [349, 351, 352] Saccharomyces cerevisiae (UNIPROT accession number: Q12505) [227, 353] Mus musculus (UNIPROT accession number: O08901) [354, 355] Homo sapiens (UNIPROT accession number: O43683) [355, 356, 357, 358, 359] Homo sapiens (UNIPROT accession number: O60566) [356, 357, 359, 360, 361] Schizosaccharomyces pombe (UNIPROT accession number: O94751) [114, 362] Saccharomyces cerevisiae (UNIPROT accession number: P41695) [363, 364] Mus musculus (UNIPROT accession number: Q9Z1S0) [360] Homo sapiens (UNIPROT accession number: O75716) [365, 366, 368] Mus musculus (UNIPROT accession number: O88697) [365, 366, 367, 368, 369] Rattus norvegicus (UNIPROT accession number: P57760) [365] Homo sapiens (UNIPROT accession number: O00311) [370, 371, 372] Saccharomyces cerevisiae (UNIPROT accession number: P06243) [373, 374, 375, 376] Schizosaccharomyces pombe (UNIPROT accession number: P50582) [114, 377] Schizosaccharomyces pombe (UNIPROT accession number: Q9UQY9) [114, 378] Mus musculus (UNIPROT accession number: Q9Z0H0) [379] Orgyia pseudotsugata multicapsid polyhedrosis virus (UNIPROT accession number: O10269) [380] Autographa californica nuclear polyhedrosis virus (UNIPROT accession number: P41415) [381, 382, 383] Heliothis zea nuclear polyhedrosis virus (UNIPROT accession number: P41719) [384] Lymantria dispar multicapsid nuclear polyhedrosis virus (UNIPROT accession number: P41720) [385] Arabidopsis thaliana (UNIPROT accession number: Q39183) [328, 329] Saccharomyces cerevisiae (UNIPROT accession number: P53323) [386, 387, 388] 19

Non-specific serine/threonine protein kinase

























20

2.7.11.1

Homo sapiens (UNIPROT accession number: Q96S44) [7] Mus musculus (UNIPROT accession number: Q99PW4) [277] Mus musculus (UNIPROT accession number: Q99mw1) [389] Homo sapiens (UNIPROT accession number: Q9BXU) [389] Saccharomyces cerevisiae (UNIPROT accession number: P32361) [28, 390, 391, 392] Schizosaccharomyces pombe (UNIPROT accession number: O13839) [114] Saccharomyces cerevisiae (UNIPROT accession number: P22209) [287, 288, 393, 394] Mycobacterium tuberculosis (UNIPROT accession number: O05871) [330, 331] Anabaena sp. (UNIPROT accession number: P54734) [332, 333] Myxococcus xanthus (UNIPROT accession number: P54736) [334] Myxococcus xanthus (UNIPROT accession number: P54737) [335] Myxococcus xanthus (UNIPROT accession number: P54738) [335] Streptomyces coelicolor (UNIPROT accession number: P54739) [336, 337] Streptomyces coelicolor (UNIPROT accession number: P54740) [336, 337] Streptomyces coelicolor (UNIPROT accession number: P54741) [336, 338, 339] Streptomyces coelicolor (UNIPROT accession number: P54742) [339] Schizosaccharomyces pombe (UNIPROT accession number: P38938) [114, 324] Saccharomyces cerevisiae (UNIPROT accession number: P53894) [226, 327] Homo sapiens (UNIPROT accession number: Q10 466) [325, 326] starfish (no sequence specified) [423] Nicotiana tabacum (UNIPROT accession number: Q8LP30) [435] Nicotiana tabacum (UNIPROT accession number: Q709M1) [449] Nicotiana tabacum (UNIPROT accession number: Q709M0) [449] Rattus norvegicus (UNIPROT accession number: Q9JJ76) [444] Rattus norvegicus (UNIPROT accession number: Q99PS2) [444] Rattus norvegicus (UNIPROT accession number: Q9JJ75) [444] Nicotiana tabacum (UNIPROT accession number: Q9SDY2) [446] Zea mays (UNIPROT accession number: Q6TQF8) [417] Homo sapiens (UNIPROT accession number: Q6SA08) [411] Homo sapiens (UNIPROT accession number: Q9BXA6) [442] Mus musculus (UNIPROT accession number: Q925K9) [442] Homo sapiens (UNIPROT accession number: Q6E0B2) [455] Mus musculus (UNIPROT accession number: A9P6P9) [464] Mus musculus (UNIPROT accession number: ABO33082) [464] Mus musculus (UNIPROT accession number: A9P6P7) [464] Mus musculus (UNIPROT accession number: A3QQR1) [464]

2.7.11.1

Non-specific serine/threonine protein kinase

3 Reaction and Specificity Catalyzed reaction ATP + a protein = ADP + a phosphoprotein ( active site structure [425]; active site and activation loop structure and conformation, Lys233, Cys250, Asp368, and Thr386 are important for activity, the substrate binding groove determines the substrate specificity via residues Val318 and Ala448 [450]; catalytic mechanism and regulation of PKN [423]; glycogen synthase kinase-3b also performs the t-protein kinase reaction, EC 2.7.11.26 [424]; Lys45 is important for PknH catalytic and autophosphorylation activity [421]; the enzyme shows Mn2+ -dependent serine/threonine kinase and Ca2+ -dependent histidine kinase, EC 2.7.13.3, activities [446]) ATP + protamine = ADP + O-phosphoprotamine Reaction type phospho group transfer Natural substrates and products S ATP + AfsR protein ( kinase AfsK specifically phosphorylates AfsR, a transcriptional activator with ATPase activity, starting a signal transduction pathway via induction of AfsS expression, thereby controlling the secondary metabolism of the bacterium, overview [436]) (Reversibility: ?) [436] P ADP + AfsR phosphoprotein S ATP + Akt ( CK2 phosphorylates and activates Akt directly [467]) (Reversibility: ?) [467] P ADP + phosphorylated Akt S ATP + BAF155 (Reversibility: ?) [469] P ADP + phosphorylated BAF155 S ATP + C/EBPa (Reversibility: ?) [434] P ADP + phosphorylated C/EBPa S ATP + C/EBPb (Reversibility: ?) [434] P ADP + phosphorylated C/EBPb S ATP + CREB ( phosphorylation by TSSK5 at Ser133 stimulates the cAMP responsive element/cAMP responsive element binding protein CRE/CREB responsive pathway in recombinant HEK293 cells [411]) (Reversibility: ?) [411] P ADP + phosphorylated CREB S ATP + Daxx ( Daxx is a protein acting in TGF- b-induced JNK activation and in apoptosis [418]) (Reversibility: ?) [418] P ADP + phosphorylated Daxx S ATP + Gli protein ( the Gli proteins are important in regulation of Hedgehog signaling, overview [460]) (Reversibility: ?) [460] P ADP + phosphorylated Gli protein S ATP + M3-muscarnic receptor (Reversibility: ?) [456] P ADP + phosphorylated M3-muscarnic receptor

21

Non-specific serine/threonine protein kinase

2.7.11.1

S ATP + PKB protein kinase ( PDK1 kinase, phosphorylates Thr308 and Ser473, which is required for activation of PKB [414]) (Reversibility: ?) [414] P ADP + phosphorylated PKB protein kinase S ATP + S6 protein of the 40s ribosomal subunit ( the mitogenactivated protein kinase plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1 [65]) (Reversibility: ?) [65] P ADP + phosphorylated S6 protein of the 40s ribosomal subunit S ATP + SC35 ( substrate prephosphorylated SC35, SC35 is a member of the SR family of serine/arginine-rich splicing factors [430]) (Reversibility: ?) [430] P ADP + phosphorylated SC35 S ATP + SIV/17E-Fr Nef protein ( phosphorylation at Ser12, the protein kinase CK2 associates with the Nef proteins from the Human immunodeficiency virus and the macrophage-tropic neurovirulent Simian immunodeficiency virus clone, SIV/17E-Fr, the Nef protein play important roles in viral disease progression [471]) (Reversibility: ?) [471] P ADP + phosphorylated SIV/17E-Fr Nef protein S ATP + SR protein ( dsk1 protein may play an important role in mitotic control by altering cellular location, degree of phosphorylation and kinase activity [311]; enzyme acts as a corepressors for homeodomain transcription factors [320]; enzyme regulates the activation and adhesion of T cells. CD43 may mediate its biologic effects through activation of a kinase cascade, resulting in the regulation of cell growth [319]; implication of KIS in the control of trafficking and/or splicing of RNAs probably through phosphorylation of associated factors [313]; enzyme plays a role as a co-repressor for homeodomain transcription factors [317]; enzyme is essential for growth [315]; enzyme is essential for eye and embryonic development [312]; enzyme may play a role in mitosis [311]) (Reversibility: ?) [311, 312, 313, 315, 317, 319, 320] P ADP + ? S ATP + [t-protein] ( t is primarily found in neurons, regulation of t phosphorylation by GSK3b via interaction with FRAT-1 and FRAT-2, i.e. frequently rearranged in advanced T-cell lymphoma proteins [433]) (Reversibility: ?) [433] P ADP + O-phospho -[t-protein] S ATP + [t-protein] ( abnormal hyperphosphorylation of tau by GSK-3 is associated with Alzheimers disease and other tauopathies leading to neuronal degeneration [431]; activity in organisms with mutated APP and tau, not in wild-type, overview [441]; phosphorylation of tau, especially at the primed epitope T231 negatively regulates tmicrotubule interactions, different effects of phosphorylation on primed 22

2.7.11.1

Non-specific serine/threonine protein kinase

T231 and unprimed S396/S404 epitopes of tau, overview [432]; substrate of GSK3b in brain [424]) (Reversibility: ?) [424, 427, 430, 431, 432, 438, 441] P ADP + O-phospho-[tau-protein] S ATP + a protein ( the enzyme is essential for viral replication [221]; not essential for cell viability [233]; Cki2, may contribute to the regulation of cell morphology [233]; the enzyme is a regulator of DNA strand-break repair [228]; enzyme is involved in the regulation of DNA repair [235]; enzyme plays a pivotal role in eukaryotic cell regulation [225]; gene disruption reveals that cki3+ is dispensable for cell viability, and cells lacking functional cki3+ exhibit no characteristic phenotype [216]; casein kinase 1 a gene is developmentally regulated and the kinase activity of the protein is induced by DNA damage. Possible requirement in mechanisms associated with DNA repair during early embryogenesis [241]; Pkn5 negatively regulates Myxococcus xanthus development [335]; AfsK plays a regulatory role in aerial mycelium formation in Streptomyces griseus [339]; expressed constitutively throughout the life cycle, with slight increases at an early stage of development [335]; the enzyme blocks the secretion of b-lactamase by phosphorylation [334]; pknA is required for both normal cellular growth and differentiation [333]; Pkn6 may be a transmembrane sensor of external signals for development [335]; involvement of afsK in the regulation of secondary metabolism [338]; the enzyme is required to promote the activity of at least six distinct transport systems for nitrogenous nutrients under conditions of nitrogen catabolite derepression [345]; the enzyme is a negative regulator of both sexual conjugation and meiosis [344]; STK1 mostly affects a loweraffinity, low-capacity polyamine transport activity [349]; enzyme is not essential for vegetative growth [350]; the SKS1 protein kinase is a multicopy suppressor of the snf3 mutation of Saccharomyces cerevisiae [353]; the enzyme enhances spermine uptake in Saccharomyces cerevisiae [352]; in some cancers displaying chromosomal instability the loss of this checkpoint is associated with the mutational inactivation of a human homologue of the yeast BUB1 gene [359]; the enzyme is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. bub1(+)function is essential to maintain correct ploidy through mitosis [362]; the enzyme is required for function of the spindle assembly checkpoint [355]; cell cycle-dependent expression, Bub1b has a putative destruction box that can target proteins 23

Non-specific serine/threonine protein kinase

P S

P S P S P S

P

24

2.7.11.1

for degradation by proteosomes during mitosis [360]; the enzyme is involved in spindle assembly during the cell cycle [363]; hBUBR1 may regulate multiple functions that include the kinetochore and the spindle midzone [361]; kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage [354]; hBub1 is an important component of the spindle checkpoint pathway. hBub1 expression is restricted to proliferating cells and appears to be involved in regulating cell cycle progression [358]; Bub1 is required for binding Bub3 [357]; huCdc7 may regulate processes of DNA replication by modulating MCM functions [372]; HsCdc7 may phosphorylate critical substrates that regulate the G1/S phase transition and/or DNA replication [371]; the enzyme is required for the initiation of mitotic DNA synthesis. It is necessary for genetic recombination during meiosis and for the formation of ascospores, involved in an error-prone DNA repair pathway [376]; the enzyme is required for inhibition of mitosis until completion of S phase, it may also be involved in proper execution of mitosis [377]; enzyme is required for the initiation of DNA synthesis during mitosis as well as for synaptonemal complex formation and commitment to recombination during meiosis [374]; the enzyme is dispensable for mitotic growth and premeiotic DNA replication, its primary role is in meiosis [378]; the enzyme is essential for normal cell life [386]; the enzyme is required for inositol phototrophy in Saccharomyces cerevisiae [392]; Ire1p is required for activation of the unfolded protein response. It senses the accumulation of unfolded proteins in the ER and transmits the signal across the membrane toward the transcription machinery, possibly by phosphorylating downstream components of the UPR pathway [390]; the enzyme is required for signaling from the ER to the nucleus [391]) (Reversibility: ?) [216, 221, 225, 228, 233, 235, 241, 333, 334, 335, 338, 339, 344, 345, 349, 350, 352, 353, 354, 355, 357, 358, 359, 360, 361, 362, 363, 371, 372, 374, 376, 377, 378, 386, 390, 391, 392, 415] ADP + a phosphoprotein ATP + adenomatous polyposis coli protein ( i.e. APC, APC is phosphorylated in vitro by CKId and CKIe [460]) (Reversibility: ?) [460] ADP + phosphorylated adenomatous polyposis coli protein ATP + a-casein ( casein kinase 1a [443]) (Reversibility: ?) [443] ADP + phosphorylated a-casein ATP + axin ( glycogen synthase kinase 3 [414]) (Reversibility: ?) [414] ADP + phosphorylated axin ATP + b-catenin ( glycogen synthase kinase 3 [414]; phosphorylation at Ser45, priming GSK3b phosphorylation [460]) (Reversibility: ?) [414, 46] ADP + phosphorylated b-catenin

2.7.11.1

Non-specific serine/threonine protein kinase

S ATP + geminin ( a cell cycle regulatory protein [456]) (Reversibility: ?) [456] P ADP + phosphorylated geminin S ATP + glycogen synthase ( glycogen synthase kinase 3 is involved in the insulin signaling pathway regulating glycogen synthesis via glycogen synthase antagonizing insulin [414]) (Reversibility: ?) [414] P ADP + phosphorylated glycogen synthase S ATP + glycogen synthase kinase 3 ( PKB protein kinase [414]) (Reversibility: ?) [414] P ADP + phosphorylated glycogen synthase kinase 3 S ATP + histone H3 ( RSK2 is a key regulator of histone H3 phosphorylation [457]) (Reversibility: ?) [457] P ADP + phosphorylated histone H3 S ATP + histone H3 ( Ark1 phosphorylates Ser10 of histone H3 in vivo [115]) (Reversibility: ?) [115] P ? S ATP + hypoxia-inducible factor-1 ( i.e. HIF-1, casein kinase 2 inhibits the activation of HIF-1 activity by phosphorylation and thereby stabilization of the protein, CK2 is involved in and influenced by hypoxia, overview [422]) (Reversibility: ?) [422] P ADP + phosphorylated hypoxia-inducible factor-1 S ATP + lamin A/C ( phosphorylation of host cell lamin by US3 kinase [468]) (Reversibility: ?) [468] P ADP + phosphorylated lamin A/C S ATP + phospholipase D ( CKII is involved in 4b-phorbol 12myristate 13-acetate-induced phospholipase D activation, of PLD1 or PLD2 activity as well as basal PLD activity [452]) (Reversibility: ?) [452] P ADP + phosphorylated phospholipase D S ATP + procaspase-2 ( phosphorylation of procaspase-2 by CK2 prevents the activation of caspase activity by inhibiting the dimerization of procaspase-2, CK2 activity is involved in the direct regulation of the caspase-8 pathway, where it can control caspase-8 activity through the phosphorylation of ARC or by preventing the ability of caspase-8 to cleave its target Bid [456]) (Reversibility: ?) [456] P ADP + phosphorylated procaspase-2 S ATP + protein ( enzyme is essential for the late nuclear division in the yeast [289]; enzyme activates the p38 pathway, SPAK may act as a novel mediator of stress-activated signals [281]; the enzyme is an effector for Cdc42Hs and is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia [285]; enzyme is required for virulence and hyphal formation of Candida albicans [279]; potentially important role in the control of the cellular architecture and/or signaling in the central nervous system [284]; PAK3 may be critical for human cognitive function [280]; the enzyme regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for effector for Cdc42 and Rac1 in proll motility [301]; 25

Non-specific serine/threonine protein kinase

P S

P S

P S

P S P S P S P S P S

P

26

2.7.11.1

the enzyme is required to link the yeast pheromone response G-protein b g subunits to downstream signalling components [295]; the enzyme is necessary for mating [296]; Pak1 protein is likely to be an effector for Cdc42p or a related GTPase, Pak1p may be involved in the maintenance of cell polarity and in mating [294]; the enzyme is involved in budding and cytokinesis and interacts with Cdc42, a GTPase required for polarized cell growth [292]; hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway. PAK kinase plays a key part in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation [300]) (Reversibility: ?) [279, 280, 281, 284, 285, 289, 292, 294, 295, 296, 298, 300, 301] ADP + phosphoprotein ATP + protein ( PKL12 may play a role in a very general cellular function, probably related with the secretory pathway [368]; EDPK plays a crucial role in intracellular signaling not only during mouse development but also in adult tissues [369]) (Reversibility: ?) [368, 37] ? ATP + protein ( GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro [19]; phosphorylates and inactivates the actin binding/depolymerizing factor cofilin and induces actin cytoskeletal changes [12]) (Reversibility: ?) [12, 19] ADP + protein tyrosine phosphate ATP + protein Cubitus interruptus ( Ci proteins are important in regulation of Hedgehog signaling, overview [460]) (Reversibility: ?) [460] ADP + phosphorylated protein Cubitus interruptus ATP + protein LRP6 ( CKIg [460]) (Reversibility: ?) [460] ADP + phosphorylated protein LRP6 ATP + protein TCF3 ( CKIe [460]) (Reversibility: ?) [460] ADP + phosphorylated protein TCF3 ATP + protein kinase Cz ( phosphorylation and activation of protein kinase Cz [34]) (Reversibility: ?) [34] ADP + phosphorylated protein kinase Cz ATP + ribosomal protein S6 ( specifically phosphorylated by S6K [417]) (Reversibility: ?) [417] ADP + phosphorylated ribosomal protein S6 ATP + tau protein ( tau in Alzheimer disease brain is highly phosphorylated and aggregates into paired helical filaments comprising characteristic neurofibrillary tangles, overview [465]) (Reversibility: ?) [465] ADP + phosphorylated t protein

2.7.11.1

Non-specific serine/threonine protein kinase

S ATP + testis-specific serine-threonine kinase 3 ( substrate of phosphoinositide-dependent kinase-1 leading to activation [420]) (Reversibility: ?) [420] P ADP + phosphorylated testis-specific serine-threonine kinase 3 S Additional information ( stress-induced activation of MAPKAPK-2, in turn, results in the phosphorylation of small heat-shock proteins [174]; LIMK may be involved in developmental or oncogenic processes through interactions with LIM-containing proteins [14]; Kiz-1 may play distinct roles in dividing cells and in differentiated neurons [16]; Williams syndrome is a complex neurodevelopmental disorder arising from a microdeletion at Chr band 7q11.23, which results in a hemizygous condition for a number of genes, LIMK1, WBSCR1, and RFC2 [18]; LIMK1 may be particularly relevant when explaining cognitive defects observed in WS patients [15]; LIM-kinase1 hemizygosity is implicated in impaired visuospatial constructive cognition [13]; the enzyme is part of a growth control pathway which is at least partially redundant with the cAMP pathway [29]; the enzyme positively regulates the progression of yeast cells through the G1 phase of the cell cycle [27]; deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy [41]; AKT1 gene is not a major contributor to susceptibility to type II diabetes mellitus in Ashkenazi Jews [39]; K+ -channel activation by all three isoforms of serum-dependent and glucocorticoid-dependent protein kinase SGK [36]; the enzyme acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression [40]; NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants [57]; the enzyme is required to repress entry into the conidiation program [55]; induction of enzymatically active Sgk functions as a key cell survival component in response to different environmental stress stimuli [35]; transcript levels are strongly altered during anisotonic and isotonic cell volume changes [45]; AKT2 may contribute to the pathogenesis of ovarian carcinomas [75]; enzyme may function in the adaptation of plant cells to cold or high-salt conditions [78]; the DBF20 mRNA is expressed at a low level and not under cell-cycle control [70]; kinase plays a part in regulating events associated with fertilization [69]; DBF2 mRNA is expressed under cell-cycle control at or near START [70]; enzyme is required for cell growth [60]; atpk1 is involved in the control of plant growth and development [80]; DBF2 is likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division [71]; the enzyme is transcriptionally regulated 27

Non-specific serine/threonine protein kinase

2.7.11.1

by serum and glucocorticoids in mammary epithelial cells, hormoneregulated protein kinase gene with a functionally defined p53 promoter recognition element [92,94,95]; induction of sgk gene may be associated with a series of axonal regenerations after brain injury, and in addition, the sgk gene may also play important roles in the development of particular groups of neurons in the postnatal brain [94]; may function in the adaptation of plant cells to cold or high-salt conditions [78]; enzyme is induced during ovarian cell differentiation [92]; TPK may have specialized functions in different areas of central nervous system. Alterations of this complex expression pattern can be responsible for the mental status impairment observed in myotonic dystrophy patients [96]; enzyme may have a role in the development of mental symptoms in severe cases of myotonic dystrophy [98]; the enzyme is highly regulated at the transcriptional level by glucocorticoid hormones [95]; SGK is a component of the phosphoinositide 3 (PI 3)-kinase signaling pathway [92]; enzyme is important for cell growth [113]; mutations in the ribosomal S6 kinase (Rsk-2) gene are associated with Coffin-Lowry syndrome, an X-linked disorder characterized by facial dysmorphism, digit abnormalities and severe psychomotor retardation [82,83,84]; ribosomal S6-kinase RSK4 is commonly deleted in patients with complex X-linked mental retardation, RSK4 plays a role in normal neuronal development. RSK4 is completely deleted in eight patients with the contiguous gene syndrome including MRX, partially deleted in a patient with DFN3 and present in patients with an Xq21 deletion and normal intellectual abilities [111]; decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy [99]; inherited defects in insulin-stimulated activation of muscle glycogen synthesis in patients with insulin-resistant NIDDM may be located further upstream of ISPK-1 in the insulin action cascade [85]; Akt2 expression is activated during cellular differentiation and suggest that it functions in the signaling pathways of some adult tissues [103]; associates with mitotic structures in a stage dependent manner and is required for chromosome segregation [115]; protein kinase is required for meiotic recombination [118]; type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation, required during the later part of each cell cycle [121]; required for chromosome synapsis and recombination [120]; enzyme plays a critical role in regulation by carbon catabolite repression [150]; the enzyme plays a role in development [142]; nim1+ is a negative regulator of the wee1+ mitotic inhibitor, another protein kinase homolog. The combined mitotic induction activities of nim1+ and cdc25+ counteract the wee1+ mitotic inhibitor in a regulatory network that appears also to involve the cdc2+ protein kinase, which is required for mitosis [152]; Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination CHK1 gene is a candidate tumor suppressor gene [128]; enzyme plays a role in germ line establishment [136]; 28

2.7.11.1

Non-specific serine/threonine protein kinase

kinase functions downstream of ATM protein in the cellular response to DNA damage [145]; in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry [127]; in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis [148]; heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the TP53 gene. hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors [147]; Unc51.1 signals the program of gene expression leading to the formation of granule cell axons [137]; required to give a normal cell cycle response to nitrogen starvation [151]; kinase-related apoptosis-inducing protein kinase 1 may play an important role in the core apoptosis program in osteoclast [123]; required for alteration of growth polarity and actin localization [124]; likely role for DCAMKL1 transmembrane protein in developing and adult brain, possibly in a pathway of cortical development [132]; Cdc25 mitotic inducer is targeted by chk1 DNA damage checkpoint kinase [162]; enzyme controls the DNA damage response [171]; enzyme mediates checkpoint pathway [161]; required for pattern formation within embryonic segments and imaginal discs, possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development [156]; Pak1 may function by modifying and partially stabilizing thermolabile DNA polymerases, perhaps during DNA repair [168]; chk1 protein kinase links the rad checkpoint pathway to cdc2 [164]; Chk1 is required for function of the DNA damage checkpoint in Saccharomyces cerevisiae [154]; important for growth polarity [155]; enzyme is essential for the derepression of catabolic repression [178]; the enzyme is required for the autophagic process in Saccharomyces cerevisiae [179]; deletion of GIN4 is not lethal but leads to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation [200]; the enzyme is a regulator of G2/M progression and cytokinesis [190]; mutations of STK11 cause Peutz-Jeghers syndrome [201]; kinase Cdr2 regulates the onset of mitosis in fission yeast [189]; Peutz-Jeghers syndrome is an autosomal-dominant disorder characterized by melanocytic macules of the lips, multiple gastrointestinal hamartomatous polyps and an increased risk for various neoplasms, including gastrointestinal cancer, germline mutations in STK11, probably in conjunction with acquired genetic defects of the second allele in somatic cells, cause the manifestations of PJ syndrome [202]; enzyme plays a major role in the regulation of lipid metabolism, be involved in the regulation of a wide range of metabolic pathways [198]; RKIN1 protein has a role in the control of carbon metabolism in endosperms of rye [194]; Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control [212]; the enzyme is essential for the derepres29

Non-specific serine/threonine protein kinase

2.7.11.1

sion of glucose repression, interacts with additional regulatory pathways and affects the expression of multiple target genes [191]; the damage/replication G2 checkpoint kinase Chk1 phosphorylates and inhibits Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophaseI-arrested oocytes [212]; Peutz-Jeghers syndrome is the first cancer-susceptibility syndrome to be identified that is due to inactivating mutations in a protein kinase, STK11 [204]; the enzyme is a key component of the DNA replication-monitoring S/G2 checkpoint system. Its primary role is to monitor DNA synthesis by interacting with DNA polymerase a and send a signal to block the onset of mitosis while DNA synthesis is in progress [199]; enzyme is involved in the synthesis of seed storage compounds during seed development [183]; may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism [207]; protein phosphorylation by the unc-51 product is important for axonal elongation and possibly for axonal guidance [206]; the enzyme is essential for vegetative growth of Dictyostelium discoideum, the a subunit is expressed constitutively like its mRNA during the life cycle [259]; enzyme is implicated in the control of cell growth and proliferation. Androgenic regulation of CK-2 gene transcription is not an early event related to androgen action, but is substantial in the prereplicative phase of prostatic cell proliferation mediated by androgen. Androgenic stimulation of the mRNA expression for the a and b subunits of CK-2 appears to be differential [251]; translational and/or post-translational mechanisms play an important role in the developmental regulation of casein kinase II activity [257]; plays a role in the translation of cell polarity into polarized growth, but not in the establishment of polarity itself [274]; key regulatory enzyme involved in many cellular processes, including the control of growth and cell division [270]; the casein kinase II gene can serve as an oncogene, and its dysregulated expression is capable of transforming lymphocytes in a two-step pathway with c-myc [273]; unbalanced expression of CK2 catalytic subunit synergizes with Ha-ras in cell transformation [267]; NH2 -terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1 [306]; enzyme is involved in pre-mRNA splicing [310]; calmodulin-binding region of titin can play a regulatory role for the enzyme [326]; the cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein [324]; enzyme may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock [341]; Nek2 protein is almost undetectable during G1 but accumulates progressively throughout S, reaching maximal levels in late G2, may function at the onset of mitosis [342]; AfsK is a Hanks-type protein kinase [436]; enzyme is involved in stress re30

2.7.11.1

Non-specific serine/threonine protein kinase

sponse and adaptation to environmental changes in mycobacteria [421]; glycogen synthase kinase-3b also performs the t-protein kinase reaction, EC 2.7.11.26, phosphorylating t protein at serine and threonine residues [424]; GSK-3 affects the t-mRNA splicing of exon 10 via phosphorylation of the splicing factors of the serine/arginine-rich splicing factor SR family, e.g. SC35, leading to priming and dislocation of the splicing factor, aberrant t splicing contributes to topathies including Alzheimers disease, overview [430]; GSK3b works coordinatedly with PKA, EC 2.7.11.11, on t phosphorylation [438]; HIPK2 regulates transforming growth factor-b-induced c-Jun NH(2)-terminal kinase activation and apoptosis in hepatoma cells, HIPK2 triggers promyelotic leukemia nuclear body disruption and release of Daxx [418]; isozyme RSK1, i.e. 14-3-3, is regulated by inhibitory binding of 14-3-3b, a RSK1-binding protein, to Lys49 of the enzyme, Lys49 is also responsible for interaction of RSK1 with Bcr, Raf, and Cbl [426]; kinase Rio2 is required for rRNA cleavage in 40S ribosomal subunit maturation [451]; mechanism of Ca2+ mediated regulation of NDR protein kinase through autophosphorylation and phosphorylation by an upstream kinase [428]; mechanism of S6K2 regulation via phosphorylation and autoinhibition with T338 playing the key role, S6K2 is regulated in a mitogen-activated protein kinase/ extracellular-signal-regulated kinase kinase MEK-dependent manner, overview [415]; naturally occurring V717I mutation of the amyloid precursor protein APP in a CT100 fragment of organisms with Alzheimers disease leads to augmented age-dependent tau phosphorylation, followed by increased activation status of mitogen-activated protein kinase family members, e.g. ERK1/2, p38, and c-Jun NH2 -terminal kinase, compared to the wild-type organism, naturally occurring V337M mutation of tau protein of patients suffering Alzheimers disease leads to age-dependent memory deficits [441]; naturally occurring V717I mutation of the amyloid precursor protein APP in a CT100 fragment of patients suffering Alzheimers disease leads to augmented age-dependent tau phosphorylation, followed by increased activation status of mitogen-activated protein kinase family members, e.g. ERK1/2, p38, and c-Jun NH2 -terminal kinase, compared to the wild-type organism, naturally occurring V337M mutation of tau protein of patients suffering Alzheimers disease leads to age-dependent memory deficits [441]; NTHK2 is an ethylene receptor protein [446]; phosphorylated tau is important in cytoskeleton assembly [427]; phosphorylation at Ser9 by PKGI and PKGII inhibits the glycogen synthase kinase-3 inducing dephosphorylation of C/EBPb, i.e. the CCAAT enhancer-binding protein b, important in regulation of gene expression during cell proliferation, differentiation, and apoptosis [434]; photoregulation of phototropin, overview [447]; PKD2 regulation involving ligand-binding domains of the enzyme, isozyme PKD2 might play a role in the nucleus after activation by G protein-coupled receptors, overview, intracellular trafficking of PKD2, overview [440]; protein kinase StkP and Mn2+ -dependent cytosolic protein phosphatase PhpP act as a functional pair in vivo [419]; regulation 31

Non-specific serine/threonine protein kinase

2.7.11.1

mechanism and biological function of PKN [423]; regulation mechanism and biological function of PKN, PKNa is involved in insulin-induced actin cytoskeleton reorganization and cell adhesion, overview, isozyme PKNa is involved in glucose transport in 3T3/L1 cells [423]; regulation mechanism and biological function of PKN, PKNa is involved in insulin-induced actin cytoskeleton reorganization and cell adhesion, overview, PKNa is involved in vesicle transport in the endoplasmic reticulum, PKNa is cleaved by caspase-3 or related proteases in apoptotic Jurkat and U-937 cells contributing to signal transduction, PKN interacts with papillomaviral oncoproteins being involved in tumorigenesis, overview [423]; regulation mechanism and biological function of PKN, PKNa is involved in insulin-induced actin cytoskeleton reorganization, overview [423]; regulation mechanism and biological function of PKN, the enzyme is involved in cell cycle control [423]; serine/ threonine protein kinase is SSTK essential for male fertility [442]; serine/threonine protein kinase SSTK is essential for male fertility, a DNA condensation defect in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins [442]; t becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain, inhibition of t hyperphosphorylation inhibits an associated loss in spatial memory [431]; the enzyme participates in Alzheimers disease [409]; WKN1 and WKN4 are involved in regulation of ion transport across diverse epithelia, WKN4 reduces the activity of Na-Cl-cotransporter activity and the potassium channel ROMK by reducing their appearance in the plasma membrane [410]; WNK1 causes a hereditary form of hypertension, pseudohypoaldosteronism type II [450]; CK2 impairs spatial memory formation through differential cross talk with PI-3 kinase signaling by activation of Akt and inactivation of SGK1 through protein phosphatase 2A [467]; CK2 is a highly conserved serine/threonine kinase involved in several intracellular pathways, which control, among others, cell cycle, proliferation, apoptosis and transformation, overview, protein kinase AKT interacts with CK2 subunits in vivo as well as in vitro enhancing AKT activity, overview [461]; CK2 is involved in phosphorylation of a large portion of the proteome, overview, increased enzyme activity in associated with a number of pathological processes, including cancer, infectious diseases, neurodegeneration, and cardiovascular pathologies playing a global anti-apoptotic role, overview [454]; CK2 phosphorylates about 300 different cellular proteins, ranging from transcription factors to proteins involved in chromatin structure and cell division, CK2 induces neoplastic growth when overexpressed [470]; CK2 plays a role in the protection of cells from apoptosis via the regulation of tumor suppressor and oncogene activity and stability, e.g. in the NF-kB or the Wnt signaling pathways, molecular mechanisms, detailed overview, CK2 activity is linked to Her-2/neu oncogene, which is overexpressed in 30% of breast cancers, regulation of apoptotic machinery by CK2 and its antiapoptotic effect, overview, role of CK2 in chromosomal DNA strand break 32

2.7.11.1

Non-specific serine/threonine protein kinase

repair and the maintenance of genomic integrity, mutational study, overview [456]; high CK2 activity occurs in solid tumors due to growthrelated functions and to suppression of cellular apoptosis, nuclear localization of domain CK2a is associated with high-grade tumors and a poor prognostic factor, overview [459]; members of the hSWI/SNF chromatin remodeling complex associate with and are phosphorylated by protein kinase B/Akt, overview [469]; multiple CKI family members play a role in both positively and negatively regulating Wnt and Hedgehog signaling, and the regulation of the stability of cytoplasmic b-catenin, detailed overview, the partial degradation of Ci is promoted by phosphorylation and by a cytoplasmic complex, which includes Costal-2, a microtubule-binding protein, and Fused, a serine-threonine protein kinase [460]; phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization, overview [464]; RSK2 is a critical serine/threonine kinase for the regulation of cell transformation, RSK is regulated by the tumor promoters epidermal growth factor EGF or 12-Otetradecanoylphorbol-13-acetate, TPA, overview [457]; SpkA is a regulator of expression of three putative pilA operons, formation of thick pili, and cell motility, DNA microarray and electron microscopy analysis, overview [462]; the enzyme alters localization of lamin A/C in infected cells, recombinantly expressed lamin as GFP-tagged protein in HepG2 cells, enzyme inactivation causes capsids to aggregate aberrantly between the inner and outer nuclear membranes within evaginations/extensions of the perinuclear space, overview, US3 kinase activity regulates HSV-1 capsid nuclear regress at least in part by phosphorylation of lamin A/C [468]; the enzyme inhibits NF-kB activity via interaction with the leucine-rich repeat domain of the Salmonella enterica serovar typhimurium-derived effector SspH1 in infected intestine-407 cells, overview [458]; the enzyme is involved in the inflammation cascade in intestine and colon epithelium [455]; the eukaryotic-type serine/threonine protein kinase StkP is a global regulator of gene expression in Streptococcus pneumoniae, and is important for the resistance of the organism to various stress conditions, StkP positively controls the transcription of a set of genes encoding functions involved in cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response, overview [463]) (Reversibility: ?) [13, 14, 15, 16, 18, 25, 27, 29, 35, 36, 39, 40, 41, 45, 55, 57, 60, 69, 70, 71, 75, 77, 78, 80, 82, 83, 84, 85, 92, 94, 95, 96, 98, 99, 103, 111, 113, 115, 118, 120, 121, 123, 124, 127, 128, 132, 136, 137, 142, 145, 147, 148, 150, 151, 152, 154, 155, 156, 161, 162, 164, 168, 171, 174, 178, 179, 183, 189, 190, 191, 194, 198, 199, 200, 201, 202, 203, 204, 206, 207, 212, 213, 251, 257, 259, 267, 270, 273, 274, 306, 310, 324, 326, 341, 342, 409, 410, 415, 418, 419, 421, 423, 424, 426, 427, 428, 430, 431, 434, 436, 438, 440, 441, 442, 446, 447, 450, 451, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 467, 468, 469, 470] P ? 33

Non-specific serine/threonine protein kinase

2.7.11.1

Substrates and products S ATP + 3-hydroxy-3-methylglutaryl-CoA reductase (Reversibility: ?) [198] P ADP + phosphorylated 3-hydroxy-3-methylglutaryl-CoA reductase S ATP + ABCA1 NBD1-R1 protein ( substrate protein is a recombinant construct of wild-type and mutant nucleotide binding domain 1 and cytoplasmic domain R1 of ATP-binding cassette transporter protein A1 expressed in Escherichia coli, phosphorylation at T1242, T1243, and S1255 [429]) (Reversibility: ?) [429] P ADP + phosphorylated ABCA1 NBD1-R1 protein S ATP + ABCA1 NBD2-R2 protein ( substrate protein is a recombinant construct of wild-type and mutant nucleotide binding domain 2 and cytoplasmic domain R2 of ATP-binding cassette transporter protein A1 expressed in Escherichia coli [429]) (Reversibility: ?) [429] P ADP + phosphorylated ABCA1 NBD2-R2 protein S ATP + AfsR ( phosphorylation at both Ser and Thr residues [338]) (Reversibility: ?) [338] P ADP + ? S ATP + AfsR protein ( kinase AfsK specifically phosphorylates AfsR, a transcriptional activator with ATPase activity, starting a signal transduction pathway via induction of AfsS expression, thereby controlling the secondary metabolism of the bacterium, overview [436]; phosphorylation of serine and threonine residues by activated kinase AfsK, and by PkaG and SCD10.09, the Streptomyces coelicolor strain A3(2) contains more than 40 enzymes phosphorylating the substrate AsfR [436]) (Reversibility: ?) [436] P ADP + AfsR phosphoprotein S ATP + Akt ( CK2 phosphorylates and activates Akt directly [467]; CK2 phosphorylates and activates Akt directly, phosphorylation at Ser473 [467]) (Reversibility: ?) [467] P ADP + phosphorylated Akt S ATP + BAF155 ( BAF155 is a hSWI/SNF protein that contains three Akt consensus sites, phosphorylation site analysis using recombinant full-length and truncated substrate versions, overview [469]) (Reversibility: ?) [469] P ADP + phosphorylated BAF155 S ATP + BAF170 ( BAF170 is a hSWI/SNF protein that contains one Akt consensus site [469]) (Reversibility: ?) [469] P ADP + phosphorylated BAF170 S ATP + Bub3 (Reversibility: ?) [364] P ADP + phosphorylated Bub3 S ATP + C/EBPa ( phosphorylation at consensus sequence (S/ T)XXX(S/T) with X representing any amino acid [434]) (Reversibility: ?) [434] P ADP + phosphorylated C/EBPa S ATP + C/EBPb ( phosphorylation at multiple sites, preferably at Thr189 and Ser185, reduced activity with mutants T189A and S185A, mu34

2.7.11.1

P S

P S P S P S

P S P S

P S P S P S P S P S P S P S P S P

Non-specific serine/threonine protein kinase

tation of either site appears to impair phosphorylation of neighbouring sites [434]) (Reversibility: ?) [434] ADP + phosphorylated C/EBPb ATP + CREB ( phosphorylation by TSSK5 at Ser133 stimulates the cAMP responsive element/cAMP responsive element binding protein CRE/CREB responsive pathway in recombinant HEK293 cells [411]; i.e. cAMP responsive element binding protein, phosphorylation by TSSK5 at Ser133, no activity with CREB S133A mutant [411]) (Reversibility: ?) [411] ADP + phosphorylated CREB ATP + Cdc25C ( Chk2 phosphorylated Cdc25C on Ser216 [148]) (Reversibility: ?) [148] ADP + ? ATP + Cdc25C ( phosphorylated Cdc25C on Ser216 [148]) (Reversibility: ?) [148, 21] ADP + phosphorylated Cdc25C ATP + Cdc42Hs ( PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain [285]) (Reversibility: ?) [285] ADP + ? ATP + DDDDVASLPGLRRR (Reversibility: ?) [245] ADP + ? ATP + Daxx ( Daxx is a protein acting in TGF-b-induced JNK activation and in apoptosis [418]; recombinant Daxx expressed as GST-fusion protein in Escherichia coli [418]) (Reversibility: ?) [418] ADP + phosphorylated Daxx ATP + GST-CREB2 protein (Reversibility: ?) [457] ADP + phosphorylated GST-CREB2 protein ATP + GST-NFAT3D4-261-365 (Reversibility: ?) [457] ADP + phosphorylated GST-NFAT3D4-261-365 ATP + Gli protein ( the Gli proteins are important in regulation of Hedgehog signaling, overview [460]) (Reversibility: ?) [460] ADP + phosphorylated Gli protein ATP + IgG2A heavy chain ( substrate protein from mouse [437]) (Reversibility: ?) [437] ADP + phosphorylated IgG2A heavy chain ATP + KKRNRRLSVA ( peptide substrate of NDR1 [428]) (Reversibility: ?) [428] ADP + KKRNRRL-phosphoserine-VA ATP + Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala (Reversibility: ?) [177] ADP + ? ATP + M3-muscarnic receptor (Reversibility: ?) [456] ADP + phosphorylated M3-muscarnic receptor ATP + MARCKS protein ( isozyme PKNa [423]) (Reversibility: ?) [423] ADP + MARCKS phosphoprotein 35

Non-specific serine/threonine protein kinase

2.7.11.1

S ATP + MB protein (Reversibility: ?) [423] P ADP + MB phosphoprotein S ATP + MCM2 ( wild-type huCdc7 protein expressed in COS7 cells phosphorylates MCM2 and MCM3 proteins in vitro [372]) (Reversibility: ?) [372] P ADP + ? S ATP + MCM3 ( wild-type huCdc7 protein expressed in COS7 cells phosphorylates MCM2 and MCM3 proteins in vitro [372]) (Reversibility: ?) [372] P ADP + ? S ATP + NKCC1-(1-260) ( i.e. Na+ -K+ -2Cl- co-transporter-1-(1260) construct, recombinant WNK1 or WNK4 with recombinant protein substrate, both expressed in Escherichia coli [416]) (Reversibility: ?) [416] P ADP + phosphorylated NKCC1-(1-260) S ATP + NS5A protein ( recombinant NS5A expressed in Sf9 cells, substrate is a hepatitis C virus protein playing a critical role in virus replication and conferring interferon resistance to the virus, the viral protein is phosphorylated by diverse serine/threonine protein kinases, phosphorylation patterns, overview [439]; recombinant NS5A expressed in Sf9 cells, substrate is a hepatitis C virus protein playing a critical role in virus replication and conferring interferon resistance to the virus, the viral protein is phosphorylated by several serine/threonine protein kinases, e.g. casein kinase 1, phosphorylation pattern, overview [439]; recombinant NS5A expressed in Sf9 cells, substrate is a hepatitis C virus protein playing a critical role in virus replication and conferring interferon resistance to the virus, the viral protein is phosphorylated by several STE or AGC type protein kinases CK2, AKT1, SGK1, 90S6K, STE, phosphorylation patterns, overview [439]) (Reversibility: ?) [439] P ADP + phosphorylated NS5A protein S ATP + ORF62 protein ( protein substrate from Varicella-Zoster virus, no activity with a truncated ORF62 mutant [437]) (Reversibility: ?) [437] P ADP + ORF62 phosphoprotein S ATP + ORF63 protein ( protein substrate from Varicella-Zoster virus [437]) (Reversibility: ?) [437] P ADP + ORF63 phosphoprotein S ATP + PKB protein kinase ( PDK1 kinase, phosphorylates Thr308 and Ser473, which is required for activation of PKB [414]; PDK1 kinase [414]) (Reversibility: ?) [414] P ADP + phosphorylated PKB protein kinase S ATP + RRSSSY ( peptide substrate of TSSK3 consisting of the consensus sequence [420]) (Reversibility: ?) [420] P ADP + phosphorylated RRSS-phosphoserine-Y S ATP + RSK peptide (Reversibility: ?) [417] P ADP + RSK phosphopeptide S ATP + RSRSRSRSRSRSPPPVSK ( SC35-derived peptide 180-197, recombinant GSK-3b [430]) (Reversibility: ?) [430] 36

2.7.11.1

Non-specific serine/threonine protein kinase

P ADP + phosphorylated RSRSRSRSRSRSPPPVSK S ATP + S6 protein of the 40s ribosomal subunit ( the mitogenactivated protein kinase plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1 [65]) (Reversibility: ?) [65] P ADP + phosphorylated S6 protein of the 40s ribosomal subunit S ATP + SC35 ( substrate prephosphorylated SC35, SC35 is a member of the SR family of serine/arginine-rich splicing factors [430]; substrate prephosphorylated SC35, SC35 is a member of the SR family of serine/arginine-rich splicing factors, recombinant GSK-3b [430]) (Reversibility: ?) [430] P ADP + phosphorylated SC35 S ATP + SF2/ASF ( SF2/ASF is a human SR splicing factor, phosphorylation in the RS domain [310]) (Reversibility: ?) [306, 31] P ADP + phosphorylated SF2/ASF S ATP + SIV/17E-Fr Nef protein ( phosphorylation at Ser12, the protein kinase CK2 associates with the Nef proteins from the Human immunodeficiency virus and the macrophage-tropic neurovirulent Simian immunodeficiency virus clone, SIV/17E-Fr, the Nef protein play important roles in viral disease progression [471]; phosphorylation at Ser12, no or poor activity with SIV/17E-Fr Nef protein mutants S12P and D15A, respectively, in transfected 293T cells, Asp15 of the substrate is important for phosphorylation [471]) (Reversibility: ?) [471] P ADP + phosphorylated SIV/17E-Fr Nef protein S ATP + SR protein ( dsk1 protein may play an important role in mitotic control by altering cellular location, degree of phosphorylation and kinase activity [311]; enzyme acts as a corepressors for homeodomain transcription factors [320]; enzyme regulates the activation and adhesion of T cells. CD43 may mediate its biologic effects through activation of a kinase cascade, resulting in the regulation of cell growth [319]; implication of KIS in the control of trafficking and/or splicing of RNAs probably through phosphorylation of associated factors [313]; enzyme plays a role as a co-repressor for homeodomain transcription factors [317]; enzyme is essential for growth [315]; enzyme is essential for eye and embryonic development [312]; enzyme may play a role in mitosis [311]) (Reversibility: ?) [311, 312, 313, 315, 317, 319, 320] P ADP + ? S ATP + STE/SPS1-related proline/alanine-rich kinase SPAK ( recombinant WNK1 or WNK4 with recombinant protein substrate, both expressed in Escherichia coli [416]) (Reversibility: ?) [416] P ADP + phosphorylated STE/SPS1-related proline/alanine-rich kinase SPAK 37

Non-specific serine/threonine protein kinase

2.7.11.1

S ATP + TEM-b-lactamase ( the enzyme is phosphorylated only at Thr residues, shifting its apparent molecular mass from 29000 Da to 44000 Da. The phosphorylated b-lactamase is unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions [334]) (Reversibility: ?) [334] P ADP + phosphorylated TEM-b-lactamase S ATP + Trx-His-S-CREB protein ( i.e. thioredoxin fusion cAMP responsive element binding protein, phosphorylation at Ser133 [464]) (Reversibility: ?) [464] P ADP + phosphorylated Trx-His-S-CREB protein S ATP + Varicella-Zoster viral gB protein ( phosphorylation at S34 in the sequence QSEDIT [437]) (Reversibility: ?) [437] P ADP + phosphorylated Varicella-Zoster viral gB protein S ATP + [FRAT-2 protein] ( i.e. frequently rearranged in advanced T-cell lymphoma protein 2, phosphorylation by GSK3b [433]) (Reversibility: ?) [433] P ADP + phosphorylated [FRAT-2 protein] S ATP + [M3 muscarinic acetylcholine receptor] ( recombinant substrate expressed in HEK-293 and CHO cells, casein kinase 1a [443]) (Reversibility: ?) [443] P ADP + phospho-[M3 muscarinic acetylcholine receptor] S ATP + [t-protein] ( tau is primarily found in neurons, regulation of tau phosphorylation by GSK3b via interaction with FRAT-1 and FRAT2, i.e. frequently rearranged in advanced T-cell lymphoma proteins [433]; phosphorylation of primed and unprimed sites by GSK3b, wild-type and recombinant tau, recombinant GSK3b S9A [433]) (Reversibility: ?) [433] P ADP + O-phospho -[t-protein] S ATP + [t-protein] ( abnormal hyperphosphorylation of tau by GSK-3 is associated with Alzheimers disease and other tauopathies leading to neuronal degeneration [431]; activity in organisms with mutated APP and t, not in wild-type, overview [441]; phosphorylation of tau, especially at the primed epitope T231 negatively regulates tmicrotubule interactions, different effects of phosphorylation on primed T231 and unprimed S396/S404 epitopes of tau, overview [432]; substrate of GSK3b in brain [409,424]; phosphorylation at the C-terminus, lower activity with C-terminally truncated tau D421 compared to the wild-type t, the truncated t protein forms sarcosyl-insoluble aggregates [427]; phosphorylation by GSK-3 at Ser404, Ser396, Ser198, Ser199, and Ser202 [431]; substrate of GSK-3 in brain [430]) (Reversibility: ?) [409, 424, 427, 430, 431, 432, 438, 441] P ADP + O-phospho-[t-protein] S ATP + a protein ( autophosphorylation [364,386]; autophosphorylation when incubated with ATP and Mg2+ [247]; strong preference 38

2.7.11.1

Non-specific serine/threonine protein kinase

for ATP over GTP [215]; Pkn5 is autophosphorylated only at Ser [335]; self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK [338]; Pkn6 is autophosphorylated both at Ser and Thr [335]; autophosphorylation on Thr [331]; the enzyme is essential for viral replication [221]; not essential for cell viability [233]; Cki2, may contribute to the regulation of cell morphology [233]; the enzyme is a regulator of DNA strand-break repair [228]; enzyme is involved in the regulation of DNA repair [235]; enzyme plays a pivotal role in eukaryotic cell regulation [225]; gene disruption reveales that cki3+ is dispensable for cell viability, and cells lacking functional cki3+ exhibit no characteristic phenotype [216]; casein kinase 1 a gene is developmentally regulated and the kinase activity of the protein is induced by DNA damage. Possible requirement in mechanisms associated with DNA repair during early embryogenesis [241]; Pkn5 negatively regulates Myxococcus xanthus development [335]; AfsK plays a regulatory role in aerial mycelium formation in Streptomyces griseus [339]; expressed constitutively throughout the life cycle, with slight increases at an early stage of development [335]; the enzyme blocks the secretion of b-lactamase by phosphorylation [334]; pknA is required for both normal cellular growth and differentiation [333]; Pkn6 may be a transmembrane sensor of external signals for development [335]; involvement of afsK in the regulation of secondary metabolism [338]; the enzyme is required to promote the activity of at least six distinct transport systems for nitrogenous nutrients under conditions of nitrogen catabolite derepression [345]; the enzyme is a negative regulator of both sexual conjugation and meiosis [344]; STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity [349]; enzyme is not essential for vegetative growth [350]; the SKS1 protein kinase is a multicopy suppressor of the snf3 mutation of Saccharomyces cerevisiae [353]; the enzyme enhances spermine uptake in Saccharomyces cerevisiae [352]; in some cancers displaying chromosomal instability the loss of this checkpoint is associated with the mutational inactivation of a human homologue of the yeast BUB1 gene [359]; the enzyme is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. bub1(+)function is essential to maintain correct ploidy through mitosis [362]; the enzyme is required for function of the spindle assembly checkpoint [355]; cell cycle-dependent expression, Bub1b has a putative destruction box that can target proteins for degradation by proteosomes during mitosis [360]; the enzyme is involved in spindle assembly during the cell cycle [363]; hBUBR1 may regulate multi39

Non-specific serine/threonine protein kinase

P S P S

P S P S P

40

2.7.11.1

ple functions that include the kinetochore and the spindle midzone [361]; kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage [354]; hBub1 is an important component of the spindle checkpoint pathway. hBub1 expression is restricted to proliferating cells and appears to be involved in regulating cell cycle progression [358]; Bub1 is required for binding Bub3 [357]; huCdc7 may regulate processes of DNA replication by modulating MCM functions [372]; HsCdc7 may phosphorylate critical substrates that regulate the G1/S phase transition and/or DNA replication [371]; the enzyme is required for the initiation of mitotic DNA synthesis. It is necessary for genetic recombination during meiosis and for the formation of ascospores, involved in an error-prone DNA repair pathway [376]; the enzyme is required for inhibition of mitosis until completion of S phase, it may also be involved in proper execution of mitosis [377]; enzyme is required for the initiation of DNA synthesis during mitosis as well as for synaptonemal complex formation and commitment to recombination during meiosis [374]; the enzyme is dispensable for mitotic growth and premeiotic DNA replication, its primary role is in meiosis [378]; the enzyme is essential for normal cell life [386]; the enzyme is required for inositol phototrophy in Saccharomyces cerevisiae [392]; Ire1p is required for activation of the unfolded protein response. It senses the accumulation of unfolded proteins in the ER and transmits the signal across the membrane toward the transcription machinery, possibly by phosphorylating downstream components of the UPR pathway [390]; the enzyme is required for signaling from the ER to the nucleus [391]) (Reversibility: ?) [28, 114, 215, 216, 221, 225, 227, 228, 233, 235, 241, 247, 331, 333, 334, 335, 338, 339, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 357, 358, 359, 360, 361, 362, 363, 364, 371, 372, 374, 376, 377, 378, 380, 381, 382, 383, 384, 385, 386, 389, 390, 391, 392, 415] ADP + a phosphoprotein ATP + acetyl-CoA carboxylase ( phosphorylation and inactivation [188]) (Reversibility: ?) [188, 2] ADP + phosphorylated acetyl-CoA carboxylase ATP + adenomatous polyposis coli protein ( i.e. APC, APC is phosphorylated in vitro by CKId and CKIe [460]) (Reversibility: ?) [460] ADP + phosphorylated adenomatous polyposis coli protein ATP + a-casein ( casein kinase 1a [443]; dephosphorylated a-casein [451]) (Reversibility: ?) [443, 45] ADP + phosphorylated a-casein ATP + axin ( glycogen synthase kinase 3 [414]) (Reversibility: ?) [414] ADP + phosphorylated axin

2.7.11.1

Non-specific serine/threonine protein kinase

S ATP + b-catenin ( glycogen synthase kinase 3 [414]; phosphorylation at Ser45, priming GSK3b phosphorylation [460]) (Reversibility: ?) [414, 46] P ADP + phosphorylated b-catenin S ATP + b-hydroxy b-methylglutaryl-coenzyme A reductase ( phosphorylation and inactivation [188]) (Reversibility: ?) [188] P ADP + phosphorylated b-hydroxy b-methylglutaryl-coenzyme A reductase S ATP + bovine serum albumin ( not [396]; mitochondrial protamine kinases [401]; not cytosolic kinase [401]; phosphorylated at 40-70% the rate of protamine [398]; phosphorylated at 48% the rate of protamine [401]) (Reversibility: ?) [396, 398, 401] P ? S ATP + casein ( substrate of CK1 and CK2 [413]; substrate of TSSK3 [420]; the chromophoric domain light-oxygen-voltage-sensing 2 LOV2 of phototropin acts as a blue light-regulated molecular switch [447]) (Reversibility: ?) [228, 245, 247, 386, 413, 420, 437, 447] P ADP + phosphorylated casein S ATP + casein ( not [396]; poor substrate [407]; not mitochondrial protamine kinases [401]; phosphorylated at 5% the rate of protamine, cytosolic kinase [401,402]) (Reversibility: ?) [396, 401, 402, 407] P ? S ATP + claudin-4 ( recombinant WNK1 or WNK4 with recombinant protein substrate, both expressed in Escherichia coli [416]) (Reversibility: ?) [416] P ADP + phosphorylated claudin-4 S ATP + eukaryotic protein synthesis initiation factor ( i.e. eIF-4E, 1 mol phosphate per mol eIF-4E [405]) (Reversibility: ?) [405] P ? S ATP + geminin ( a cell cycle regulatory protein [456]) (Reversibility: ?) [456] P ADP + phosphorylated geminin S ATP + glycogen synthase ( glycogen synthase kinase 3 [414]; glycogen synthase kinase 3 is involved in the insulin signaling pathway regulating glycogen synthesis via glycogen synthase antagonizing insulin [414]) (Reversibility: ?) [414] P ADP + phosphorylated glycogen synthase S ATP + glycogen synthase ( from rabbit skeletal muscle [401]; mitochondrial protamine kinases [401]; not cytosolic kinase [401]; phosphorylated at 15% the rate of protamine [398,401]) (Reversibility: ?) [398, 4] P ? S ATP + glycogen synthase kinase 3 ( PKB protein kinase [414]; PKB protein kinase, phosphorylation at S21 and S9 [414]) (Reversibility: ?) [414] 41

Non-specific serine/threonine protein kinase

2.7.11.1

P ADP + phosphorylated glycogen synthase kinase 3 S ATP + histone ( histones from calf thymus, phosphorylation on Ser [331]) (Reversibility: ?) [331, 410, 421, 451] P ADP + phosphorylated histone S ATP + histone H1 ( PDK2 [440]; substrate from calf, substrate of SSTK [442]; substrate of TSSK3 [420]) (Reversibility: ?) [50, 370, 420, 440, 442] P ADP + phosphorylated histone H1 S ATP + histone H1 ( mitochondrial protamine kinases [401]; not microsomal kinases [402]; not cytosolic [401]; phosphorylated at 80% the rate of protamine [398]) (Reversibility: ?) [398, 401, 402] P ? S ATP + histone H2A ( recombinant human substrate, substrate of SSTK [442]) (Reversibility: ?) [442] P ADP + phosphorylated histone H2A S ATP + histone H2AX ( recombinant human substrate, substrate of SSTK [442]) (Reversibility: ?) [442] P ADP + phosphorylated histone H2AX S ATP + histone H2B ( recombinant human substrate, substrate of SSTK [442]) (Reversibility: ?) [442] P ADP + phosphorylated histone H2B S ATP + histone H3 ( RSK2 is a key regulator of histone H3 phosphorylation [457]) (Reversibility: ?) [457] P ADP + phosphorylated histone H3 S ATP + histone H3 ( Ark1 phosphorylates Ser10 of histone H3 in vivo [115]) (Reversibility: ?) [115] P ? S ATP + histone H3 ( Ark1 phosphorylates Ser10 of histone H3 in vivo [115]) (Reversibility: ?) [115] P ADP + phosphorylated histone S ATP + histone H4 ( recombinant substrate from Xenopus laevis, substrate of SSTK [442]) (Reversibility: ?) [442] P ADP + phosphorylated histone H4 S ATP + histone IIA ( poor substrate for cAMP-independent protamine kinase [399]) (Reversibility: ?) [399] P ? S ATP + histone IIB ( phosphorylated at 8% the rate of protamine, cytosolic kinase [401,402]; not mitochondrial protamine kinases [401]) (Reversibility: ?) [401, 4] P ? S ATP + histone III (Reversibility: ?) [196] P ADP + phosphorylated histone III S ATP + histone f1 ( lysine-rich histone, phosphorylated at a specific serine residue [397]) (Reversibility: ?) [397] P ? S ATP + histone fraction II ( slightly lysine-rich, from trout testes [407]) (Reversibility: ?) [407] 42

2.7.11.1

Non-specific serine/threonine protein kinase

P ? S ATP + hypoxia-inducible factor-1 ( i.e. HIF-1, casein kinase 2 inhibits the activation of HIF-1 activity by phosphorylation and thereby stabilization of the protein, CK2 is involved in and influenced by hypoxia, overview [422]; i.e. HIF-1 [422]) (Reversibility: ?) [422] P ADP + phosphorylated hypoxia-inducible factor-1 S ATP + lamin A/C ( phosphorylation of host cell lamin by US3 kinase [468]; phosphorylation of host cell lamin by US3 kinase at multiple sites in vitro, eventhough lamin A/C contains only one putative consensus sequence [468]) (Reversibility: ?) [468] P ADP + phosphorylated lamin A/C S ATP + mPer1 ( CK1e-1 [444]; CKIe-3 [444]) (Reversibility: ?) [444] P ADP + phosphorylated mPer1 S ATP + maltose-binding protein (Reversibility: ?) [437] P ADP + phosphorylated maltose-binding protein S ATP + myelic basic protein ( phosphorylation of serine and threonine residues by PknB [425]) (Reversibility: ?) [425] P ADP + myelic basic phosphoprotein S ATP + myelin basic protein ( myelin basic proteins from bovine brain, phosphorylation on Ser [331]; PknB, PknD, PknE, and PknF [412]; recombinant WNK1 or WNK4 expressed in Escherichia coli [416]; substrate of TSSK3 [420]) (Reversibility: ?) [311, 331, 412, 416, 420, 421, 442, 451, 455, 464] P ADP + phosphorylated myelin basic protein S ATP + oxidative stress response kinase 1 OSR1 ( recombinant WNK1 or WNK4 with recombinant protein substrate, both expressed in Escherichia coli, phosphorylation at Ser325 and Thr185, the latter is required for activation of OSR1 [416]) (Reversibility: ?) [416] P ADP + phosphorylated oxidative stress response kinase 1 OSR1 S ATP + RRRDDDSDDD ( a CK2-specific synthetic peptide substrate [461]) (Reversibility: ?) [461] P ADP + RRRDDDphosphoSDDD S ATP + phospho-glycogen synthase peptide (Reversibility: ?) [433] P ADP + ? S ATP + phosphoglucosamine mutase (Reversibility: ?) [419] P ADP + phosphorylated phosphoglucosamine mutase S ATP + phosphoglycogen synthase peptide 2 (Reversibility: ?) [431, 432, 438] P ADP + phosphorylated phosphoglycogen synthase peptide 2 S ATP + phospholipase D ( CKII is involved in 4b-phorbol 12myristate 13-acetate-induced phospholipase D activation, of PLD1 or PLD2 activity as well as basal PLD activity [452]) (Reversibility: ?) [452] P ADP + phosphorylated phospholipase D S ATP + phosvitin (Reversibility: ?) [245, 25] P ADP + ?

43

Non-specific serine/threonine protein kinase

2.7.11.1

S ATP + plant ribosomal proteins ( two plant ribosomal proteins of 14000 Da and 16000 Da can be phosphorylated by the Atpk1 protein kinase [79]) (Reversibility: ?) [79] P ADP + phosphorylated plant ribosomal proteins S ATP + procaspase-2 ( phosphorylation of procaspase-2 by CK2 prevents the activation of caspase activity by inhibiting the dimerization of procaspase-2, CK2 activity is involved in the direct regulation of the caspase-8 pathway, where it can control caspase-8 activity through the phosphorylation of ARC or by preventing the ability of caspase-8 to cleave its target Bid [456]) (Reversibility: ?) [456] P ADP + phosphorylated procaspase-2 S ATP + protamine ( preferred substrate [395, 398, 401, 407]; phosphorylates threonine residues [395]; phosphorylates serine [395,396,400]; transfers terminal phosphoryl group from ATP into O-phosphoseryl linkages in the acceptor molecule [407]; no substrates are rabbit muscle glycogen phosphorylase b, human g-globulin [396]; no substrates are histone H4, ovalbumin, synthetic peptide poly(Glu,Tyr) (4:1) [401]; phosphorylates little, if any activity with branched-chain a-keto acid dehydrogenase, pyruvate dehydrogenase, casein, ovalbumin or histone H2B [398]; no substrates are GTP [401, 402, 407]; no substrates are GTP or UTP [407]; no substrates are acidic proteins, e.g. casein, phosvitin [395, 396, 397, 400, 408]; phosphorylates histone fraction I (lysine-rich) and histone fractions III and IV (arginine-rich), from trout testes [407]) (Reversibility: ?) [395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408] P ADP + O-phosphoprotamine [395, 396, 397, 400, 407] S ATP + protein (Reversibility: ?) [365, 366, 367, 368, 369] P ADP + a phosphoprotein S ATP + protein ( PKL12 may play a role in a very general cellular function, probably related with the secretory pathway [368]; EDPK plays a crucial role in intracellular signaling not only during mouse development but also in adult tissues [369]) (Reversibility: ?) [368, 37] P ? S ATP + protein ( GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro [19]; phosphorylates and inactivates the actin binding/depolymerizing factor cofilin and induces actin cytoskeletal changes [12]) (Reversibility: ?) [12, 19] P ADP + protein tyrosine phosphate S ATP + protein ( autophosphorylation [57, 104, 106, 171, 281, 311, 313]; blue light-dependent autophosphorylating [56]; Ser916 is an in vivo autophosphorylation site [104]; autophosphorylates exclusively serines within its COOH-terminal region in an intermolecular fashion [116]; 44

2.7.11.1

P S

P S P S P S P S

Non-specific serine/threonine protein kinase

autophosphorylated in vitro in its PS domain [138]; minimum sequence required for efficient phosphorylation is Xaa-Xaa-HydXaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (in decreasing order), Phe, Leu, Val, Ala, MAPKAP kinase-2 can not tolerate a proline residue at position n + 1 [177]; autophosphorylation of Thr192 [210]; Ser/Thr kinase [314]; phosphotyrosine as well as phosphoserine/threonine are found in autophosphorylation [311]; enzyme is essential for the late nuclear division in the yeast [289]; enzyme activates the p38 pathway, SPAK may act as a novel mediator of stress-activated signals [281]; the enzyme is an effector for Cdc42Hs and is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia [285]; enzyme is required for virulence and hyphal formation of Candida albicans [279]; potentially important role in the control of the cellular architecture and/or signaling in the central nervous system [284]; PAK3 may be critical for human cognitive function [280]; the enzyme regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for effector for Cdc42 and Rac1 in proll motility [301]; the enzyme is required to link the yeast pheromone response G-protein b g subunits to downstream signalling components [295]; the enzyme is necessary for mating [296]; Pak1 protein is likely to be an effector for Cdc42p or a related GTPase, Pak1p may be involved in the maintenance of cell polarity and in mating [294]; the enzyme is involved in budding and cytokinesis and interacts with Cdc42, a GTPase required for polarized cell growth [292]; hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway. PAK kinase plays a key part in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation [300]) (Reversibility: ?) [56, 57, 104, 106, 116, 138, 171, 177, 210, 279, 280, 281, 284, 285, 289, 292, 294, 295, 296, 298, 300, 301, 311, 313, 314] ADP + phosphoprotein ATP + protein Cubitus interruptus ( Ci proteins are important in regulation of Hedgehog signaling, overview [460]; i.e. Ci, e.g. Ci-75 or Ci-155 [460]) (Reversibility: ?) [460] ADP + phosphorylated protein Cubitus interruptus ATP + protein INI ( INI is a hSWI/SNF protein [469]) (Reversibility: ?) [469] ADP + phosphorylated INI ATP + protein LRP6 ( CKIg [460]) (Reversibility: ?) [460] ADP + phosphorylated protein LRP6 ATP + protein TCF3 ( CKIe [460]) (Reversibility: ?) [460] ADP + phosphorylated protein TCF3 ATP + protein kinase C ( isozyme PKNa [423]) (Reversibility: ?) [423] 45

Non-specific serine/threonine protein kinase

2.7.11.1

P ADP + phosphorylated protein kinase C S ATP + protein kinase Cz ( phosphorylation and activation of protein kinase Cz [34]) (Reversibility: ?) [34] P ADP + phosphorylated protein kinase Cz S ATP + ribosomal protein S6 ( specifically phosphorylated by S6K [417]) (Reversibility: ?) [417] P ADP + phosphorylated ribosomal protein S6 S ATP + several 40S ribosomal polypeptides ( e.g. S6, 2.5 mol phosphate per mol S6 [402]) (Reversibility: ?) [402] P ? S ATP + stathmin ( phosphorylates on Ser [313]) (Reversibility: ?) [313] P ADP + phosphorylated stathmin S ATP + synaptotagmin 2 ( WNK1 specific substrate [450]) (Reversibility: ?) [450] P ADP + phosphorylated synaptotagmin 2 S ATP + synthetic peptide ( Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala [402]) (Reversibility: ?) [402] P ? S ATP + t protein ( t in Alzheimer disease brain is highly phosphorylated and aggregates into paired helical filaments comprising characteristic neurofibrillary tangles, overview [465]; determination of several phosphorylation sites, e.g. Ser258, Ser289, Ser262, and Ser356 within the microtubule-binding repeats or at Ser184 and Ser185 of the central region, for casein kinase I, casein kinase 2, and glycogen synthase kinase-3b in insoluble tau, PHF-t, extracted from Alzheimer brain and of tau from control healthy brain by mass spectrometry, overview [465]) (Reversibility: ?) [465] P ADP + phosphorylated t protein S ATP + testis-specific serine-threonine kinase 3 ( substrate of phosphoinositide-dependent kinase-1 leading to activation [420]; substrate of recombinant Myc-tagged catalytic subunit of PDK1 leading to activation [420]) (Reversibility: ?) [420] P ADP + phosphorylated testis-specific serine-threonine kinase 3 S ATP + tomato mosaic virus movement protein ( recombinant CK2 catalytic subunit, substrate is phosphorylated at S261 and T256 of the C-terminus [435]) (Reversibility: ?) [435] P ADP + phosphorylated tomato mosaic virus movement protein S ATP + vimentin ( isozyme PKNa [423]) (Reversibility: ?) [423] P ADP + phosphorylated vimentin S RRRDDDSDDD + ATP (Reversibility: ?) [260] P ? + ADP S casein + ATP (Reversibility: ?) [251, 253, 254, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 270, 272, 273, 274] P phosphorylated casein + ADP S casein + GTP (Reversibility: ?) [257, 26] 46

2.7.11.1

Non-specific serine/threonine protein kinase

P phosphorylated casein + GDP S Additional information ( interaction between RAC-PK and protein kinase C [81]; guanylate kinase activity [131]; stress-induced activation of MAPKAPK-2, in turn, results in the phosphorylation of small heat-shock proteins [174]; the 41000 Da polypeptide of the enzyme and the 32000 Da polypeptide both incorporate phosphate during autophosphorylation [268]; enzyme undergoes autophosphorylation [257]; LIMK may be involved in developmental or oncogenic processes through interactions with LIMcontaining proteins [14]; Kiz-1 may play distinct roles in dividing cells and in differentiated neurons [16]; Williams syndrome is a complex neurodevelopmental disorder arising from a microdeletion at Chr band 7q11.23, which results in a hemizygous condition for a number of genes, LIMK1, WBSCR1, and RFC2 [18]; LIMK1 may be particularly relevant when explaining cognitive defects observed in WS patients [15]; LIM-kinase1 hemizygosity is implicated in impaired visuospatial constructive cognition [13]; the enzyme is part of a growth control pathway which is at least partially redundant with the cAMP pathway [29]; the enzyme positively regulates the progression of yeast cells through the G1 phase of the cell cycle [27]; deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy [41]; AKT1 gene is not a major contributor to susceptibility to type II diabetes mellitus in Ashkenazi Jews [39]; K+ -channel activation by all three isoforms of serum-dependent and glucocorticoid-dependent protein kinase SGK [36]; the enzyme acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression [40]; NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants [57]; the enzyme is required to repress entry into the conidiation program [55]; induction of enzymatically active Sgk functions as a key cell survival component in response to different environmental stress stimuli [35]; transcript levels are strongly altered during anisotonic and isotonic cell volume changes [45]; AKT2 may contribute to the pathogenesis of ovarian carcinomas [75]; enzyme may function in the adaptation of plant cells to cold or high-salt conditions [78]; the DBF20 mRNA is expressed at a low level and not under cell-cycle control [70]; kinase plays a part in regulating events associated with fertilization [69]; DBF2 mRNA is expressed under cell-cycle control at or near START [70]; enzyme is required for cell growth [60]; atpk1 is involved in the control of plant growth and development [80]; DBF2 is likely to encode a protein kinase that may function in in47

Non-specific serine/threonine protein kinase

2.7.11.1

itiation of DNA synthesis and also in late nuclear division [71]; the enzyme is transcriptionally regulated by serum and glucocorticoids in mammary epithelial cells, hormone-regulated protein kinase gene with a functionally defined p53 promoter recognition element [92,94,95]; induction of sgk gene may be associated with a series of axonal regenerations after brain injury, and in addition, the sgk gene may also play important roles in the development of particular groups of neurons in the postnatal brain [94]; may function in the adaptation of plant cells to cold or high-salt conditions [78]; enzyme is induced during ovarian cell differentiation [92]; T-PK may have specialized functions in different areas of central nervous system. Alterations of this complex expression pattern can be responsible for the mental status impairment observed in myotonic dystrophy patients [96]; enzyme may have a role in the development of mental symptoms in severe cases of myotonic dystrophy [98]; the enzyme is highly regulated at the transcriptional level by glucocorticoid hormones [95]; SGK is a component of the phosphoinositide 3 (PI 3)-kinase signaling pathway [92]; enzyme is important for cell growth [113]; mutations in the ribosomal S6 kinase (Rsk-2) gene are associated with Coffin-Lowry syndrome, an Xlinked disorder characterized by facial dysmorphism, digit abnormalities and severe psychomotor retardation [82,83,84]; ribosomal S6-kinase RSK4 is commonly deleted in patients with complex X-linked mental retardation, RSK4 plays a role in normal neuronal development. RSK4 is completely deleted in eight patients with the contiguous gene syndrome including MRX, partially deleted in a patient with DFN3 and present in patients with an Xq21 deletion and normal intellectual abilities [111]; decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy [99]; inherited defects in insulin-stimulated activation of muscle glycogen synthesis in patients with insulin-resistant NIDDM may be located further upstream of ISPK-1 in the insulin action cascade [85]; Akt2 expression is activated during cellular differentiation and suggest that it functions in the signaling pathways of some adult tissues [103]; associates with mitotic structures in a stage dependent manner and is required for chromosome segregation [115]; protein kinase is required for meiotic recombination [118]; type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation, required during the later part of each cell cycle [121]; required for chromosome synapsis and recombination [120]; enzyme plays a critical role in regulation by carbon catabolite repression [150]; the enzyme plays a role in development [142]; nim1+ is a negative regulator of the wee1+ mitotic inhibitor, another protein kinase homolog. The combined mitotic induction activities of nim1+ and cdc25+ counteract the wee1+ mitotic inhibitor in a regulatory network that appears also to involve the cdc2+ protein kinase, which is required for mitosis [152]; Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination CHK1 48

2.7.11.1

Non-specific serine/threonine protein kinase

gene is a candidate tumor suppressor gene [128]; enzyme plays a role in germ line establishment [136]; kinase functions downstream of ATM protein in the cellular response to DNA damage [145]; in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry [127]; in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis [148]; heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the TP53 gene. hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors [147]; Unc51.1 signals the program of gene expression leading to the formation of granule cell axons [137]; required to give a normal cell cycle response to nitrogen starvation [151]; kinase-related apoptosis-inducing protein kinase 1 may play an important role in the core apoptosis program in osteoclast [123]; required for alteration of growth polarity and actin localization [124]; likely role for DCAMKL1 transmembrane protein in developing and adult brain, possibly in a pathway of cortical development [132]; Cdc25 mitotic inducer is targeted by chk1 DNA damage checkpoint kinase [162]; enzyme controls the DNA damage response [171]; enzyme mediates checkpoint pathway [161]; required for pattern formation within embryonic segments and imaginal discs, possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development [156]; Pak1 may function by modifying and partially stabilizing thermolabile DNA polymerases, perhaps during DNA repair [168]; chk1 protein kinase links the rad checkpoint pathway to cdc2 [164]; Chk1 is required for function of the DNA damage checkpoint in Saccharomyces cerevisiae [154]; important for growth polarity [155]; enzyme is essential for the derepression of catabolic repression [178]; the enzyme is required for the autophagic process in Saccharomyces cerevisiae [179]; deletion of GIN4 is not lethal but leads to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation [200]; the enzyme is a regulator of G2/M progression and cytokinesis [190]; mutations of STK11 cause Peutz-Jeghers syndrome [201]; kinase Cdr2 regulates the onset of mitosis in fission yeast [189]; PeutzJeghers syndrome is an autosomal-dominant disorder characterized by melanocytic macules of the lips, multiple gastrointestinal hamartomatous polyps and an increased risk for various neoplasms, including gastrointestinal cancer, germline mutations in STK11, probably in conjunction with acquired genetic defects of the second allele in somatic cells, cause the manifestations of PJ syndrome [202]; enzyme plays a major role in the regulation of lipid metabolism, be involved in the regulation of a wide range of metabolic pathways [198]; RKIN1 protein has a role in the control of carbon metabolism in endosperms of rye [194]; 49

Non-specific serine/threonine protein kinase

2.7.11.1

Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control [212]; the enzyme is essential for the derepression of glucose repression, interacts with additional regulatory pathways and affects the expression of multiple target genes [191]; the damage/replication G2 checkpoint kinase Chk1 phosphorylates and inhibits Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophaseI-arrested oocytes [212]; Peutz-Jeghers syndrome is the first cancer-susceptibility syndrome to be identified that is due to inactivating mutations in a protein kinase, STK11 [204]; the enzyme is a key component of the DNA replication-monitoring S/G2 checkpoint system. Its primary role is to monitor DNA synthesis by interacting with DNA polymerase a and send a signal to block the onset of mitosis while DNA synthesis is in progress [199]; enzyme is involved in the synthesis of seed storage compounds during seed development [183]; may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism [207]; protein phosphorylation by the unc-51 product is important for axonal elongation and possibly for axonal guidance [206]; the enzyme is essential for vegetative growth of Dictyostelium discoideum, the a subunit is expressed constitutively like its mRNA during the life cycle [259]; enzyme is implicated in the control of cell growth and proliferation. Androgenic regulation of CK-2 gene transcription is not an early event related to androgen action, but is substantial in the prereplicative phase of prostatic cell proliferation mediated by androgen. Androgenic stimulation of the mRNA expression for the a and b subunits of CK-2 appears to be differential [251]; translational and/or post-translational mechanisms play an important role in the developmental regulation of casein kinase II activity [257]; plays a role in the translation of cell polarity into polarized growth, but not in the establishment of polarity itself [274]; key regulatory enzyme involved in many cellular processes, including the control of growth and cell division [270]; the casein kinase II gene can serve as an oncogene, and its dysregulated expression is capable of transforming lymphocytes in a two-step pathway with c-myc [273]; unbalanced expression of CK2 catalytic subunit synergizes with Ha-ras in cell transformation [267]; NH2 -terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1 [306]; enzyme is involved in pre-mRNA splicing [310]; calmodulin-binding region of titin can play a regulatory role for the enzyme [326]; the cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein [324]; enzyme may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock [341]; Nek2 protein is almost undetectable during G1 but accumulates progressively throughout S, reaching maximal le50

2.7.11.1

Non-specific serine/threonine protein kinase

vels in late G2, may function at the onset of mitosis [342]; AfsK is a Hanks-type protein kinase [436]; enzyme is involved in stress response and adaptation to environmental changes in mycobacteria [421]; glycogen synthase kinase-3b also performs the t-protein kinase reaction, EC 2.7.11.26, phosphorylating t protein at serine and threonine residues [424]; GSK-3 affects the t-mRNA splicing of exon 10 via phosphorylation of the splicing factors of the serine/arginine-rich splicing factor SR family, e.g. SC35, leading to priming and dislocation of the splicing factor, aberrant t splicing contributes to tauopathies including Alzheimers disease, overview [430]; GSK3b works coordinatedly with PKA, EC 2.7.11.11, on t phosphorylation [438]; HIPK2 regulates transforming growth factor-b-induced c-Jun NH(2)-terminal kinase activation and apoptosis in hepatoma cells, HIPK2 triggers promyelotic leukemia nuclear body disruption and release of Daxx [418]; isozyme RSK1, i.e. 14-3-3, is regulated by inhibitory binding of 14-3-3b, a RSK1-binding protein, to Lys49 of the enzyme, Lys49 is also responsible for interaction of RSK1 with Bcr, Raf, and Cbl [426]; kinase Rio2 is required for rRNA cleavage in 40S ribosomal subunit maturation [451]; mechanism of Ca2+ mediated regulation of NDR protein kinase through autophosphorylation and phosphorylation by an upstream kinase [428]; mechanism of S6K2 regulation via phosphorylation and autoinhibition with T338 playing the key role, S6K2 is regulated in a mitogen-activated protein kinase/ extracellular-signal-regulated kinase kinase MEK-dependent manner, overview [415]; naturally occurring V717I mutation of the amyloid precursor protein APP in a CT100 fragment of organisms with Alzheimers disease leads to augmented age-dependent tau phosphorylation, followed by increased activation status of mitogen-activated protein kinase family members, e.g. ERK1/2, p38, and c-Jun NH2 -terminal kinase, compared to the wild-type organism, naturally occurring V337M mutation of tau protein of patients suffering Alzheimers disease leads to age-dependent memory deficits [441]; naturally occurring V717I mutation of the amyloid precursor protein APP in a CT100 fragment of patients suffering Alzheimers disease leads to augmented age-dependent tau phosphorylation, followed by increased activation status of mitogen-activated protein kinase family members, e.g. ERK1/2, p38, and c-Jun NH2 -terminal kinase, compared to the wild-type organism, naturally occurring V337M mutation of tau protein of patients suffering Alzheimers disease leads to age-dependent memory deficits [441]; NTHK2 is an ethylene receptor protein [446]; phosphorylated t is important in cytoskeleton assembly [427]; phosphorylation at Ser9 by PKGI and PKGII inhibits the glycogen synthase kinase-3 inducing dephosphorylation of C/EBPb, i.e. the CCAAT enhancer-binding protein b, important in regulation of gene expression during cell proliferation, differentiation, and apoptosis [434]; photoregulation of phototropin, overview [447]; PKD2 regulation involving ligand-binding domains of the enzyme, isozyme PKD2 might play a role in the nucleus after activation by G protein-coupled receptors, overview, intracellular trafficking of PKD2, overview [440]; 51

Non-specific serine/threonine protein kinase

2.7.11.1

protein kinase StkP and Mn2+ -dependent cytosolic protein phosphatase PhpP act as a functional pair in vivo [419]; regulation mechanism and biological function of PKN [423]; regulation mechanism and biological function of PKN, PKNa is involved in insulin-induced actin cytoskeleton reorganization and cell adhesion, overview, isozyme PKNa is involved in glucose transport in 3T3/L1 cells [423]; regulation mechanism and biological function of PKN, PKNa is involved in insulin-induced actin cytoskeleton reorganization and cell adhesion, overview, PKNa is involved in vesicle transport in the endoplasmic reticulum, PKNa is cleaved by caspase-3 or related proteases in apoptotic Jurkat and U-937 cells contributing to signal transduction, PKN interacts with papillomaviral oncoproteins being involved in tumorigenesis, overview [423]; regulation mechanism and biological function of PKN, PKNa is involved in insulin-induced actin cytoskeleton reorganization, overview [423]; regulation mechanism and biological function of PKN, the enzyme is involved in cell cycle control [423]; serine/threonine protein kinase is SSTK essential for male fertility [442]; serine/ threonine protein kinase SSTK is essential for male fertility, a DNA condensation defect in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins [442]; tau becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain, inhibition of tau hyperphosphorylation inhibits an associated loss in spatial memory [431]; the enzyme participates in Alzheimers disease [409]; WKN1 and WKN4 are involved in regulation of ion transport across diverse epithelia, WKN4 reduces the activity of NaCl-cotransporter activity and the potassium channel ROMK by reducing their appearance in the plasma membrane [410]; WNK1 causes a hereditary form of hypertension, pseudohypoaldosteronism type II [450]; AfsK, PkaG, and SCD10 perform autophosphorylation [436]; CKII phosphorylates serine or threonine residues with an acidic amino acid in the +2 or +3 position [437]; enzyme assay under dim red light conditions, phototropin performs autophosphorylation [447]; enzyme performs autophosphorylation, PKN binds several associated proteins via its ACC domain [423]; enzyme performs autophosphorylation, PKN binds several associated proteins via its ACC domain, i.e. RhoA, a small GTPase, which binds to the ACC domain of PKNa forming a catalytic active site [423]; enzyme performs autophosphorylation, PKN binds several associated proteins via its ACC domain, i.e. RhoA, a small GTPase, which binds to the ACC domain of PKNa forming a catalytic active site, isozyme PRK2/PKNg/PAK-2 binds to the large non-transmembrane protein Tyr phosphatase PTP-BL involved in the modulation of cytoskeleton, mediated by PSD-95 [423]; GSK-3 catalyzes tau phosphorylation in the brain, while it performs phosphorylation of glycogen synthase and other proteins in other tissues [431]; GSK-3ab catalyzes tau phosphorylation in brain, but phosphorylation of glycogen synthase and other proteins, EC 2.7.11.1, in different tissues [441]; 52

2.7.11.1

Non-specific serine/threonine protein kinase

GSK3b catalyzes tau phosphorylation in brain, EC 2.7.11.26, but phosphorylation of glycogen synthase and other proteins in different tissues [432,438]; GSK3b catalyzes tau phosphorylation, EC 2.7.11.1, in brain, but phosphorylation of glycogen synthase and other proteins in different tissues [433]; GSK3b catalyzes tau phosphorylation, EC 2.7.11.26, in brain, but phosphorylation of glycogen synthase and other proteins in different tissues [427]; NDR1 performs autophosphorylation at S281, T444, and T74 dependent on intracellular Ca2+ [428]; PK1 performs autophosphorylation [449]; PK2 performs autophosphorylation [449]; PKD2 performs autophosphorylation [440]; PknB performs autophosphorylation [425]; PknB, PknD, PknE, and PknF perform autophosphorylation [412]; PknH performs autophosphorylation [421]; Rio2 performs autophosphorylation [451]; StkP performs autophosphorylation, StkP is dephosphorylated by PhpP, a PP2C-type protein phosphatase [419]; substrate ABC transporter protein structure, phosphorylation of the ABCA1 transporter reduces its flippase activity, overview [429]; substrate specificity of full-length enzyme and truncated mutant enzymes, overview, NTHK2 kinase performs autophosphorylation, the enzyme shows Mn2+ -dependent serine/ threonine kinase and Ca2+ -dependent histidine kinase, EC 2.7.13.3, activities [446]; substrate specificity of GSK3b [409]; substrate specificity of PDK1, PDK1 binds the hydrophobic motif of substrates via its PIF pocket, GSK3 substrates need to bind tightly to the enzyme for phosphorylation [414]; substrate specificity with diverse peptide substrates, overview, SSTK forms stable complexes with heat shock signal proteins HSP90-1b, HSC70, and HSP70-1, which seems to be necessary for activity [442]; substrate specificity with diverse peptide substrates, overview, SSTK forms stable complexes with heat shock signate proteins HSP90-1b, HSC70, and HSP70-1, which seems to be necessary for activity [442]; the minimal consensus sequence of ORF47 is S/T-X-D/E-D/E, no activity of ORF47 with glutathione S-transferase, or Varicella-Zoster viral gB protein [437]; TSSK3 performs autophosphorylation [420]; WKN1 and WKN4 perform autophosphorylation [410]; WNK1 interacts with OSR1 and SPAK, overview [416]; WNK1 performs autophosphorylation at S382 for activation [450]; CK2 impairs spatial memory formation through differential cross talk with PI-3 kinase signaling by activation of Akt and inactivation of SGK1 through protein phosphatase 2A [467]; CK2 is a highly conserved serine/threonine kinase involved in several intracellular pathways, which control, among others, cell cycle, proliferation, apoptosis and transformation, overview, protein kinase AKT interacts with CK2 subunits in vivo as well as in vitro enhancing AKT activity, overview [461]; CK2 is involved in phosphorylation of a large portion of the proteome, overview, increased enzyme activity in associated with a number of pathological processes, including cancer, infectious diseases, neurodegeneration, and cardiovascular pathologies playing a global anti-apoptotic role, overview [454]; CK2 phosphorylates about 300 different cellular proteins, ranging from transcrip53

Non-specific serine/threonine protein kinase

2.7.11.1

tion factors to proteins involved in chromatin structure and cell division, CK2 induces neoplastic growth when overexpressed [470]; CK2 plays a role in the protection of cells from apoptosis via the regulation of tumor suppressor and oncogene activity and stability, e.g. in the NF-kB or the Wnt signaling pathways, molecular mechanisms, detailed overview, CK2 activity is linked to Her-2/neu oncogene, which is overexpressed in 30% of breast cancers, regulation of apoptotic machinery by CK2 and its antiapoptotic effect, overview, role of CK2 in chromosomal DNA strand break repair and the maintenance of genomic integrity, mutational study, overview [456]; high CK2 activity occurs in solid tumors due to growthrelated functions and to suppression of cellular apoptosis, nuclear localization of domain CK2a is associated with high-grade tumors and a poor prognostic factor, overview [459]; members of the hSWI/SNF chromatin remodeling complex associate with and are phosphorylated by protein kinase B/Akt, overview [469]; multiple CKI family members play a role in both positively and negatively regulating Wnt and Hedgehog signaling, and the regulation of the stability of cytoplasmic b-catenin, detailed overview, the partial degradation of Ci is promoted by phosphorylation and by a cytoplasmic complex, which includes Costal-2, a microtubule-binding protein, and Fused, a serine-threonine protein kinase [460]; phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization, overview [464]; RSK2 is a critical serine/threonine kinase for the regulation of cell transformation, RSK is regulated by the tumor promoters epidermal growth factor EGF or 12-Otetradecanoylphorbol-13-acetate, TPA, overview [457]; SpkA is a regulator of expression of three putative pilA operons, formation of thick pili, and cell motility, DNA microarray and electron microscopy analysis, overview [462]; the enzyme alters localization of lamin A/C in infected cells, recombinantly expressed lamin as GFP-tagged protein in HepG2 cells, enzyme inactivation causes capsids to aggregate aberrantly between the inner and outer nuclear membranes within evaginations/extensions of the perinuclear space, overview, US3 kinase activity regulates HSV-1 capsid nuclear regress at least in part by phosphorylation of lamin A/C [468]; the enzyme inhibits NF-kB activity via interaction with the leucine-rich repeat domain of the Salmonella enterica serovar typhimurium-derived effector SspH1 in infected intestine-407 cells, overview [458]; the enzyme is involved in the inflammation cascade in intestine and colon epithelium [455]; the eukaryotic-type serine/threonine protein kinase StkP is a global regulator of gene expression in Streptococcus pneumoniae, and is important for the resistance of the organism to various stress conditions, StkP positively controls the transcription of a set of genes encoding functions involved in cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response, overview [463]; CK2 contains a consensus sequence in which a carboxylic acid side chain at position n+3 relative to the target serine/threo54

2.7.11.1

Non-specific serine/threonine protein kinase

nine residue plays a crucial role for activity, CK2 shows a broad substrate specificity of diverse proteins [454]; CK2 inhibition increases SGK1 phosphorylation at Ser422 [467]; members of the hSWI/SNF chromatin remodeling complex associate with and are phosphorylated by protein kinase B/Akt, however, no Akt consensus sequences are found on BRG1, hBrm, BAF60, BAF57, or on INI1, overview [469]; only splicing variant TSSK5a exhibits kinase activity [464]; protein kinase AKT interacts with CK2 subunits in vivo as well as in vitro enhancing AKT activity, overview [461]; splicing variant TSSK5b is inactive [464]; splicing variant TSSK5d is inactive [464]; splicing variant TSSK5g is inactive [464]; StkP performs autophosphorylation [453]; the catalytic kinase domain, residues 561-942, of PKN1 is active with out the regulatory domain [458]; the pleiotropic protein kinase CK2 possesses many protein interaction sites [470]) (Reversibility: ?) [13, 14, 15, 16, 18, 25, 27, 29, 35, 36, 39, 40, 41, 45, 55, 57, 60, 69, 70, 71, 75, 77, 78, 80, 81, 82, 83, 84, 85, 92, 94, 95, 96, 98, 99, 103, 111, 113, 115, 118, 120, 121, 123, 124, 127, 128, 131, 132, 136, 137, 142, 145, 147, 148, 150, 151, 152, 154, 155, 156, 161, 162, 164, 168, 171, 174, 178, 179, 183, 189, 190, 191, 194, 198, 199, 200, 201, 202, 203, 204, 206, 207, 212, 213, 251, 257, 259, 267, 268, 270, 273, 274, 306, 310, 324, 326, 341, 342, 409, 410, 412, 414, 415, 416, 418, 419, 420, 421, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 436, 437, 438, 440, 441, 442, 446, 447, 449, 450, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 467, 468, 469, 470] P ? Inhibitors (5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid ( i.e. IQA [454, 456]) [454, 456] 1-oleoyl-2-acetylglycerol ( inhibits phorbol ester binding [105]) [105] 14-3-3b ( a p90 ribosomal S6 kinase isoform 1-binding protein that negatively regulates RSK1 kinase activity, interaction via Lys49 of 14-3-3b and S154 of phosphorylated RSK1, phosphorylation of RSK1 is required for interaction, no binding of mutant 14-3-3b K49Q or K49A [426]) [426] 2-aminopurine [422] 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole ( i.e DMAT [454]; i.e. DMAT [456]) [454, 456] 3,8-dibromo-7-hydroxy-4-methyl-2H-chromen-2-one ( i.e. DBC [454]) [454] 4,5,6,7-tetrabromo-1H-benzotriazole ( i.e. TBB [454,456]) [454, 456] 4,5,6,7-tetrabromobenzimidazole ( i.e. TBBz or TBI [456]) [456] 8-hydroxy-4-methyl-9-nitro-2H-benzo[g]chromen-2-one ( i.e. NBC [454]) [454] AMP-PNP ( ATP analogue adenosine 5-(b,g-imino)triphosphate [409,425]) [409, 425] apigenin ( a CKII-specific inhibitior [452]) [452, 456]

55

Non-specific serine/threonine protein kinase

2.7.11.1

CKI-7 ( half-maximally at 0.0112 mM [245]) [215, 238, 245, 460] Ca2+ ( not [402]; inhibits phosphorylation of myelin basic proteins [331]; mitochondrial kinase, not cytosolic [401]) [331, 398, 401, 402] Catalytic subunit of protein phosphatase 2A ( NaCl, diphosphate, phosphate, NaF or ATP protects [404]; Mg2+ , Mn2+ , Ca2+ , EDTA, EGTA or vanadate does not [404]) [402, 404] Co2+ ( above 30 mM, activating below 20 mM [396]) [396] DRB [456] EDTA [446] ellagic acid [454, 456] fostriecin ( protein phosphatase 2A inhibitor fostriecin reverses the memory-impairing effect of CK2aWT, it also reverses the effect of CK2aWT in decreasing SGK1 phosphorylation [467]) [467] H-7 [421] heparin ( inhibition of recombinant casein kinase I d when phosvitin is the substrate [245]; inhibition with casein and phosvitin as substrate [247]; recombinant GST-CK2 catalytic subunit [435]; strongly inhibits casein kinase II [437]) [245, 247, 257, 264, 435, 437] K-252a [436] KCl ( not [401,402]; eIF-4E as substrate [405]) [401, 402, 405] kaempferol ( an inhibitor of RSK2 invitro and in vivo, and suppresses proliferation and EGF-induced transformation in JB6 Cl41 cells, overview [457]) [457] Mg2+ ( above 30 mM, activating below 20 mM [396]) [396] Mn2+ ( above 30 mM, activating below 20 mM [396]) [396] NaCl ( not [401,402]; eIF-4E [405]; bovine serum albumin, not protamine as substrate [398]) [398, 401, 402, 405] O-phosphoprotamine ( bovine serum albumin, not protamine as substrate [398]) [398] protein phosphatase 1b ( weak [403]) [403] protein phosphatase 2A ( 2A1 and 2 [402]; specific protamine kinase inhibitors [402,403]; okadaic acid, microcystin-LR [402,403]; ATP [402,403]; phosphate or NaF prevents [402]; Mn2+ enhances inactivation rate [403]; protein phosphatase inhibitor 2 does not protect [403]; rate of inactivation is unaffected by EDTA, EGTA, Mg2+ , Ca2+ [403]; diphosphate [403]; eIF-4E, 40S ribosomes or protamine as substrate [405]) [402, 403, 405] Ro 31-8220 [53] spermine ( bovine serum albumin, not protamine as substrate [398]) [398] staurosporine ( inhibits phosphorylation of myelin basic proteins [331]) [53, 331, 419, 421, 436] TBCA [456]

56

2.7.11.1

Non-specific serine/threonine protein kinase

alsterpaullone ( inhibition of AMPK, GSK3b, S6K1, PKBa, PDK1, MAPKAP-K2 [413]; weak inhibition of CK1, inhibition of CK2, and slightly of MAPKAP-K1a [413]) [413] emodin ( a CKII-specific inhibitior [452]) [452, 456] indirubin-3’-monoxime ( inhibition of AMPK, GSK3b, S6K1, PKBa, PDK1, MAPKAP-K2 [413]; inhibition of CK1 and MAPKAP-K1a [413]) [413] kenpaullone ( slight inhibition of AMPK, GSK3b, PKBa, PDK1 [413]; slight inhibition of MAPKAP-K1a [413]) [413] lithium ( after inhibition of GSK-3 in cortical neurons, the splicing factor SC35 is nuclearly redistributed and enriched in nuclear speckles and colocalizes with the kinase [430]) [430, 434] purvalanol ( inhibition of AMPK, slight inhibition of GSK3b and S6K1, PKBa [413]; slight inhibition of CK1 and MAPKAP-K1a [413]) [413] roscovitine ( inhibition of CK1 and MAPKAP-K1a [413]; slight inhibition of PKBa and MAPKAP-K2 [413]) [413] Additional information ( no inhibition by cAMP [395,401]; cGMP, heat-stable inhibitor of cAMP-dependent protein kinase [401]; heparin, Ca2+ /calmodulin, phosphatidylserine and/or diolein (in the absence or presence of Ca2+ ) [398,401,402]; protein inhibitor of cAMP-dependent protein kinase (protamine as substrate), monoclonal antibodies to catalytic domain of protein kinase C from rat brain [398]; EGTA, calmodulin [401,402]; protein phosphatase 2B, 2C, catalytic subunit of protein phosphatase 1, protein tyrosine phosphatases (PTPase 1B and T-cell PTPase), pyruvate dehydrogenase phosphatase [403]; enzyme expression level is decreased under low pH and heat shock conditions [421]; GSK3 phosphorylation and inhibition by PKB protein kinase at S21 and S9 [414]; no inhibition of most serine/threonine kinases by roscovitine [413]; no inhibition of S6K2 by wortmannin or rapamycin, S6K2 phosphorylation by MEK of S410, S417, and S423 of the autoinhibition domain leads to inhibition of the enzyme [415]; ORF47 kinase is resistant to heparin, host cell pretreatment prior to virus infection with d,l-a-difluoromethylornithine reduces intracellular polyamine concentration and thus inhibits the viral enzyme ORF47 [437]; phosphorylation at Ser9 by PKGI and PKGII inhibits the glycogen synthase kinase 3 [434]; phosphorylation of GSK3b at S9 inhibits the enzyme [427, 432, 433]; PKN possesses an autoinhibitory site at the C-terminal C2 domain, which also binds activating arachidonic acid [423]; the chromophoric domain light-oxygen-voltage-sensing 2 LOV2 of phototropin acts as an intramolecular inhibitor to substrate protein phosphorylation [447]; the pleckstrin homology PH domain is a negative regulator of PKD2 [440]; WKN4 possesses an autoinhibitory domain, residues 444-518, the isolated autoinhibitory domainin 1-443 inhibits WKN1 [410]; CDPKs contain an autoinhibitory junction region whose calcium-dependent interaction with the tethered regulatory calmodulin-like domain activates the catalytic kinase domain [466]; in vivo inhibition strategies in the mouse model, valida57

Non-specific serine/threonine protein kinase

2.7.11.1

tion of CK2 targets identification of bona fide targets, overview [456]) [395, 398, 401, 402, 403, 410, 413, 414, 415, 421, 423, 427, 432, 433, 434, 437, 440, 447, 456, 466] Cofactors/prosthetic groups 3-phosphoinositide ( enzyme is dependent on [26, 34]) [26, 34] ATP ( binding site structure [409]; binding site structure involving Lys233 [450]; binding structure involving residues K168, S53, and D166, overview [451]; dependent on, cannot be substituted by other nucleotide triphosphates like CTP or GTP [420]) [409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 448, 449, 450, 451, 452, 453, 455, 456, 457, 458, 460, 461, 464, 465, 466, 467, 468, 469, 471] FMN ( apoprotein noncovalently binds FMN to form the holoprotein nph1 [56]) [56] flavin ( flavoprotein [58]; LOV1 and LOV2 may be flavinbinding domains that regulate kinase activity in response to blue light-induced redox changes [58]) [57,58] Activating compounds 2-Mercaptoethanol ( activation, can replace DTT [407]) [407] 3’,5’-AMP ( not [395, 396, 400, 401]; i.e. cAMP, activation (cAMP-dependent kinase) [399]; i.e. cAMP, activation [397,401,408]) [395, 396, 397, 399, 400, 401, 407, 408] 5’-AMP ( stimulates [185, 187]; activates [185,197]) [185, 187, 197] AMP ( dependent on AMP [69]) [69] arachidonic acid ( activates in vitro, binds at the autoinhibitory site at the C-terminal C2-domain [423]; activates in vitro, binds at the autoinhibitory site at the C-terminus [423]) [423] cardiolipin ( activates isozyme PKNa [423]) [423] DTT ( activation [400]) [400] FRAT-2 ( i.e. frequently rearranged in advanced T-cell lymphoma protein 2, activates phosphorylation of primed sites of tau protein about 8fold, no effect on unprimed site phosphorylation [433]) [433] fatty acids ( activate PKN1 [458]) [458] heparin ( with the peptide substrate DDDDVASLPGLRRR, 4-5fold activation of recombinant casein kinase I d, half-maximal activation at 0.0095 mg/ml. A truncated form of casein kinase I d, lacking the COOH-terminal 111 amino acids, is no longer activated by heparin [245]; activation with peptide substrate [247]) [245, 247] insulin ( activated endogenous protein kinase B a1 2fold in L6 myotubes, while after transfection into 293 cells PKBa is activated 20fold and 50fold in response to insulin and IGF-1 respectively. In both cells, the 58

2.7.11.1

Non-specific serine/threonine protein kinase

activation of PKBa is accompanied by its phosphorylation at Thr308 and Ser473 [47]) [47] KbpA ( an AfsK-binding protein activates and modulates the enzyme activity [436]) [436] linoleic acid [423] lysophosphatidic acid ( activates isozyme PKNa [423]) [423] lysophosphatidylinositol ( activates isozyme PKNa [423]) [423] PDK1 interacting fragment PIF ( required for activity of PDK1 [414]) [414] phorbol esters ( bind to the cysteine-rich domain C1b [440]) [440] phosphatidylcholine ( stimulation [196]) [196] phosphatidylinositol ( stimulation [196]) [196] phosphatidylinositol 4,5-bisphosphate ( activates isozyme PKNa [423]) [423] putrescine ( aliphatic, positively charged polyamine, required for in vitro activity of ORF47, polyamine depletion leads to 80% reduced activity [437]) [437] Rac1 ( GTPase, binds and activates PKN [423]) [423] Rac2 ( GTPase, binds and activates PKN [423]) [423] Rho ( small GTPase, binds and activates PKN [423]; activates PKN1 [458]) [423, 458] Rho1 ( small GTPase, binds GTP-dependently and activates PKN [423]) [423] RhoA ( small GTPase, binds to the ACC domain and activates isozymes PKNa and PRK2/PKNg/PAK-2 [423]; small GTPase, binds to the ACC domain of PKNa forming a catalytic active site [423]) [423] Serum ( ,activation of Akt is associated with tyrosine phosphorylation of Akt [37]) [37] spermidine ( aliphatic, positively charged polyamine, required for in vitro activity of ORF47, polyamine depletion leads to 80% reduced activity [437]) [437] spermine ( aliphatic, positively charged polyamine, required for in vitro activity of ORF47, polyamine depletion leads to 80% reduced activity [437]) [437] gastrin ( bind to the cysteine-rich domain C1b, activates, triggers nuclear accumulation of PPKD2 in AGS-B cancer cells [440]) [440] kenpaullone ( slight activation of S6K1 [413]) [413] neurabin ( by way of its PDZ domain, the neuronal-specific neurabin may target p70(S6k) to nerve terminals [65]) [65] pervanadate ( activation of Akt is associated with tyrosine phosphorylation of Akt [37]) [37] phosphatidylinositol 3,4,5-trisphosphate ( activates isozyme PKNa [423]; required by PDK1 for phosphorylation and activation of PKB [414]) [414, 423] protein 14-3-3 ( a dimeric scaffold protein, protein is absolutely required for connection by simultaneous binding of glycogen synthase kinase-3 59

Non-specific serine/threonine protein kinase

2.7.11.1

b to tau within a brain microtubule-associated tau phosphorylation complex [424]) [424] small GTPase Rho ( small GTPase, binds and activates PKN [423]) [423] Additional information ( activation of serum-regulated and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 and PDK2 [42]; activation of SGK by IGF-1 or hydrogen peroxide is initiated by a PtdIns(3,4, 5)P3-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the PtdIns(3,4,5)P3-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1 [43]; PKBa becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s) [47]; transcriptionally regulated by serum and glucocorticoids in mammary epithelial cells [92]; activated in response to ionizing radiation [145]; Chk2 is rapidly phosphorylated and activated in response to replication blocks and DNA damage, the response to DNA damage occurrs in an ataxia telangiectasia mutated ATM-dependent manner [148]; the enzyme is directly activated by GTP-Rac1 or GTP-Cdc42 [290]; no activation by cGMP [401]; activation loop structure [409]; activation loop structure and conformation, WNK1 performs autophosphorylation at S382 for activation [450]; autophosphorylation patterns for activation of PknB, PknD, PknE, and PknF, e.g. PknB phosphorylation at Thr171 and Thr173 of the activation loop and at Thr294 of the juxtamembrane domain are necessary for activity [412]; forskolin has no effect on GSK-3 [438]; insulin induces the phosphorylation of TSSK3, TSSK3 activation by phosphoinositide-dependent kinase-1 PDK1-dependent signalling, phosphorylation at Thr168 activates [420]; insulin stimulates GSK3 phosphorylation at Ser21 and Ser9 by PKB protein kinase [414]; NDR1 is activated by phosphorylation at S281, T444, and T74 [428]; no activation by calcium/phosphatidylserine/diolein, phosphorylation of PKN is required for full activation [423]; NTHK2 is an inducible enzyme [446]; phosphorylation of PKN is required for full activation [423]; PKD2 is activated via G protein-coupled receptors [440]; RSK1 stimulation by EGF via CREB, a cAMP-response element-binding protein, autophosphorylation at Ser363 and Ser380 of RSK1 and membrane translocation are required for full RSK1 activation [426]; S6K is activated by phosphorylation at Thr468 during seed germination, the process can be induced by an insulin signaling transduction pathway and blocked by rapamycin in germinating axes [417]; S6K2 is phosphorylated at T228, T338, and S370 leading to activation, T338 plays the key role [415]; tau becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain [431]; WKN4 needs to be activated for activity [410]; CDPKs contain an autoinhibitory junction region whose calcium-dependent interaction with the tethered regulatory calmodulin-like domain activates the catalytic kinase domain, activation mechanism of CDPK, overview [466]; expression of the intestinal inflam60

2.7.11.1

Non-specific serine/threonine protein kinase

mation-associated colonic epithelial SPAK isozyme is upregulated by the proinflammatory cytokine IFN-g, but not by TGF-b and TNF-a, overview [455]; RSK2 is activated by phosphorylation by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones [457]; splicing variant TSSKa is stimulated at the CREB/CRE responsive pathway [464]; splicing variant TSSKb is not stimulated at the CREB/CRE responsive pathway [464]; splicing variant TSSKd is not stimulated at the CREB/CRE responsive pathway [464]; splicing variant TSSKg is not stimulated at the CREB/ CRE responsive pathway [464]) [42, 43, 47, 92, 145, 148, 290, 401, 409, 410, 412, 414, 415, 417, 420, 423, 426, 428, 431, 438, 440, 446, 450, 455, 457, 464, 466] Metals, ions Ca2+ ( dependent on [466]; enzyme is dependent on Ca2+ [183,184]; autophosphorylation activity is repressed upon addition of Ca2+ /calmodulin [116]; activity is dependent on [160]; enzyme contains a neural visinin-like calcium-binding domain [175]; 3-6fold stimulation [196]; N-terminal Ca2+ , calmodulin-dependent protein kinase sequence [209]; can partly substitute for Mn2+ , activates [448]; can substitute for Mn2+ in serine/threonine kinase activity [446]; has regulatory function for NDR protein kinase, mechanism, overview [428]) [116, 160, 175, 183, 184, 196, 209, 428, 446, 448, 466] Co2+ ( not [407]; requirement, 1-10 mM, can replace Mg2+ to some extent, inhibits above 30 mM [396]) [396, 407] High ionic strength ( activation, e.g. NaCl, NH4 Cl, KCl, sodium acetate, essential for maximal phosphate incorporation with protamine as substrate, histone phosphorylation is suppressed [407]) [407] Mg2+ ( activates [448]; requirement [395, 396, 399, 401, 407]; activation [400]; 10 mM [400]; reaction prefers Mn2+ to Mg2+ [210]; , autophosphorylation when incubated with ATP and Mg2+ [247]; inhibits above 30 mM [396]; Km -values: 1.8 mM (cytosolic kinase) and 0.04 mM (mitochondrial kinase) [401]; 1 mM (eIF-4E as substrate) [405]; 1.5 mM (mitochondrial kinase) [398, 401]; 10 mM (cytosolic kinase) [401]; binding site structure [409]; Mg2+ or Mn2+ , dependent on [421]) [210, 247, 395, 396, 398, 399, 400, 401, 402, 405, 407, 409, 410, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 426, 427, 428, 429, 430, 431, 432, 433, 434, 436, 438, 439, 440, 441, 443, 447, 448, 449, 450, 451, 453, 455, 456, 457, 458, 464, 465, 466, 467, 468, 471] Mn2+ ( required [331]; can replace Mg2+ to some extent [396]; reaction prefers Mn2+ to Mg2+ [210]; inhibits above 30 mM [396]; can replace Mg2+ with 20% efficiency [402]; requirement, 1-10 mM [396]; can replace Mg2+ with 8% efficiency [407]; dependent on, required for serine/threonine kinase activity [446]; Mg2+ or Mn2+ , dependent on [421]; preferred

61

Non-specific serine/threonine protein kinase

2.7.11.1

cation, optimal at 10 mM [420]; preferred cation, StkP [419]; strictly required, activates [448]) [210, 331, 396, 402, 407, 412, 419, 420, 421, 425, 426, 446, 448, 449, 452] Zn2+ ( bound to the zinc finger core at residues 105-147 and coordinated by Cys109, Cys114, Cys137 ans Cys140, of the b subunit dimer [470]) [470] Additional information ( no activation by Ca2+ or Zn2+ [407]; divalent cations are required for activity [420]; no activity with cations other than Mg2+ or Mn2+ [421]; no activity with Mg2+ alone, no activity with Cu2+ or Zn2+ [448]; the enzyme shows Mn2+ dependent serine/threonine kinase and Ca2+ -dependent histidine kinase, EC 2.7.13.3, activities [446]) [407, 420, 421, 446, 448] Specific activity (U/mg) 0.04 ( fusion of an amino-terminally truncated AK1 to the Cterminus of glutathione S-transferase, histone III as substrate [196]) [196] 0.07 [407] 3 ( substrate: RRRDDDSDDD [260]) [260] 5.11 [398] 7.53 [402] 17.38 [401] Additional information ( lamin disassembly assay, overview [468]) [408, 430, 468] Km-Value (mM) 0.005 (ATP) [396] 0.0093 (Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala) [176] 0.145 (myelin basic protein, pH 7.4, 30 C, recombinant TSSK3 [420]) [420] Ki-Value (mM) 0.00002 (ellagic acid) [456] 0.00004 (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole) [456] 0.000077 (TBCA) [456] 0.00017 ((5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid) [456] 0.0004 (4,5,6,7-tetrabromo-1H-benzotriazole) [456] 0.0007 (4,5,6,7-tetrabromobenzimidazole) [456] 0.00074 (Apigenin) [456] 0.00185 (emodin) [456] 0.0045 (DRB) [456] pH-Optimum 5.7-9 ( broad, protamine as substrate [401]) [401] 6-8 [396] 6-9 [402] 6.1-8.2 ( protamine as substrate [398]) [398] 6.7 ( bovine serum albumin as substrate [398]) [398]

62

2.7.11.1

Non-specific serine/threonine protein kinase

7 ( assay at [412,417,425]; catalytic unit of cAMPdependent protein kinase [395]) [395, 412, 417, 425] 7-7.6 ( eIF-4E as substrate [405]) [405] 7.2 ( assay at [415,439]) [415, 439] 7.4 ( assay at [418, 421, 430, 431, 438, 440, 443, 448, 452]; TSSK3 [420]) [418, 420, 421, 430, 431, 438, 440, 443, 448, 452] 7.5 ( assay at [413, 416, 422, 426, 427, 428, 432, 433, 449, 451, 453, 455, 464, 465]; PDK1 assay at [420]) [413, 416, 420, 422, 426, 427, 428, 432, 433, 449, 451, 453, 455, 464, 465] 7.6 ( assay at [446,450]) [446, 450] 7.8 ( assay at [447]) [447] 7.8-8 ( at high salt concentration [407]) [407] 8 ( assay at [410]; cAMP-independent protamine kinase [395]) [395, 400, 410] 9 ( assay at [468]) [468] pH-Range 3-9 ( StkP [419]) [419] 5.8-8.8 ( about 85% of maximal activity at pH 5.8 and about 80% of maximal activity at pH 8.8, protamine as substrate [398]) [398] 6.4-8.6 ( about half-maximal activity at pH 6.4 and about 65% of maximal activity at pH 8.6, at high salt concentration [407]) [407] 6.6-6.8 ( about half-maximal activity at pH 6.6 and pH 6.8, bovine serum albumin as substrate [398]) [398] 6.8-8.2 ( TSSK3 shows a broad range pH-optimum [420]) [420] Temperature optimum ( C) 20 ( assay at [407]) [407] 21 ( assay at room temperature [413]) [413] 22 ( assay at [429, 446]; assay at room temperature [425, 453]) [425, 429, 446, 453] 24 ( assay at [417]) [417] 30 ( assay at [396, 398, 399, 401, 402, 403, 404, 405, 410, 412, 415, 416, 426, 427, 428, 431, 432, 433, 438, 439, 440, 447, 449, 455, 457, 464, 465, 468]; assay at, in vitro [418]; TSSK3, and phosphoinositide-dependent kinase-1 assay at [420]) [396, 398, 399, 401, 402, 403, 404, 405, 410, 412, 415, 416, 418, 420, 426, 427, 428, 431, 432, 433, 438, 439, 440, 447, 449, 455, 457, 464, 465, 468] 33 ( assay at with histone as substrate [399]) [399] 37 ( assay at [397, 421, 430, 443, 448, 451, 452]; assay at, in vivo [418]) [397, 418, 421, 430, 443, 448, 451, 452]

63

Non-specific serine/threonine protein kinase

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4 Enzyme Structure Molecular weight 30000 ( gel filtration [396]) [396] 34000 [221] 43000 ( gel filtration [401]) [247, 401] 45000 ( gel filtration [398,402]) [398, 402] 45500 [247] 49700 [247] 62000 [225] 81000 ( cAMP-independent protamine kinase, gel filtration [399]) [399] 105000 [237] 202000 ( recombinant phosphorylated, full-length enzyme, native PAGE [453]) [453] Additional information ( complete nucleotide sequence of vmil [4]; c-Rmil gene encodes two proteins of 94000 Da and 95 000 Da, resulting from an alternative splicing mechanism [21]; molecular weights of several cAMP-dependent protamine kinases [399]) [4, 21, 399] Subunits ? ( x * 73132, calculation from nucleotide sequence [5]; x * 52554, calculation from nucleotide sequence [80]; x * 59109 [67]; x * 56160 [66]; x * 49000 [95]; x * 70000 [97, 331]; x * 40100, calculation from nucleotide sequence [172]; x * 58000 [166]; x * 41200, calculation from nucleotide sequence [172]; x * 57175, calculation from nucleotide sequence [160]; x * 72645, calculation from nucleotide sequence [196]; x * 47300, calculation from nucleotide sequence [238]; x * 49121, calculation from nucleotide sequence [245]; axbx, x * 39833 + x * 24700, calculation from nucleotide sequence [272]; x * 29000 + x * 42000 [257]; x * 102000 [296]; x * 52000, calculation from nucleotide sequence [344]; x * 86000, calculation from nucleotide sequence [345]; x * 58370, calculation from nucleotide sequence [377]; x * 64000, calculation from nucleotide sequence [371]; x * 126983, calculation from nucleotide sequence [392]; x * 39200, PK1, calculated from amino acid sequence [449]; x * 39500, PK2, calculated from amino acid sequence [449]; x * 61805, amino acid sequence calculation, x * 55000, recombinant cytoplasmic domain, SDS-PAGE, x * 8000085000, recombinant full-length PknI, migrates probably as a doublet, SDSPAGE [448]; x * 62000, S6K, SDS-PAGE [417]; x * 97000, recombinant GST-fusion PknH, SDS-PAGE, x * 68000, non-tagged PknH, SDS-PAGE [421]; x * 52600, about, sequence calculation, x * 53000, in vitro expressed enzyme, SDS-PAGE, x * 60000, about, enzyme expressed in Caco-2BBE cells, SDS-PAGE [455]) [5, 66, 67, 80, 95, 97, 160, 166, 172, 196, 238, 245, 257, 272, 296, 331, 344, 345, 371, 377, 392, 417, 421, 448, 449, 455]

64

2.7.11.1

Non-specific serine/threonine protein kinase

dimer ( homodimerization in vivo, recombinant enzyme [453]) [453] monomer ( 1 * 45000, SDS-PAGE [398, 401, 402, 404]; crystal structure, modelling of the activated monomer to account for the intra-molecular recognition of the two domains, overview [466]) [398, 401, 402, 404, 466] tetramer ( 2 * a or 2 * a + 2 * b [261]; heterotetramer, the dimer of dimers shows a a2 b2 structure, the a subunit CK2a ist a catalytic heterodimer, the b subunit CK2b is a regulatory compact globular homodimer, but also functions as a multisubstrate docking platform for several other binding partners, CK2b contains a Zn2+ bound to the zinc finger core at residues 105-147, the N-terminus of CK2b contains ligand and protein interaction sites, e.g. the juxta-dimer interface region containing residues Asp105, Arg111, Glu115, Lys134, Asp142, and Lys147, determination by the optimal docking area method, organization and interaction motifs of CK2b, overview [470]) [261, 470] Additional information ( p70 S6 kinase a I with a calculated MW of 58946 Da consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the p70 S6 kinase a II with a calculated MW of 56153 Da. Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. The slightly longer a I polypeptide exhibits anomalously slow mobility on SDS-PAGE, migrating at an apparent MW of 90000 Da probably because of the presence of six consecutive Arg residues immediately following the initiator methionine [72]; 3 polypeptides detected by SDS-PAGE: a - 45000 Da, a - 40000 Da, b - 26000 Da [260]; the b-subunit Ckb1 is a positive regulator of the enzyme activity, it plays a role in mediating the interaction of casein kinase II with downstream targets and/or with additional regulators [270]; Saccharomyces cerevisiae contains two distinct a subunits which must be encoded by separate genes, a 42000 Da polypeptide and a 35000 Da polypeptide. The 41000 Da is the b subunit, the 32000 Da polypeptide may be the b’-subunit [268]; Ire1p oligomerizes in response to the accumulation of unfolded proteins in the ER [390]; enzyme docking sequences and targeting, overview [414]; isozyme structure analysis, PKN kinase contains a catalytic domain homologous to that of protein kinase C, as well as a unique regulatory region containing antiparallel coiled-coil domain, the ACC domain, and an autoinhibitory C2 domain binding activators [423]; kinase domain structure of WNK1 [450]; phototropin has 2 chromophoric domains, the light-oxygen-voltage-sensing 1 and 2, i.e. LOV1 and LOV2, in the N-terminal half, and a Ser/Thr kinase motif in the C-terminal half [447]; PknB, PknD, PknE, and PknF possess an N-terminal kinase domain, a juxtamembrane domain of varying length, and an activation loop [412]; PknH contains 11 subdomains, schematic structure, overview [421]; SSTK is a small enzyme consisting only of the C- and N-terminal lobe of a protein kinase catalytic do65

Non-specific serine/threonine protein kinase

2.7.11.1

main [442]; the kinase domain is tightly connected to a wHTH domain [451]; TSSK3 contains a T-loop structure [420]; outside of the kinase domain, CKI family members fall into subfamilies that have little homology to each other and differ in the length and amino acid sequence of their N- and C-terminal extensions [460]; splicing variant b has an insertion of ten amino acid residues, RLTPSLSAAG, in region VIb of the HisArgAsp domain [464]; splicing variant d has an insertion of ten amino acid residues, RLTPSLSAAG, in region VIb of the HisArgAsp domain [464]; splicing variant g has an insertion of ten amino acid residues, RLTPSLSAAG, in region VIb of the HisArgAsp domain [464]; the autoinhibitory junction region bound to the calmodulin-like domain and Ca2+ forms symmetric dimers with domain-swap interactions, in which the J region of one protomer interacts extensively with the carboxy-terminal EF- hand domain, C-lobe, of the partner protomer [466]; TSSK5a has no insertion of ten amino acid residues in region VIb of the HisArgAsp domain [464]) [72, 260, 268, 270, 390, 412, 414, 420, 421, 423, 442, 447, 450, 451, 460, 464, 466] Posttranslational modification phosphoprotein ( autophosphorylation [281,311,313]; GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro [19]; phosphorylation is an essential regulatory feature of LIM kinase, threonine 508 and the adjacent basic insert sequences of the activation loop are required for this process [12]; Akt3 is phosphorylated in response to insulin [52]; two splice variants of protein kinase B g have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site Ser472 in the carboxyl-terminal hydrophobic domain [51]; regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain, Thr305 and Ser472, phosphorylation of both sites is required for full activity [54]; Akt-3 also possess a C-terminal tail that contains a phosphorylation site Ser472 thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appears to contribute to the activation of Akt-3 [53]; in SKOV3 ovarian carcinoma cells that exhibit high basal levels of Akt activity, Akt is tyrosine-phosphorylated in the basal state, and this phosphorylation is further enhanced by both pervanadate and insulin-like growth factor-1, Tyr474 is the site of tyrosine phosphorylation [37]; regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain, Thr305 and Ser472 [51]; two splice variants of protein kinase B g have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site Ser472 in the carboxyl-terminal hydrophobic domain, activation of PKB g 1 requires phosphorylation at a single regulatory site Thr305 [51]; modified by phosphorylation [145]; phosphorylation of Dun1 increases in response to DNA

66

2.7.11.1

Non-specific serine/threonine protein kinase

damage in a Dun1-dependent manner, suggesting an increase in autophosphorylation activity [171]; Chk2 is rapidly phosphorylated and activated in response to replication blocks and DNA damage [148]; Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine [212]; recombinant MAPKAP kinase 2 is phosphorylated and activated by MAP kinase in vitro [176]; key mode of Cdc2-cyclin B regulation is the inhibitory phosphorylation of Cdc2 on Tyr15 [189]; the 41000 Da polypeptide of the enzyme and the 32000 Da polypeptide both incorporate phosphate during autophosphorylation [268]; phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein [260]; phosphoserine is the major phosphorylated amino acid [311]; autophosphorylation on Thr [331]; phosphorylated mainly at Thr [337]; phosphorylated at both Ser and Thr residues [334]; phosphorylated mainly at Thr and slightly at Ser [337]; self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK [338]; autophosphorylation only at Ser [335]; the activity is potentially regulated by phosphorylation of the CDC7 protein [374]; Ire1p oligomerizes in response to the accumulation of unfolded proteins in the ER and is phosphorylated in trans by other Ire1p molecules as a result of oligomerization [390]; AfsK is autophosphorylated at serine and threonine residues leading to its activation, PkaG and SCD10.09 are autophosphorylated at serine and threonine residues [436]; autophosphorylation of PK1 [449]; autophosphorylation of PK2 [449]; autophosphorylation of recombinant GST-CK2 catalytic subunit [435]; autophosphorylation of WKN1 and WKN4 [410]; e.g. phosphorylation of isozyme PRK2/PKNg/PAK-2 at Ser473 and Thr308, phosphorylation is required for full activation [423]; insulin-induced GSK3 phosphorylation and inhibition by PKB protein kinase at S21 and S9, phosphorylation at Thr308 and Ser473 of PKB leading to activation of PKB by PDK1 [414]; mapping of autophosphorylation sites, NDR1 is activated by phosphorylation at S281, T444, and T74 dependent on intracellular Ca2+ [428]; phosphorylation at Ser9 by PKGI and PKGII inhibits the glycogen synthase kinase 3 inducing dephosphorylation of C/EBPb important in regulation of gene expression during cell proliferation, differentiation, and apoptosis [434]; phosphorylation is required for full activation [423]; phosphorylation of GSK3b at S9 inhibits the enzyme [427,432,433]; phosphorylation of S6K2 is regulated via interdependent steps, overview, S6K2 is phosphorylated at T228 by PDK1 and at T338, playing the key role, and at S370, leading to activation, phosphorylation of S410, S417, and S423 of the autoinhibition domain by MEK leads to inhibition of the enzyme [415]; phototropin performs autophosphorylation [447]; PKD2 performs autophosphorylation [440]; PknB is autophosphorylated [425]; PknB, PknD, PknE, and PknF are autophosphorylated at multiple sites, phosphorylation patterns, overview, the enzymes are dephosphorylated by the cognate mycobacterial phosphatase PstP [412]; PknH performs autophosphorylation, mutant K45M is inactive [421]; Rio2 performs autophosphorylation [451]; RSK1 is autophos67

Non-specific serine/threonine protein kinase

2.7.11.1

phorylated at Ser363 and Ser380 [426]; StkP performs autophosphorylation [419,453]; the NTHK2 kinase performs serine/threonine autophosphorylation [446]; TSSK3 performs autophosphorylation, TSSK3 is phosphorylated at Thr168 of the T-loop by recombinant Myc-tagged catalytic subunit of phosphoinositide-dependent kinase-1 leading to activation of TSSK3, TSSK3 can also be phosphorylated by cAMP-dependent protein kinase PKA, EC 2.7.11.11, but not by protein kinase B [420]; WNK1 performs autophosphorylation at S382 for activation [450]; phosphorylation at Thr774 of the activation loop is required for activity in vivo, PKN1 is phosphorylated during Salmonella enterica infection independent of SspH1 [458]; RSK2 is activated by phosphorylation by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones [457]) [12, 19, 37, 51, 52, 53, 54, 145, 148, 171, 176, 189, 212, 247, 260, 268, 281, 311, 313, 331, 334, 335, 337, 338, 374, 390, 410, 412, 414, 415, 419, 420, 421, 423, 425, 426, 427, 428, 432, 433, 434, 435, 436, 440, 446, 447, 449, 450, 451, 453, 457, 458] proteolytic modification ( PKNa is cleaved by caspase-3 or related proteases in apoptotic Jurkat and U-937 cells contributing to signal transduction [423]) [423]

5 Isolation/Preparation/Mutation/Application Source/tissue 3T3 cell ( isozyme PKNa [423]) [423] 3T3-L1 cell ( isozyme PKNa [423]) [423] A2780-DX3 cell ( lymphoid cell line, specific expression of isozyme PRK2/PKNg/PAK-2 [423]) [423] Dami cell ( leukemia cell line, cells undergo terminal differentiation after treatment with phorbol ester, hBub1 expression in this cell line is down-regulated rapidly [358]) [358] HEK-293 cell [429, 454] HeLa cell ( isozymes PRK2/PKNg/PAK-2, and PKNb [423]) [361, 423, 437, 459, 469] Hep-3B cell [418] Hep-G2 cell [422] JB6 Cl41 cell [457] JURKAT cell ( isozyme PRK2/PKNg/PAK-2 [423]) [423] K-562 cell ( chromic myelogenous leukemia cell line, isozyme PKNb [423]) [423] MEF cell [457] MeWo cell ( melanoma cell line [437]) [437] Neuro-2A cell [438] SH-SY5Y cell ( neuroblastoma cell line [443]) [443] SKOV-3 cell [37] T-lymphocyte [261] U-937 cell ( isozyme PKNa [423]) [423] 68

2.7.11.1

Non-specific serine/threonine protein kinase

UMR-106 cell ( primary osteoblast cells [434]) [434] WE-480 cell ( colorectal adenocarcinoma cell line, isozyme PKNb [423]) [423] adipose tissue ( very low expression level of WNK1 [416]) [416] axe ( embryonic, germinating, enzyme activity increases during seed germination due to increased enzyme activation by phosphorylation at Thr468 [417]) [417] brain ( fetal and adult [132]; fetal [133]; in adult brain Kiz-1 is expressed exclusively in neurons, not in astrocytes or oligodendrocytes. In the developing embryo, Kiz-1 is expressed in all tissues [16]; the cells which strongly express the sgk gene are in the deep layers of the cortex and in the corpus callosum. It is likely that the sgk transcript is expressed by oligodendrocytes after brain injury. Neurons in layers I and II of the cortex, lateroposterior and laterodorsal thalamic nucleus, and ventral posterolateral and posteromedial thalamic nucleus strongly express sgk mRNA at postnatal day 1 and day 7, but these neurons show no expression in fetal or adult brain. Induction of sgk gene may be associated with a series of axonal regenerations after brain injury, and in addition, the sgk gene may also play important roles in the development of particular groups of neurons in the postnatal brain [94]; different expression of the myotonin protein kinase gene in discrete areas of brain [96]; four splicing variants of KIAA0369: KIAA0369-AS-type A, short version, KIAA0369-AL-type A, long version, KIAA0369-BS-type B, short version, and KIAA0369-BL-type B, long version. KIAA0369-B, which lacks the DC domain and maintains the kinase domain, is expressed in adult as well as fetal brain, but the variants that included the DC domain, KIAA0369-A, is expressed predominantly in fetal brain. In the adult brain, KIAA0369 is expressed in all 15 different regions examined, more intensely in cerebral cortex, occipital pole, frontal lobe, amygdala, and hippocampus, and less intensely in corpus callosum and thalamus [134]; preferentially expressed in brain and pancreas [281]; epithelial cells of brain [283]; distribution in brain regions, overview [441]; expression of CK1e-1, CK1e-2, CK1e-3 [444]; isozyme PKNa [423]; very low expression level of WNK1 [416]; from Alzheimer patients, isozyme CKId [465]) [16, 19, 20, 43, 50, 94, 96, 98, 106, 111, 132, 133, 134, 135, 141, 185, 209, 231, 247, 281, 283, 284, 290, 291, 309, 395, 400, 409, 416, 423, 424, 427, 430, 431, 432, 433, 438, 441, 444, 445, 465, 467] brain cortex [430] cell culture ( HeLa cells [86, 117, 140, 306]; hepatoma cell line [45]; HepG2 cell [14]; cell lines MCF- 7 and WI38 [49,76]; SKOV3 ovarian carcinoma cell [37]; ovarian carcinoma cell lines [75]; Con8.hd6 rat mammary tumor cell line [95]; rat BUB1 mRNA accumulation correlates with the proliferation status of cells in culture [355]) [14, 37, 45, 49, 75, 76, 86, 95, 117, 140, 306, 355, 460] central nervous system [284] 69

Non-specific serine/threonine protein kinase

2.7.11.1

cerebellum ( expression of CK1e-2, CK1e-1 [444]) [444, 445] cerebral cortex ( expression of CK1e-3 [444]) [444] choroid plexus [283] coleoptile [184] colon ( epithelium, inflamed and non-inflamed tissue [455]) [455] commercial preparation ( purified recombinant GSK-3b [430]) [430] embryo ( embryo kidney 293 cells [92]; at early cleavage-stages Dmnk transcripts are transiently present throughout the embryo, but become restricted to the posterior pole and then to the newly-formed primordial germ cells by the blastoderm stage. The transcripts are sustained in the pole cells during gastrulation [136]; present during early embryogenesis [241]; by embryonic day 15, the transcript is localized to cells that will eventually become exocrine in nature, high levels of expression in the choroid plexus, the developing myocardium, kidney, CNS, dorsal root ganglia, and testes [282]) [34, 92, 136, 210, 241, 257, 282, 284, 369, 417, 423, 460] epithelium ( transporting epithelial cells of brain [283]) [283, 423, 455] erythroleukemia cell [109] fibroblast ( IR fibroblasts [423]) [50, 355, 423, 457] gastrointestinal tract [367] heart ( very low expression level of WNK1 [416]; high expression level of splicing variant a of the testis specific serine/threonine kinase [464]; high expression level of splicing variant b of the testis specific serine/threonine kinase [464]; high expression level of splicing variant d of the testis specific serine/threonine kinase [464]; high expression level of splicing variant g of the testis specific serine/threonine kinase [464]) [50, 141, 185, 247, 416, 464] hematopoietic cell [319] hepatoma cell [68, 418, 422] hippocampus ( expression of CK1e-3, gyrus of the hippocampus [444]) [423, 431, 444] intestine [455] intestine-407 cell [458] keratinocyte ( isozyme PRK2/PKNg/PAK-2 [423]) [423] kidney ( cortex [401]; distal tubule and collecting duct [283]; expression of CK1e-3 [444]) [43, 111, 141, 185, 247, 283, 398, 401, 402, 403, 404, 405, 444] larva [257] leaf [435] liver ( not highly expressed [53]; expression of CK1e-1, CK1e-3 [444]; isozyme PRK2/PKNg/ PAK-2 [423]; very low expression level of WNK1 [416]) [43, 53, 66, 72, 141, 185, 247, 397, 408, 416, 423, 444] lung ( high activity [95]; expression of CK1e-1, CK1e-2 [444]) [50, 95, 141, 185, 247, 444] 70

2.7.11.1

Non-specific serine/threonine protein kinase

mature ovarian follicle [93] medulla oblongata ( CKIe-3, nucleus of the trapezoid body [444]) [444] melanoma cell [437] mitotic cell ( actively expressed in mitotically dividing cells [394]) [394] muscle ( vascular smooth muscle cells [256]) [247, 256] nervous system ( developing [17]; high expression levels in the spinal cord and the cranial nerve and dorsal root ganglia [19]) [17, 19, 284] neuron ( isozyme PKNa [423]) [206, 283, 423, 430, 433] oocyte ( isozyme PRK2/PKNg/PAK-2 [423]) [136, 258, 423] osteoblast [434] osteoclast ( cultured on ivory [123]) [123] ovary ( high activity [95]) [59, 95] pancreas ( preferentially expressed in brain and pancreas [281]; low expression level of WNK1 [416]) [43, 141, 281, 282, 416] pancreatic b cell line [281] petal [449] pistil [449] placenta [85, 141] pollen [449] pollen tub [449] prostate adenocarcinoma cell [459] prostate gland ( overexpression in malignant prostate glandular cells [459]) [459] prostate gland ventral lobe [251] root [184] salivary gland [283] schizont ( stage-specific expression in the parasite, in the decreasing order: trophozoite, trophozoite in ring form, schizont [215]) [215, 265] seed ( developing [183]) [183] skeletal muscle ( not highly expressed [53]) [53, 102, 141] somatic embryo [182] spermatid ( head of spermatid [442]) [442] spermatozoon ( transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide is present in mature spermatozoa [69]) [69] spinal cord [19, 284] spleen ( very low expression level of WNK1 [416]) [360, 416, 423, 464] stomach ( parietal cells [283]; expression of CK1e-3 [444]) [283, 444] 71

Non-specific serine/threonine protein kinase

2.7.11.1

testis ( most abundant in [442]; accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes, localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes [128]; transcribed most abundantly in testis [372]; testis-specific serine-threonine kinase 3, i.e. TSSK3 [420]; TSSK5, absolutely testis-specific [411]; WNK4, high expression level of WNK1 [416]; high expression level of splicing variant a of the testis specific serine/threonine kinase [464]; high expression level of splicing variant b of the testis specific serine/threonine kinase [464]; high expression level of splicing variant d of the testis specific serine/threonine kinase [464]; high expression level of splicing variant g of the testis specific serine/threonine kinase [464]) [34, 81, 128, 243, 245, 247, 260, 367, 372, 407, 411, 416, 420, 423, 442, 464] thymus ( high activity [95]) [95, 113, 231, 359, 423] thyroid gland [399] trophoblast ( giant cell [17]) [17] trophozoite ( stage-specific expression in the parasite, in the decreasing order: trophozoite, trophozoite in ring form, schizont [215]) [215] zona glomerulosa [283] Additional information ( SGK3 is expressed in all tissues examined, but SGK2 mRNA is only present at significant levels in liver, kidney and pancreas and, at lower levels, in the brain [43]; expressed widely [53]; present during early embryogenesis and in adult females [241]; bovine lymphocytes transformed by Theileria parva [265]; activity is negligible in liver and skeletal muscle [283]; the enzyme is expressed preferentially during sporulation [286]; hBub1 mRNA level is abundantly expressed in tissues or cells with a high mitotic index. The hBub1 protein level is low in G1 and remains relatively constant in S, G2, and M phases [358]; no expression in nondividing tissues [360]; actively expressed in mitotically dividing cells [394]; expressed in many normal tissues, but overexpressed in certain tumor types and all transformed cell lines examined. In some of the tumors tested, CDC7Hs expression correlates with expression of a proliferation marker, the histone H3 gene. In other cases, no such correlation was observed. CDC7Hs expression may be associated hyperproliferation in some tumors and neoplastic transformation in others [370]; developmental expression, overview [449]; enzyme expression level under stress conditions, overview [421]; isozymes show different tissue distribution [423]; PK1 is not expressed in other tissues, developmental expression in pollen, overview [449]; splicing form CK1e-1 shows tissue-specific expression pattern, overview [444]; splicing form CKIe2 shows tissue-specific expression pattern, overview [444]; splicing form CKIe-3 shows tissue-specific expression pattern, overview [444]; SSTK is ubiquitously expressed in human tissue [442]; ubiquitous ex72

2.7.11.1

Non-specific serine/threonine protein kinase

pression of isozyme PKNa, isozymes show different tissue distribution [423]; ubiquitous expression of isozyme PKNa, isozymes show different tissue distribution, PKNb is mainly expressed in cancer cells in adults [423]; elevated CK2 activity occurs with the malignant transformation of several tissues and is associated with aggressive tumor behaviour [456]; high CK2 activity in solid tumors [459]; no activity of the intestinal inflammation-associated colonic epithelial isozyme in liver, spleen, brain, prostate, and kidney [455]) [43, 53, 241, 265, 283, 286, 358, 360, 370, 394, 421, 423, 442, 444, 449, 455, 456, 459] Localization Golgi apparatus ( Brefeldin A-sensitive Golgi compartment [116]) [116] Golgi membrane ( coexpression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization [285]) [285] centromere ( mitotic, activation of the checkpoint results in the recruitment of Bub1 to centromeres, Bub1 is recruited to kinetochores during the early stages of mitosis. A pool of Bub1 remains centromere-associated at metaphase and even until telophase [362]) [362] centrosome ( presence in the centrosome appears to be enhanced during osmotic stress [116]) [116] chloroplast [447] cytoplasm ( in COS cells transfected with Kiz-1 complementary DNA and in the immortalized olfactory epithelial cells, Kiz-1 is found mainly in the cytoplasm, but in neurons of the adult brain, it resides also in the nucleus [16]; full-length SPAK is expressed in the cytoplasm in transfected cells [281]; in G2-arrested cells, dsk1 locates in the cytoplasm [311]; distributed diffusibly in cytoplasm in the mitotic phase [372]; Ire1p spans the ER membrane or the nuclear membrane with which the ER is continuous, with its kinase domain localized in the cytoplasm or in the nucleus [390]; the cytoplasmic C-terminal portion of the enzyme carries an essential protein kinase activity [391]; CK1e-1 [444]; CKa in normoxia [422]; enzyme contains a cytoplasmic domain [448]) [16, 116, 124, 233, 241, 281, 311, 319, 335, 372, 390, 391, 422, 426, 440, 444, 448] cytoskeleton ( Triton X-100-insoluble [283]) [283, 424] cytosol ( low activity [441]; two mitochondrial kinases I and II and a cytosolic kinase [401]; juxtanuclear, isozyme PKNa, no isozyme PKNb [423]) [140, 167, 257, 283, 395, 399, 401, 402, 403, 404, 405, 408, 423, 437, 441, 449] endoplasmic reticulum ( isozyme PKNa [423]) [423] endosome ( isozyme PKNa [423]) [423] kinetochore ( activation of the checkpoint results in the recruitment of Bub1 to centromeres, Bub1 is recruited to kinetochores during the early stages of mitosis. A pool of Bub1 remains centromere-asso-

73

Non-specific serine/threonine protein kinase

2.7.11.1

ciated at metaphase and even until telophase [362]; kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage [354]) [354, 357, 358, 361, 362] membrane ( bound to [225]; transmembrane protein [132]; phosphorylation of the hydrophobic motif at the extreme C terminus of PKB g may facilitate translocation of the kinase to the membrane [51]; the enzyme contains membrane localization signals [105]; distinct lateral and/or basal plasma membrane domains in different epithelial cell types [131]; membrane-associated protein, synaptic plasma membranes [209]; Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane [334]; the Pkn6-PhoA fusion protein in Escherichia coli has a single transmembrane domain with the N-terminal domain in the cytoplasm and the C-terminal domain outside the cytoplasmic membrane [335]; deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring [331]; a transmembrane enzyme [390,391]; Ire1p spans the ER membrane or the nuclear membrane with which the ER is continuous, with its kinase domain localized in the cytoplasm or in the nucleus [390]; the kinase contains a membrane-spanning domain [392]; mainly [441]; CK2b in hypoxia [422]; isozyme PKNa [423]; PknH is a transmembrane protein [421]; postnuclear fraction [423]; membrane anchored kinase domain [453]) [51, 105, 131, 132, 209, 225, 331, 334, 335, 338, 390, 391, 392, 412, 419, 421, 422, 423, 441, 448, 453] microsome ( glycosylated N-terminal portion is located inside microsomes [391]; microsomal kinase is a form of the cytosolic kinase [402]) [391, 402] microtubule [427, 430, 431, 432, 438, 441] mitochondrion ( two mitochondrial kinases I and II and a cytosolic kinase [401]) [398, 401] nuclear body ( HIPK2 colocalizes with Daxx in promyelotic leukemia nuclear bodies [418]) [418] nucleus ( predominantly localized in [370]; in neurons of the adult brain it resides in cytoplasm and also in the nucleus [16]; localized to the nuclei in a speckled pattern. Dmnk proteins become detectable in both somatic and germ line cell nuclei upon their arrival at the periplasm of the syncytial embryo, but then disappear from the somatic cell nuclei. Consistent with mRNA expression, Dmnk proteins in pole cell nuclei are sustained during gastrulation [136]; in a mutant corresponding to caspase-cleaved SPAK the enzyme is expressed predominantly in the nucleus [281]; mainly localized in the nucleus [317]; HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles [320]; in mitotically arrested cells, nuclear stain is intense, in wild-type cells, nuclear stain is seen only in mitotic cells [311]; overproduced Bub1 is 74

2.7.11.1

Non-specific serine/threonine protein kinase

found to localize to the cell nucleus [364]; the enzyme is preferentially present in [378]; the enzyme is localized primarily in nuclei in interphase [372]; Ire1p spans the ER membrane or the nuclear membrane with which the ER is continuous, with its kinase domain localized in the cytoplasm or in the nucleus [390]; GSK-3 after cell treatment with lithium [430]; isozyme PKNb [423]; SAST124 is specifically localized in the nuclei of cerebellar granule neurons [445]; immunohistochemic detection of CK2 catalytic domain CK2a, nuclear localization is associated with high-grade tumors and a poor prognostic factor, overview [459]) [16, 116, 136, 281, 306, 311, 317, 319, 320, 364, 370, 372, 378, 390, 423, 430, 440, 445, 457, 459] perinuclear space ( isozyme PKNb [423]) [423] plasma membrane ( membrane translocation is required for full RSK1 activation [426]) [232, 426] ribosome [79, 80, 415, 417] soluble [215, 395, 396, 398, 399, 401, 402, 403, 404, 405, 408] spindle ( throughout G2 Ark1 is concentrated in one to three nuclear foci that are not associated with the spindle pole body/centromere complex. Following commitment to mitosis Ark1 associated with chromatin and is particularly concentrated at several sites including kinetochores/centromeres. Kinetochore/centromere association diminishes during anaphase A, after which it is distributed along the spindle. The protein becomes restricted to a small central zone that transiently enlarges as the spindle extends [115]) [115] vesicle ( isozyme PKNa [423]) [423] Additional information ( treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus [92]; activity is excluded from the nucleus [167]; the enzyme is colocalized with the septins at the mother-bud neck [200]; localization pattern of dsk1 protein strikingly alters depending on cell cycle stages [311]; hBub1 protein colocalizes with a centromere-kinetochore antigen CREST in interphase, mitotic prophase, and nocodazole-treated cells [358]; nucleocytoplasmic shuttling regulated by a CRm-1-dependent mechanism, involving the C1b domain, and the C1a domain containing a nuclear export signal sequence [440]; PKNa translocates from the cytosol to the nucleus in response to various stresses, PRK2/PKNg/PAK-2 translocates from the cytosol to germinal vesicles during meiotic maturation in oocytes [423]; PknB, PknD, PknE, and PknF possess an N-terminal kinase domain, a juxtamembrane domain of varying length, and an activation loop [412]; subcellular localization of different epitope labeled forms of StkP, overview [453]) [92, 167, 200, 311, 358, 412, 423, 440, 453] Purification (recombinant wild-type and mutant GST-fusion TSSK3s by glutathione affinity chromatography, recombinant Myc-tagged catalytic subunit of phosphoinositide-dependent kinase-1 from human 293T cells) [420]

75

Non-specific serine/threonine protein kinase

2.7.11.1

(recombinant CK2 from insect Sf9 cells to homogeneity) [429] (recombinant Leu-Glu-His6-tagged GSK3b from Escherichia coli) [409] (recombinant wild-type and mutant GST-fusion NDR1 from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography) [428] [395] (PKN from testis to homogeneity by immuno affinity) [423] (WNK1 partially from testis, recombinant GST-fusion truncated WNK1 comprising residues 1-661 with claudin-4, wild-type and mutant oxidative stress response kinase 1 OSR1, STE/SPS1-related proline/alanine-rich kinase SPAK, and Na+ -K+ -2Cl- co-transporter-1-(1-260) construct NKCC1-(1-260) from Escherichia coli by glutathione affinity chromatography) [416] (partial) [408] (recombinant GST-fusion WNK4 and WNK1 kinases from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography) [410] (recombinant His-tagged low activity WNK1 kinase domain mutant S382A, comprising residues 194-483, wild-type and selenomethionine-labeled, from Escherichia coli by chelating affinity and ion exchange chromatography, and gel filtration) [450] (partial, several cAMP-dependent protamine and histone phosphorylating activities) [399] (partial) [396] (cytosolic) [401] (isozyme PKNa from brain membrane) [423] (microsomal) [402] (mitochondrial kinase) [398] [400] (recombinant GST-fusion LOV2 and kinase domain LV2-KD of phototropin from Escherichia coli by glutathione affinity chromatography) [447] (recombinant GST-fusion StkP from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, the GST-tag is cleaved off by factor Xa, no binding of His-tagged recombinant enzyme to a nickel chelating matrix can be achieved) [419] (recombinant His-tagged N-terminal catalytic domain of PknB and His-tagged residues 1-279 construct from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration) [425] (recombinant His-tagged PknB, PknD, PknE, and PknF rom Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration) [412] (recombinant His-tagged full-length and cytoplasmic domain of PknI from Escherichia coli strain BL21 by nickel affinity chromatography) [448] (recombinant wild-type and mutant GST-fusion PknH from Escherichia coli by glutathione affinity chromatography) [421] (recombinant Rio2 from Escherichia coli by adsorption chromatography and gel filtration) [451] (cAMP-Sepharose affinity chromatography) [406] (partial) [407]

76

2.7.11.1

Non-specific serine/threonine protein kinase

(recombinant GST-tagged enzyme by glutathione affinity chromatography) [468] [50] [160] (fusion protein of an amino-terminally truncated AK1 to the C-terminus of glutathione S-transferase) [196] [215] (truncation mutant of CKI d lacking the C-terminal autoinhibitory region) [246] (recombinant enzyme, partial) [247] (recombinant enzyme, partial) [247] (recombinant enzyme, partial) [247] [252] [260] [268] [341] [341] (recombinant enzyme is purified as a fusion protein with gluthatione S-transferase) [331] (recombinant GST-fusion catalytic subunit of CK2 from Escherichia coli by glutathione affinity chromatography, the GST-tag is cleaved off) [435] (recombinant GST-fusion PK1 from Escherichia coli strain BL21 by glutathione affinity chromatography, the GST-tag is cleaved off) [449] (recombinant GST-fusion PK2 from Escherichia coli strain BL21 by glutathione affinity chromatography, the GST-tag is cleaved off) [449] (recombinant GST-fusion full-length and truncated NTHK2 kinase wild-type and mutants from Escherichia coli by glutathione affinity chromatography) [446] (recombinant GST-fusion TSSK5 from HEK-293 cells by glutathione affinity chromatography) [411] Renaturation (refolding of recombinant enzyme) [448] Crystallization (purified recombinant Leu-Glu-His6-tagged GSK3b in complex with ATP or ATP analogue AMP-PNP, 10 mg/ml protein, 2 mM ATP or AMP-PNP, 1214% w/v PEG 6000, 100 mM NaCl, 5 mM MgCl2 , 10% v/v glycerol, in 100 mM HEPES-NaOH, pH 7.5, hanging drop vapor diffusion method, 4 C, several day, soaking in 0.1 mM ethylmercuric thiosalyclate at pH 7.5 for 1 h, cryoprotection by 30% w/v d-sorbitol, X-ray diffraction structure determination and analysis at 1.7-2.6 A resolution) [409] (purified recombinant low activity WNK1 mutant S382A, wild-type and selenomethionine-labeled, hanging drop vapour diffusion method, 16 C, mixing of 0.002 ml of protein solution and of well solution, the latter containing 24% PEG monomethyl ester 2000, 0.3 M NaCl, 0.1 M HEPES, pH 7.0-8.0, 3 days, stabilization with 15% glycerol, X-ray diffraction structure determination and analysis at 1.8 A resolution) [450] 77

Non-specific serine/threonine protein kinase

2.7.11.1

(purified recombinant His-tagged N-terminal catalytic domain of PknB, residues 1-331, hanging drop vapour diffusion method, 0.001 ml protein solution containing 5 mg/ml protein mixed with equal volume of 0.1 M HEPES, pH 7.5, 30 mM MgCl2 , 0.15 mM inhibitor AMP-PNP, 27% PEG 400, and 4% 1,3-butanediol, 19 C, versus 1 ml of a solution containing 0.1 M HEPES, pH 7.5, 30 mM MgCl2 , and 27% PEG 400, 2-3 weeks, X-ray diffraction structure determination and analysis at 2.2 A resolution) [425] (purified recombinant wild-type and selenomethionine-labeled Rio2, hanging drop vapour diffusion method, equal volumes of protein solution, containing 5-12% PEG 900, and 0.1 M phosphate citrate buffer, pH 3.6-4.1, and reservoir solution, versus 1 ml reservoir solution, purified recombinant Rio2 complexed with ATP using 0.1 M Tris, pH 7.5, 20 mM ATP, 20 mM MgCl2 , 20 mM adenosine 5’-(b,g-imino)triphosphate, and 20% ethylene glycol, 20 C, 2-4 days, X-ray diffraction structure determination and analysis at 2.0 A resolution) [451] [130] (autoinhibitory junction region bound to the calmodulin-like domain and Ca2+ , 20 mg/ml protein in 20 mM Tris buffer, pH 7.0, 10 mM CaCl2 , 10 mM DTT, mixed with an equal volume of precipitating agent composed of 15% w/v PEG 400, 0.1 M calcium chloride, and 0.1 M sodium acetate at pH 7.6, equilibration against a reservoir of precipitating agent by vapour diffusion at 20 C, X-ray diffraction structure determination and anaylsis at 2.0 A resolution, multiple-wavelength anomalous dispersion) [466] [234] (truncation mutant of CKI d lacking the C-terminal autoinhibitory region) [246] Cloning (overexpression of CKI in Drosophila Kc cell line, the sole Drosophila CKId/e gene, but not CKIa, activates a wingless transcriptional reporter in the absence of ligand) [460] (PKNa gene mapps to 19p12-p13.1 of chromosome 8 situated at the prostanoid receptor gene locus) [423] (ectopic overexpression of RSK2 in MEF cells induces anchorage-independent cell transformation) [457] (expression of wild-type and mutant GST-fusion TSSK3s in Escherichia coli strain BL21, expression of Myc-tagged catalytic subunit of phosphoinositide-dependent kinase-1 in human 293T cells) [420] (DNA sequence determination of isozyme PKNb) [423] (co-expression of Akt and hSWI/SNF proteins in Spodoptera frugiperda Sf9 cells using the baculovirus infection system, complex formation between the enzyme and the substrate proteins occurs, overview) [469] (expresion of wild-type and mutant GST-fusion NDR1 in Escherichia coli strain BL21(DE3), expression of HA-tagged NDR1 in COS-1 cells) [428] (expression of CK2 a and b subunits in Spodoptera frugiperda Sf9 cells via baculovirus transfection system) [429]

78

2.7.11.1

Non-specific serine/threonine protein kinase

(expression of EGFP- or FLAG-tagged wild-type or domain-deletion mutants of PKD2 in HEK-293 cells, gastric cancer AGS-B cells, and in PANC-1 cells) [440] (expression of FLAG-tagged full-length PKN1 and of its isolated catalytic kinase domain, residues 561-942, in CHO-K1 cells, co-expression with Salmonella enterica SspH1, SspH2, YopM and IpaH9.8, all HA-tagged, in HeLa cells, interaction study, overview) [458] (expression of GSK3b in Escherichia coli in fusion with a Leu-Glu-His6tag at the C-terminus) [409] (expression of His-tagged CK2 in Escherichia coli strain BL21) [459] (expression of subunits CK2a and CK2b, and of the CK2 holoenzyme in Escherichia coli, expression of Myc-tagged CK2b in GV10.15 cells, expression of the human Drosophila homologue CK2ff.-Timekeeper) [461] (overexpression of wild-type and mutant FLAG-tagged HIPK2 in 293T cells, coexpression of wild-type and mutant HIPK2 with HA-tagged JNK1 in 293 cells) [418] (stable 20fold overexpression of K46R mutant CK1a in SH-SY5Y neuroblastoma cells via adenovirus infection system) [443] (targeted overexpression of both CK2 and either c-Myc or Tal-1 in Tcells, as well as in tumor suppressor p53 (-/-) mice or murine BALB/c 3T3 fibroblasts) [456] (transient co-expression of human tau long isoform, HA-tagged GSK3b, and GST-tagged GFP-fusion FRAT-2 protein in HEK-293 cells) [433] (transient expression of CKII a and b subunits in human U87 astroglioma cells) [452] (transient expression of HA-tagged wild-type and mutant enzymes in CHO cells) [427, 432] (cloning, DNA and amino acid sequence determination, and analysis of SAST and splicing variant SAST124, expression of His-tagged SAST124 in mice, expression of SAST124 in HEK-293 cells) [445] (expression of GST-fusion WNK4 and WNK1 kinases in Escherichia coli strain BL21(DE3), expression of WNK4 and WNK1 kinases, and of the autoinhibitory domain of WNK4, residues 444-518 and 444-563, as well as the Nterminal domain 1-443 in HEK-293T cells) [410] (expression of His-tagged low activity WNK1 kinase domain mutant S382A, comprising residues 194-483, in Escherichia coli) [450] (expression of WNK1 and truncated WNK4 comprising residues 1-593 in HEK-293 cells, co-expression of truncated WNK1 comprising residues 1661 with claudin-4, oxidative stress response kinase 1 OSR1, STE/SPS1-related proline/alanine-rich kinase SPAK, and the Na+ -K+ -2Cl- co-transporter1-(1-260) construct NKCC1-(1-260) in Escherichia coli strain BL21 as GSTfusion proteins) [416] (gene mapping, DNA sequence determination and analysis of multiple isozymes) [423] (in vitro transcription and translation of RSK1, expression of RSK1 in PS127A cells or in HEK-293 cells, coexpression and colocalization in cytoplasm of FLAG-tagged RSK1 and 14-3-3b in COS-7 cells) [426] 79

Non-specific serine/threonine protein kinase

2.7.11.1

(co-expression of glycogen synthase kinase-3 b, t, and 14-3-3 in COS-7 cells and in HEK-293 cells) [424] (expression of GST-fusion LOV2 and kinase domain LV2-KD of phototropin in Escherichia coli) [447] (expression of His-tagged StkP and of GST-fusion StkP in Escherichia coli strain BL21(DE3)) [419] (expression of full-length and truncated StkP forms in Escherichia coli, overview) [453] (gene stkP, wild-type and mutant expression analysis, analysis of expression of the competence regulon, quantitative RT-PCR and microarray method, overview) [463] (expression of His-tagged PknB, PknD, PknE, and PknF in Escherichia coli strain BL21(DE3)) [412] (expression of N-terminal catalytic domain of PknB, comprising residues 1-331, and of a construct comprising residues 1-279 in Escherichia coli BL21(DE3) as His-tagged proteins) [425] (gene Rv1266c or pknH, overexpression of wild-type and mutant GSTfusion PknH in Escherichia coli) [421] (gene pknI, DNA and amino acid sequence determination and analysis, NaCl-inducible expression of N-terminally His-tagged full-length and cytoplasmic domain of PknI in Escherichia coli strain BL21 in inclusion bodies) [448] (expression of AsfK and AsfR in Escherichia coli, expression of PkaG and SCD10.09 in Escherichia coli) [436] (gene spkA, expression analysis) [462] (expression of Rio2 in Escherichia coli) [451] (expression of GST-tagged enzyme) [468] (expression in COS cells) [11] (identification of cDNA) [14] (in COS cells transfected with Kiz-1 complementary DNA Kiz-1 is found mainly in the cytoplasm) [16] (located at the distal end of mouse chromosome 5) [19] (isolation of cDNA) [20] [22] [30] [33] [34] [45] [48, 49] (characterization of the gene) [39] (expression in COS cells) [50] [53, 54] [55] (isolation of cDNA) [78] [85, 86] [48, 50] (expression in Cos-1 cells) [97] 80

2.7.11.1

Non-specific serine/threonine protein kinase

[86] (expression in COS cells) [102] [86] (isolation of cDNA) [103] (bacterially expressed catalytic domain of PKD) [105] [106] (DNA sequences encoding the rat Rsk-1 S6 kinase modified by insertion of a peptide epitope at the polypeptide aminoterminus, expressed in COS cells) [107] [109, 110] [54] [117] [118] [123] [131] (determination of genomic structure) [133] (expression in COS7 cells) [138] [139] [141] [142] [144] (expression in Escherichia coli) [175] [177] (isolation of cDNA) [182] [184] [184] [187, 188] (a fusion protein of an amino-terminally truncated AK1 to the C-terminus of glutathione S-transferase is expressed in Escherichia coli) [196] (expression of autoinhibitory junction region bound to the calmodulin-like domain, residues Lys428-Gly591, in Escherichia coli) [466] [210] [211] [144] [212] [215] [217] (isolation of cDNA) [225] (isolation of cDNA) [225] [236] [236] [237] [239] [240] [238] [241] [243] 81

Non-specific serine/threonine protein kinase

2.7.11.1

(expressed in both eukaryotic and prokaryotic systems) [244] [245] (expression in Escherichia coli) [245] (truncation mutant of CKI d lacking the C-terminal autoinhibitory region is expressed in Escherichia coli) [246] (expressed as active enzyme in Escherichia coli) [247] (expressed as active enzyme in Escherichia coli) [247] (expressed as active enzyme in Escherichia coli) [247] [249] [250] (a-subunit and b-subunit) [272] (a and b subunit) [251] (isolation of the a subunit of casein kinase II) [253] [261, 263] (intronless gene) [254] [255] (a and b subunit) [256] [258] (a-subunit) [259] [262] [262] (expression in Escherichia coli) [264] (expression in Escherichia coli) [264] [265] [276] (a’-subunit) [267] (a’-subunit) [269] (catalytic a-subunit and b-subunit) [270] (expression in Saccharomyces cerevisiae) [279] [282, 283] [284] [290] [297] (microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells) [301] [284] [290] [305] [306] [312] (isolation and characterization of cDNA) [317] [319] (isolation and characterization of cDNA) [317] [340] [340] [341] [341] 82

2.7.11.1

Non-specific serine/threonine protein kinase

[348] [358] (genomic intron-exon structure of the human BUB1 gene) [356] (genomic intron-exon structure of the human BUB1 gene) [356] [366] [366, 367, 368, 369] (expressed in COS7 cells) [372] (isolation of cDNA) [371] [377] [329] (expression in Escherichia coli) [386] [392] [393] (expression in Escherichia coli) [331] (expression in Escherichia coli under a T7 promoter) [334] (expression in Escherichia coli) [335] (Pkn6-PhoA fusion protein expressed in Escherichia coli) [335] (overexpression in Escherichia coli) [337] (overexpression in Escherichia coli) [337] (expression in Escherichia coli) [338] (DNA sequence determination, expression of the catalytic subunit of CK2 as GST-fusion protein in Escherichia coli) [435] (DNA and amino acid sequence determination and analysis of protein kinase PK1, expression as GST-fusion protein in Escherichia coli strain BL21) [449] (DNA and amino acid sequence determination and analysis of protein kinase PK2, expression as GST-fusion protein in Escherichia coli strain BL21) [449] (DNA sequence determination and analysis of splicing form CK1e-1, gene CK1e is located on chromosome 7q34, transient expression of CK1e-1 in COS-7 cells) [444] (DNA sequence determination and analysis of splicing form CKIe-3, gene Ck1e is located on chromosome 7q34, transient expression of CKIe-3 in COS-7 cells, expression of GST-fusion CKIe-3 in Escherichia coli) [444] (NTHK2 DNA and amino acid sequence determination and analysis, expression of GST-fusion in Escherichia coli, expression of full-length and truncated NTHK2 kinase wild-type and mutants in Schizosaccharomyces pombe strain SP-Q01) [446] (ZmS6K gene DNA and amino acid sequence determination and analysis) [417] (DNA and amino acid sequence determination and analysis of TSSK5, expression of His-tagged CREB in Escherichia coli, expression of GST-fusion TSSK5 in HEK-293 cells, co-expression of TSSK5 and CREB in Saccharomyces cerevisiae using the two-hydrid system) [411] (SSTK gene, DNA and amino acid sequence determination and analysis, transient expression of wild-type and mutant Myc-tagged SSTK in 293T cells) [442] 83

Non-specific serine/threonine protein kinase

2.7.11.1

(SSTK gene, DNA and amino acid sequence determination and analysis, transient expression of Myc-tagged SSTK in 293T cells) [442] (DNA and amino acid sequence determination and analysis, in vitro transcription and translation, overexpression of the SPAK isozyme in Caco-2BBE cells) [455] (gene tssk4, localization on chromosome 14qC3, DNA and amino acid sequence determination and analysis of splicing variant g, expression of wildtype Myc-tagged TSSKd in HEK-293T cells) [464] (gene tssk4, localization on chromosome 14qC3, DNA and amino acid sequence determination and analysis of splicing variant TSSKa, expression of wild-type and mutant Myc-tagged TSSKa in HEK-293T cells) [464] (gene tssk4, localization on chromosome 14qC3, DNA and amino acid sequence determination and analysis of splicing variant b, expression of wildtype Myc-tagged TSSKb in HEK-293T cells) [464] (gene tssk4, localization on chromosome 14qC3, DNA and amino acid sequence determination and analysis of splicing variant g, expression of wildtype Myc-tagged TSSKg in HEK-293T cells) [464] Engineering D173N ( the dominant-negative form of CKIg inhibits Wnt signaling in a reporter assay in 293T cells, and gish RNAi partially blocks transcription induced by Wg in SL2 cells [460]) [460] E268Q ( site-directed mutagenesis, residue is probably important in enzyme stabilization since the mutant enzyme cannot be expressed recombinantly [450]) [450] H384Q ( site-directed mutagenesis, the mutant cannot perform autophosphorylation of the receiver domain, but still phosphorylates its kinase domain [446]) [446] I119A ( site-directed mutagenesis of a PIF pocket residue of PDK1, mutant shows reduced activity compared to the wild-type enzyme [414]) [414] I34L ( site-directed mutagenesis, inactive mutant of the intestinal inflammation-associated colonic epithelial isozyme [455]) [455] K221A ( site-directed mutagenesis of HIPK2, mutant shows reduced interaction and colocalization with Daxx [418]) [418] K39R ( site-directed mutagenesis, inactive mutant [420]) [420] K41M ( site-directed mutagenesis, inactive mutant, no interaction with heat shock proteins [442]) [442] K42R ( site-directed mutagenesis, mutant StkP activity is reduced by 87% compared to the wild-type enzyme [419]) [419] K45M ( site-directed mutagenesis, inactive mutant [421]) [421] K46R ( site-directed mutagenesis, inactive, dominant negative mutants of CK1a, overexpression in SH-SY5Y cells does not influence endogenous basal and metacholine-stimulated M3 muscarinic acetylcholine receptor phosphorylation and desensitization by the endogenous wild-type GRK6 [443]) [443]

84

2.7.11.1

Non-specific serine/threonine protein kinase

K54M ( site-directed mutagenesis of splicing variant TSSKa [464]) [464] K644E ( site-directed mutagenesis, inactive mutant, PKN1 depletion causes an increase in NF-kB-dependent reporter gene expression [458]) [458] K73R ( the dominant-negative form of CKIg inhibits Wnt signaling in a reporter assay in 293T cells, and gish RNAi partially blocks transcription induced by Wg in SL2 cells [460]) [460] K81I ( mutation totally abolishes kinase activity [210]) [210] M24L ( mutation in the a I polypeptide suppresses synthesis of the a II polypeptides [72]) [72] M24T ( mutation in the a I polypeptide suppresses synthesis of the a II polypeptides [72]) [72] Q150A ( site-directed mutagenesis of a PIF pocket residue of PDK1, mutant shows reduced activity compared to the wild-type enzyme [414]) [414] R96A ( site-directed mutagenesis of GSK3b, the mutant shows reduced phosphorylation activity at primed epitopes and increased activity at unprimed epitopes of tau protein substrate compared to the wild-type GSK3b [432]) [432] S154A ( site-directed mutagenesis, mutation modulates the enzyme activity [426]) [426] S166A ( site-directed mutagenesis, mutation of a T-loop residue, highly reduced kinase activity compared to the wild-type TSSK3, no autophosphorylation [420]) [420] S166D ( site-directed mutagenesis, mutation of a T-loop residue, reduced kinase activity compared to the wild-type TSSK3, no autophosphorylation [420]) [420] S166G ( site-directed mutagenesis, mutation of a T-loop residue, highly reduced kinase activity compared to the wild-type TSSK3, no autophosphorylation [420]) [420] S281A ( site-directed mutagenesis, mutant shows abolished autophosphorylation at T444 [428]) [428] S370A ( site-directed mutagenesis, S6K2 mutant is inactive [415]) [415] S370D ( site-directed mutagenesis, S6K2 mutant is inactive [415]) [415] S382A ( site-directed mutagenesis, mutant remains in the low activity form [450]) [450] S442X ( mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation [92]) [92] S9A ( site-directed mutagenesis, mutant cannot be inhibited by phosphorylation at position 9 and is thus constitutively active [427, 432, 433]) [427, 432, 433] T168A ( site-directed mutagenesis, mutation of a T-loop residue, inactive TSSK3 mutant, no autophosphorylation [420]) [420]

85

Non-specific serine/threonine protein kinase

2.7.11.1

T168D ( site-directed mutagenesis, mutation of a T-loop residue, unaltered kinase activity and reduced autophosphorylation compared to the wild-type TSSK3 [420]) [420] T228A ( site-directed mutagenesis, S6K2 mutant shows reduced activation compared to the wild-type enzyme [415]) [415] T228A/T338E ( site-directed mutagenesis, S6K2 mutant is inactive [415]) [415] T228E ( site-directed mutagenesis, S6K2 mutant shows highly reduced activation compared to the wild-type enzyme [415]) [415] T256A ( the conditional IPTG inducible expression of wild type Sgk, but not of the kinase dead mutant T256A Sgk, protects Con8 mammary epithelial tumor cells from serum starvation-induced apoptosis [35]) [35] T256A/S422A ( unlike the wild-type enzyme the phosphorylation site mutant has no effect on cell survival [35]) [35] T256X ( mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation [92]) [92] T338E ( site-directed mutagenesis, S6K2 mutant is fully active and rapamycin-resistant, while the S6K1 mutant is inactive [415]) [415] T444A ( site-directed mutagenesis, mutant shows almost unaltered autophosphorylation at S281 compared to the wild-type enzyme [428]) [428] T474F ( 55% inhibition of Akt activation [37]) [37] T508V ( activity is abolished [12]) [12] T74A ( site-directed mutagenesis, mutant shows only slightly reduced kinase activity and autophosphorylation compared to the wild-type enzyme [428]) [428] V318E/A448K ( site-directed mutagenesis, the mutant shows 50-60% reduced activity compared to the wild-type WNK1 [450]) [450] V66A/I174A ( site-directed mutagenesis, an engineered TBB inhibitor-resistant CK2 mutant CK2aR [456]) [456] V67A/I175A ( site-directed mutagenesis, an inhibitor-resistant CK2 mutant CK2aR [456]) [456] Additional information ( replacement of Thr508 with 2 glutamic acid residues results in a fully active enzyme [12]; mutation of residue D460 within the IHRDL motif abolishes kinase activity [19]; phosphorylation of Ser472 and phosphorylation of Thr305 appears to contribute to the activation of Akt-3 because mutation of both these residues to aspartate increases the catalytic activity of Akt-3, whereas mutation to alanine inhibits activation [53]; a CTG triplet repeat undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population, unaffected individuals have been 5 and 27 copies, myotonic dystrophy patients are minimally affected have at least 50 repeats, more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs [101]; R114W mutation falls just outside the N-terminal ATP-binding site in a highly conserved region of the protein and may lead to structural changes since tryptophan has an aromatic side chain whereas arginine is a 5 carbon basic amino acid, missense mutation R729Q, 2bp deletion (AG) of 86

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Non-specific serine/threonine protein kinase

bases 451 and 452 creates a frameshift that results in a stop codon 25 amino acids downstream, thereby producing a truncated protein. This deletion also falls within the highly conserved amino-catalytic domain of the protein. Nonsense mutation (C2065T) which results in a premature stop codon, thereby producing a truncated protein. These mutations further confirm Rsk-2 as the gene involved in CLS and may help in understanding the structure and function of the protein [82]; a mutant cki3 gene in which a highly conserved lysine residue in the kinase subdomain II is substituted to arginine, loses the ability to recover the growth defect in the srs1 mutant, indicating that catalytic activity is necessary for suppression. Gene disruption reveals that cki3+ is dispensable for cell viability, and cells lacking functional cki3+ exhibit no characteristic phenotype [216]; dbtS and dbtL mutations, which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase [217]; construction of HIPK2 knockout mutants in Hep-3B cells [418]; construction of several NTHK2 kinase truncation mutants, overview [446]; construction of SSTK knockout mice showing impaired sperm motility and morphology resulting in male sterility, a DNA condensation defect in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins [442]; mutation of Leu155 and Lys115, residues of the PIF pocket of PDK1, results in 4fold higher PDK1 activity [414]; naturally occurring V717I mutation of the amyloid precursor protein APP in a CT100 fragment of organisms with Alzheimers disease leads to augmented age-dependent t phosphorylation, followed by increased activation status of mitogen-activated protein kinase family members, e.g. ERK1/2, p38, and c-Jun NH2 -terminal kinase, compared to the wild-type organism, naturally occurring V337M mutation of tau protein of patients suffering Alzheimers disease leads to age-dependent memory deficits [441]; naturally occurring V717I mutation of the amyloid precursor protein APP in a CT100 fragment of patients suffering from Alzheimers disease leads to augmented age-dependent tau phosphorylation, followed by increased activation status of mitogen-activated protein kinase family members, e.g. ERK1/2, p38, and c-Jun NH2 -terminal kinase, compared to the wild-type organism, naturally occurring V337M mutation of tau protein of patients suffering from Alzheimers disease leads to age-dependent memory deficits [441]; changes, increase or decrease, in gene expression of genes pilA5, pilA6, pilA1, pilA2, pilA10, pilA9, and pilA11 induced by mutation of the spkA, overview [462]; CK2 induces neoplastic growth when overexpressed [470]; comparison of the whole-genome expression profiles of the wild-type strain and DstkP mutant strain, constructed by deletion of the stkP gene, by microarray technology, the competence regulon is deregulated, inactivation of stkP leads to overexpression of competence genes, mutant phenotype, overview [463]; deletion of the extracellular domain of StkP negatively affects the stability of a core kinase domain [453]; HeLa cells are depleted of CK2 subunits by treatment with CK2 antisense oligodeoxynucleotides directed against the catalytic a/a and regulatory b subunits prior induction of apoptosis [461]; injection of 87

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kinase-dead CKIe into early Xenopus embryos causes defects in convergent extension during gastrulation [460]; knockdown of RSK2 by siRNA expression in NIH3T3 cells blocks foci formation, overview [457]; overexpression of the SPAK isozyme leads to leading to nuclear translocation of an N-terminal fragment of the SPAK isozyme, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability [455]; PKN1 depletion by siRNA causes an increase in NF-kB-dependent reporter gene expression [458]; targeted overexpression of both CK2 and either c-Myc or Tal-1 in T-cells, as well as the overexpression of CK2 in tumor suppressor p53 (-/-) mice, results in an increase in oncogenic activity and progression of tumorigenesis, a cooperative increase in oncogenic activity is also observed in BALB/c 3T3 fibroblasts co-expressing H-Ras and CK2a, overview, knockdown studies of CK2 using RNAi shows that CK2 negatively regulate apoptosis in IR damage and affects the cell cycle progression in HeLa cells [456]; transfection of the dominant negative mutant of CK2, CK2aA156, to the CA1 area, but not to the DG area, decreases CK2 activity but enhances spatial memory formation, it increases SGK1 phosphorylation at Ser422, decreased Akt phosphorylation at Ser473, and increases cAMP response element-binding protein phosphorylation at Ser133, transfection significantly decreases CK2 activity in CA1 area by about 38%, compared to the control group, and downregulates CK2 signaling in CA1 neurons, while SGK1 phosphorylation at Ser422 is increased, overview [467]) [12, 19, 53, 82, 101, 216, 217, 414, 418, 441, 442, 446, 453, 455, 456, 457, 458, 460, 461, 462, 463, 467, 470] Application drug development ( protein kinases are major targets for drug development and design in the pharmaceutical industry [414]; CK2 is a target for drug development and design of selective inhibitors [454]) [414, 454] medicine ( GSK-3 is a target for the therpeutical treatement of Alzheimers disease and other tauopathies [431]) [431] pharmacology ( CK2 is a potential therapeutic target and a target for inhibitor design, e.g. in anti-cancer therapy [456]) [456]

6 Stability pH-Stability 7.5 ( at 50 C, t1=2 : 10 min [396]) [396] Temperature stability 50 ( at pH 7.5, t1=2 : 10 min [396]) [396] General stability information , Triton X-100 stabilizes during freeze-thawing procedures [402] , diluted solutions are unstable [398] , repeated freeze-thawing inactivates [398]

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, the extracellular domain of StkP is required for the stability of a core kinase domain, while the membrane-anchored kinase domain and the fulllength form of StkP are stable and capable of autophosphorylation [453] , freeze-thawing inactivates [407] Storage stability , -20 C, at least 2 months [396] , -20 C, in 0.05 M imidazole chloride, pH 7.3, 10% glycerol, 1 mM DTT, protease inhibitors, 1 mM EDTA, at least 6 months [398] , -70 C, in 0.025 M Tris-HCl, pH 7.3, 10% glycerol, 1 mM EDTA, 1 mM benzamidine, 0.1 mM PMSF, 14 mM 2-mercaptoethanol, 0.1% Triton X-100, 12 months [402] , 0-4 C, in 0.05 M imidazole chloride, pH 7.3, 10% glycerol, 1 mM DTT, protease inhibitors, 1 mM EDTA, up to 3 weeks [398] , 0-4 C, partially purified preparation, several weeks [407]

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[406] Jergil, B.; Mosbach, K.: Cyclic AMP: purification of protamine kinase. Methods Enzymol., 34, 261-264 (1974) [407] Jergil, B.; Dixon, G.H.: Protamine kinase from rainbow trout testis. Partial purification and characterization. J. Biol. Chem., 245, 425-434 (1970) [408] Baggio, B.; Pinna, L.A.; Moret, V.; Siliprandi, N.: A simple procedure for the purification of rat liver phosvitin kinase. Biochim. Biophys. Acta, 207, 515-517 (1970) [409] Aoki, M.; Yokota, T.; Sugiura, I.; Sasaki, C.; Hasegawa, T.; Okumura, C.; Ishiguro, K.; Kohno, T.; Sugio, S.; Matsuzaki, T.: Structural insight into nucleotide recognition in t-protein kinase I/glycogen synthase kinase 3 b. Acta Crystallogr. Sect. D, 60, 439-446 (2004) [410] Wang, Z.; Yang, C.L.; Ellison, D.H.: Comparison of WNK4 and WNK1 kinase and inhibiting activities. Biochem. Biophys. Res. Commun., 317, 939944 (2004) [411] Chen, X.; Lin, G.; Wei, Y.; Hexige, S.; Niu, Y.; Liu, L.; Yang, C.; Yu, L.: TSSK5, a novel member of the testis-specific serine/threonine kinase family, phosphorylates CREB at Ser-133, and stimulates the CRE/CREB responsive pathway. Biochem. Biophys. Res. Commun., 333, 742-749 (2005) [412] Duran, R.; Villarino, A.; Bellinzoni, M.; Wehenkel, A.; Fernandez, P.; Boitel, B.; Cole, S.T.; Alzari, P.M.; Cervenansky, C.: Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases. Biochem. Biophys. Res. Commun., 333, 858-867 (2005) [413] Bain, J.; McLauchlan, H.; Elliott, M.; Cohen, P.: The specificities of protein kinase inhibitors: an update. Biochem. J., 371, 199-204 (2003) [414] Biondi, R.M.; Nebreda, A.R.: Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions. Biochem. J., 372, 1-13 (2003) [415] Phin, S.; Kupferwasser, D.; Lam, J.; Lee-Fruman, K.K.: Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1. Biochem. J., 373, 583-591 (2003) [416] Vitari, A.C.; Deak, M.; Morrice, N.A.; Alessi, D.R.: The WNK1 and WNK4 protein kinases that are mutated in Gordon’s hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases. Biochem. J., 391, 17-24 (2005) [417] Reyes de la Cruz, H.; Aguilar, R.; Sanchez de Jimenez, E.: Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K). Biochemistry, 43, 533-539 (2004) [418] Hofmann, T.G.; Stollberg, N.; Schmitz, M.L.; Will, H.: HIPK2 regulates transforming growth factor-b-induced c-Jun NH(2)-terminal kinase activation and apoptosis in human hepatoma cells. Cancer Res., 63, 8271-8277 (2003) [419] Novakova, L.; Saskova, L.; Pallova, P.; Janecek, J.; Novotna, J.; Ulrych, A.; Echenique, J.; Trombe, M.C.; Branny, P.: Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates. FEBS J., 272, 1243-1254 (2005) 119

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[420] Bucko-Justyna, M.; Lipinski, L.; Burgering, B.M.; Trzeciak, L.: Characterization of testis-specific serine-threonine kinase 3 and its activation by phosphoinositide-dependent kinase-1-dependent signalling. FEBS J., 272, 6310-6323 (2005) [421] Sharma, K.; Chandra, H.; Gupta, P.K.; Pathak, M.; Narayan, A.; Meena, L.S.; D’Souza, R.C.J.; Chopra, P.; Ramachandran, S.; Singh, Y.: PknH, a transmembrane Hank’s type serine/threonine kinase from Mycobacterium tuberculosis is differentially expressed under stress conditions. FEMS Microbiol. Lett., 233, 107-113 (2004) [422] Mottet, D.; Ruys, S.P.; Demazy, C.; Raes, M.; Michiels, C.: Role for casein kinase 2 in the regulation of HIF-1 activity. Int. J. Cancer, 117, 764-774 (2005) [423] Mukai, H.: The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC. J. Biochem., 133, 17-27 (2003) [424] Agarwal-Mawal, A.; Qureshi, H.Y.; Cafferty, P.W.; Yuan, Z.; Han, D.; Lin, R.; Paudel, H.K.: 14-3-3 connects glycogen synthase kinase-3 b to tau within a brain microtubule-associated t phosphorylation complex. J. Biol. Chem., 278, 12722-12728 (2003) [425] Ortiz-Lombardia, M.; Pompeo, F.; Boitel, B.; Alzari, P.M.: Crystal structure of the catalytic domain of the PknB serine/threonine kinase from Mycobacterium tuberculosis. J. Biol. Chem., 278, 13094-13100 (2003) [426] Cavet, M.E.; Lehoux, S.; Berk, B.C.: 14-3-3b is a p90 ribosomal S6 kinase (RSK) isoform 1-binding protein that negatively regulates RSK kinase activity. J. Biol. Chem., 278, 18376-18383 (2003) [427] Cho, J.H.; Johnson, G.V.: Glycogen synthase kinase 3b phosphorylates tau at both primed and unprimed sites. Differential impact on microtubule binding. J. Biol. Chem., 278, 187-193 (2003) [428] Tamaskovic, R.; Bichsel, S.J.; Rogniaux, H.; Stegert, M.R.; Hemmings, B.A.: Mechanism of Ca2+ -mediated regulation of NDR protein kinase through autophosphorylation and phosphorylation by an upstream kinase. J. Biol. Chem., 278, 6710-6718 (2003) [429] Roosbeek, S.; Peelman, F.; Verhee, A.; Labeur, C.; Caster, H.; Lensink, M.F.; Cirulli, C.; Grooten, J.; Cochet, C.; Vandekerckhove, J.; Amoresano, A.; Chimini, G.; Tavernier, J.; Rosseneu, M.: Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter. J. Biol. Chem., 279, 37779-37788 (2004) [430] Hernandez, F.; Perez, M.; Lucas, J.J.; Mata, A.M.; Bhat, R.; Avila, J.: Glycogen synthase kinase-3 plays a crucial role in t exon 10 splicing and intranuclear distribution of SC35. Implications for Alzheimer’s disease. J. Biol. Chem., 279, 3801-3806 (2004) [431] Liu, S.J.; Zhang, J.Y.; Li, H.L.; Fang, Z.Y.; Wang, Q.; Deng, H.M.; Gong, C.X.; Grundke-Iqbal, I.; Iqbal, K.; Wang, J.Z.: t becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain. J. Biol. Chem., 279, 50078-50088 (2004) [432] Cho, J.H.; Johnson, G.V.: Glycogen synthase kinase 3 b induces caspasecleaved tau aggregation in situ. J. Biol. Chem., 279, 54716-54723 (2004) 120

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[433] Stoothoff, W.H.; Cho, J.H.; McDonald, R.P.; Johnson, G.V.: FRAT-2 preferentially increases glycogen synthase kinase 3 b-mediated phosphorylation of primed sites, which results in enhanced tau phosphorylation. J. Biol. Chem., 280, 270-276 (2005) [434] Zhao, X.; Zhuang, S.; Chen, Y.; Boss, G.R.; Pilz, R.B.: Cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein b functions through inhibition of glycogen synthase kinase-3. J. Biol. Chem., 280, 32683-32692 (2005) [435] Matsushita, Y.; Ohshima, M.; Yoshioka, K.; Nishiguchi, M.; Nyunoya, H.: The catalytic subunit of protein kinase CK2 phosphorylates in vitro the movement protein of Tomato mosaic virus. J. Gen. Virol., 84, 497-505 (2003) [436] Horinouchi, S.: AfsR as an integrator of signals that are sensed by multiple serine/threonine kinases in Streptomyces coelicolor A3(2). J. Ind. Microbiol. Biotechnol., 30, 462-467 (2003) [437] Kenyon, T.K.; Homan, E.; Storlie, J.; Ikoma, M.; Grose, C.: Comparison of varicella-zoster virus ORF47 protein kinase and casein kinase II and their substrates. J. Med. Virol., 70 Suppl 1, S95-102 (2003) [438] Zhang, Y.; Li, H.L.; Wang, D.L.; Liu, S.J.; Wang, J.Z.: A transitory activation of protein kinase-A induces a sustained tau hyperphosphorylation at multiple sites in N2a cells-imply a new mechanism in Alzheimer pathology. J. Neural Transm., 2, 1-11 (2006) [439] Coito, C.; Diamond, D.L.; Neddermann, P.; Korth, M.J.; Katze, M.G.: Highthroughput screening of the yeast kinome: identification of human serine/ threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein. J. Virol., 78, 3502-3513 (2004) [440] Auer, A.; von Blume, J.; Sturany, S.; von Wichert, G.; Van Lint, J.; Vandenheede, J.; Adler, G.; Seufferlein, T.: Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling. Mol. Biol. Cell, 16, 4375-4385 (2005) [441] Lambourne, S.L.; Sellers, L.A.; Bush, T.G.; Choudhury, S.K.; Emson, P.C.; Suh, Y.H.; Wilkinson, L.S.: Increased tau phosphorylation on mitogen-activated protein kinase consensus sites and cognitive decline in transgenic models for Alzheimer’s disease and FTDP-17: evidence for distinct molecular processes underlying tau abnormalities. Mol. Cell. Biol., 25, 278-293 (2005) [442] Spiridonov, N.A.; Wong, L.; Zerfas, P.M.; Starost, M.F.; Pack, S.D.; Paweletz, C.P.; Johnson, G.R.: Identification and characterization of SSTK, a serine/threonine protein kinase essential for male fertility. Mol. Cell. Biol., 25, 4250-4261 (2005) [443] Willets, J.M.; Mistry, R.; Nahorski, S.R.; Challiss, R.A.: Specificity of G protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell M3 muscarinic acetylcholine receptor signaling. Mol. Pharmacol., 64, 1059-1068 (2003) [444] Takano, A.; Hoe, H.S.; Isojima, Y.; Nagai, K.: Analysis of the expression, localization and activity of rat casein kinase 1e-3. NeuroReport, 15, 14611464 (2004) 121

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[445] Yano, R.; Yap, C.C.; Yamazaki, Y.; Muto, Y.; Kishida, H.; Okada, D.; Hashikawa, T.: Sast124, a novel splice variant of syntrophin-associated serine/ threonine kinase (SAST), is specifically localized in the restricted brain regions. Neuroscience, 117, 373-381 (2003) [446] Zhang, Z.G.; Zhou, H.L.; Chen, T.; Gong, Y.; Cao, W.H.; Wang, Y.J.; Zhang, J.S.; Chen, S.Y.: Evidence for serine/threonine and histidine kinase activity in the tobacco ethylene receptor protein NTHK2. Plant Physiol., 136, 29712981 (2004) [447] Matsuoka, D.; Tokutomi, S.: Blue light-regulated molecular switch of Ser/ Thr kinase in phototropin. Proc. Natl. Acad. Sci. USA, 102, 13337-13342 (2005) [448] Gopalaswamy, R.; Narayanan, P.R.; Narayanan, S.: Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv. Protein Expr. Purif., 36, 82-89 (2004) [449] Dissanayake, K.; Castillo, C.; Takasaki, T.; Nakanishi, T.; Norioka, N.; Norioka, S.: Molecular cloning, functional expression and characterization of two serine/threonine-specific protein kinases from Nicotiana tabacum pollen. Sex. Plant Reprod., 17, 165-175 (2004) [450] Min, X.; Lee, B.H.; Cobb, M.H.; Goldsmith, E.J.: Crystal structure of the kinase domain of WNK1, a kinase that causes a hereditary form of hypertension. Structure, 12, 1303-1311 (2004) [451] LaRonde-LeBlanc, N.; Wlodawer, A.: Crystal structure of A. fulgidus Rio2 defines a new family of serine protein kinases. Structure, 12, 1585-1594 (2004) [452] Ahn, B.H.; Min, G.; Bae, Y.S.; Min do, S.: Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells. Exp. Mol. Med., 38, 55-62 (2006) [453] Pallova, P.; Hercik, K.; Saskova, L.; Novakova, L.; Branny, P.: A eukaryotictype serine/threonine protein kinase StkP of Streptococcus pneumoniae acts as a dimer in vivo. Biochem. Biophys. Res. Commun., 355, 526-530 (2007) [454] Pagano, M.A.; Cesaro, L.; Meggio, F.; Pinna, L.A.: Protein kinase CK2: a newcomer in the druggable kinome. Biochem. Soc. Trans., 34, 1303-1306 (2006) [455] Yan, Y.; Nguyen, H.; Dalmasso, G.; Sitaraman, S.V.; Merlin, D.: Cloning and characterization of a new intestinal inflammation-associated colonic epithelial Ste20-related protein kinase isoform. Biochim. Biophys. Acta, 1769, 106-116 (2007) [456] Duncan, J.S.; Litchfield, D.W.: Too much of a good thing: The role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2. Biochim. Biophys. Acta, 1784, 33-47 (2008) [457] Cho, Y.Y.; Yao, K.; Kim, H.G.; Kang, B.S.; Zheng, D.; Bode, A.M.; Dong, Z.: Ribosomal S6 kinase 2 is a key regulator in tumor promoter induced cell transformation. Cancer Res., 67, 8104-8112 (2007) [458] Haraga, A.; Miller, S.I.: A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1. Cell. Microbiol., 8, 837-846 (2006) 122

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[459] Laramas, M.; Pasquier, D.; Filhol, O.; Ringeisen, F.; Descotes, J.L.; Cochet, C.: Nuclear localization of protein kinase CK2 catalytic subunit (CK2a) is associated with poor prognostic factors in human prostate cancer. Eur. J. Cancer, 43, 928-934 (2007) [460] Price, M.A.: CKI, there’s more than one: casein kinase I family members in Wnt and Hedgehog signaling. Genes Dev., 20, 399-410 (2006) [461] Guerra, B.: Protein kinase CK2 subunits are positive regulators of AKT kinase. Int. J. Oncol., 28, 685-693 (2006) [462] Panichkin, V.B.; Arakawa-Kobayashi, S.; Kanaseki, T.; Suzuki, I.; Los, D.A.; Shestakov, S.V.; Murata, N.: Serine/threonine protein kinase SpkA in Synechocystis sp. strain PCC 6803 is a regulator of expression of three putative pilA operons, formation of thick pili, and cell motility. J. Bacteriol., 188, 7696-7699 (2006) [463] Saskova, L.; Novakova, L.; Basler, M.; Branny, P.: Eukaryotic-type serine/ threonine protein kinase StkP is a global regulator of gene expression in Streptococcus pneumoniae. J. Bacteriol., 189, 4168-4179 (2007) [464] Wei, Y.; Fu, G.; Hu, H.; Lin, G.; Yang, J.; Guo, J.; Zhu, Q.; Yu, L.: Isolation and characterization of mouse testis specific serine/threonine kinase 5 possessing four alternatively spliced variants. J. Biochem. Mol. Biol., 40, 749-756 (2007) [465] Hanger, D.P.; Byers, H.L.; Wray, S.; Leung, K.Y.; Saxton, M.J.; Seereeram, A.; Reynolds, C.H.; Ward, M.A.; Anderton, B.H.: Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis. J. Biol. Chem., 282, 23645-23654 (2007) [466] handran, V.; Stollar, E.J.; Lindorff-Larsen, K.; Harper, J.F.; Chazin, W.J.; Dobson, C.M.; Luisi, B.F.; Christodoulou, J.: Structure of the regulatory apparatus of a calcium-dependent protein kinase (CDPK): a novel mode of calmodulin-target recognition. J. Mol. Biol., 357, 400-410 (2006) [467] Chao, C.C.; Ma, Y.L.; Lee, E.H.: Protein kinase CK2 impairs spatial memory formation through differential cross talk with PI-3 kinase signaling: activation of Akt and inactivation of SGK1. J. Neurosci., 27, 6243-6248 (2007) [468] Mou, F.; Forest, T.; Baines, J.D.: US3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin A/C in infected cells. J. Virol., 81, 6459-6470 (2007) [469] Foster, K.S.; McCrary, W.J.; Ross, J.S.; Wright, C.F.: Members of the hSWI/ SNF chromatin remodeling complex associate with and are phosphorylated by protein kinase B/Akt. Oncogene, 25, 4605-4612 (2006) [470] Bolanos-Garcia, V.M.; Fernandez-Recio, J.; Allende, J.E.; Blundell, T.L.: Identifying interaction motifs in CK2b - a ubiquitous kinase regulatory subunit. Trends Biochem. Sci., 31, 654-661 (2006) [471] Caples, M.J.; Clements, J.E.; Barber, S.A.: Protein kinase CK2 phosphorylates the Nef protein from a neurovirulent simian immunodeficiency virus. Virology, 348, 156-164 (2006)

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

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1 Nomenclature EC number 2.7.11.2 Systematic name ATP:[pyruvate dehydrogenase (acetyl-transferring)] phosphotransferase Recommended name [pyruvate dehydrogenase (acetyl-transferring)] kinase Synonyms PDH kinase [27, 42, 51] PDHK [27, 42, 54, 63, 64, 68] PDHK2 [41, 44, 57] PDHK4 [56] PDK [19, 28, 29, 30, 31, 32, 33, 34, 35, 38, 40, 49, 50, 51, 55, 58, 59, 61, 65, 66, 67, 69, 70] PDK-2 [50] PDK-4 [50] PDK1 [37, 47, 48, 55] PDK2 [37, 43, 47, 48, 52, 60] PDK3 [37, 46, 47, 48] PDK4 [37, 39, 45, 47, 48, 53, 60] kinase (phosphorylating), pyruvate dehydrogenase pyruvate dehydrogenase kinase [38, 39, 40, 43, 47, 48, 50, 51, 52, 54, 55, 58, 59, 61, 63, 64, 65, 66, 67, 68, 69, 70] pyruvate dehydrogenase kinase (phosphorylating) pyruvate dehydrogenase kinase 2 [41, 43, 44, 57] pyruvate dehydrogenase kinase 3 [46] pyruvate dehydrogenase kinase 4 [37, 45, 53] pyruvate dehydrogenase kinase activator protein pyruvate dehydrogenase kinase isoenzyme 4 [56] Additional information ( PDK isozymes and the related branched-chain dehydrogenase kinase form a unique family of serine kinases [30,36]; enzyme possibly belongs to the ATPase/kinase family [24]; enzyme belongs to the PDK family [40]) [24, 30, 36, 40] CAS registry number 9074-01-5

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2 Source Organism Mammalia (no sequence specified) [30, 31, 35] Mus musculus (no sequence specified) [23, 37, 39, 45, 56, 59] Homo sapiens (no sequence specified) [28, 30, 31, 35, 37, 39, 41, 42, 43, 46, 47, 48, 49, 50, 51, 52, 55, 58, 60, 63, 64, 65, 66, 67, 68, 70] Rattus norvegicus (no sequence specified) [5, 18, 21, 23, 24, 27, 29, 30, 31, 32, 33, 34, 35, 37, 39, 42, 48, 53, 57, 60, 69] Sus scrofa (no sequence specified) [3,17] Saccharomyces cerevisiae (no sequence specified) [62] Bos taurus (no sequence specified) [1,2,3,6,7,8,9,10,11,12,13,14,15,20,30] Oryctolagus cuniculus (no sequence specified) [4] Pisum sativum (no sequence specified) [16] Zea mays (no sequence specified) [30] Arabidopsis thaliana (no sequence specified) [26, 30, 38] Xenopus laevis (no sequence specified) [40] Caenorhabditis elegans (no sequence specified) [25] Homo sapiens (UNIPROT accession number: Q16654) [19] Rattus norvegicus (UNIPROT accession number: Q63065) [33, 36] Ascaris suum (UNIPROT accession number: O02623) [22, 25] Arabidopsis thaliana (UNIPROT accession number: O82657) [54] Homo sapiens (UNIPROT accession number: L4245) [44] Rhinolophus ferrumequinum (UNIPROT accession number: Q1KM R4) [61]

3 Reaction and Specificity Catalyzed reaction ATP + [pyruvate dehydrogenase (acetyl-transferring)] = ADP + [pyruvate dehydrogenase (acetyl-transferring)] phosphate ( mechanism [8, 15, 26, 32]; molecular modeling of the catalytic domain, structure [24]; structure of the nucleotide binding pocket with responsible His115 residue and of the catalytic site [32]; His121 is involved in the catalytic reaction [26]) ATP + [pyruvate dehydrogenase (acetyl-transferring)] = ADP + [pyruvate dehydrogenase (acetyl-transferring)]phosphate ( allosteric R-T equilibrium as the mechanism for L2-stimulated PDK3 activity, the active cleft undergoes a conformational change from closed to open conformation upon stimulation by L2 binding releasing ADP [46]; ordered reaction mechanism with ATP binding before E1 [43]; ordered reaction mechanism with ATP binding first, overview [43]) Reaction type phospho group transfer

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2.7.11.2

Natural substrates and products S ATP + Pda1p subunit ( the enzyme is the kinase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex, which negatively regulates the complex by reversible phosphorylation of its Pda1p subunit, overview [62]) (Reversibility: ?) [62] P ADP + phosphorylated Pda1p subunit S ATP + [pyruvate dehydrogenase (lipoamide)] ( the enzyme is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex [26, 35]; regulatory role [2, 6]; b-subunit harbors a regulatory role [11]; enzyme regulation in the heart depends on thyroid hormone and lipid status [27]; tissue-specific regulation of the pyruvate dehydrogenase complex in order to adjust glucose consumption [30, 34, 35]; enzyme regulates glucose oxidation by pyruvate dehydrogenase complex, isozyme PDHK1 is of more potential importance in adult heart than the other isozymes [27]; catalyzes inactivation through phosphorylation of pyruvate dehydrogenase complex EC 1.2.4.1 [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 33, 34, 35, 36]; high-fat feeding increases the expression of isozyme PDK2, but not of PDK4, hyperthyroidism increases the expression of both isozymes, physiological implications [34, 35]; isozyme PDK3 has a putative regulatory role of the pyruvate dehydrogenase complex in sperm [30]; involved in regulation of mitochondrial pyruvate dehydrogenase complex [16]) (Reversibility: ir) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36] P ADP + [pyruvate dehydrogenase (lipoamide)] phosphate [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36] S ATP + pyruvate dehydrogenase ( a small pocket in the N-terminal region of PDHK2 is involved in enzyme regulation, the pocket is formed by residues L53, Y157, Y80, S83, I111, R112, H115, S153, R154, I157, R158, I161 [44]; enzyme has an important role in control of glucose homeostasis [40]; PDHK plays a key role in controlling the balance between glucose and lipid oxidation according to substrate supply, PDHK inhibition leads to increased PDH activity increasing glucose oxidation [42]; PDK is involved in fatty acid metabolism [51]; phosphorylation by PDK inhibits the pyruvate dehydrogenase complex, PDK plays a regulatory role in glucose metabolism, PDK4 expression is regulated by hepatic nuclear factor 4 and peroxisome proliferator-activated receptor g coactivator, PGC-1a [53]; phosphorylation of E2-bound E1 [43]; pyruvate dehydrogenase kinase is a negative regulator in the mitochondrial pyruvate dehydrogenase complex and plays a pivotal role in controlling TCA cycle and cell respiration [54]) (Reversibility: ir) [38, 40, 41, 42, 43, 44, 46, 48, 50, 51, 53, 54, 55] P ADP + phosphorylated pyruvate dehydrogenase

126

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

S ATP + pyruvate dehydrogenase complex ( PDHK4 inhibits the pyruvate dehydrogenase complex by phosphorylation during starvation, regulation mechanism, overview [56]) (Reversibility: ?) [56, 57] P ADP + phosphorylated pyruvate dehydrogenase complex S Additional information ( model of specific interactions and signal translation within the pyruvate dehydrogenase complex and between pyruvate dehydrogenase kinase and subunits, differences between the isozymes, mechanisms [30]; fibrates induction of PDK4 might be coupled to an decrease in serum triglycerides and fatty acid levels which can cause protein degradation in muscles, PDK4 induction is increased in acute rhabdomyolysis [45]; PDK regulates the pyruvate dehydrogenase multienzyme complex activity [44]; PDK4 is critically important in the starved state because it helps prevent hypoglycemia, the enzyme is part of the pyruvate dehydrogenase complex PDC, complex regulation and the molecular mechanisms [37]; PDK4 is critically important in the starved state because it helps prevent hypoglycemia, the enzyme is part of the pyruvate dehydrogenase complex PDC, detailed overview and modeling of the complex regulation and the molecular mechanisms [37]; peroxisome proliferator-activated receptors and insulin have regulatory functions in expression of isozymes PDK2 and PDK4, regulation mechanism [47]; pyruvate dehydrogenase kinase is part of the pyruvate dehydrogenase complex, regulation and component interactions, PDK2 binds the inner lipoyl domain L2, preferably in dimeric form, overview [52]; rapid upregulation of isozyme PDK4 in skeletal muscle after prolonged exercise, PDK activity is increased during prolonged exercise, physiologic/metabolic state, overview [50]; regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor, mechanism [39]; the PDK/PDH pathway is reduced by 73% in non small cell lung carcinoma contributing to hypoxia-inducible factor-1 stability and aerobic glycolysis [55]; a homodimer of pyruvate dehydrogenase kinase is an integral part of the pyruvate dehydrogenase complex, PDC, to which it is anchore primarily through the inner lipoyl-bearing domains L2 of transacetylase component, binding structure, catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the symmetric and asymmetric modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively, overview [68]; hypoxia-inducible factor HIF-1, inducible e.g. by CoCl2 , mediates the expression of PDK1, which inhibits the pyruvate dehydrogenase in the tricarboxylic cycle by phosphorylation, high PDK1 activity increases the ATP levels and prevents hypoxia-induced reactive oxygen species generation and apoptosis [59]; PDC activation also triggers apoptosis in cancer cells that selectively convert glucose to lactate, regulation of the pyruvate dehydrogenase complex, PDK4 overexpression in association with type I diabetes [60]; PDHK2 is required for binding to the inner lipoyl domain L2 of the dihydrolipoyl acetyltransferase of the pyruvate dehydrogenase complex [64]; PDK isozymes are molecular switches that downregulate the pyruvate dehydrogenase complex 127

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

PDC by reversible phosphorylation in mitochondria, L2 domain binding structure of isozyme PDK3, overview [70]; PDK4 expression is specifically regulated by retinoic acids, via retinoid receptors, and trichostatin A, an inhibitor of histone deacetylase [58]; PDK4 is involved in metabolic changes after induction by high-fat/low carbohydrate diet, overview [67]; phosphorylation of the pyruvate dehydrogenase complex PDC by the pyruvate dehydrogenase kinases PDK2 and PDK4 inhibits PDC activity, expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA, transcriptional regulation of the PDK4 gene by the estrogen-related receptors ERRa and ERRg, the ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle, overview [66]; pyruvate dehydrogenase kinase isozymes are the molecular switch that down-regulates activity of the pyruvate dehydrogenase complex through reversible phosphorylation [65]) (Reversibility: ?) [30, 37, 39, 44, 45, 47, 50, 52, 55, 58, 59, 60, 64, 65, 66, 67, 68, 70] P ? Substrates and products S ADP + pyruvate dehydrogenase ( phosphorylation of serine residues of the E1 PDC component [41]) (Reversibility: ir) [41] P ADP + phosphorylated pyruvate dehydrogenase S ATP + Ac-YHGHSMSDPGVSYR ( recombinant enzyme [29]; synthetic peptide substrate [29]) (Reversibility: ?) [29] P ADP + [Ac-YHGHSMSDPGVSYR]phosphate S ATP + Pda1p subunit ( the enzyme is the kinase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex, which negatively regulates the complex by reversible phosphorylation of its Pda1p subunit, overview [62]) (Reversibility: ?) [62] P ADP + phosphorylated Pda1p subunit S ATP + [pyruvate dehydrogenase (lipoamide)] ( highly specific for the substrate [1,9,10,11]; pyruvate dehydrogenase complex substrate is inactivated by ATP-dependent phosphorylation of 3 serine residues on the E1 subunit [29,31,35]; incorporates g-phosphate from ATP into E1-component of pyruvate dehydrogenase-complex a-subunit [3, 9, 10, 20]; the 3 serine phosphorylation sites of the E1 subunit are specifically and with different activity phosphorylated by the 4 isozymes, overview: site 1 is preferably utilized by PDK2, site 2 by PDK3, and site 3 is exclusively utilized by PDK1 [31]; optimum activity within a small range of ionic strength of 0.03-0.05 M [17]; recombinant hybrid enzyme of PDK1 and PDK2 phosphorylates site 3 with lower activity than the PDK1 homodimer [33]; serine phosphorylation site 3 of subunit E1 is exclusively phosphorylated by isozyme PDK1, not by PDK2, which prefers site 1 over site 2 [33]; isozyme PDK2 can phosphorylate free pyruvate dehydrogenase complex but bound dihydrolipoyl transacetylase enhances the rate up to 5000fold [35]; substrate is kinase-depleted pyruvate dehydro-

128

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P

S P S

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

genase complex from Zea mays [26]; phosphorylation sites are 3 Serresidues in the a-subunit, i.e. E1, MW 41000, of pyruvate dehydrogenase [1, 9, 10]; the enzyme is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex [26,35]; regulatory role [2,6]; b-subunit harbors a regulatory role [11]; enzyme regulation in the heart depends on thyroid hormone and lipid status [27]; tissue-specific regulation of the pyruvate dehydrogenase complex in order to adjust glucose consumption [30, 34, 35]; enzyme regulates glucose oxidation by pyruvate dehydrogenase complex, isozyme PDHK1 is of more potential importance in adult heart than the other isozymes [27]; catalyzes inactivation through phosphorylation of pyruvate dehydrogenase complex EC 1.2.4.1 [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 33, 34, 35, 36]; high-fat feeding increases the expression of isozyme PDK2, but not of PDK4, hyperthyroidism increases the expression of both isozymes, physiological implications [34, 35]; isozyme PDK3 has a putative regulatory role of the pyruvate dehydrogenase complex in sperm [30]; involved in regulation of mitochondrial pyruvate dehydrogenase complex [16]) (Reversibility: ir) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36] ADP + [pyruvate dehydrogenase (lipoamide)] phosphate [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36] ATP + casein ( kidney enzyme, low activity [6]) (Reversibility: ?) [6] ADP + casein phosphate [6] ATP + pyruvate dehydrogenase ( a small pocket in the N-terminal region of PDHK2 is involved in enzyme regulation, the pocket is formed by residues L53, Y157, Y80, S83, I111, R112, H115, S153, R154, I157, R158, I161 [44]; enzyme has an important role in control of glucose homeostasis [40]; PDHK plays a key role in controlling the balance between glucose and lipid oxidation according to substrate supply, PDHK inhibition leads to increased PDH activity increasing glucose oxidation [42]; PDK is involved in fatty acid metabolism [51]; phosphorylation by PDK inhibits the pyruvate dehydrogenase complex, PDK plays a regulatory role in glucose metabolism, PDK4 expression is regulated by hepatic nuclear factor 4 and peroxisome proliferator-activated receptor g coactivator, PGC-1a [53]; phosphorylation of E2-bound E1 [43]; pyruvate dehydrogenase kinase is a negative regulator in the mitochondrial pyruvate dehydrogenase complex and plays a pivotal role in controlling TCA cycle and cell respiration [54]; PDH inactivation by phosphorylation at serine residues of the E1a component of the complex [37]; PDK activity as component of the pyruvate dehydrogenase complex PDC binding the lipoyl domain of E2 [48]; phosphorylation of 3 serine residues in the pyruvate dehydrogenase domain of the pyruvate dehydrogenase complex [40]; phosphoryla129

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

P S

P S

130

2.7.11.2

tion of 3 serine residues of the E2 domain, isozyme-specific activity [42]; phosphorylation of E2-bound E1, higher activity with reduced E2, enzyme stimulation reduces the amount of bound ADP [43]; phosphorylation of serine residues of the E1 component of pyruvate dehydrogenase complex PDC [46]; phosphorylation of serine residues of the E1 PDC component [41]; the pyruvate dehydrogenase is a component of the pyruvate dehydrogenase complex, PDC [43]) (Reversibility: ir) [37, 38, 40, 41, 42, 43, 44, 46, 48, 50, 51, 53, 54, 55] ADP + phosphorylated pyruvate dehydrogenase ATP + pyruvate dehydrogenase complex ( PDHK4 inhibits the pyruvate dehydrogenase complex by phosphorylation during starvation, regulation mechanism, overview [56]; PDHK2 is an integral component of pyruvate dehydrogenase complex tightly bound to the inner lipoyl-bearing domains L2 of the dihydrolipoyl transacetylase component E2 of pyruvate dehydrogenase complex [57]) (Reversibility: ?) [56, 57] ADP + phosphorylated pyruvate dehydrogenase complex Additional information ( no activity with histones of calf thymus type II-A [6]; little, if any activity with casein of bovine kidney [11]; no activity with histones of calf thymus type II-A, VI-S and VIII-S [11]; performs pH-dependent autophosphorylation on serine residues [26]; no autophosphorylation [22,25]; activity depends on the buffer system, the reduction status of the lipoyl groups and on the serine phosphorylation site of the E1 subunit of the pyruvate dehydrogenase complex used as substrate [31]; performs autophosphorylation [29]; binding of homodimers of PDK1 and PDK2, respectively and the heterodimer of PDK1+PDK2 to the pyruvate dehydrogenase complex via dihydrolipoyl transacetylase [33]; no activity with glycogen synthase a and rabbit skeletal muscle phosphorylase b [1,9,10,11]; model of specific interactions and signal translation within the pyruvate dehydrogenase complex and between pyruvate dehydrogenase kinase and subunits, differences between the isozymes, mechanisms [30]; fibrates induction of PDK4 might be coupled to an decrease in serum triglycerides and fatty acid levels which can cause protein degradation in muscles, PDK4 induction is increased in acute rhabdomyolysis [45]; PDK regulates the pyruvate dehydrogenase multienzyme complex activity [44]; PDK4 is critically important in the starved state because it helps prevent hypoglycemia, the enzyme is part of the pyruvate dehydrogenase complex PDC, complex regulation and the molecular mechanisms [37]; PDK4 is critically important in the starved state because it helps prevent hypoglycemia, the enzyme is part of the pyruvate dehydrogenase complex PDC, detailed overview and modeling of the complex regulation and the molecular mechanisms [37]; peroxisome proliferator-activated receptors and insulin have regulatory functions in expression of isozymes PDK2 and PDK4, regulation mechanism [47]; pyruvate dehydrogenase kinase is part of the pyruvate dehydrogenase complex, regulation and component interactions, PDK2 binds the inner lipoyl domain L2, preferably in dimeric form,

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

overview [52]; rapid upregulation of isozyme PDK4 in skeletal muscle after prolonged exercise, PDK activity is increased during prolonged exercise, physiologic/metabolic state, overview [50]; regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor, mechanism [39]; the PDK/PDH pathway is reduced by 73% in non small cell lung carcinoma contributing to hypoxia-inducible factor-1 stability and aerobic glycolysis [55]; a homodimer of pyruvate dehydrogenase kinase is an integral part of the pyruvate dehydrogenase complex, PDC, to which it is anchore primarily through the inner lipoyl-bearing domains L2 of transacetylase component, binding structure, catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the symmetric and asymmetric modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively, overview [68]; hypoxia-inducible factor HIF-1, inducible e.g. by CoCl2 , mediates the expression of PDK1, which inhibits the pyruvate dehydrogenase in the tricarboxylic cycle by phosphorylation, high PDK1 activity increases the ATP levels and prevents hypoxia-induced reactive oxygen species generation and apoptosis [59]; PDC activation also triggers apoptosis in cancer cells that selectively convert glucose to lactate, regulation of the pyruvate dehydrogenase complex, PDK4 overexpression in association with type I diabetes [60]; PDHK2 is required for binding to the inner lipoyl domain L2 of the dihydrolipoyl acetyltransferase of the pyruvate dehydrogenase complex [64]; PDK isozymes are molecular switches that downregulate the pyruvate dehydrogenase complex PDC by reversible phosphorylation in mitochondria, L2 domain binding structure of isozyme PDK3, overview [70]; PDK4 expression is specifically regulated by retinoic acids, via retinoid receptors, and trichostatin A, an inhibitor of histone deacetylase [58]; PDK4 is involved in metabolic changes after induction by high-fat/low carbohydrate diet, overview [67]; phosphorylation of the pyruvate dehydrogenase complex PDC by the pyruvate dehydrogenase kinases PDK2 and PDK4 inhibits PDC activity, expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA, transcriptional regulation of the PDK4 gene by the estrogen-related receptors ERRa and ERRg, the ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle, overview [66]; pyruvate dehydrogenase kinase isozymes are the molecular switch that down-regulates activity of the pyruvate dehydrogenase complex through reversible phosphorylation [65]; interaction of PDK with L2 within the pyruvate dehydrogenase complex, overview [65]; ligand binding by isozyme PDK1 involves the conserved Ser75 [70]; modelling of the molecular mechanisms of recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2, residues L140, K173, I176, E179 are essential for recognition, and to a lesser extent also D164, D172, and A174, PDHK2 residues forming interfaces with L2, i.e. K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of 131

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

enhanced PDHK2 activity in the E2-bound state [57]; PD kinase isozymes PDK1, PDK2, PDK3 and PDK4, reduce the active form of pyruvate dehydrogenase complex, PDC, via binding to the inner lipoyl domain L2 of the dihydrolipoyl acetyltransferase E2, PDK rapidly access their E2bound PD substrate. The E2-enhanced activity of the widely distributed PDK2 is limited by dissociation of ADP from its C-terminal catalytic domain, and this is further slowed by pyruvate binding to the N-terminal regulatory domain, via the reverse of the PDC reaction, NADH and acetyl-CoA reductively acetylate lipoyl group of L2, which binds to the R domain and stimulates PDK2 activity by speeding up ADP dissociation, overall reaction of the pyruvate dehydrogenase complex, overview [60]) (Reversibility: ?) [1, 6, 9, 10, 11, 22, 25, 26, 29, 30, 31, 33, 37, 39, 44, 45, 47, 50, 52, 55, 57, 58, 59, 60, 64, 65, 66, 67, 68, 70] P ? Inhibitors (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropioamide ( strong inhibition, isozymes PDK1 and PDK2, inhibition mechanism [29]) [29] 2-chloroisohexanoate ( weak [4]) [4] 2-oxobutyrate [6] ADP ( competitive to ATP [6,7,9,10,16,24]; isozyme PDK2: synergistic with phosphate [35]; recombinant homodimers of PDK1 and PDK2 and heterodimers of PDK1 + PDK2, synergism with dichloroacetate [33]; inhibition only together with pyruvate, kinetics [16]; isozyme PDK3: synergistic with phosphate [30]; isozyme PDK2, wild-type and mutants G284A and G319A [24]; inhibition only in the presence of monovalent cations [1,7,9,10]; isozyme PDK4: K+ and dichloroacetate increase the inhibitory effect [30]; 50-60% inhibition of isozyme PDK3, in presence of dihydrolipoyl transacetylase 70% [28]; synergism with pyruvate [16, 30, 35]; Mg2+ does not protect [7]; binding site structure, involves Gly319 and Phe318, and K+ ions [44]; product inhibition, competitive to ATP, synergistic with pyruvate, PDK2, ADP, and pyruvate form a dead-end complex [43]; product inhibition, L2 binding increases affinities for both ADP and ATP [46]; product inhibition, synergistic with pyruvate [48]; binding kinetics, ATP or ADP plus pyruvate at low concentration of about 0.1 mM cause PDHK2 dimer to associate to a tetramer. These changes make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate, overview [64]; synergism with dichloroacetate [70]) [1, 6, 7, 9, 10, 16, 24, 28, 30, 33, 35, 43, 44, 46, 48, 60, 64, 70] ATP ( above 0.5 mM, substrate inhibition, only in the presence of K+ , Mg2+ does not protect [7]) [7] AZ12 ( i.e. N-[4-([ethylanilino]sulfonyl)2-methylphenyl]-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide, binding structure, requires K+ for inhibition [44]) [44] AZD7545 ( noncompetitive to ATP [42]; noncompetitive to ATP, IC50 values for isozymes PDHK1, PDHK2, and PDHK4 [42]; an

132

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

amide of trifluoro-2-hydroxy-2-methylpropionic acid, a tight binding inhibitor [60]; compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity [57]; structural mechanisms for inhibition of pyruvate dehydrogenase kinase isozymes, binding structure analysis, overview, when the E2p/E3BP core is absent, AZD7545 stimulates scaffold-free basal PDK1 and PDK3 activities to 1.3fold and 10fold, respectively [70]) [42, 57, 60, 70] adenosine 5’-[b,g-imido]triphosphate [43] butyryl-CoA ( at high concentrations [12]) [12] CaCl2 ( no inhibition [6]) [6, 14] chymotrypsin ( proteolysis of kinase a-, not b-subunit, no inactivation by trypsin-mediated proteolysis of b-subunit [11]) [11] Cl- ( 40% inhibition at 80 mM, K+ -independent inhibition [17]) [17] CoA [30] DTNB ( most potent at 0.001 mM [10]) [1, 10] decanoyl-CoA [12] dichloroacetate ( noncompetitive [8]; highly specific [36]; isozyme PDK2: ADP and K+ increase the inhibitory effect [30]; noncompetitive to ATP [42]; recombinant homodimers of PDK1 and PDK2 and heterodimers of PDK1 + PDK2, synergism with dichloroacetate [33]; potent and highly specific synthetic allosteric inhibitor mimicking pyruvate, inhibition mechanism [21]; isozyme PDK4: ADP, K+ and Cl- increase the inhibitory effect [30]; inhibition of isozyme PDK3 is independent of dihydrolipoyl transacetylase, while isozyme PDK2 is more sensitive to inhibition when bound to it [28]; pyruvate analog, synergism with ADP, K+ or phosphate, kinetics [8]; binds at the pyruvate binding site, binding structure, involves e.g. Arg154 [44]; binding kinetics [64]; R114, S83, I157 and, to some extent, H115 are essential for DCA binding by PDHK, Y80 and D117 are required for the communication between the dichloroacetate-binding site and active site of PDHK2, overview [63]; synergism with ADP, binding promotes conformational changes at the active-site cleft, structural mechanisms for inhibition of pyruvate dehydrogenase kinase isozymes, binding structure analysis, overview [70]) [5, 8, 21, 28, 29, 30, 33, 36, 42, 44, 63, 64, 70] dihydrolipoic acid ( inhibits the activity of PDK1 towards the reconstituted PD complex, inhibition mechanism [48]; inhibits the activity of PDK3 towards the reconstituted PD complex slightly, but the activity towards E1 alone completely, inhibition mechanism [48]) [48] disulfides ( thiols reverse [1,10]) [1, 10] HPO24- ( enhances inhibition by pyruvate or dichloroacetate [8]; within physiological range, only in the presence of K+ , not in its absence [17]; noncompetitive to ATP in the range of 1-10 mM [17]) [8, 17] hexanoyl-CoA [12] insulin ( blockage of the expression of isozyme PDK4 via insulinactivated pathway [35]; decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRa and ERRg [66]) [35, 66]

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

K+ ( synergism with ADP [7]; pyruvate or dichloroacetate [8]) [7, 8] linolenoyl-CoA [12] linoleoyl-CoA [12] MnCl2 [14] myristoyl-CoA [12] NAD+ [30] NEM [1, 10] Na+ ( above 50 mM, alone and synergism with ADP [7]) [7] Nov3r ( i.e. (4-[(2,5)-dimethyl-4-(3,3,3-trifluoro-2-hydroxy-2methyl-propanoyl)piperazinyl]carbonyl)benzonitrile, binding structure, requires K+ for inhibition [44]; an amide of trifluoro-2-hydroxy-2methylpropionic acid, a tight binding inhibitor, a mimic of the acetyl-dihydrolipoyl group, inhibits PDK2 [60]) [44, 60] Oleoyl-CoA [12] Pfz3 ( i.e. N-(2-aminoethyl)-2-(3-chloro-4-[(4-isopropylbenzyl)oxy]phenyl)acetamide, binding site structure, involves e.g. the R domain, allosteric inhibition mechanism, overview [44]) [44] phosphate ( isozyme PDK2, synergistically with ADP and pyruvate [35]; enhances inhibition by pyruvate [30]) [30, 35] pyruvamide ( inhibition of isozymes PDK1, PDK2, and PDK4 [48]) [48] pyruvate ( dead-end inhibitor [16]; kinetics [8, 16]; very weak inhibition [35]; isozyme PDK3 [30, 35]; isozyme PDK2: ADP and K+ increase the inhibitory effect [30]; synergism with ADP [6, 8, 9, 10, 16, 30]; synergism with K+ or phosphate [8]; inhibits at concentrations above 0.1 mM, activates below 0.05 mM [30]; isozyme PDK2: synergistic with phosphate [35]; isozyme PDHK4 is less sensitive than PDHK1 and PDHK2 [27]; noncompetitive to ATP [8,16]; product inhibition, synergistic with ADP [48]; product inhibition, synergistic with ADP, PDK2, ADP, and pyruvate form a dead-end complex [43]; slight product inhibition, synergistic with ADP [48]; binding kinetics, ATP or ADP plus pyruvate at low concentration of about 0.1 mM cause PDHK2 dimer to associate to a tetramer. These changes make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate, overview [64]) [1, 3, 5, 6, 8, 9, 10, 16, 27, 30, 35, 43, 48, 60, 64] R-lipoic acid ( inhibits isozyme PDK3 activity in the reconstituted PD complex, but not towards E1 alone, inhibition mechanism [48]; inhibits isozymes PDK1, PDK2, and PDK4 activities in the reconstituted PD complex and towards E1 alone, inhibition mechanism, overview [48]) [48] rapamycin ( inhibits PDK2 and PDK4, has no effect on insulincaused downregulation of the isozymes [47]) [47] S-lipoic acid ( inhibits isozyme PDK3 activity in the reconstituted PD complex, but not towards E1 alone, inhibition mechanism [48]; inhibits isozymes PDK1, PDK2, and PDK4 activities in the reconstituted PD complex and towards E1 alone, inhibition mechanism, overview [48]) [48] 134

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

SO24- ( with the same effect as HPO24- [17]) [17] stearoyl-CoA [12] thiamine diphosphate ( kinetics [15]; non- or uncompetitively inhibition of K+ -stimulated activity [14,15]; 2-oxoisopentanoate protects, not pyruvate [14]; in the presence of pyruvate [16]) [10, 14, 15, 16, 28] trypsin [25] adenosine 5’-[b,g,imido]triphosphate ( isozymes PDK1 and PDK2, inhibition mechanism [29]) [29] compound K ( noncompetitive to ATP, IC50 values for isozymes PDHK1, PDHK2, and PDHK4 [42]; an amide of trifluoro-2-hydroxy-2methylpropionic acid, a tight binding inhibitor [60]) [42, 60] dichloroacetophenone ( isozymes PDK1 and PDK2, inhibition mechanism [29]) [29] lactone derivative of dichloroacetophenone ( isozymes PDK1 and PDK2, inhibition mechanism [29]) [29] oximes of triterpenes ( with 17b hydroxyl and abietane derivatives, several, overview [23]) [23] radicicol ( binding site structure, structural mechanisms for inhibition of pyruvate dehydrogenase kinase isozymes, binding structure analysis, overview [70]) [70] triterpenes ( isozymes PDK1 and PDK2, inhibition mechanism [29]) [29] Additional information ( no inhibition by cAMP [6, 9, 10, 11]; isozyme PDK3 undergoes self-association in absence of dihydrolipoyl transacetylase domain L2 leading to a decrease in activity [35]; starvation and diabetes reduce the expression of isozyme PDK2 [35]; carnitine, acetylcarnitine, malate, spermine, and calcium have no effect on isozyme PDK3 in presence of dihydrolipoyl transacetylase [28]; no inhibition by 2-oxoglutarate [6]; model of specific interactions and signal translation within the pyruvate dehydrogenase complex and between pyruvate dehydrogenase kinase and subunits, differences between the isozymes, mechanisms [30]; not affected by calmodulin with or without Ca2+ [11]; no inhibition by succinyl-CoA, tiglyl-CoA, crotonyl-CoA, glutarylCoA, dl-3-hydroxy-3-methylglutaryl-CoA, acetylcarnitine or 3-hydroxybutyryl-CoA [12]; increase of ionic strength inhibits, changes of osmolarity of assay medium do not affect activity [17]; the effects of mono- and divalent ions vary greatly between the isozymes [30]; no inhibition by cGMP [9,10,11]; effects of recombinant E2 component-derived deletion constructs on PDK activity, overview [41]; high-fat diet with unaltered n-3 fatty acid levels leads to increase in PDK activity and decrease in PDH activity, high-fat diet with elevated levels of n-3 fatty acids attenuates the increase in PDH kinase activity and decreases PDH activity, overview [51]; insulin regulate the basal expression level of PDK4 by inhibiting the glucocorticoid-dependent stimulation of PDK4 expression via inactivation of FOXO proteins, FOXO proteins bind to the insulin response element which is required for the glucocorticoid response [37]; insulin reverses the stimu135

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

lation effect of reduced glucose levels increasing expression levels of PDK2 and PDK4, insulin alone decreases the enzyme expression levels below basal values, insulin effects on the enzyme are inhibited by phosphoatidyl 3-kinase inhibitors wortmannin and LY294002, i.e. 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one [47]; isozyme-specific inhibition by AZD7545 analogues [42]; pyruvamide is a poor inhibitor of PDK3 [48]; insulin downregulates expression of PDK4, but not of PDK2, after high-fat and control diets, but does not regulate the PDK4 protein [67]; PDK2 inhibition mechanism, overview [60]) [6, 9, 10, 11, 12, 17, 28, 30, 35, 37, 41, 42, 47, 48, 51, 60, 67] Cofactors/prosthetic groups ATP ( dependent on [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36]; binding site structure, involves Gly317 and Tyr320, and K+ ions [44]; causes a decrease in PDHK2 affinity for the L2 domain [41]; L2 binding increases affinities for both ADP and ATP [46]; ordered reaction mechanism with ATP [43]; binding kinetics, ATP or ADP plus pyruvate at low concentration of about 0.1 mM cause PDHK2 dimer to associate to a tetramer. These changes make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate, overview [64]; the ATP-binding loop in one PDHK3 subunit adopts an open conformation, implying that the nucleotide loading into the active site is mediated by the inactive pre-insertion binding mode [68]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 62, 64, 65, 66, 68, 70] ATPgS ( dissociation constants as ATP in binding to PDK3 [46]) [46] Additional information ( no activation by cAMP [6,9,10,11]; no activation by succinyl-CoA, tiglyl-CoA, crotonyl-CoA, glutaryl-CoA, dl-3hydroxy-3-methylglutaryl-CoA, acetylcarnitine or 3-hydroxybutyryl-CoA [12]; no activation by Ca2+ /calmodulin or calmodulin alone [11]; no activation by cGMP [9, 10, 11]; no activity with GTP [41]) [6, 9, 10, 11, 12, 41] Activating compounds 2-oxoisopentanoate ( only in the presence of K+ and thiamine diphosphate, kinetics [15]) [1, 9, 10, 12, 15] AZD7545 ( structural mechanisms for inhibition of pyruvate dehydrogenase kinase isozymes, binding structure analysis, overview, when the E2p/E3BP core is absent, AZD7545 stimulates scaffold-free basal PDK1 and PDK3 activities to 1.3fold and 10fold, respectively [70]) [70] acetoacetyl-CoA ( slight activation [12]) [12] acetyl-CoA ( involved in the regulation of enzyme activity regulating pyruvate dehydrogenase complex [20, 21]; activates the enzyme, especially isozyme PDK2 [35]; kinase bound to transacetylase core [22, 25]; domain-specific binding, isozymes PDK2 and PDK3 [28, 35]; high stimulation through acetylation of the transacety136

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

lase-catalyzing inner core portion of the dihydrolipoyl acetyltransferase [20]; isozyme PDK2: addition of K+ and Cl- required, phosphate increases the stimulating effect [30]; synergism with NADH [12, 22, 25, 28, 30]; stimulation by acetyl-CoA requires both K+ and at least one anion, phosphate or chloride, mechanisms for stimulation of PDK2, via the reverse of the PDC reaction, NADH and acetyl-CoA reductively acetylate lipoyl group of L2, which binds to the R domain and stimulates PDK2 activity by speeding up ADP dissociation, overview [60]) [1, 9, 10, 12, 20, 21, 22, 25, 28, 30, 35, 60] benzoyl-CoA ( slight activation [12]) [12] dihydrolipoyl transacetylase ( requirement [29]; domaine-specific binding, isozymes PDK2 and PDK3, the latter binding more tightly to the L2 domain [35]; degree of interaction and mechanism differ for the 4 different isozymes [30,31,35]; 3-5fold stimulation [9]; involved in the regulation of enzyme activity regulating pyruvate dehydrogenase complex [20]; isozyme PDK2 can phosphorylate free pyruvate dehydrogenase complex but bound dihydrolipoyl transacetylase enhances the rate up to 5000fold [35]; isozyme PDK2, activation mechanism, binding structure [32]; acts as a direct allosteric agent in altering the regulatory kinase activity, serves as an anchoring scaffold [30]; stimulation rates in different buffers, stimulating domains for the isozyme PDK2 and PDK3 differ, overview [28]; binding and activation mechanism [20,35]; rate-limiting in the holo-complex [3]; activation in presence of a binding protein, referred to as dihydrolipoamide dehydrogenase-binding protein [31]; pyruvate dehydrogenase-complex transacetylase core [13, 22, 25]; activation depends on the buffer system, the isozyme and the reduction status of the lipoyl groups [31]; lipoylation is required for binding, structural mutants stimulate less [28]; dynamic, effector-modified interactions of the regulatory isozymes with the flexibly held outer domains of the core-forming dihydrolipoyl acetyl transferase component of pyruvate dehydrogenase complex to adapt the complex activity, regulatory mechanism [35]) [3, 6, 9, 10, 11, 13, 20, 21, 22, 25, 28, 29, 30, 31, 32, 35] l-methylmalonyl-CoA ( slight activation [12]) [12] L2 domain ( inner lipoyl domain of the E2 component of PDC, activates PDK2 [43]) [43] L2 domain of pyruvate dehydrogenase complex ( binding to the inner lipoyl domains L2 of E2 component activate the enzyme by conformational changes and disrupting the ATP lid and eliminating product inhibition by ADP, binding structure, overview, L2 binding increases affinities for both ADP and ATP [46]) [46] lipid ( selective increase in amount of isozyme PDHK4 protein in both hyperthyroidism and high-fat feeding [27]; enhance expression of hepatic isozyme PDK2 during high-fat feeding [34]; in muscle, reversed by insulin [30]) [27, 30, 34] malonyl-CoA [12]

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

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NADH ( involved in the regulation of enzyme activity regulating pyruvate dehydrogenase complex [20,21]; activates the enzyme, especially isozyme PDK2 [35]; kinase bound to transacetylase core [22,25]; domain-specific binding, isozymes PDK2 and PDK3 [28,35]; isozyme PDK2: addition of K+ and Cl- required, phosphate increases the stimulating effect [30]; activation, in the presence of NH+4 [1,9,10]; synergism with acetyl-CoA [22,25,28]; activation, in the presence of K+ [1,9,10,12]; stimulation by NADH requires both K+ and at least one anion, phosphate or chloride, mechanisms for stimulation of PDK2, via the reverse of the PDC reaction, NADH and acetyl-CoA reductively acetylate lipoyl group of L2, which binds to the R domain and stimulates PDK2 activity by speeding up ADP dissociation, overview [60]) [1, 9, 10, 12, 20, 21, 22, 25, 28, 30, 35, 60] octanoate ( in muscle, reversed by insulin [30]) [30] oleate ( activates PDK2 and PDK4 nearly 2fold at 0.1 mM, not synergistic with palmitate, stimulation is completely inhibited by insulin at 0.001 mM [47]) [47] palmitate ( activates PDK2 and PDK4 nearly 2fold at 0.1 mM, not synergistic with oleate, stimulation is completely inhibited by insulin at 0.001 mmM [47]) [47] phosphate ( isozyme PDK43, direct inhibition and elevation of the Km for ATP [28]) [28] propionyl-CoA ( slight activation at low concentrations, synergism with NADH plus NAD+ [12]) [12] pyruvate ( at low concentration, with the thiamine diphosphate containing pyruvate dehydrogenase complex, stimulation mechanism [20]; inhibits at concentrations above 0.1 mM, activates below 0.05 mM, dependent on thiamine diphosphate [30]) [20, 30] thyroid hormones ( selective increase in amount of isozyme PDHK4 protein in both hyperthyroidism and high-fat feeding [27]) [27] WY-14,643 ( activator of peroxisome proliferator-activated receptora [30,34]; in liver, specifically increases PDK4 expression [34]; in gastrocnemius muscle, specifically increases PDK4 expression [30]) [30, 34] dibutyryl cAMP ( in muscle, reversed by insulin [30]) [30] free fatty acids ( leads to overexpression of isozyme PDK4 via mechanism involving peroxisome proliferator-activated receptor-a [35]) [35] glucocorticoids ( leads to overexpression of isozyme PDK4 via mechanism involving peroxisome proliferator-activated receptor-a [35]; regulate the basal expression level of PDK4, bind to a glucorticoid response element GRE located in the proximal promotor requiring binding of FOXO proteins, FOXO protein binding to the GRE can be inhibited by insulin, glucocorticoids act synergistically with retinoic acid [37]) [35, 37] inner lipoyl-bearing domain 2 ( i.e. L2, binding to the inner lipoyl domain L2 of E2 component of the PDC enzyme complex activates and regulates the enzyme by conformational changes and disrupting the ATP lid and eliminating product inhibition by ADP, mechanism, L2 binding strongly de-

138

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

creases PDHK2 affinity for ATP, strongest activation occurrs in presence of E1, E1-binding domain, and E2, overview [41]) [41] lipoyl domain L2 ( L2 of the PDK3-containing pyruvate dehydrogenase complex induces a cross-tail conformation in PDK3, resulting in an opening of the active site cleft and the stimulation of kinase activity [65]) [65] peroxisome proliferator-activated receptor-a ( involved in mechanism to enhance expression of isozyme PDK4, but not isozyme PDK2, during starvation [34]) [34] Additional information ( during hyperthyroidism, the expression of hepatic isozymes PDK2 and PDK4 is increased [34]; model of specific interactions and signal translation within the pyruvate dehydrogenase complex and between pyruvate dehydrogenase kinase and subunits, differences between the isozymes, mechanisms [30]; starvation increases expression of isozyme PDK4 [34,35]; agonist activation of peroxisome proliferator-activated receptors a and d specifically upregulates PDK4 transcription, while activation of peroxisome proliferator-activated receptor g specifically downregulates PDK2 transcription, reduced glucose levels increase expression levels of PDK2 and PDK4, the stimulation effect is reversed by insulin, insulin alone decreases the enzyme expression levels below basal values [47]; effects of recombinant E2 component-derived deletion constructs on PDK activity, overview [41]; farnesoid X receptor agonist GW4064 stimulates PDK4 expression in liver in vivo [39]; farnesoid X receptor agonists stimulate PDK4 expression in hepatocytes in vitro [39]; fasting largely increases PDK4 expression, increases PDK3 expression, but effects PDK2 and PDK1 only slightly in skeletal muscle, overview, peroxisome proliferator-activated receptor-a PPAR-a protein and forkhead homologue FKHR in rhabdomyosarcoma are putative transcriptional activators of the enzyme [49]; glucocorticoids and farnesoid X receptor agonists, dexamethasone or WY14643, stimulate PDK4 expression in hepatoma cells in vitro [39]; glucocorticoids and insulin regulate the basal expression level of PDK4, starvation and diabetes mellitus increase PDK4 expression in most tissues, no effects by dexamethasone [37]; histone acetylation and transcription activation of gene PDK4 by retinoic acid and trichostatin A, retinoic acid acts synergistically with glucocorticoids, induction by dexamethasone can be inhibited by RU486 [37]; no effects on PDK4 expression level by dexamethasone [37]; PDK4 expression is induced by hepatic nuclear factor 4, HNF4, determination of binding sites on the PDK4 promoter [53]; PDK4 mRNA is very rapidly induced by fibrates, e.g. bezafibrate, and statins, e.g. statin, pravastatin, or simvastatin, and anti-bacterial drugs, e.g. quinolon, ofloxacin, and norfloxacin, in a tissue-specific manner, overview [45]; rapid upregulation of isozyme PDK4 in skeletal muscle after prolonged exercise, PDK activity is increased 2.5fold during 240 min of exercise [50]; the enzyme is stimulated 3.45fold by NADH/NAD+ and/or acetyl-Co via reduction and reductive acetylation of the lipoyl moieties of pyruvate dehydrogenase complex component E2 dihydrolipoyl acetyltransferase requiring elevated levels of Cl- and K+ , activation increases the kcat and Km for ATP [43]; the enzyme is stimulated by reduction or 139

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reduction/acetylation of the lipoyl moieties of pyruvate dehydrogenase complex component E2 [48]; E2-dependent PDHK2 activation, molecular modeling and mechanism [57]; expression of isozymes PDK2 and PDK4 is induced by free fatty acids, overview [69]; hypoxia-inducible factor HIF-1 induces PDK1 expression, PDK1 is a direct target gene of HIF-1 [59]; PDK4 expression is 2fold induced in high-fat/low carbohydrate diet and is blunted under basal and clamp conditions after high-fat diet, overview [67]; PDK4 expression is induced by retinoic acids and trichostatin A [58]; the peroxisome proliferator-activated receptor g coactivator, PGC1g, as well as estrogen-related receptors ERRa and ERRg stimulate the expression of isozymes PDK2 and PDK4, the latter being inhibited by insulin, expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA, overview [66]) [30, 34, 35, 37, 39, 41, 43, 45, 47, 48, 49, 50, 53, 57, 58, 59, 66, 67, 69] Metals, ions Cl- ( activates [43,60]; required for stimulation of PDK2 by reduction and reductive acetylation of E2 [43]) [43, 60] Divalent cations ( requirement [5]) [5] K+ ( activates [43]; activation [19]; inhibits in presence of ADP [7]; K+ -dependent activation, inhibited by thiamine diphosphate [14]; 2.2fold activation at 20 mM K+ , not pH- and buffer concentration-dependent [17]; binds via Gly319, involved in inhibition by ADP, Nov3r, and AZ12, and in binding of ATP [44]; required for stimulation of PDK2 by reduction and reductive acetylation of E2 [43]; activates, the Km for ATP is decreased and ADP inhibition is enhanced by elevating K+ ion levels, ligand-induced changes in K+ binding, overview [60]) [7, 14, 17, 19, 43, 44, 60, 65] KCl [51] Mg2+ ( required [52]; activates [60]; requirement [1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 22, 24, 30]; actual substrate: MgATP2- [3, 6, 7, 9, 10, 22]) [1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 24, 25, 26, 28, 30, 31, 33, 36, 38, 43, 44, 46, 48, 51, 52, 56, 57, 59, 60, 65, 69, 70] Mn2+ ( requirement [1, 6, 9, 10]; can replace Mg2+ to some extent [1, 6, 9, 10]) [1, 6, 9, 10] NH+4 ( activation in the absence of ADP, inhibits in presence of ADP [7]) [7] Na+ ( hinders interaction between PDK2 and the inner lipoyl domain L2 [52]) [52] phosphate ( activates [60]) [60] Additional information ( no activation by Ca2+ [6]; no activation by NaCl, LiCl [14]; no activation by Na+ [17]) [6, 14, 17] Turnover number (min–1) 0.0004 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant E238A [38]) [38]

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0.0005 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant E238H [38]) [38] 0.0014 (pyruvate dehydrogenase, PDK activity in skeletal muscle after short 10 min or no exercise [50]) [50] 0.0032 (pyruvate dehydrogenase, PDK activity in skeletal muscle after prolonged exercise of 240 min [50]) [50] 0.0383 ([pyruvate dehydrogenase (lipoamide)], phosphorylation site 3 of subunit E1, isozyme PDK1, pH 7.0, 30 C [31]) [31] 0.07 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant L234A [38]) [38] 0.0933 ([pyruvate dehydrogenase (lipoamide)], phosphorylation site 2 of subunit E1, isozyme PDK3, pH 7.0, 30 C [31]) [31] 0.108 ([pyruvate dehydrogenase (lipoamide)], phosphorylation site 2 of subunit E1, isozyme PDK4, pH 7.0, 30 C [31]) [31] 0.11 ([pyruvate dehydrogenase (lipoamide)], phosphorylation site 1 of subunit E1, isozyme PDK1, pH 7.0, 30 C [31]) [31] 0.12 (ATP, pH 7.4, 30 C, with oxidized E2 [43]) [43] 0.17 (ATP, pH 7.4, 30 C, with oxidized E2, in presence of 2 mM DTT [43]) [43] 0.21 (ATP, pH 7.4, 30 C, with oxidized E2, in presence of NADH/ NAD+ [43]) [43] 0.277 ([pyruvate dehydrogenase (lipoamide)], phosphorylation site 1 of subunit E1, isozyme PDK2, pH 7.0, 30 C [31]) [31] 0.28 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant H233A [38]) [38] 0.39 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant L234H [38]) [38] 0.512 (ATP) [13] 0.533 (ATP) [1] 0.55 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant wildtype PDK [38]) [38] 0.9-4.7 (ATP, pH 7.4, 30 C, PDK2, with substrate E2-bound E1, dependent on the assay conditions concerning buffer composition, overview [43]) [43] 0.91 (ATP, pH 7.4, 30 C, with reduced E2 [43]) [43] 1.3 (ATP, pH 7.4, 30 C, with reduced E2, in presence of NADH/ NAD+ [43]) [43] 3.15 (ATP, pH 7.4, 30 C, with reduced E2, in presence of NADH/ NAD+ and acetyl-CoA [43]) [43] Specific activity (U/mg) 0.006 ( PDK3 activity with E1 [48]) [48] 0.008 ( purified recombinant enzyme [19]) [19] 0.018 ( purified dihydrolipoyl transacetylase-protein X-pyruvate dehydrogenase kinase subcomplex [13]) [13] 0.02 ( about, with E2 bound to E1, 0.2 mM ATP [43]) [43] 0.027 ( PDK2 activity with E1 [48]) [48]

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0.03 ( about, with E2 bound to E1 in presence of NADH/NAD+ and acetyl-CoA, 0.2 mM ATP [43]) [43] 0.031 ( PDK1 activity with E1 [48]) [48] 0.032 ( PDK4 activity with E1 [48]) [48] 0.037 ( about, with free E2, 0.2 mM ATP [43]) [43] 0.038 ( PDK4 activity with reconstituted PDC [48]) [48] 0.056 ( PDK1 activity with reconstituted PDC [48]) [48] 0.064 ( PDK3 activity with reconstituted PDC [48]) [48] 0.066 ( about, with free E2 in presence of NADH/NAD+ and acetylCoA, 0.2 mM ATP [43]) [43] 0.087 ( PDK2 activity with reconstituted PDC [48]) [48] 0.276 ( fed rat, purified enzyme [18]) [18] 0.33 [10, 11] 0.332 ( purified enzyme, in presence of dihydrolipoyl transacetylase [9,10]) [9, 10] 0.44 ( isozyme PDK3, pH 7.3, 30 C [28]) [28] 0.69 ( isozyme PDK2, pH 7.3, 30 C [28]) [28] 0.74 ( purified recombinant enzyme, in presence of NADH and acetyl-CoA [25]) [25] 0.92 ( purified recombinant enzyme, in presence of NADH and acetyl-CoA [25]) [25] 1.1 ( purified isozyme PDK2 [52]) [52] 1.24 ( starved rat, purified enzyme [18]) [18] 1.9 ( purified enzyme, in presence of dihydrolipoyl transacetylase, reconstituted enzyme complex [3]) [3] Additional information ( activity depends on the buffer system, the reduction status of the lipoyl groups and on the serine phosphorylation site of the E1 subunit of the pyruvate dehydrogenase complex used as substrate [31]; PDH activity in zucker diabetic fatty rats, i.e. ZDF rats, as indicator for the degree of PDHK inhibition [42]; PDK activity in different dietary states [51]; pyruvate dehydrogenase complex activities of wild-type and recombinant strains [62]) [22, 27, 31, 42, 43, 50, 51, 62] Km-Value (mM) 0.0006 (pyruvate dehydrogenase, pH 7.5, 30 C [6]; pH 7.0, 30 C [9,10]; in presence of dihydrolipoyl transacetylase [6,9,10]) [6, 9, 10] 0.006 (ATP, isozyme PDK2, 30 C, pH 7.3, MOPS-K+ buffer [28]; isozyme PDK3, 30 C, pH 7.3, Tris-HEPES buffer [28]) [28] 0.007 (ATP, recombinant His-tagged isozyme PDK2, 37 C, pH 7.8 [33]) [33] 0.0077 (ATP, pH 7.4, 30 C, with oxidized E2 [43]) [43] 0.01 (ATP, at 0.2 M buffer concentration [17]) [17] 0.0109 (ATP, pH 7.4, 30 C, with oxidized E2, in presence of NADH/ NAD+ [43]) [43] 0.0113 (ATP, pH 7.4, 30 C, with oxidized E2, in presence of 2 mM DTT [43]) [43]

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0.013 (ATP, isozyme PDK3, 30 C, pH 7.3, MOPS-K+ buffer [28]) [28] 0.016 (ATP, 37 C, recombinant isozyme PDK2 wild-type [24]) [24] 0.02 (ATP, kidney enzyme [6,9,10]; pH 7.5, 30 C [6]; pH 7.5, 25 C [16]; pH 7.0, 30 C [9,10]; pyruvate dehydrogenase complex [6,9,10]) [6, 9, 10, 16] 0.02 (Mg2+ , kidney enzyme [6,9,10]; pH 7.5, 30 C [6]; pH 7.0, 30 C [9,10]; pyruvate dehydrogenase complex [6,9,10]) [1, 6, 9, 10] 0.02 (pyruvate dehydrogenase, pH 7.5, 30 C [6]; pH 7.0, 30 C [9,10]; in absence of dihydrolipoyl transacetylase [6,9,10]) [6, 9, 10] 0.023 (ATP, isozyme PDK2, 30 C, pH 7.3, Tris-HEPES buffer [28]) [28] 0.025 (ATP, at 0.04 M buffer concentration [17]) [8, 17] 0.025-0.22 (ATP, pH 7.4, 30 C, PDK2, with substrate E2-bound E1, dependent on the assay conditions concerning buffer composition, overview [43]) [43] 0.026 (ATP) [16] 0.028 (ATP, 37 C, recombinant mutant G284A of isozyme PDK2 [24]) [24] 0.029 (ATP, isozyme PDK3, 30 C, pH 7.3, phosphate buffer [28]) [28] 0.034 (ATP, recombinant His-tagged isozyme PDK1, 37 C, pH 7.8 [33]) [33] 0.039 (ATP, pH 7.4, 30 C, with reduced E2 [43]) [43] 0.04 (ATP, isozyme PDK2, 30 C, pH 7.3, phosphate buffer [28]) [28] 0.047 (ATP, pH 7.4, 30 C, with reduced E2, in presence of NADH/ NAD+ [43]) [43] 0.05 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant L234H [38]; pH 7.5, 37 C, recombinant wild-type PDK [38]) [38] 0.075 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant L234A [38]) [38] 0.2 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant H233A [38]) [38] 0.28 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant E238A [38]) [38] 0.4 (pyruvate dehydrogenase, pH 7.5, 37 C, recombinant PDK mutant E238Q [38]) [38] 1.16 (ATP, pH 7.4, 30 C, with reduced E2, in presence of NADH/ NAD+ and acetyl-CoA [43]) [43] 1.5 (ATP, 37 C, recombinant mutant G319A of isozyme PDK2 [24]) [24] 3.5 (pyruvate dehydrogenase, about, pH 7.5, 37 C, recombinant PDK mutant E238H [38]) [38] Additional information ( kinetics [38,43]; the Km value for ADP depends on the presence and concentration of K+ , effect of K+ on kinetic parameters [7]; changes in ionic strength [17]; dissocia143

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

tion constants for ADP and ATP binding, L2 binding increases affinities for both ADP and ATP [46]; pyruvate dehydrogenase complex components interaction dissociation constants and kinetics [52]; binding of wildtype and mutant PDHK2 proteins to the unaltered L2 domain, kinetics [57]; kinetics and thermodynamics of PDK binding to L2 within the pyruvate dehydrogenase complex [65]) [7, 17, 38, 43, 46, 52, 57, 65] Ki-Value (mM) 0.0031 (adenosine 5’-[b,g-imido]triphosphate, pH 7.4, 30 C, versus ATP [43]) [43] 0.1 (ADP, pH 7.5, 30 C [6]) [6] 0.14-0.52 (ADP, pH 7.4, 30 C, versus ATP, dependent on the assay conditions concerning buffer composition, overview [43]) [43] 0.2 (ADP, 37 C, recombinant isozyme PDK2 wild-type [24]) [24] 0.2 (dichloroacetate, pH 7.4, 37 C [21]; isozyme PDHK2, 30 C [42]) [21, 42] 0.21 (ADP, pH 7.5, 25 C [16]) [16] 0.27 (dichloroacetate, pH 7.2, 30 C [8]; kinetic constant from binding study [8]) [8] 0.27 (pyruvate, pH 7.2, 30 C [8]; kinetic constant from binding study [8]) [8] 0.3 (ADP, 37 C, recombinant mutant G284A of isozyme PDK2 [24]) [24] 0.325 (pyruvate, pH 7.5, 25 C [16]) [16] 0.5 (dichloroacetate, isozyme PDHK4, 30 C [42]) [42] 1 (dichloroacetate, isozyme PDHK1, 30 C [42]) [42] 1.6 (ADP, 37 C, recombinant mutant G319A of isozyme PDK2 [24]) [24] 10 (HPO24-, pH 7.8, 30 C [17]) [17] Additional information ( isozyme PDK4: K+ reduces the Ki for ADP and therefor enhances the inhibitory affect [30]; Ki value for ADP depends on the presence and concentration of monovalent cations, e.g. NH+4 or K+ [7]; inhibition kinetics for thiamine diphosphate [15]; inhibition kinetics [43]; dichloroacetate inhibition kinetics and EC50 values with wild-type and mutant PDHK2s [63]) [7, 15, 30, 43, 63] pH-Optimum 7 ( assay at [3,9,10,11,13,48]) [3, 9, 10, 11, 13, 48] 7-7.2 ( in presence of Mg2+ or Mn2+ [6]) [6] 7-7.4 ( assay at [27,28]) [27, 28] 7.2 ( assay at [8]) [8] 7.2-8 ( broad, at 0.15 M buffer concentration [17]) [17] 7.3 ( assay at [12,65]; autophosphorylation [26]) [12, 26, 65] 7.4 ( assay at [21, 22, 25, 36, 43, 51, 69]) [21, 22, 25, 36, 43, 51, 69] 7.5 ( assay at [16, 19, 52, 64, 70]; assay at, pH-dependency of wild-type and mutant PDKs, overview [38]) [16, 19, 38, 52, 64, 70] 144

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

7.8 ( assay at [33,57]) [33, 57] Additional information ( different buffer systems [31]; optimal activity within a range of 0.03 M and 0.05 M buffer concentrations [17]) [17, 31] pH-Range 5.5-8.5 ( about 50% or 60% of maximal activity at pH 5.5 and about 65% or 50% of maximal activity at pH 8.5, in the presence of Mg2+ or Mn2+ , respectively [6]) [6] 6.2-9 ( about half-maximal activity at pH 6.2 and 9 [17]) [17] Temperature optimum ( C) 22 ( assay at room temperature [65]; room temperature, assay at [19]) [19, 65] 25 ( assay at [16, 26, 64, 70]) [16, 26, 64, 70] 30 ( assay at [3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 22, 27, 28, 31, 42, 43, 48, 51, 52]; phosphate buffer [28]) [3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 22, 27, 28, 31, 42, 43, 48, 51, 52] 37 ( assay at [4, 21, 24, 25, 33, 57]; assay at, temperature-dependency of wild-type and mutant PDKs, overview [38]) [4, 21, 24, 25, 33, 38, 57]

4 Enzyme Structure Molecular weight 76000 ( recombinant wild-type, gel filtration [25]) [25] 80000 ( about, recombinant enzyme, gel filtration [22]) [22] 86000 ( approximately, recombinant enzyme with maltose-binding protein cleaved off, gel filtration [26]) [26] 90220 ( isozyme PDK2, sedimentation equilibrium centrifugation analysis [52]) [52] 92000 ( recombinant hybrid dimer of PDK1 and PDK2, gel filtration [33]) [33] 100000 [1] 136000 ( pyruvate dehydrogenase, gel filtration [3]) [3] Additional information ( sedimentation equilibrium centrifugation analysis of the pyruvate dehydrogenase complex and components [52]) [52] Subunits ? ( x * 45000, free pyruvate dehydrogenase kinase, SDSPAGE [18]; x * 47500, recombinant His-tagged enzyme, SDS-PAGE [19]; x * 48000, native enzyme, SDS-PAGE [36]; x * 36000, recombinant enzyme, SDS-PAGE [36]; x * 45066, isozyme PDK2, DNA sequence determination [30]; x * 45806, recombinant isozyme PDK2, detagged, amino acid determination [28]; x * 48391, isozyme PDK1, DNA sequence determination [30]; x * 46230, isozyme PDK4, DNA sequence determination [30]; x * 46504, recombinant isozyme PDK3, detagged,

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

amino acid determination [28]; x * 45883, isozyme PDK3, DNA sequence determination [30]) [18, 19, 28, 30, 36] dimer ( homodimer [63]; dimer of monomers built of 2 different domains, crystal structure [32]; 1 * 48000, a + 1 * 45000, b, SDS-PAGE [1,9,10,11]; 2 * 45000, recombinant wild-type, SDS-PAGE [25]; 2 * 43000-43470, mature protein, SDS-PAGE and DNA sequence determination [22]; 2 * 44000, recombinant enzyme with maltose-binding protein cleaved off, SDS-PAGE [26]; 2 * 45816, sedimentation equilibrium centrifugation analysis in presence of 1% ethylene glycol, 0.1% Pluronics F68, and 0.15 M NaCl [52]; PDHK2 is a component of the pyruvate dehydrogenase complex, complex structure [44]; PDK is a component of the pyruvate dehydrogenase complex bound to the inner lipoyl domains, i.e. L2, of the E2 component, the C-terminus of one PDK subunit constitutes an integral part of the lipoyl-binding pocket in the N-terminus of the opposing other subunit [46]) [1, 9, 10, 11, 22, 25, 26, 32, 44, 46, 52, 63] dimer or tetramer ( effects of ligand binding on distal structure of PDHK2, analytical ultracentrifugation, structure, overview [64]) [64] Additional information ( subunit composition and complex structure [3,13,30]; kinase activity resides in a-subunit [1,11]; PDK is a component of the pyruvate dehydrogenase complex [43,55]; PDK is a component of the pyruvate dehydrogenase complex PDC binding the lipoyl domain of E2 [43,48]; PDK is a component of the pyruvate dehydrogenase complex, i.e. PDC, the E2 component of PDC comprises the transacetylase domain, the E1-binding domain, and two lipoylbearing domains L1 and L2 [41]; a homodimer of pyruvate dehydrogenase kinase is an integral part of the pyruvate dehydrogenase complex, PDC, to which it is anchore primarily through the inner lipoyl-bearing domains L2 of transacetylase component, the PDHK3 subunits have distinct conformations: one subunit exhibits open and the other closed configuration of the putative substrate-binding cleft, domain organization, binding structure, modeling, overview [68]; components and organization of the mammalian pyruvate dehydrogenase complex, including the enzyme [60]; PDHK2 is an integral component of pyruvate dehydrogenase complex tightly bound to the inner lipoyl-bearing domains L2 of the dihydrolipoyl transacetylase component E2 of pyruvate dehydrogenase complex, residues L140, K173, I176, E179 are essential for recognition, and to a lesser extent also D164, D172, and A174, PDHK2 residues forming interfaces with L2, i.e. K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state, enzyme structure, overview [57]; pyruvate dehydrogenase complex L2 domain binding structure of isozyme PDK3, overview [70]; structural determinants for cross-talk between PDK3 and lipoyl domain 2 of the pyruvate dehydrogenase complex, overview [65]; the molecular organization of PDH is influenced by Yil042cp, encoding the enzyme, which is the kinase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex, epitope tagging, overview [62]) [1, 3, 11, 13, 30, 41, 43, 48, 55, 57, 60, 62, 65, 68, 70]

146

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

Posttranslational modification phosphoprotein ( PDK2 performs autophosphorylation in presence of R-lipoic acid [48]) [48]

5 Isolation/Preparation/Mutation/Application Source/tissue 293T cell [37] C2C12 cell ( myoblast cells [45]) [45] Fao cell ( subclone of the hepatoma HIIE cell line [45]) [45] H4IIE cell ( hepatoma cell line, determination of PDK4 expression level in presence or absence of farnesoid X receptor agonists [39]) [39] Hep-G2 cell [37] L-6 cell ( muscle cell [37]) [37] Morris hepatoma 7800C1 cell [37] P493-6 cell [59] T-lymphocyte [37] adenocarcinoma ( high PDK expression [55]) [55] adipose tissue ( mainly isozyme PDK2 [60]) [60] brain ( isozymes PDK2 and PDK3 [30]; higher activity in cerebral cortex and hippocampus than in hypothalamus, pons, medulla and olfactory bulbs [5]; low expression level of PDK4 [61]) [5, 30, 61] brown adipose tissue ( isozyme PDK2 [30]) [30] cell culture [47] cerebral cortex [5] egg [40] fibroblast ( embryonic fibroblast cell line [59]; in the non small cell lung tumor-supporting stroma, reduced expression of PDK1, high PDH activity [55]) [55, 59] gastrocnemius ( overexpression of isozyme PDK4 during starvation or diabetes, reversible by insulin [30]) [30] heart ( appearance of isozyme PDK1 is limited to the heart, isozyme PDK2 [30]; developing and adult heart, the latter contains 3 isozymes: PDHK1, PDHK2 and PDHK4, clear differences in protein expression patterns of the isoforms dependent on developmental stage, overview [27]) [1, 2, 3, 6, 9, 14, 15, 23, 27, 30, 35, 36, 40, 42, 60, 61, 66] hepatocyte ( primary [53]; primary, determination of PDK4 expression level in presence or absence of farnesoid X receptor agonists [39]) [39, 53] hepatoma cell ( Fao cell line [45]; PDK4 [37]) [37, 39, 45, 66] hippocampus [5] kidney ( cortex [17]; PDK2-like isozyme very tightly bound to dihydrolipoyl transacetylase, possibly 2 forms of isozyme PDK2 exist [30]; isozymes PDK2, PDK3 and PDK4 [30]; PDK2 [37]) [1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 17, 20, 30, 35, 37, 40, 42, 45] 147

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

leaf ( green leaf tissue [16]) [16, 54] liver ( a complex signalling mechanism mediates the regulation of the isozymes PDK2 and PDK4 in response to the feeding status of the organism, overview [34]; isozymes PDK2 and PDK4 are both overexpressed under conditions of starvation, diabetes, or high-fat feeding, low-carbohydrate diet, or due to artificially elevated cAMP or 3,5,3-triidothyronine levels, reversible by insulin or high-carbohydrate diet [30]; determination of PDK4 expression level in presence or absence of farnesoid X receptor agonists [39]; PDK2 [37]; isozymes PDK2 and PDK4 [60]; isozymes PDK2 and PDK4 expression analysis in liver at different developmental stages in diabetic and healthy, starved or fed, rats, overview [69]; isozymes PDK2 and PDK4, obese Zucker rats show levels of expression of PDK2 and PDK4 in liver and skeletal muscle similar to those found in lean rats [60]; low expression level of PDK4 [61]) [4, 18, 30, 34, 37, 39, 40, 42, 45, 53, 60, 61, 69] lung ( isozymes PDK3 and PDK4, low levels of isozyme PDK2 [30]; PDK1 and PDH expression patterns [55]) [30, 40, 55] mammary gland ( lactating, isozyme PDK2 [30]) [30] muscle ( adult [22]) [22, 23, 25] muscle cell [47] myoblast [47] myoblast cell [45] myocyte ( cardiac [53]) [53] myotube ( differentiation of myoblasts to [45]) [45, 47] non-small cell lung cancer ( i.e. NSCLC, reduced PDK/PDH activity, PDK1 and PDH expression patterns [55]) [55] oocyte ( oocyte-specific PDK isoform [40]) [40] pancreatic b cell [42] seed [54] seedling [16] silique [54] skeletal muscle ( isozyme PDK2 [30]; isozyme PDK1PDK4, with isozymes PDK2 and PDK4 being the most abundant, expression analysis in normal and fasted muscle [49]; isozymes PDK2 and PDK4 [60]; isozymes PDK2 and PDK4, obese Zucker rats show levels of expression of PDK2 and PDK4 in liver and skeletal muscle similar to those found in lean rats [60]) [30, 37, 42, 45, 49, 50, 51, 56, 60, 61, 66, 67] skin [40] sperm ( isozyme PDK3 with unique E1 subunit probably encoded on the Y-chromosome in contrary to the normal E1 subunit which is encoded on the X-chromosome, regulatory role [30]) [30] spleen ( isozyme PDK2, low level [30]) [30, 40] squamous cell carcinoma ( high PDK expression [55]) [55] testis [40] tibialis anterior [42] white adipose tissue ( isozyme PDK2 [30]) [30, 61]

148

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

Additional information ( isozyme PDK4 is overexpressed in many tissues during diabetes and starvation, particularly important in skeletal and heart muscle [30]; selective increase in amount of isozyme PDHK4 protein in both hyperthyroidism and high-fat feeding [27]; tissue-specific expression, regulation mechanisms [30,31]; differential tissue-specific expression of isozymes, distribution overview [40]; starvation and diabetes mellitus increase PDK4 expression in most tissues, PDK2 especially in liver and kidney [37]; tissue-specific induction of PDK4 by statins, fibrates, and anti-bacterial drugs, overview [45]; tissue-specific isozyme distribution [42]; quantitative PDK4 tissue expression analysis [61]) [27, 30, 31, 37, 40, 42, 45, 61] Localization mitochondrial inner membrane [10] mitochondrial matrix [10] mitochondrion ( inner membrane matrix compartment [1,10]; enzyme contains a 18-amino acid mitochondrial import signal sequence [22]; purified pyruvate dehydrogenase-complex contains 3 molecules of kinase, but one molecule of dihydrolipoyl transacetylase-protein x-subcomplex of pyruvate dehydrogenase activates more than 15 molecules of kinase [13]; tightly bound to dihydrolipoamide acetyltransferase of pyruvate dehydrogenase complex [1,2,9,10]; protein x serves to anchor the kinase to the core of the complex [13]) [1, 2, 3, 6, 9, 10, 11, 13, 16, 18, 22, 26, 27, 32, 33, 35, 40, 41, 43, 44, 50, 51, 53, 54, 57, 59, 60, 61, 62, 63, 65, 66, 70] Purification (recombinant His-tagged PDK2, E2, and E3 by nickel affinity chromatography, the His-tag is removable) [43] (recombinant His-tagged PDK3 from Escherichia coli strain BL21) [48] (recombinant His-tagged isozymes PDK2 and PDK3 from Escherichia coli BL21(DE3)) [28] (free pyruvate dehydrogenase kinase, separable from pyruvate dehydrogenase complex by gel filtration, to homogeneity) [18] (recombinant His-tagged PDK2 expressed in Escherichia coli) [21, 33] (recombinant His-tagged isozymes PDK1 and PDK2 expressed in Escherichia coli BL21(DE3)) [29] (recombinant His-tagged isozymes PDK1, PDK2, and PDK4 from Escherichia coli strain BL21) [48] (recombinant from Escherichia coli BL21(DE3) as His-tagged protein, to homogeneity) [32] (recombinant hybrid dimer of T7-tagged PDK1 and His-tagged PDK2) [33] (recombinant wild-type and mutants as His-tagged proteins from Escherichia coli BL21(DE3)) [24] [3] (recombinant c-Myc-tagged or HA-tagged enzyme from strain BY4741 mitochondria by tandem affinity chromatography) [62] 149

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

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[2, 14] (2700fold to homogeneity) [11] (kidney, from highly purifed pyruvate dehydrogenase-complex) [1, 3, 9, 10, 11, 13] (removed from the dihydrolipoyl transacetylase by treatment with p-hydroxymercuriphenylsulfonate) [3, 13] (recombinant His6-tagged wild-type and mutant PDKs from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration) [38] (recombinant from Escherichia coli as a fusion protein with the maltose-binding protein, to near homogeneity) [26] (recombinant wild-type and truncated form from Escherichia coli as His-tagged and maltose-binding fusion protein, respectively, to homogeneity) [25] (recombinant His-tagged protein from Sf9 insect cells, to near homogeneity) [19] (recombinant from Escherichia coli as His-tagged isozyme PDK1) [33] (recombinant from Escherichia coli, large scale) [36] (recombinant hybrid dimer of T7-tagged PDK1 and His-tagged PDK2) [33] (recombinant from Escherichia coli as His-tagged protein to homogeneity) [22, 25] (recombinant N-terminally His-tagged residues A8-T399 of isozyme PDHK2 from insect cells by nickel affinity chromatography, the His-tag is cleaved off by thrombin) [44] Renaturation (reconstitution of the active pyruvate dehydrogenase complex PDC) [48] (reconstitution of the active pyruvate dehydrogenase complex PDC) [48] (reconstitution after solubilization with 6 M urea) [36] Crystallization (isozyme PDHK3 in an asymmetric complex with L2, hanging-drop vapor diffusion technique, mixing of 0.002 ml of protein solution containing 3-5 mg/ml of the PDHK3/L2 complex, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM DTT, and 2.5% v/v ethylene glycol with an equal volume of reservoir solution containing 100 mM histidine, pH 6.4, 30 mM EDTA, and 7% v/v 2,4methyl penthane diol, X-ray diffraction structure determination and analysis at 2.5 A resolution, molecular replacement, modelling) [68] (isozyme PDK1 in complex with inhibitors AZD7545 and dichloroacetate, hanging-drop vapor-diffusion method, mixing of 2 ml of PDK1 solution with 50 mg/ml untagged PDK1 in 50 mM potassium phosphate, pH 7.5, 250 mM KCl, 800 mM lysine, 5% glycerol, and 20 mM DTT with 2 ml of well solution containing 0.42 M Na-K-tartrate, 0.1 M Na citrate, pH 5.6,at 20 C, 1 week, X-ray diffraction structure determination and analysis at 1.9-2.6 A resolution) [70] (pyruvate dehydrogenase kinase 3 bound to ATP and the lipoyl domain 2 of human pyruvate dehydrogenase complex, X-ray structure determination 150

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

and analysis at 1.0 A resolution, modeling of closed and open conformation) [46] (vapor diffusion method, protein 10 mg/ml in 100 mM imidazole or 100 mM MES, pH 6.5-7.0, 0.5 M KCL, 3.5 mM 3-iodopropionic acid, 3.5 mM ADP, 7 mM MgCl2 , 6.5% polyethylene glycol 6000, 1.0% ethylene glycol, 1 week, cryoprotection with 25% glycerol, X-ray diffraction structure determination) [32] (phosphorylated and nonphosphorylated components of the pyruvate dehydrogenase complex from heart and kidney) [2] (purified recombinant residues A8-T399 of isozyme PDHK2 free or in complex with ADP and chloroacetate, ATP, Nov3r, Pfz3, or AZ12, hanging drop vapour diffusion method, protein solution contains 10 mg/ml protein, 20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, and 1 mM DTT, versus 0.1 M sodium acetate, pH 5.6-5.8, 6-9% 2-propanol, and 75-125 mM MgCl2 , 4 C, 2 weeks, complexing with ligands by soaking of PDHK2 crystals in solutions containing 10 mM ATP, 10 mM ADP and 100 mM dichloroacetate, or 1 mM of Nov3r, Pfz3, or AZ12, cryoprotection by 30-35% glycerol, X-ray diffraction structure determination and analysis at 2.2-3.0 A resolution) [44] Cloning (4 isozymes: PDK1, PDK2, PDK3, PDK4) [30] (the gene encoding PDK4 is located on chromosome 6, DNA sequence analysis, genomic organization, possible regulatory important elements are Sp1 and CBF sites) [37] (His-tagged PDK3 overexpression in Escherichia coli strain BL21) [48] (PDK4 gene is located in q21.3 region of chromosome 7, DNA sequence analysis, genomic organization, DNA sequence determination of the partial proximal promotor) [37] (PDK4, expression of PDK2 and PDK4 in 29T cells, transient expression of PDK4 and PDK4 promoter in CV-1 cells, expression is regulated by retinoic acids and trichostatin A, the proximal promoter contains two retinoic acid response elements binding retinoid X receptor a and retinoid acid receptor a leading to transcriptional activation, the PDK4 expression activation by all components is prevented by interaction of p300/CBP with E1A, expression analysis, overview) [58] (co-expression of GST-tagged PDHK2 with E1 and E2 components of PDC in Escherichia coli strain BL21(DE3)) [41] (co-expression of PDK2, E2, and E3 as His-tagged proteins) [43] (expression analysis and mRNA level determination for isozymes PDK2 and PDK4 in response to several effectors) [47] (expression of isozyme PDK4 in Escherichia coli, unmodified and modified enzyme) [35] (expression of isozyme PDK4-luciferase in McA-RH7777 cells using the adenovirus transfection method, PDK2 and PDK4 expression analysis, overview) [66] (expression of wild-type and mutant PDHK2 isozymes) [63]

151

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

(isozymes PDK2 and PDK3, expression in Escherichia coli BL21(DE3) as His-tagged proteins) [28] (overexpression of isozyme PDK3 in Escherichia coli BL21(DE3) as Histagged protein) [31] (DNA sequence determination and analysis of the PDK4 gene promoter, analysis of transcriptional activation mechanism, expression analysis, overview, transient expression of PDK4 promoter mutants in hepatocytes) [53] (coexpression of T7-tagged PDK1 and His-tagged PDK2 polypeptides in Escherichia coli, formation of a hybrid dimer) [33] (expression of His-tagged isozymes PDK1 and PDK2 in Escherichia coli BL21(DE3)) [29] (expression of isozyme PDK2 as T7-tagged protein in Escherichia coli) [33] (expression of isozyme PDK2 in Escherichia coli as His-tagged enzyme) [21, 33] (expression of isozyme PDK2 in Escherichia coli as His-tagged protein) [32] (expression of wild-type and mutant PDHK2s in Escherichia coli) [57] (expression of wild-type isozyme PDK2 and mutants in Escherichia coli as His-tagged enzymes) [24] (individual overexpressions of His-tagged isozymes PDK1, PDK2, and PDK4 in Escherichia coli strain BL21) [48] (isozymes PDK2 and PDK4 expression analysis in liver at different developmental stages) [69] (overexpression of isozymes PDK1, PDK2, and PDK4 in Escherichia coli BL21(DE3) as His-tagged proteins) [31] (the gene encoding PDK4 is located on chromosome 4, DNA sequence analysis, genomic organization, possible regulatory important elements are Sp1 and CBF sites) [37] (gene YIL042c, overexpression of c-Myc-tagged or HA-tagged enzyme in strain BY4741) [62] (PDK DNA sequence determination and analysis, expression of His6tagged wild-type and mutant PDKs in Escherichia coli strain BL21(DE3)) [38] (functional expression as maltose-binding protein fusion protein in Escherichia coli) [26] (DNA sequence determination and analysis of different isozymes, phylogenetic analysis, expression analysis of different isozymes) [40] (expression of truncated enzyme forms, comprising residues 284-402 and 1-334, respectively, as maltose-binding-protein fusion proteins in Escherichia coli JM109) [25] (functional expression in Escherichia coli as His-tagged protein) [25] (gene pdk4, DNA and amino sequence determination and analysis, genomic organisation, functional expression of the His-tagged enzyme in Spodoptera frugiperda Sf9 cells via bacuovirus infection system) [19] (DNA and amino acid sequence determination, functional expression as soluble protein in Escherichia coli strain HMS 174 (DE3)) [36] 152

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

(coexpression of T7-tagged PDK1 and His-tagged PDK2 polypeptides in Escherichia coli, formation of a hybrid dimer) [33] (expression of T7-tagged and His-tagged isozyme PDK1 in Escherichia coli) [33] (DNA sequence determination and analysis) [22] (functional expression in Escherichia coli strain BL21(DE3) as Histagged protein) [22, 25] (expression of N-terminally His-tagged residues A8-T399 of isozyme PDHK2 in Trichoplusia ni TN5B1-4 insect cells using the baculovirus infection system) [44] (isozyme PDK4, DNA and amino acid sequence determination and analysis, expression analysis) [61] Engineering D117A ( site-directed mutagenesis, the mutant enzyme shows no inhibition by dichloroacetate in contrast to the wild-type PDHK2 [63]) [63] D164A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is slightly reduced compared to the wild-type isozyme PDK3 [65]) [65] D172A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is slightly reduced compared to the wild-type isozyme PDK3 [65]) [65] D282A ( isozyme PDK2, site-directed mutagenesis, mutation of conserved amino acid, no activity, not able to bind ATP but the protein substrate [24]) [24] E162A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is slightly reduced compared to the wild-type isozyme PDK3 [65]) [65] E170A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is reduced compared to the wild-type isozyme PDK3 [65]) [65] E179A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is reduced compared to the wild-type isozyme PDK3 [65]) [65] E238A ( site-directed mutagenesis, highly reduced activity compared to the wild-type PDK [38]) [38] E238H ( site-directed mutagenesis, highly reduced activity compared to the wild-type PDK [38]) [38] E238Q ( site-directed mutagenesis, highly reduced activity compared to the wild-type PDK [38]) [38] E389A ( site-directed mutagenesis, the mutant shows unaltered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] F168A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] F28A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] F31A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57]

153

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

F32A ( site-directed mutagenesis of isozyme PDK3 lipoyl-binding pocket, the mutant has lost its L2 binding and L2-stimulated PDK3 activity [65]) [65] F35A ( site-directed mutagenesis of isozyme PDK3 lipoyl-binding pocket, the mutant has lost its L2 binding and L2-stimulated PDK3 activity [65]) [65] F44A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] F48A ( site-directed mutagenesis of isozyme PDK3 lipoyl-binding pocket, the mutant has lost its L2 binding and L2-stimulated PDK3 activity [65]) [65] G284A ( isozyme PDK2, site-directed mutagenesis, mutation of conserved amino acid, properties similar to wild-type [24]) [24] G286A ( isozyme PDK2, site-directed mutagenesis, mutation of conserved amino acid, no activity, not able to bind ATP but the protein substrate [24]) [24] G319A ( isozyme PDK2, site-directed mutagenesis, mutation of conserved amino acid, catalytically active, but very poor binding of ATP [24]) [24] H115A ( site-directed mutagenesis, the mutant enzyme shows reduced inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63] H121A ( site-directed mutagenesis, 50% decreased trans- and autophosphorylation activity [26]) [26] H121Q ( site-directed mutagenesis, 50% decreased trans- and autophosphorylation activity [26]) [26] H233A ( site-directed mutagenesis, reduced activity compared to the wild-type PDK [38]) [38] I111A ( site-directed mutagenesis, the mutant enzyme shows reduced inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63] I157A ( site-directed mutagenesis, the mutant enzyme shows increased inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63] I161A ( site-directed mutagenesis, the mutant enzyme shows unaltered inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63] I167A ( site-directed mutagenesis, the mutant shows slightly altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] K17A ( site-directed mutagenesis, the mutant shows slightly altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] K241A ( site-directed mutagenesis, highly reduced activity compared to the wild-type PDK [38]) [38]

154

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

K368A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] K391A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] L140A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is reduced compared to the wild-type isozyme PDK3 [65]) [65] L160A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] L234A ( site-directed mutagenesis, reduced activity compared to the wild-type PDK [38]) [38] L234H ( site-directed mutagenesis, reduced activity compared to the wild-type PDK [38]) [38] L23A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] L27A ( site-directed mutagenesis of isozyme PDK3 lipoyl-binding pocket, the mutant has lost its L2 binding and L2-stimulated PDK3 activity [65]) [65] L45A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] L53A ( site-directed mutagenesis, the mutant enzyme shows reduced inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63] N247A ( isozyme PDK2, site-directed mutagenesis, mutation of conserved amino acid, no activity, not able to bind ATP but the protein substrate [24]) [24] P142A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is unaltered compared to the wild-type isozyme PDK3 [65]) [65] P22A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] Q47A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] R112A ( site-directed mutagenesis, the mutant enzyme shows unaltered inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63] R114A ( site-directed mutagenesis, the mutant enzyme shows no inhibition by dichloroacetate in contrast to the wild-type PDHK2 [63]) [63] R154A ( site-directed mutagenesis, the mutant enzyme shows unaltered inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63]

155

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

R158A ( site-directed mutagenesis, the mutant enzyme shows unaltered inhibition by dichloroacetate compared to the wild-type PDHK2 [63]) [63] R196A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is slightly reduced compared to the wild-type isozyme PDK3 [65]) [65] R372A ( site-directed mutagenesis, the mutant shows altered L2 component binding within the pyruvate dehydrogenase complex compared to the wild-type PDHK2 [57]) [57] S83A ( site-directed mutagenesis, the mutant enzyme shows no inhibition by dichloroacetate in contrast to the wild-type PDHK2 [63]) [63] T175A ( site-directed mutagenesis of isozyme PDK3, the L2 binding is unaltered compared to the wild-type isozyme PDK3 [65]) [65] W383F ( site-directed mutagenesis of isozyme PDHK2, the mutant shows unaltered catalytic activity compared to the wild-type isozyme PDHK2, but altered ligand binding and higher sensitivity to inhibition by pyruvate and ADP [64]) [64] Y80A ( site-directed mutagenesis, the mutant enzyme shows no inhibition by dichloroacetate in contrast to the wild-type PDHK2 [63]) [63] Additional information ( construction of truncated enzyme forms, the N-terminally truncated form, residues 284-402, is catalytically inactive, the C-terminally reduced form, residues 1-334, shows reduced activity [25]; construction of PDK4 gene promoter deletion and point mutants for functional analysis, overview [53]; construction of several E2 component-derived domains, overview [41]; construction of transgenic plants with silenced PDHK via expression of an antisense construct driven by both constitutive and seed-specific promoters leading to increased respiration rate in leaves, seed oil content, and seed weight in both cases, mutant phenotypes, overview [54]; enzyme-deficient PDHK4-/- mice show lower blood glucose levels and increased pyruvate dehydrogenase complex activity in kidney, gastrocnemius muscle, diaphragm and heart, but not in the liver, compared to the wild-type mice, the mutant mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis, phenotype, overview, heterozygous mutant mice show about 50% reduced enzyme activity in skeletal muscle, genotype distribution, overview [56]; overexpression of PDK1 increases the ATP levels and prevents hypoxia-induced reactive oxygen species generation and apoptosis, hypoxia-inducible factor HIF-1 knockout embryonic fibroblasts fail to activate PDK1 and undergo apoptosis with a high rise in reactive oxygen species, hypoxic P493-6 cells with reduced PDK1 levels show impaired growth [59]; serial truncations of the isozyme PDK3 C-terminal tail region either impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-enhanced kinase activity, overview [65]) [25, 41, 53, 54, 56, 59, 65] Application medicine ( increased glucose oxidation by inhibition of PDHK activity may be effective for increasing glucose utilization in diabetes treat-

156

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[Pyruvate dehydrogenase (acetyl-transferring)] kinase

ment [42]; PDHK inhibition provides a route for therapeutic intervention in diabetes and cardiovascular disorders [44]) [42, 44] pharmacology ( target for development of specific inhibitors of PDK isozymes to regulate glucose levels in the blood [30]) [30]

6 Stability General stability information , kinase has tendency to aggregate in other buffers than 0.01 M imidazole-asparagine, pH 7.3, 0.1 mM MgCl2 , 0.01 M EDTA [9] , labile to freeze-thawing [10, 11] Storage stability , 4 C, 0.02% NaN3 , t1=2 : 1 month [10, 11]

References [1] Reed, L.J.; Damuni, Z.; Merryfield, M.L.: Regulation of mammalian pyruvate and branched-chain a-keto acid dehydrogenase complexes by phosphorylation-dephosphorylation. Curr. Top. Cell. Regul., 27, 41-49 (1985) [2] Linn, T.C.; Pelley, J.W.; Pettit, F.H.; Hucho, F.; Randall, D.D.; Reed, L.J.: aKeto acid dehydrogenase complexes. XV. Purification and properties of the component enzymes of the pyruvate dehydrogenase complexes from bovine kidney and heart. Arch. Biochem. Biophys., 148, 327-342 (1972) [3] Kerbey, A.L.; Randle, P.J.: Pyruvate dehydrogenase kinase activity of pig heart pyruvate dehydrogenase (E1 component of pyruvate dehydrogenase complex). Biochem. J., 231, 523-529 (1985) [4] Harris, R.A.; Paxton, R.; DePaoli-Roach, A.: Inhibition of branched chain a-ketoacid dehydrogenase kinase activity by a-chloroisocaproate. J. Biol. Chem., 257, 13915-13918 (1982) [5] Sheu, K.F.R.; Lai, J.C.K.; Blass, J.P.: Properties and regional distribution of pyruvate dehydrogenase kinase in rat brain. J. Neurochem., 42, 230-236 (1984) [6] Hucho, F.; Randall, D.D.; Roche, T.E.; Burgett, M.W.; Pelley, J.W.; Reed, L.J.: a-Keto acid dehydrogenase complexes. XVII. Kinetic and regulatory properties of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase from bovine kidney and heart. Arch. Biochem. Biophys., 151, 328340 (1972) [7] Roche, T.E.; Reed, L.J.: Monovalent cation requirement for ADP inhibition of pyruvate dehydrogenase kinase. Biochem. Biophys. Res. Commun., 59, 1341-1348 (1974) [8] Pratt, T.E.; Roche, T.E.: Mechanism of pyruvate inhibition of kidney pyruvate dehydrogenase kinase and synergistic inhibition by pyruvate and ADP. J. Biol. Chem., 254, 7191-7196 (1979)

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[9] Pettit, F.H.; Yeaman, S.J.; Reed, L.J..: Pyruvate dehydrogenase kinase from bovine kidney. Methods Enzymol., 90, 195-201 (1982) [10] Pettit, F.H.; Yeaman, S.J.; Reed, L.J..: Pyruvate dehydrogenase kinase from bovine kidney. Methods Enzymol., 99, 331-336 (1983) [11] Stepp, L.R.; Pettit, F.H.; Yeaman, S.J.; Reed, L.J..: Purification and properties of pyruvate dehydrogenase kinase from bovine kidney. J. Biol. Chem., 258, 9454-9458 (1983) [12] Rahmatullah, M.; Roche, T.E.: Modification of bovine kidney pyruvate dehydrogenase kinase activity by CoA esters and their mechanism of action. J. Biol. Chem., 260, 10146-10152 (1985) [13] Rahmatullah, M.; Jilka, J.M.; Radke, G.A.; Roche, T.E.: Properties of the pyruvate dehydrogenase kinase bound to and separated from the dihydrolipoyl transacetylase-protein X subcomplex and evidence for binding of the kinase to protein X. J. Biol. Chem., 261, 6515-6523 (1986) [14] Robertson, J.G.; Barron, L.L.; Olson, M.S.: Effects of a-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase. J. Biol. Chem., 261, 76-81 (1986) [15] Robertson, J.G.; Barron, L.L.; Olson, M.S.: Bovine heart pyruvate dehydrogenase kinase stimulation by a-ketoisovalerate. J. Biol. Chem., 265, 1681416820 (1990) [16] Schuller, K.A.; Randall, D.D.: Mechanism of pyruvate inhibition of plant pyruvate dehydrogenase kinase and synergism with ADP. Arch. Biochem. Biophys., 278, 211-216 (1990) [17] Pawelczyk, T.; Olson, M.S.: Regulation of pyruvate dehydrogenase kinase activity from pig kidney cortex. Biochem. J., 288, 369-373 (1992) [18] Priestman, D.A.; Mistry, S.C.; Kerbey, A.L.; Randle, P.J.: Purification and partial characterization of rat liver pyruvate dehydrogenase kinase activator protein (free pyruvate dehydrogenase kinase). FEBS Lett., 308, 83-86 (1992) [19] Rowles, J.; Scherer, S.W.; Xi, T.; Majer, M.; Nickle, D.C.; Rommens, J.M.; Popov, K.M.; Harris, R.A.; Riebow, N.L.; et al.: Cloning and characterization of PDK4 on 7q21.3 encoding a fourth pyruvate dehydrogenase kinase isoenzyme in human. J. Biol. Chem., 271, 22376-22382 (1996) [20] Ravindran, S.; Radke, G.A.; Guest, J.R.; Roche, T.E.: Lipoyl domain-based mechanism for the integrated feedback control of the pyruvate dehydrogenase complex by enhancement of pyruvate dehydrogenase kinase activity. J. Biol. Chem., 271, 653-662 (1996) [21] Popov, K.M.: Regulation of mammalian pyruvate dehydrogenase kinase. FEBS Lett., 419, 197-200 (1997) [22] Chen, W.; Huang, X.; Komuniecki, P.R.; Komuniecki, R.: Molecular cloning, functional expression, and characterization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum. Arch. Biochem. Biophys., 353, 181-189 (1998) [23] Aicher, T.D.; Damon, R.E.; Koletar, J.; Vinluan, C.C.; Brand, L.J.; Gao, J.; Shetty, S.S.; Kaplan, E.L.; Mann, W.R.: Triterpene and diterpene inhibitors of pyruvate dehydrogenase kinase (PDK). Bioorg. Med. Chem. Lett., 9, 2223-2228 (1999) 158

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[24] Bowker-Kinley, M.; Popov, K.M.: Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily. Biochem. J., 344, 47-53 (1999) [25] Chen, W.; Komuniecki, P.R.; Komuniecki, R.: Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex. Biochem. J., 339, 103-109 (1999) [26] Thelen, J.J.; Miernyk, J.A.; Randall, D.D.: Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues. Biochem. J., 349, 195-201 (2000) [27] Sugden, M.C.; Langdown, M.L.; Harris, R.A.; Holness, M.J.: Expression and regulation of pyruvate dehydrogenase kinase isoforms in the developing rat heart and in adulthood: role of thyroid hormone status and lipid supply. Biochem. J., 352, 731-738 (2000) [28] Baker, J.C.; Yan, X.; Peng, T.; Kasten, S.; Roche, T.E.: Marked differences between two isoforms of human pyruvate dehydrogenase kinase. J. Biol. Chem., 275, 15773-15781 (2000) [29] Mann, W.R.; Dragland, C.J.; Vinluan, C.C.; Vedananda, T.R.; Bell, P.A.; Aicher, T.D.: Diverse mechanisms of inhibition of pyruvate dehydrogenase kinase by structurally distinct inhibitors. Biochim. Biophys. Acta, 1480, 283-292 (2000) [30] Roche, T.E.; Baker, J.C.; Yan, X.; Hiromasa, Y.; Gong, X.; Peng, T.; Dong, J.; Turkan, A.; Kasten, S.A.: Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog. Nucleic Acid Res. Mol. Biol., 70, 33-75 (2001) [31] Korotchkina, L.G.; Patel, M.S.: Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase. J. Biol. Chem., 276, 37223-37229 (2001) [32] Steussy, C.N.; Popov, K.M.; Bowker-Kinley, M.M.; Sloan, R.B., Jr.; Harris, R.A.; Hamilton, J.A.: Structure of pyruvate dehydrogenase kinase. Novel folding pattern for a serine protein kinase. J. Biol. Chem., 276, 3744337450 (2001) [33] Boulatnikov, I.; Popov, K.M.: Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase. Biochim. Biophys. Acta, 1645, 183-192 (2003) [34] Holness, M.J.; Bulmer, K.; Smith, N.D.; Sugden, M.C.: Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone. Biochem. J., 369, 687-695 (2003) [35] Roche, T.E.; Hiromasa, Y.; Turkan, A.; Gong, X.; Peng, T.; Yan, X.; Kasten, S.A.; Bao, H.; Dong, J.: Essential roles of lipoyl domains in the activated function and control of pyruvate dehydrogenase kinases and phosphatase isoform 1. Eur. J. Biochem., 270, 1050-1056 (2003) [36] Popov, K.M.; Kedishvili, N.Y.; Zhao, Y.; Shimomura, Y.; Crabb, D.W.; Harris, R.A.: Primary structure of pyruvate dehydrogenase kinase establishes a new family of eukaryotic protein kinases. J. Biol. Chem., 268, 26602-26606 (1994) 159

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

[37] Kwon, H.-S.; Harris, R.A.: Mechanisms responsible for regulation of pyruvate dehydrogenase kinase 4 gene expression. Adv. Enzyme Regul., 44, 109121 (2004) [38] Tovar-Mendez, A.; Hirani, T.A.; Miernyk, J.A.; Randall, D.D.: Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase. Arch. Biochem. Biophys., 434, 159-168 (2005) [39] Savkur, R.S.; Bramlett, K.S.; Michael, L.F.; Burris, T.P.: Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor. Biochem. Biophys. Res. Commun., 329, 391-396 (2005) [40] Terazawa, Y.; Tokmakov, A.A.; Shirouzu, M.; Yokoyama, S.: Molecular cloning and expression analysis of PDK family genes in Xenopus laevis reveal oocyte-specific PDK isoform. Biochem. Biophys. Res. Commun., 338, 17981804 (2005) [41] Tuganova, A.; Popov, K.M.: Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity. Biochem. J., 387, 147153 (2005) [42] Mayers, R.M.; Leighton, B.; Kilgour, E.: PDH kinase inhibitors: a novel therapy for Type II diabetes?. Biochem. Soc. Trans., 33, 367-370 (2005) [43] Bao, H.; Kasten, S.A.; Yan, X.; Roche, T.E.: Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP. Biochemistry, 43, 13432-13441 (2004) [44] Knoechel, T.R.; Tucker, A.D.; Robinson, C.M.; Phillips, C.; Taylor, W.; Bungay, P.J.; Kasten, S.A.; Roche, T.E.; Brown, D.G.: Regulatory roles of the Nterminal domain based on crystal structures of human pyruvate dehydrogenase kinase 2 containing physiological and synthetic ligands. Biochemistry, 45, 402-415 (2006) [45] Motojima, K.; Seto, K.: Fibrates and statins rapidly and synergistically induce pyruvate dehydrogenase kinase 4 mRNA in the liver and muscles of mice. Biol. Pharm. Bull., 26, 954-958 (2003) [46] Kato, M.; Chuang, J.L.; Tso, S.C.; Wynn, R.M.; Chuang, D.T.: Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex. EMBO J., 24, 1763-1774 (2005) [47] Abbot, E.L.; McCormack, J.G.; Reynet, C.; Hassall, D.G.; Buchan, K.W.; Yeaman, S.J.: Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells. FEBS J., 272, 3004-3014 (2005) [48] Korotchkina, L.G.; Sidhu, S.; Patel, M.S.: R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase. Free Radic. Res., 38, 1083-1092 (2004) [49] Spriet, L.L.; Tunstall, R.J.; Watt, M.J.; Mehan, K.A.; Hargreaves, M.; Cameron-Smith, D.: Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting. J. Appl. Physiol., 96, 2082-2087 (2004) [50] Watt, M.J.; Heigenhauser, G.J.; LeBlanc, P.J.; Inglis, J.G.; Spriet, L.L.; Peters, S.J.: Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise. J. Appl. Physiol., 97, 12611267 (2004)

160

2.7.11.2

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

[51] Turvey, E.A.; Heigenhauser, G.J.; Parolin, M.; Peters, S.J.: Elevated n-3 fatty acids in a high-fat diet attenuate the increase in PDH kinase activity but not PDH activity in human skeletal muscle. J. Appl. Physiol., 98, 350-355 (2005) [52] Hiromasa, Y.; Roche, T.E.: Facilitated interaction between the pyruvate dehydrogenase kinase isoform 2 and the dihydrolipoyl acetyltransferase. J. Biol. Chem., 278, 33681-33693 (2003) [53] Ma, K.; Zhang, Y.; Elam, M.B.; Cook, G.A.; Park, E.A.: Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor g coactivator. J. Biol. Chem., 280, 29525-29532 (2005) [54] Marillia, E.-F.; Micallef, B.J.; Micallef, M.; Weninger, A.; Pedersen, K.K.; Zou, J.; Taylor, D.C.: Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase. J. Exp. Bot., 54, 259-270 (2003) [55] Koukourakis, M.I.; Giatromanolaki, A.; Sivridis, E.; Gatter, K.C.; Harris, A.L.: Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma. Neoplasia, 7, 1-6 (2005) [56] Jeoung, N.H.; Wu, P.; Joshi, M.A.; Jaskiewicz, J.; Bock, C.B.; Depaoli-Roach, A.A.; Harris, R.A.: Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation. Biochem. J., 397, 417-425 (2006) [57] Tuganova, A.; Klyuyeva, A.; Popov, K.M.: Recognition of the inner lipoylbearing domain of dihydrolipoyl transacetylase and of the blood glucoselowering compound AZD7545 by pyruvate dehydrogenase kinase 2. Biochemistry, 46, 8592-8602 (2007) [58] Kwon, H.; Huang, B.; Jeoung, N.H.; Wu, P.; Steussy, C.N.; Harris, R.A.: Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression. Biochim. Biophys. Acta, 1759, 141-151 (2006) [59] Kim, J.W.; Tchernyshyov, I.; Semenza, G.L.; Dang, C.V.: HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab., 3, 177-185 (2006) [60] Roche, T.E.; Hiromasa, Y.: Pyruvate dehydrogenase kinase regulatory mechanisms and inhibition in treating diabetes, heart ischemia, and cancer. Cell. Mol. Life Sci., 64, 830-849 (2007) [61] Chen, J.; Sun, M.; Liang, B.; Xu, A.; Zhang, S.; Wu, D.: Cloning and expression of PDK4, FOXO1A and DYRK1A from the hibernating greater horseshoe bat (Rhinolophus ferrumequinum). Comp. Biochem. Physiol. B, 146B, 166-171 (2007) [62] Krause-Buchholz, U.; Gey, U.; Wuenschmann, J.; Becker, S.; Roedel, G.: YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. FEBS Lett., 580, 25532560 (2006)

161

[Pyruvate dehydrogenase (acetyl-transferring)] kinase

2.7.11.2

[63] Klyuyeva, A.; Tuganova, A.; Popov, K.M.: Amino acid residues responsible for the recognition of dichloroacetate by pyruvate dehydrogenase kinase 2. FEBS Lett., 581, 2988-2992 (2007) [64] Hiromasa, Y.; Hu, L.; Roche, T.E.: Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2. J. Biol. Chem., 281, 12568-12579 (2006) [65] Tso, S.C.; Kato, M.; Chuang, J.L.; Chuang, D.T.: Structural determinants for cross-talk between pyruvate dehydrogenase kinase 3 and lipoyl domain 2 of the human pyruvate dehydrogenase complex. J. Biol. Chem., 281, 2719727204 (2006) [66] Zhang, Y.; Ma, K.; Sadana, P.; Chowdhury, F.; Gaillard, S.; Wang, F.; McDonnell, D.P.; Unterman, T.G.; Elam, M.B.; Park, E.A.: Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression. J. Biol. Chem., 281, 39897-39906 (2006) [67] Chokkalingam, K.; Jewell, K.; Norton, L.; Littlewood, J.; van Loon, L.J.; Mansell, P.; Macdonald, I.A.; Tsintzas, K.: High-fat/low-carbohydrate diet reduces insulin-stimulated carbohydrate oxidation but stimulates nonoxidative glucose disposal in humans: An important role for skeletal muscle pyruvate dehydrogenase kinase 4. J. Clin. Endocrinol. Metab., 92, 284-292 (2007) [68] Devedjiev, Y.; Steussy, C.N.; Vassylyev, D.G.: Crystal structure of an asymmetric complex of pyruvate dehydrogenase kinase 3 with lipoyl domain 2 and its biological implications. J. Mol. Biol., 370, 407-416 (2007) [69] Bajotto, G.; Murakami, T.; Nagasaki, M.; Qin, B.; Matsuo, Y.; Maeda, K.; Ohashi, M.; Oshida, Y.; Sato, Y.; Shimomura, Y.: Increased expression of hepatic pyruvate dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima Fatty rats: induction by elevated levels of free fatty acids. Metab. Clin. Exp., 55, 317-323 (2006) [70] Kato, M.; Li, J.; Chuang, J.L.; Chuang, D.T.: Distinct structural mechanisms for inhibition of pyruvate dehydrogenase kinase isoforms by AZD7545, dichloroacetate, and radicicol. Structure, 15, 992-1004 (2007)

162

Dephospho-[reductase kinase] kinase

2.7.11.3

1 Nomenclature EC number 2.7.11.3 Systematic name ATP:dephospho[[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] phosphotransferase Recommended name dephospho-[reductase kinase] kinase Synonyms AMP-activated protein kinase kinase hydroxymethylglutaryl coenzyme A reductase kinase kinase hydroxymethylglutaryl coenzyme A reductase kinase kinase (phosphorylating) reductase kinase kinase CAS registry number 72060-33-4

2 Source Organism Homo sapiens (no sequence specified) [6] Rattus norvegicus (no sequence specified) [1, 2, 3, 4, 5, 7, 8, 9]

3 Reaction and Specificity Catalyzed reaction ATP + dephospho-[[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] = ADP + [[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] Reaction type phospho group transfer Natural substrates and products S ATP + dephospho-[[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] ( bicyclic phosporylation system, enzyme is believed to be involved in protecting cells against ATP depletion due to environmental stress by inactivating several key biosynthetic enzymes [7]; involved in regulation cascade of hydroxymethylglutaryl-CoA reductase, 163

Dephospho-[reductase kinase] kinase

2.7.11.3

EC 1.1.1.34 [3]; important for the responses of cells to metabolic stresses such as lack of cell nutrients, hypoxia, ischemia and muscular exercise [9]; phosphorylation activates EC 2.7.1.109 [1, 2, 3, 4, 5, 6, 7, 8]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9] P ADP + [[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] [2, 3, 4, 9] Substrates and products S ATP + casein (Reversibility: ?) [3] P ? S ATP + dephospho-[[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] ( i.e. AMP-activated protein kinase [4]; No substrate is hydroxymethylglutaryl-CoA reductase (NADPH) [3]; phosphorylates catalytic subunit of EC 2.7.11.31 [4]; bicyclic phosporylation system, enzyme is believed to be involved in protecting cells against ATP depletion due to environmental stress by inactivating several key biosynthetic enzymes [7]; involved in regulation cascade of hydroxymethylglutaryl-CoA reductase, EC 1.1.1.34 [3]; important for the responses of cells to metabolic stresses such as lack of cell nutrients, hypoxia, ischemia and muscular exercise [9]; phosphorylation activates EC 2.7.1.109 [1,2,3,4,5,6,7,8]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9] P ADP + [[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] [2, 3, 4, 9] S ATP + histone 2A ( poor substrate [3]) (Reversibility: ?) [3] P ? S ATP + phosvitin ( slight activity [3]) (Reversibility: ?) [3] P ? Inhibitors adenosine(5’)tetraphospho(5’)adenosine ( i.e. AP4A, inhibits in the presence of AMP [4]) [4] poly(Arg) ( casein as substrate [3]) [3] Additional information ( no inhibition by inhibitor of cAMP-dependent protein kinase [1]) [1] Cofactors/prosthetic groups AMP ( allosteric activator, the allosteric effect and the promotion of phosphorylation and activation by the kinase kinase are due to binding of AMP to a single site on the kinase [4]) [4] Activating compounds 5’-AMP [8] 8-aza-9-deaza-AMP ( activation, i.e. formycin A-5-monophosphate, can replace AMP [4]) [4] 8-aza-9-deaza-IMP ( slight activation, i.e. formycin B-5-monophosphate [4]) [4] mevalonolactone ( activation, in vitro and in vivo [5]) [5] Additional information ( cAMP-independent enzyme [1,3]; no activation by formycin A or B [4]) [1, 3, 4] 164

2.7.11.3

Dephospho-[reductase kinase] kinase

Metals, ions Mg2+ ( requirement, actual substrate: MgATP [1,3,4,5,6]) [1, 3, 4, 5, 6] Specific activity (U/mg) 101 [8] Km-Value (mM) 0.2 (ATP, pH 7.4, 37 C [2]) [2] Ki-Value (mM) 3-4 (sodium fluoride, pH 7.4, 37 C [2]) [2] pH-Optimum 7 ( assay at [3,5]) [3, 5] 7.4 ( assay at [1,6]) [1, 6] Temperature optimum ( C) 30 ( assay at [3]) [3, 4] 37 ( assay at [3]) [3, 5, 6]

4 Enzyme Structure Molecular weight 196000 [8] 380000 ( gel filtration [3]) [3] Subunits ? ( ? * 58000 + ?, catalytic a subunit, SDS-PAGE [8]) [8]

5 Isolation/Preparation/Mutation/Application Source/tissue liver [1, 2, 3, 4, 5, 6, 7, 8] Localization cytosol ( predominant [1,5]) [1, 3, 4, 5] microsome [2, 3, 6] Additional information ( subcellular distribution [1]) [1] Purification (partial) [6] (cytosolic enzyme) [3] (partial) [1, 3, 4, 5, 8] Cloning (bacterially expressed recombinant a1 subunit proteins) [9] Engineering T172D ( site-directed mutagenesis [9]) [9] 165

Dephospho-[reductase kinase] kinase

2.7.11.3

6 Stability Storage stability , -20 C, can be stored in buffer containing 50% glycerol for up to a month [8]

References [1] Ingebritsen, T.S.; Parker, R.A.; Gibson, D.M.: Regulation of liver hydroxymethylglutaryl-CoA reductase by a bicyclic phosphorylation system. J. Biol. Chem., 256, 1138-1144 (1981) [2] Ingebritsen, T.S.; Lee, H.-S.; Parker, R.A.; Gibson, D.M.: Reversible modulation of the activities of both liver microsomal hydroxymethylglutaryl coenzyme A reductase and its inactivating enzyme. Evidence for regulation by phosphorylation-dephosphorylation. Biochem. Biophys. Res. Commun., 81, 1268-1277 (1978) [3] Beg, Z.H.; Stonik, J.A.; Brewer, B.: Characterization and regulation of reductase kinase, a protein kinase that modulates the enzymic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proc. Natl. Acad. Sci. USA, 76, 4375-4379 (1979) [4] Weekes, J.; Hawley, S.A.; Corton, J.; Shugar, D.; Hardie, D.G.: Activation of rat liver AMP-activated protein kinase by kinase kinase in a purified, reconstituted system. Effects of AMP and AMP analogues. Eur. J. Biochem., 219, 751757 (1994) [5] Beg, Z.H.; Stonik, J.A.; Brewer, B.: In vivo modulation of rat liver 3-hydroxy3-methylglutaryl-coenzyme A reductase, reductase kinase, and reductase kinase kinase by mevalonolactone. Proc. Natl. Acad. Sci. USA, 81, 7293-7297 (1984) [6] Beg, Z.H.; Stonik, J.A.; Brewer, B.: Human hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase: evidence for the regulation of enzymic activity by a bicyclic phosphorylation cascade. Biochem. Biophys. Res. Commun., 119, 488-498 (1984) [7] Hawley, S.A.; Selbert, M.A.; Goldstein, E.G.; Edelman, A.M.; Carling, D.; Hardie, D.G.: 5’-AMP activates the AMP-activated protein kinase cascade, and Ca2+ /calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J. Biol. Chem., 270, 2718627191 (1995) [8] Hawley, S.A.; Davison, M.; Woods, A.; Davies, S.P.; Beri, R.K.; Carling, D.; Hardie, D.G.: Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J. Biol. Chem., 271, 2787927887 (1996) [9] Hamilton, S.R.; O’Donnell, J.B., Jr.; Hammet, A.; Stapleton, D.; Habinowski, S.A.; Means, A.R.; Kemp, B.E.; Witters, L.A.: AMP-activated protein kinase kinase: detection with recombinant AMPK a1 subunit. Biochem. Biophys. Res. Commun., 293, 892-898 (2002) 166

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

2.7.11.4

1 Nomenclature EC number 2.7.11.4 Systematic name ATP:[3-methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] phosphotransferase Recommended name [3-methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase Synonyms BCK BCKD kinase [29, 30, 31] BCKDH kinase BDK [32] branched-chain 2-oxo acid dehydrogenase kinase branched-chain a-keto acid decarboxylase/dehydrogenase kinase [29] branched-chain a-keto acid dehydrogenase kinase [32] branched-chain a-ketoacid dehydrogenase kinase [30, 31] branched-chain keto acid dehydrogenase kinase kinase, branched-chain oxo acid dehydrogenase (phosphorylating) Additional information ( kinase activity is an intrinsic activity of branched-chain oxo acid dehydrogenase complex [7]) [7] CAS registry number 82391-38-6

2 Source Organism Mus musculus (no sequence specified) [20, 21, 31] Rattus norvegicus (no sequence specified) [1, 3, 4, 6, 7, 11, 14, 15, 16, 17, 19, 22, 23, 24, 25, 26, 27, 28, 29, 32] Sus scrofa (no sequence specified) [30] Bos taurus (no sequence specified) [1, 2, 4, 8, 9, 10, 15, 18] Oryctolagus cuniculus (no sequence specified) [1, 3, 4, 5, 11, 12, 13, 15]

167

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

2.7.11.4

3 Reaction and Specificity Catalyzed reaction ATP + [3-methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] = ADP + [3-methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] phosphate Reaction type phospho group transfer Natural substrates and products S ATP + [3-methyl-2-oxobutanoate dehydrogenase (lipoamide)] ( branched-chain amino acid metabolism [3, 24]; regulatory enzyme of branched-chain 2-oxoacid dehydrogenase complex [2, 24]; phosphorylation inactivates EC 1.2.4.4 [1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 24, 26]) (Reversibility: ?) [1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 24, 26] P ADP + [3-methyl-2-oxobutanoate dehydrogenase (lipoamide)] phosphate S ATP + branched-chain a-keto acid decarboxylase/dehydrogenase ( inactivation of the substrate enzyme [29]; the catalyzed reversible specific phosphorylation of the BCKD subunit regulates the first committed step in the pathway for leucine, isoleucine, and valine catabolism [31]; the enzyme catalyzes the regulatory inactivation of the rate limiting enzyme in branched-chain amino acid catabolism [32]) (Reversibility: ?) [29, 31, 32] P ADP + phosphorylated branched-chain a-keto acid decarboxylase/dehydrogenase S Additional information ( BCKD kinase transcription regulation, overview [30]) (Reversibility: ?) [30] P ? Substrates and products S ATP + [3-methyl-2-oxobutanoate dehydrogenase (lipoamide)] ( phosphorylates a-subunit of multienzyme complex component E1 [8,9]; 2 Ser-residues in E1-a-subunit [1,3,4,15]; incorporates 0.8 mol phosphate/mol a-subunit [9]; tight binding to multienzyme complex is required for phosphorylation, free enzyme is inactive [26]; Ser-residues of MW 46000-subunit [7,8]; GTP cannot replace ATP [5]; phosphorylates a-subunit of multienzyme complex component E1 and additional sites not associated with inactivation of the enzyme [10]; phosphorylates exclusively MW 47000 subunit of substrate [5]; incorporates 0.75 mol phosphate per mol phosphorylation site and 1.5 mol/mol a-subunit [4]; branched-chain amino acid metabolism [3,24]; regulatory enzyme of branched-chain 2-oxoacid dehydrogenase complex [2, 24]; phosphorylation inactivates EC 1.2.4.4 [1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 24, 26]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 22, 24, 26, 27, 28]

168

2.7.11.4

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

P ADP + [3-methyl-2-oxobutanoate dehydrogenase (lipoamide)] phosphate [1, 3, 4, 7, 8, 9, 10, 15] S ATP + branched-chain a-keto acid decarboxylase/dehydrogenase ( inactivation of the substrate enzyme [29]; the catalyzed reversible specific phosphorylation of the BCKD subunit regulates the first committed step in the pathway for leucine, isoleucine, and valine catabolism [31]; the enzyme catalyzes the regulatory inactivation of the rate limiting enzyme in branched-chain amino acid catabolism [32]; phosphorylation of the branched-chain a-keto acid dehydrogenase complex leads to its inactivation, BDK itself is regulated via protein-protein interaction with the BCKD complex [32]; recombinant human wildtype and mutant substrate proteins, overview, phosphorylation at Ser301 and Ser302 in the phosphorylation loop of decarboxylase E1b component of the large branched-chain a-keto acid dehydrogenase complex, loop structure, overview [29]) (Reversibility: ?) [29, 31, 32] P ADP + phosphorylated branched-chain a-keto acid decarboxylase/dehydrogenase S ATP + histone II-S (Reversibility: ?) [16, 27] P ? S Additional information ( R288A mutant of E1 is not phosphorylated by the enzyme [6]; enzyme has also ATPase activity in absence of E1 [23]; BCKD kinase transcription regulation, overview [30]) (Reversibility: ?) [6, 23, 30] P ? Inhibitors 2-(N-morpholino)propane sulfonate buffer [15] 2-chloroisohexanoate ( 50% inhibition at 0.014 mM [27]; i.e. 2-chloro-4-methylpentanoate, strong [13]; (R)(+)-isomer is twice as effective as (S)(-)-isomer [3]; 50% inhibition at 0.014 mM, no inhibition with histone II-S as substrate [16]; site-specific inhibitor [3]; potassium phosphate increases sensitivity to this inhibitor [3]; ATP does not protect [13]; enhanced by monovalent cations and further enhanced by phosphate [14]; no inhibition by (R)(-)-2-chloroisopentanoate [3]) [3, 4, 12, 13, 14, 16, 27] 2-oxo-3-methylpentanoate ( more effective than 2-oxoisopentanoate [3]) [3, 4, 12] 2-oxobutanoate [4] 2-oxohexanedioate [4, 15] 2-oxohexanoate [4, 12] 2-oxoisopentanoate ( less effective than 2-oxoisohexanoate and 2-oxo-3-methylpentanoate [3]) [3, 4, 12] 2-oxopentanoate ( kinetics [12]) [4, 12, 15] 2-oxoisocaproate ( kinetics, 40% inhibition at 0.065 mM [12]; more effective than 2-oxo-3-methylpentanoate and 2-oxoisopentanoate [3]) [3, 4, 8, 12] 3-methyl-2-oxobutanoate [8]

169

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

2.7.11.4

4-(2-thienyl)-2-oxo-3-butenoate ( 2 mM [2]) [2] 4-(3-thienyl)-2-oxo-3-butenoate ( 2 mM [2]) [2] 4-hydroxyphenylacetate [11] 4-hydroxyphenylpyruvate ( very weak: 3-hydroxyphenylpyruvate [11]) [4, 11] 4-methyl-2-oxopentanoate [8] 4-hydroxyphenyllactate ( weak [11]) [11] ADP ( competitive [4]; product inhibition [23]; kinetics [5]; 50% inhibition at 0.4 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1, 4, 5, 8, 15, 23] ATP ( 50% inhibition at 0.2 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1, 7] acetate ( weak, in vivo and in vitro [11]) [11] acetoacetyl-CoA ( 40% inhibition at 0.01 mM [12]) [4, 12, 15] acyl-CoA [1] branched-chain 2-oxo acids [1, 8] CDP ( 50% inhibition at 0.4 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1] CTP ( 50% inhibition at 0.25 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1] Ca2+ ( weak [4]) [4, 5] clofibric acid ( in vivo and in vitro [11]) [4, 11] CoA [1] dexamethasone ( decreases enzyme expression level in renal tubule cells [30]) [30] dichloroacetate ( weak [3]; ATP slightly protects [5]; 50% inhibition at 1.8 mM [16,27]) [3, 4, 5, 11, 13, 16, 27] diphosphate [8] furfurylidenepyruvate ( 1.85 mM [2]) [2] GDP ( 50% inhibition at 0.2 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1] GTP ( 50% inhibition at 0.06 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1] heparin ( 50% inhibition at 0.002 mM [1]; 40% inhibition at 0.012 mg/ml [12]) [1, 4, 12] isobutyryl-CoA [4, 12] isovaleryl-CoA [4, 12] malonyl-CoA [4, 12] methylmalonyl-CoA ( 40% inhibition at 0.2 mM [12]) [4, 12, 15] Mg2+ ( at concentrations above 1.5 mM, activation below [4]) [4] MgATP2- [8] NADP+ ( 40% inhibition at 1.5 mM [12]) [4, 12] phenylacetate ( strong [11]) [4, 11] phenyllactate ( strong [11]) [4, 11] phenylpyruvate ( in vivo and in vitro [11]) [11] pyruvate ( weak [11]) [4, 11, 13] thiamine [8] 170

2.7.11.4

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

thiamine diphosphate ( inhibits phosphorylation of wild-type E1, mutant E1-S303A and mutant E1-D296A/S303A, but not phosphorylation of mutant E1-H292A [6]) [1, 6, 8, 15] UDP ( 50% inhibition at 0.25 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1] UTP ( 50% inhibition at 0.1 mM, inhibition can be reversed by 2 mM Mg2+ [1]) [1] a-chloroisocaproate ( inhibits the enzyme by releasing it from the BCKD complex via dissociation [32]) [32] a-ketoisocaproate ( inhibits the enzyme by releasing it from the BCKD complex via dissociation [32]) [32] a-ketoisovalerate ( inhibits the enzyme by releasing it from the BCKD complex via dissociation [32]) [32] n-octanoate ( 40% inhibition at 0.5 mM [12]) [4, 12, 15] Additional information ( no inhibition by GTP [5]; no inhibition by lactate [11,12]; no inhibition by isovaleryl-CoA [1]; no inhibition by NADH, NAD+ 1 mM each [12]; no inhibition by dl-leucine [13]; no inhibition by 2-chloropropionate [3]; no inhibition by acetyl-CoA [8,12]; no inhibition by acetate [12]; no inhibition by coenzyme A [1,8]; no inhibition by methylcrotonyl-CoA, bhydroxy-b-methylglutaryl-CoA, crotonyl-CoA, octanoyl-CoA, succinyl-CoA, propionyl-CoA, 0.1 mM each, propionate, b-hydroxybutyrate, acetoacetate, malonate, a-ketomalonate, succinate, citrate, oxaloacetate, FAD+, NADPH, 2 mM [12]; binding of thiamin diphosphate cofactor to branched-chain a-keto acid decarboxylase/dehydrogenase, which induces a phosphorylation loop conformation change, inhibits the phosphorylation of the protein by the BCKD kinase, no inhibition of phosphorylation of mutant R287A, D295A, Y300F, and R301A E1B components [29]; clofibric acid and thiamine diphosphate do not affect the protein-protein interaction of BDK with the BCKD complex [32]) [1, 3, 5, 8, 11, 12, 13, 29, 32] Cofactors/prosthetic groups ATP [29, 31, 32] calmodulin ( activation [5]) [5] Activating compounds histone H3 ( 1.5 to 3fold [1]) [1] poly-l-arginine ( 1.5 to 3fold [1]) [1] poly-l-lysine ( 1.5 to 3fold [1]) [1] protamine ( 1.5 to 3fold [1]) [1] Additional information ( tissue-specific translation of branchedchain a-ketoacid dehydrogenase kinase mRNA is dependent upon an upstream open reading frame in the 5-untranslated region [31]) [31] Metals, ions EGTA ( 0.1 mM [5]; activation, presumably by chelation of Ca2+ [4]) [4, 5] K+ ( activation, 0.1 M [14]) [14]

171

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

2.7.11.4

Mg2+ ( requirement, actual substrate: MgATP2- [5,8,9,14,15]; maximum activity at 1.5 mM, inhibits above 1.5 mM [4]; Km -value: 0.025 mM [5]) [4, 5, 8, 9, 14, 15, 29] Rb+ ( activation [14]) [14] Additional information ( no activation by Ca2+ [5]; no activation by Li+ , Na+ [14]) [5, 14] Turnover number (min–1) 0.0542 (phosphate, 25 C, recombinant enzyme alone [19]) [19] 0.475 (phosphate, 25 C, reconstituted with lipoylated recombinant E2 [19]) [19] Specific activity (U/mg) 0.0247 ( without added salt [14]) [14] 0.0268 ( liver enzyme [16,27]) [16, 27] 0.0357 ( heart enzyme [16]) [16] 0.0357 ( heart enzyme, depending on purification method [27]) [27] 0.05 ( recombinant enzyme [27]) [27] Additional information ( various assay methods [15]) [15] Km-Value (mM) 0.004 (ATP) [14] 0.0126 (MgATP2-, pH 7.5, 30 C [8]) [8] 0.013 (MgATP2-, pH 7.5, 30 C [15]) [15] 0.025 (ATP, pH 7.5, 30 C [5]; pH 7.35, 20 C [4]) [4, 5] Ki-Value (mM) 0.00027 (ADP, pH 7.5, 30 C [8]) [8] 0.00048 (4-methyl-2-oxopentanoate, pH 7.5, 30 C [8]) [8] 0.00092 (2-oxoisocaproate, pH 7.5, 30 C [8]) [8] 0.004 (diphosphate, pH 7.5, 30 C [8]) [8] 0.0059 (thiamine, pH 7.5, 30 C [8]) [8] 0.0089 (4-methyl-2-oxopentanoate, pH 7.5, 30 C [8]) [8] 0.13 (ADP, pH 7.5, 30 C [5]; pH 7.35, 20 C [4]) [4, 5] 0.27 (ADP, pH 7.5, 30 C [15]) [15] 0.5 (2-chloroisohexanoate, 37 C [3]) [3] 4.5 (furfurylidenepyruvate) [2] pH-Optimum 7.1 [5] 7.4 ( assay at [24,29]) [24, 29] 7.5 [4] Additional information ( in decreasing order of activity: HEPES, potassium phosphate, imidazole, 3-(N-morpholino)ethane buffer [4]; HEPES-potassium buffer promotes higher activity than imidazole-chloride, 4-morpholinopropanesulfonic acid-potassium or potassium phosphate buffer [5]) [4, 5] pH-Range 6.5-8.3 ( about half-maximal activity at pH 6.5 and 8.3 [5]) [5] 172

2.7.11.4

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

Temperature optimum ( C) 22 ( assay at room temperature [29]) [29] 30 ( assay at [8,16,24]) [8, 16, 24] 37 ( assay at [2,5,7,12,13,14]) [2, 5, 7, 12, 13, 14]

4 Enzyme Structure Molecular weight 43280 ( calculated from amino acid sequence [17]) [17] 44000-45000 ( SDS-PAGE [27]) [27] 460000 ( gel filtration [1]) [1] 2000000 ( above 2000000, gel filtration [4,5]) [4, 5] Subunits ? ( x * 44000, SDS-PAGE [16]) [16] dimer ( dimerizes through direct interaction of two opposing nucleotide-binding domains, crystallographic data [23]) [23] monomer ( 1 * 43000, uncomplexed kinase, SDS-PAGE [18]) [18]

5 Isolation/Preparation/Mutation/Application Source/tissue BNL CL.2 cell ( liver cell line [31]) [31] C2C12 cell ( undifferentiated myoblast cell line [31]) [31] LLC-PK1-GR101 cell ( LLC-PK1 renal tubule cell line expressing the Rattus norvegicus glucocorticoid receptor GR [30]) [30] NIH-3T3 cell ( fibroblast cell line [31]) [31] adipocyte [15] brain [4, 20, 21] embryo [21] fibroblast [31] heart [4, 11, 15, 16, 17, 20, 21, 23, 27, 28] hepatocyte [3] kidney ( cortex [4]) [1, 4, 7, 8, 9, 10, 15, 18, 19, 20, 21, 28, 30] liver ( enzyme activity is 3-5fold higher in female than in male rats [24]; malnutrition results in changed amounts of enzyme level [28]) [2, 3, 4, 5, 11, 12, 13, 14, 15, 16, 20, 24, 26, 27, 28, 31, 32] lung [20] muscle [20, 21] myoblast [31] myotube [31] renal tubule [30] skeletal muscle ( enzyme content decreases 0.7fold after running exercise for 5 weeks [22]) [4, 22, 24, 31]

173

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

2.7.11.4

testis [20] uterus [20] Additional information ( enzyme expression analysis [30]; tissue distribution, tissue-specific translation of branched-chain a-ketoacid dehydrogenase kinase mRNA is dependent upon an upstream open reading frame in the 5-untranslated region, thus mRNA and protein level do not correlate [31]) [30, 31] Localization mitochondrial matrix ( 2 forms: first form is bound to E2, second form is free and seems to be inactive [26]) [14, 26] mitochondrion ( part of intramitochondrial branchedchain 2-oxoacid dehydrogenase complex [4]) [1, 2, 4, 7, 8, 9, 15, 17, 18, 20, 21, 25, 28, 30, 31] Purification [4, 19] (a-ketoacid dehydrogenase complex) [7] (from liver and heart, homogeneity) [15] (from purified branched-chain a-keto acid dehydrogenase complex) [16] (liver enzyme, heart enzyme and recombinant enzyme expressed in Escherichia coli) [27] (partially from mitochondria) [30] [8] (5000fold) [18] (copurifies with EC 1.2.4.4) [9] [11] (a-ketoacid dehydrogenase complex) [4, 5] Renaturation (reconstitution with lipoylated recombinant E2) [19] Crystallization (vapor diffusion method) [23] Cloning [20, 21] (DNA sequence determination and analysis, promotor region footprinting, expression analysis, mechanism of the enzyme expression regulation) [31] [27] (cloned and expressed in Escherichia coli) [17] (fragments of the enzyme cloned into firefly luciferase plasmid) [25] (fusion protein with maltose-binding protein) [19, 23] (BCKD kinase transcription regulation, overview) [30]

174

2.7.11.4

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

6 Stability pH-Stability 7 ( loss of activity during purification at pH-values below 7 [8]) [8] General stability information , precipitation of branched-chain oxo acid dehydrogenase enzyme complex at acid pH-values, especially below 6.5, results in specific loss of kinase activity [15] Storage stability , labile enzyme, best stored at -70 C in the presence of DTT [16]

References [1] Reed, L.J.; Damuni, Z.; Merryfield, M.L.: Regulation of mammalian pyruvate and branched-chain a-keto acid dehydrogenase complexes by phosphorylation-dephosphorylation. Curr. Top. Cell. Regul., 27, 41-49 (1985) [2] Lau, K.S.; Cooper, A.J.L.; Chuang, D.T.: Inhibition of the bovine branchedchain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates. Biochim. Biophys. Acta, 1038, 360-366 (1990) [3] Harris, R.A.; Kuntz, M.J.; Simpson, R.: Inhibition of branched-chain a-keto acid dehydrogenase kinase by a-chloroisocaproate. Methods Enzymol., 166, 114-123 (1988) [4] Paxton, R.: Branched-chain a-keto acid dehydrogenase and its kinase from rabbit liver and heart. Methods Enzymol., 166, 313-320 (1988) [5] Paxton, R.; Harris, R.A.: Isolation of rabbit liver branched chain a-ketoacid dehydrogenase and regulation by phosphorylation. J. Biol. Chem., 257, 14433-14439 (1982) [6] Hawes, J.W.; Schnepf, R.J.; Jenkins, A.E.; Shimomura, Y.; Popov, K.M.; Harris, R.A.: Roles of amino acid residues surrounding phosphorylation site 1 and branched-chain a-ketoacid dehydrogenase (BCKDH) in catalysis and phosphorylation site recognition by BCKDH kinase. J. Biol. Chem., 270, 31071-31076 (1995) [7] Odessey, R.: Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity. Biochem. J., 204, 353-356 (1982) [8] Lau, K.S.; Fatania, H.R.; Randle, P.J.: Regulation of the branched chain 2oxoacid dehydrogenase kinase reaction. FEBS Lett., 144, 57-62 (1982) [9] Lawson, R.; Cook, K.G.; Yeaman, S.J.: Rapid purification of bovine kidney branched-chain 2-oxoacid dehydrogenase complex containing endogenous kinase activity. FEBS Lett., 157, 54-58 (1982) [10] Cook, K.G.; Lawson, R.; Yeaman, S.J.: Multi-site phosphorylation of bovine kidney branched-chain 2-oxoacid dehydrogenase complex. FEBS Lett., 157, 59-62 (1982)

175

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

2.7.11.4

[11] Paxton, R.; Harris, R.A.: Clofibric acid, phenylpyruvate, and dichloroacetate inhibition of branched-chain a-ketoacid dehydrogenase kinase in vitro and in perfused rat heart. Arch. Biochem. Biophys., 231, 58-66 (1984) [12] Paxton, R.; Harris, R.A.: Regulation of branched-chain a-ketoacid dehydrogenase kinase. Arch. Biochem. Biophys., 231, 48-57 (1984) [13] Harris, R.A.; Paxton, R.; DePaoli-Roach, A.: Inhibition of branched chain a-ketoacid dehydrogenase kinase activity by a-chloroisocaproate. J. Biol. Chem., 257, 13915-13918 (1982) [14] Shimomura, Y.; Kuntz, M.J.; Suzuki, M.; Ozawa, T.; Harris, R.A.: Monovalent cations and inorganic phosphate alter branched-chain a-ketoacid dehydrogenase-kinase activity and inhibitor sensitivity. Arch. Biochem. Biophys., 266, 210-218 (1988) [15] Espinal, J.; Beggs, M.; Randle, P.J.: Assay of branched-chain a-keto acid dehydrogenase kinase in mitochondrial extracts and purified branchedchain a-keto acid dehydrogenase complexes. Methods Enzymol., 166, 166175 (1988) [16] Shimomura, Y.; Nanaumi, N.; Suzuki, M.; Popov, K.M.; Harris, R.A.: Purification and partial characterization of branched-chain a-ketoacid dehydrogenase kinase from rat liver and rat heart. Arch. Biochem. Biophys., 283, 293-299 (1990) [17] Popov, K.M.; Zhao, Y.; Shimomura, Y.; Kuntz, M.J.; Harris, R.A.: Branchedchain a-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases. J. Biol. Chem., 267, 13127-13130 (1992) [18] Lee, H.Y.; Hall, T.B.; Kee, S.M.; Tung, H.Y.L.; Reed, L.J.: Purification and properties of branched-chain a-keto acid dehydrogenase kinase from bovine kidney. BioFactors, 3, 109-112 (1991) [19] Davie, J.R.; Wynn, R.M.; Meng, M.; Huang, Y.S.; Aalund, G.; Chuang, D.T.; Lau, K.S.: Expression and characterization of branched-chain a-ketoacid dehydrogenase kinase from the rat. Is it a histidine-protein kinase?. J. Biol. Chem., 270, 19861-19867 (1995) [20] Doering, C.B.; Coursey, C.; Spangler, W.; Danner, D.J.: Murine branched chain a-ketoacid dehydrogenase kinase; cDNA cloning, tissue distribution, and temporal expression during embryonic development. Gene, 212, 213219 (1998) [21] Doering, C.B.; Danner, D.J.: Expression of murine branched-chain a-keto acid dehydrogenase kinase. Methods Enzymol., 324, 491-497 (2000) [22] Fujii, H.; Shimomura, Y.; Murakami, T.; Nakai, N.; Sato, T.; Suzuki, M.; Harris, R.A.: Branched-chain a-keto acid dehydrogenase kinase content in rat skeletal muscle is decreased by endurance training. Biochem. Mol. Biol. Int., 44, 1211-1216 (1998) [23] Machius, M.; Chuang, J.L.; Wynn, R.M.; Tomchick, D.R.; Chuang, D.T.: Structure of rat BCKD kinase: nucleotide-induced domain communication in a mitochondrial protein kinase. Proc. Natl. Acad. Sci. USA, 98, 1121811223 (2001) [24] Nakai, N.; Kobayashi, R.; Popov, K.M.; Harris, R.A.; Shimomura, Y.: Determination of branched-chain a-keto acid dehydrogenase activity state and 176

2.7.11.4

[25] [26] [27] [28]

[29]

[30] [31]

[32]

[3-Methyl-2-oxobutanoate dehydrogenase (acetyl-transferring)] kinase

branched-chain a-keto acid dehydrogenase kinase activity and protein in mammalian tissues. Methods Enzymol., 324, 48-62 (2000) Nellis, M.M.; Doering, C.B.; Kasinski, A.; Danner, D.J.: Insulin increases branched-chain a-ketoacid dehydrogenase kinase expression in Clone 9 rat cells. Am. J. Physiol., 283, E853-E860 (2002) Obayashi, M.; Sato, Y.; Harris, R.A.; Shimomura, Y.: Regulation of the activity of branched-chain 2-oxo acid dehydrogenase (BCODH) complex by binding BCODH kinase. FEBS Lett., 491, 50-54 (2001) Popov, K.M.; Shimomura, Y.; Hawes, J.W.; Harris, R.A.: Branched-chain aketo acid dehydrogenase kinase. Methods Enzymol., 324, 162-178 (2000) Popov, K.M.; Zhao, Y.; Shimomura, Y.; Jaskiewicz, J.; Kedishvili, N.Y.; Irwin, J.; Goodwin, G.W.; Harris, R.A.: Dietary control and tissue specific expression of branched-chain a-ketoacid dehydrogenase kinase. Arch. Biochem. Biophys., 316, 148-154 (1995) Li, J.; Wynn, R.M.; Machius, M.; Chuang, J.L.; Karthikeyan, S.; Tomchick, D.R.; Chuang, D.T.: Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain a-keto acid decarboxylase/dehydrogenase. J. Biol. Chem., 279, 32968-32978 (2004) Wang, X.; Price, S.R.: Differential regulation of branched-chain a-ketoacid dehydrogenase kinase expression by glucocorticoids and acidification in LLC-PK1-GR101 cells. Am. J. Physiol. Renal. Physiol., 286, F504-508 (2004) Muller, E.A.; Danner, D.J.: Tissue-specific translation of murine branchedchain a-ketoacid dehydrogenase kinase mRNA is dependent upon an upstream open reading frame in the 5’-untranslated region. J. Biol. Chem., 279, 44645-44655 (2004) Murakami, T.; Matsuo, M.; Shimizu, A.; Shimomura, Y.: Dissociation of branched-chain a-keto acid dehydrogenase kinase (BDK) from branchedchain a-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors. J. Nutr. Sci. Vitaminol., 51, 48-50 (2005)

177

[Isocitrate dehydrogenase (NADP+ )] kinase

2.7.11.5

1 Nomenclature EC number 2.7.11.5 Systematic name ATP:[isocitrate dehydrogenase (NADP+ )] phosphotransferase Recommended name [Isocitrate dehydrogenase (NADP+ )] kinase Synonyms ICDH [14] ICDH kinase/phosphatase [8] IDH kinase [1] IDH kinase/phosphatase [4, 5] IDH-K/P [12] IDHK/P [9, 11] [isocitrate dehydrogenase (NADP+ )] kinase isocitrate dehydrogenase kinase (phosphorylating) isocitrate dehydrogenase kinase/phosphatase [7, 9, 10, 11] CAS registry number 83682-93-3

2 Source Organism Salmonella typhimurium (no sequence specified) [1] Escherichia coli (no sequence specified) ( large subunit [7,8]) [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14]

3 Reaction and Specificity Catalyzed reaction ATP + [isocitrate dehydrogenase (NADP+ )] = ADP + [isocitrate dehydrogenase (NADP+ )] phosphate Reaction type phospho group transfer

178

2.7.11.5

[Isocitrate dehydrogenase (NADP+ )] kinase

Natural substrates and products S ATP + [isocitrate dehydrogenase (NADP+ )] ( reversible phosphorylation of isocitrate dehydrogenase plays a major role in the control of the Krebs cycle and glyoxylate pathways [1, 5, 6]; phosphorylation of isocitrate dehydrogenase during growth on acetate is to render this enzyme rate-limiting in the citric acid cycle, this should cause an increase in the level of isocitrate and divert the flux of carbon through the glyoxylate bypass [3, 13]; controls the oxidative metabolism, exibits a high intrinsic ATPase activity [9]) (Reversibility: r) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] P ADP + [isocitrate dehydrogenase (NADP+ )] phosphate [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] Substrates and products S ATP + Bacillus subtilis [isocitrate dehydrogenase (NADP+ )] ( BsIDH is a much poorer substrate for the enzyme than EcIDH [12,13]) (Reversibility: ?) [12, 13] P ADP + Bacillus subtilis [isocitrate dehydrogenase (NADP+ )] phosphate S ATP + [isocitrate dehydrogenase (NADP+ )] ( reversible phosphorylation of isocitrate dehydrogenase plays a major role in the control of the Krebs cycle and glyoxylate pathways [1, 5, 6]; phosphorylation of isocitrate dehydrogenase during growth on acetate is to render this enzyme rate-limiting in the citric acid cycle, this should cause an increase in the level of isocitrate and divert the flux of carbon through the glyoxylate bypass [3,13]; controls the oxidative metabolism, exibits a high intrinsic ATPase activity [9]) (Reversibility: r) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] P ADP + [isocitrate dehydrogenase (NADP+ )] phosphate [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] S ATP + [isocitrate dehydrogenase (NADP+ )]IS (Reversibility: ?) [10] P ADP + [isocitrate dehydrogenase (NADP+ )]IS phosphate S ATP + [isocitrate dehydrogenase (NADP+ )]N115L (Reversibility: ?) [10] P ADP + [isocitrate dehydrogenase (NADP+ )]N115L phosphate S Additional information ( uses only ATP, no other nucleoside triphospates as only very poor phosphate donors for the kinase activity, GTP and UTP can activate the phosphatase activity to some extent [3]) (Reversibility: ?) [3] P ? Inhibitors 2-oxoglutarate ( inhibits kinase activity [3]) [3] 5,5’-dithio-bis(2-nitrobenzoic acid) [9] 8-azido-ATP [4] ADP ( kinase hyperbolically inhibited [3]) [3] AMP ( kinase hyperbolically inhibited [3]) [3, 10] dl-isocitrate ( inhibits only kinase activity [3,10]) [3, 10] glyoxylate ( in combination with oxaloacetate [1]) [1, 3] 179

[Isocitrate dehydrogenase (NADP+ )] kinase

2.7.11.5

NADPH ( inhibits both IDH kinase and IDH phosphatase [10]) [3, 10] oxaloacetate ( inhibits kinase activity [3]) [1, 3] phosphoenolpyruvate ( kinase hyperbolically inhibited [3]) [3] pyruvate ( inhibits kinase activity [3]) [1, 3, 10] [isocitrate dehydrogenase (NADP+ )] phosphate ( wild-type [6]) [6] cupric 1,10 phenanthrolinate [9] Cofactors/prosthetic groups ADP ( isocitrate dehydrogenase phosphatase requires a nucleotide for activity [3]) [3] ATP ( isocitrate dehydrogenase phosphatase requires a nucleotide for activity [3]) [3] Activating compounds acetate [1] ethanol [1] a-methylglucoside [1] deoxyglucose [1] Metals, ions Mg2+ ( absolute requirement, isocitrate dehydrogenase phosphatase responds hyperbolically to Mg2+ ions [3]) [3, 11] Additional information ( Mn2+ or Ca2+ cannot replace Mg2+ [3]) [3] Specific activity (U/mg) 0.038 [2] 0.11 [5] Km-Value (mM) 0.00023 ([isocitrate dehydrogenase (NADP+ )], pH 7.5, 37 C, wildtype and mutant AceK4, kinase activity [6]) [6] 0.00025 ([isocitrate dehydrogenase (NADP+ )], pH 7.5, 37 C, mutant AceK3, kinase activity [6]) [6] 0.00035 ([isocitrate dehydrogenase (NADP+ )], pH 7.3, 37 C, kinase activity [3]) [3] 0.00078 ([isocitrate dehydrogenase (NADP+ )], pH 7.5, 37 C, kinase activity at saturating ATP [13]) [13] 0.0009 ([isocitrate dehydrogenase (NADP+ )]N15L, pH 7.5, 37 C [10]) [10] 0.0017 ([isocitrate dehydrogenase (NADP+ )], pH 7.5, 37 C [10]) [10] 0.0049 (Bacillus subtilis [isocitrate dehydrogenase (NADP+ )], pH 7.5, 37 C [13]) [13] 0.0059 ([isocitrate dehydrogenase (NADP+ )], pH 7.5, 37 C [13]) [13] 0.0069 (ATP, pH 7.5, 37 C, wild-type [11]) [11] 0.0087 (ATP, pH 7.5, 37 C, mutant D403A [11]) [11] 0.0098 (ATP, pH 7.5, 37 C, mutant Glu439Ala [11]) [11]

180

2.7.11.5

[Isocitrate dehydrogenase (NADP+ )] kinase

0.0147 (ATP, pH 7.5, 37 C, mutant Asn377Ala [11]) [11] 0.016 (ATP, pH 7.5, 37 C, wild-type, kinase activity [6]) [6] 0.02 ([isocitrate dehydrogenase (NADP+ )]IS, pH 7.5, 37 C [10]) [10] 0.0582 (Bacillus subtilis [isocitrate dehydrogenase (NADP+ )], pH 7.5, 37 C, kinase activity at saturating ATP [13]) [13] 0.088 (ATP, pH 7.3, 37 C [3]) [3] 0.1 (ATP, pH 7.5, 37 C, mutant AceK3, kinase activity [6]) [6] 0.32 (ATP, pH 7.5, 37 C, mutant AceK4, kinase activity [6]) [6] Ki-Value (mM) 0.008 (AMP, pH 7.5, 37 C [10]) [10] 0.011 (isocitrate, pH 7.5, 37 C, mutant AceK4, kinase activity [10]) [10] 0.015 (isocitrate, pH 7.5, 37 C, mutant AceK3, kinase activity [10]) [10] 0.016 (isocitrate, pH 7.5, 37 C, wild-type, kinase activity [10]) [10] 0.02 (AMP, pH 7.5, 37 C, mutant AceK4, kinase activity [10]) [10] 0.023 (dl-isocitrate, pH 7.3, 37 C [3]) [3] 0.042 (NADPH, pH 7.3, 37 C [3]) [3] 0.056 (AMP, pH 7.3, 37 C [3]) [3] 0.058 (NADPH, pH 7.5, 37 C, mutant AceK4, kinase activity [10]) [10] 0.073 (NADPH, pH 7.5, 37 C, mutant AceK3, kinase activity [10]) [10] 0.082 (NADPH, pH 7.5, 37 C, wild-type, kinase activity [10]) [10] 0.17 (AMP, pH 7.5, 37 C, mutant AceK3, kinase activity [10]) [10] 0.2 (pyruvate, pH 7.5, 37 C, wild-type, kinase activity [10]) [10] 0.45 (ADP, pH 7.3, 37 C [3]) [3] 0.55 (phosphoenolpyruvate, pH 7.3, 37 C [3]) [3] 1 (3-phosphoglycerate, pH 7.5, 37 C, wild-type, kinase activity [10]) [10] 1 (pyruvate, pH 7.5, 37 C, mutant AceK4, kinase activity [10]) [10] 4 (3-phosphoglycerate, pH 7.5, 37 C, mutant AceK4, kinase activity [10]) [10] 4 (pyruvate, pH 7.5, 37 C, mutant AceK3, kinase activity [10]) [10] 20 (3-phosphoglycerate, pH 7.5, 37 C, mutant AceK3, kinase activity [10]) [10] pH-Optimum 8-8.5 ( kinase activity [3]) [3]

4 Enzyme Structure Molecular weight 130000 ( recombinant enzyme, gel filtration [7]) [7] 135000 ( gel filtration, glycerol density gradient centrifugation [2]) [2] 181

[Isocitrate dehydrogenase (NADP+ )] kinase

2.7.11.5

Subunits dimer ( 2 * 66000, homodimer, SDS-PAGE [2,9,11]; 2 * 68800, homodimer, theoretical molecular mass [9]) [2, 7, 9, 11] Posttranslational modification phosphoprotein ( reversible inactivation of the enzyme is due to reversible phosphorylation catalyzed by ICDH kinase/phosphatase. This activity requires ATP. Phosphorylation of the Ser113 residue renders the enzyme catalytically inactive as it prevents isocitrate from binding to the active site. This is a consequence of the negative charge carried on phosphoserine 113 and the conformational change associated with it. The ICDH molecule readily undergoes domain shift and/or induced-fit conformational changes to accomodate the binding changes of ICDH kinase/phosphatase [14]) [14]

5 Isolation/Preparation/Mutation/Application Purification [6, 7, 9] (partial, bifunctional protein) [2, 4, 5] Cloning (aceK gene) [6] (aceK gene of Escherichia coli K-12 cloned in pQE30 expression vector to overproduce the protein in Escherichia coli JM105) [7] (bifunctional protein, expressed from the aceK gene) [5] (recombinant wild-type IDHK/P on overproducing plasmid pJCD4, expressed in Escherichia coli JM109) [9] Engineering D403A ( site-directed mutagenesis [11]) [11] E439A ( site-directed mutagenesis [11]) [11] N377A ( site-directed mutagenesis [11]) [11]

6 Stability Storage stability , -20 C, stable for at least 3 months [2] , 4 C, can be stored for several days without significant loss of activity [2]

References [1] Wang, J.Y.J.; Koshland, D.E.: The reversible phosphorylation of isocitrate dehydrogenase of Salmonella typhimurium. Arch. Biochem. Biophys., 218, 59-67 (1982)

182

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[Isocitrate dehydrogenase (NADP+ )] kinase

[2] Nimmo, G.A.; Borthwick, A.C.; Holms, W.H.; Nimmo, H.G.: Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308. Eur. J. Biochem., 141, 401-408 (1984) [3] Nimmo, G.A.; Nimmo, H.G.: The regulatory properties of isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase from Escherichia coli ML308 and the roles of these activities in the control of isocitrate dehydrogenase. Eur. J. Biochem., 141, 409-414 (1984) [4] Varela, I.; Nimmo, H.G.: Photoaffinity labelling shows that Escherichia coli isocitrate dehydrogenase kinase/phosphatase contains a single ATP-binding site. FEBS Lett., 231, 361-365 (1988) [5] Ikeda, T.P.; Houtz, E.; LaPorte, D.C.: Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity. J. Bacteriol., 174, 1414-1416 (1992) [6] Miller, S.P.; Karschnia, E.J.; Ikeda, T.P.; LaPorte, D.C.: Isocitrate dehydrogenase kinase/phosphatase. Kinetic characteristics of the wild-type and two mutant proteins. J. Biol. Chem., 271, 19124-19128 (1996) [7] Rittinger, K.; Negre, D.; Divita, G.; Scarabel, M.; Bonod-Bidaud, C.; Goody, R.S.; Cozzone, A.J.; Cortay, J.C.: Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues. Eur. J. Biochem., 237, 247-254 (1996) [8] El-Mansi, E.M.T.: Control of metabolic interconversion of isocitrate dehydrogenase between the catalytically active and inactive forms in Escherichia coli. FEMS Microbiol. Lett., 166, 333-339 (1998) [9] Oudot, C.; Jaquinod, M.; Cortay, J.C.; Cozzone, A.J.; Jault, J.M.: The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer. Eur. J. Biochem., 262, 224-229 (1999) [10] Miller, S.P.; Chen, R.; Karschnia, E.J.; Romfo, C.; Dean, A.; LaPorte, D.C.: Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem., 275, 833-839 (2000) [11] Oudot, C.; Cortay, J.C.; Blanchet, C.; Laporte, D.C.; Di Pietro, A.; Cozzone, A.J.; Jault, J.M.: The “catalytic“ triad of isocitrate dehydrogenase kinase/ phosphatase from E. coli and its relationship with that found in eukaryotic protein kinases. Biochemistry, 40, 3047-3055 (2001) [12] Singh, S.K.; Matsuno, K.; LaPorte, D.C.; Banaszak, L.J.: Crystal structure of Bacillus subtilis isocitrate dehydrogenase at 1.55 A. Insights into the nature of substrate specificity exhibited by Escherichia coli isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem., 276, 26154-26163 (2001) [13] Singh, S.K.; Miller, S.P.; Dean, A.; Banaszak, L.J.; LaPorte, D.C.: Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem., 277, 7567-7573 (2002) [14] Cozzone, A.J.; El-Mansi, M.: Control of isocitrate dehydrogenase catalytic activity by protein phosphorylation in Escherichia coli. J. Mol. Microbiol. Biotechnol., 9, 132-146 (2005) 183

[Tyrosine 3-monooxygenase] kinase

2.7.11.6

1 Nomenclature EC number 2.7.11.6 Systematic name ATP:[tyrosine-3-monoxygenase] phosphotransferase Recommended name [tyrosine 3-monooxygenase] kinase Synonyms kinase, tyrosine 3-monooxygenase (phosphorylating) CAS registry number 103537-12-8

2 Source Organism Rattus norvegicus (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction ATP + [tyrosine 3-monooxygenase] = ADP + phospho-[tyrosine 3-monooxygenase] Reaction type phospho group transfer Natural substrates and products S ATP + [tyrosine 3-monooxygenase] (Reversibility: ?) [1, 2] P ADP + [tyrosine 3-monooxygenase] phosphate Substrates and products S ATP + [tyrosine 3-monooxygenase] ( specific, incorporates 1 mol phosphate per mol enzyme tetramer [2]; phosphorylation site: Ser-40 [2]) (Reversibility: ?) [1, 2] P ADP + [tyrosine 3-monooxygenase] phosphate Inhibitors Additional information ( no inhibition by EGTA [1]) [1]

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[Tyrosine 3-monooxygenase] kinase

Activating compounds Additional information ( no requirement for cAMP [1,2]) [1, 2] Metals, ions Mg2+ ( requirement [1]) [1] Additional information ( no requirement for Ca2+ [1,2]) [1, 2] pH-Optimum 7.2 ( assay at [1,2]) [1, 2] Temperature optimum ( C) 30 ( assay at [1,2]) [1, 2]

5 Isolation/Preparation/Mutation/Application Source/tissue pheochromocytoma cell ( from adrenal gland [1,2]) [1, 2] Purification (partial, during purification the kinase remains associated with its substrate) [1]

References [1] Pigeon, D.; Drissi-Daoudi, R.; Gros, F.; Thibault, J.: Copurification of tyrosine hydroxylase from rat pheochromocytoma, with a protein kinase activity. C. R. Acad. Sci. Paris Ser.3, 302, 435-438 (1986) [2] Pigeon, D.; Ferrara, P.; Gros, F.; Thibault, J.: Rat pheochromocytoma tyrosine hydroxylase is phosphorylated on serine 40 by an associated protein kinase. J. Biol. Chem., 262, 6155-6158 (1987)

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1 Nomenclature EC number 2.7.11.7 Systematic name ATP:[myosin heavy-chain] O-phosphotransferase Recommended name myosin-heavy-chain kinase Synonyms MHCK [14, 16, 17, 18, 25, 26] MHCK A [27, 28] MIHC kinase [20] MIHCK [19, 21, 22, 23, 24] kinase (phosphorylating), myosin heavy chain myosin I heavy-chain kinase myosin II heavy chain kinase [29] myosin II heavy-chain kinase myosin heavy chain kinase myosin heavy chain kinase A [27, 28] Additional information ( member of the p21-activated kinase family [22,23,24]) [22, 23, 24] CAS registry number 64763-54-8

2 Source Organism Gallus gallus (no sequence specified) [11, 12] Bos taurus (no sequence specified) [13] Dictyostelium discoideum (no sequence specified) [2, 3, 4, 6, 14, 18, 27, 29] Acanthamoeba castellanii (no sequence specified) [5, 7, 8, 9, 10, 17, 20, 21] Dictyostelium discoideum (UNIPROT accession number: P42527) [1, 25, 26, 28] Dictyostelium discoideum (UNIPROT accession number: P90648) [1, 15, 25] Dictyostelium discoideum (UNIPROT accession number: P34125) [1, 16]

186

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Myosin-heavy-chain kinase

Dictyostelium discoideum (UNIPROT accession number: O41467) [19] Dictyostelium discoideum (UNIPROT accession number: Q94488) [22] Acanthamoeba castellanii (UNIPROT accession number: Q93107) [23, 24] Dictyostelium discoideum (UNIPROT accession number: O76739) [25] Dictyostelium discoideum (UNIPROT accession number: Q8MY12) [14]

3 Reaction and Specificity Catalyzed reaction ATP + [myosin heavy-chain] = ADP + [myosin heavy-chain] phosphate ( catalytic domain: amino acid residues 31-259 [14]; catalytic domain structure, modeling [21]; residues Ser627, Thr631 and Thr632 are essential for catalytic activity [21]; activation of the enzyme by autophosphorylation is necessary for full catalytic activity [5]; enzyme contains the C-terminal WD-domain, responsible for substrate binding, the catalytic domain, and the N-terminal coiled-coil translocation domain [15,26]; major phosphorylation site: Ser8 [22]) Reaction type phospho group transfer Natural substrates and products S ATP + [myosin-heavy-chain] (Reversibility: ?) [27, 28] P ADP + [myosin-heavy-chain]phosphate S ATP + myosin I heavy chain ( reaction in regulatory contractile activity in Dictyostelium discoideum [2]; involved in regulation of myosin II filament assembly [3, 4]; increased activity during chemotaxis [4]) (Reversibility: ?) [2, 3, 4, 5, 6, 7, 8, 10, 17, 19, 20, 21, 22, 23, 24] P ADP + myosin I heavy chain phosphate [2, 3, 4, 5, 6, 7, 8, 10, 17, 19, 20, 21, 22, 23, 24] S ATP + myosin II heavy chain ( involved in regulation of myosin II filament assembly [14, 15]; key role in regulating myosin localization [18, 26]; the enzymes anterior localization is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events via direct binding to F-actin [26]) (Reversibility: ?) [6, 7, 9, 14, 15, 18, 25, 26] P ADP + myosin II heavy chain phosphate [6, 7, 9, 14, 15, 18, 26] S ATP + myosin heavy chain (Reversibility: ?) [11, 12, 13] P ADP + myosin heavy chain phosphate [11, 12, 13] S ATP + myosin heavy chain kinase ( intramolecular autophosphorylation [16]; increased activity during chemotaxis [16]) (Reversibility: ?) [16] P ADP + myosin heavy chain kinase phosphate [16] S Additional information ( biochemical mechanism for the spatial regulation of myosin II filament disassembly acts via MHCK A activa187

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tion by actin [28]; C-terminal phosphorylation of myosin heavy chain is required for regulation of myosin II filament assembly, regulation mechanism [27]; C-terminal phosphorylation of myosin heavy chain is required for regulation of myosin II filament assembly, the 3 isozymes MHCK A, MHCK B, and MHCK C show differential localization patterns in living cells [29]) (Reversibility: ?) [27, 28, 29] P ? Substrates and products S ATP + AKRVSMMR ( peptide derived from myosin I heavy chain kinase amino acid residues 4-11, exchange at position 4: Ser to Ala [22]) (Reversibility: ?) [22] P ADP + AKRVS(-phosphate)MMR ( phophorylation site is Ser8 [22]) [22] S ATP + GRGRSSVYS ( synthetic peptide with a sequence corresponding to the phosphorylation site of myosin IC [5, 8, 21, 23, 24]) (Reversibility: ?) [5, 8, 17, 20, 21, 23, 24] P ADP + GRGRSS(-phosphate)VYS [5, 8] S ATP + GRSARVSTYA ( peptide derived from Dictyostelium myosin ID [22]) (Reversibility: ?) [22] P ADP + GRSARVS(-phosphate)TYA [22] S ATP + RKKFGESEKTKTKEFL ( isozyme MHCK B [15]; synthetic peptide MH1 [15]) (Reversibility: ?) [15] P ? S ATP + RKKFGESEKTKTKEFL-amide ( low activity [25]; catalytic domain [25]; isozyme myosin II heavy chain kinase A [6]; synthetic peptide MH-3 [6,25]) (Reversibility: ?) [6, 25] P ? S ATP + YAYDTRYRR ( consensus sequence [25]; catalytic domain of the enzyme [25]) (Reversibility: ?) [25] P ? S ATP + [myosin-heavy-chain] (Reversibility: ?) [27, 28] P ADP + [myosin-heavy-chain]phosphate S ATP + caldesmon ( catalytic domain [25]) (Reversibility: ?) [25] P ADP + caldesmon phosphate S ATP + casein ( no activity [2,4]; poor substrate [7]; catalytic domain, low activity [25]) (Reversibility: ?) [2, 4, 7, 13, 25] P ADP + casein phosphate S ATP + chicken gizzard myosin light chain ( no activity [12]; poor substrate [13]) (Reversibility: ?) [12, 13] P ADP + chicken gizzard myosin light chain phosphate S ATP + histone 2A ( no activity [13]; low activity [2]) (Reversibility: ?) [2, 7, 13] P ADP + histone 2A phosphate

188

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S ATP + myelin basic protein ( catalytic domain [25]) (Reversibility: ?) [25] P ADP + myelin basic protein phosphate S ATP + myosin I heavy chain ( substrate: myosin IB, phosphorylation site is Ser315 [7,8]; higher activity with membranebound substrate myosin I [17]; myosin light chains are no substrates [2,4,10]; substrate myosin IA: phosphorylation of a single threonine [8]; 4 mol phosphate per mol of myosin [4]; phosphorylates serine-residues [7]; phosphorylation of residues Thr1833 and Thr2029 [3,6]; substrate is heavy chain of myosin IC [8,23,24]; incorporation of 0.9-1.0 mol phosphate per mol of heavy chain myosin [7]; phosphorylates threonine residues [2,3,4,5,6]; 1.9 mol phosphate per mol myosin [2]; 10 mol phosphate per mol of kinase subunit [6]; 20 mol phosphate per mol of kinase subunit, kinetics [4]; myosin I from intestinal brush border [8]; major site of phosphorylation is Ser8 [22]; 35 kDa trypsin fragment of the C-terminus of the maximally activated, phosphorylated enzyme is fully catalytically active and contains 2 thirds of the autophosphorylation sites of the native enzyme [20]; bovine muscle myosin is no substrate [4]; contains two myosin heavy chain kinases: one for myosin I and one for myosin II [7]; substrate: myosin IC, phosphorylation site is Ser311 [8]; no substrates are human platelet myosin, ovalbumin and smooth muscle myosin from turkey [2]; a basic amino acid is essential on aminoterminal side of phosphorylation site, two are preferable, and a Tyr-residue is essential two residues away on the COOH-terminal side [8]; 68 mol phosphate per mol of enzyme [6]; substrates are heavy chains of myosin IA and IB [7,8,17]; substrate: myosin ID [19,22]; reaction in regulatory contractile activity in Dictyostelium discoideum [2]; involved in regulation of myosin II filament assembly [3,4]; increased activity during chemotaxis [4]) (Reversibility: ?) [2, 3, 4, 5, 6, 7, 8, 10, 17, 19, 20, 21, 22, 23, 24] P ADP + myosin I heavy chain phosphate [2, 3, 4, 5, 6, 7, 8, 10, 17, 19, 20, 21, 22, 23, 24] S ATP + myosin II heavy chain ( myosin light chains are no substrates [9]; contains two myosin heavy chain kinases: one for myosin I and one for myosin II [7]; specific isozyme A for the heavy chain of myosin II [6,18]; incorporation of 3 mol phosphate per mol of myosin II heavy chain, phosphorylation sites are mainly identical in vitro and in vivo [9]; isozyme MHCK B [15]; phosphorylates serine residues [7,9]; drives filament disassembly in vitro [15,18]; catalytic domain [25]; phosphorylates primarly threonine residues [18,25]; involved in regulation of myosin II filament assembly [14,15]; key role in regulating myosin localization [18,26]; the enzymes anterior localization is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events via direct binding to F-actin [26]) (Reversibility: ?) [6, 7, 9, 14, 15, 18, 25, 26] 189

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P ADP + myosin II heavy chain phosphate [6, 7, 9, 14, 15, 18, 25, 26] S ATP + myosin heavy chain ( substrate specificity [12]; phosphorylates threonine residues [11,12]; rabbit skeletal myosin heavy chains are phosphorylated [12]; 0.7 mol phosphate per mol of heavy chain [12]; best substrate: chicken intestine brush border myosin [11,12]; protamine is no substrate [13]; no activity with myosins from gizzard, cardiac or skeletal muscle, but active with the isolated 20 kDa gizzard myosin light chain [13]; substrate: bovine brain myosin [13]; specific for myosin heavy chains, no phosphorylation of light chains [11,12,13]) (Reversibility: ?) [11, 12, 13] P ADP + myosin heavy chain phosphate [11, 12, 13] S ATP + myosin heavy chain kinase ( 35 kDa trypsin fragment of the C-terminus of the maximally activated, phosphorylated enzyme is fully catalytically active and contains 2 thirds of the autophosphorylation sites of the native enzyme [20]; intermolecular autophosphorylation with vesicle-bound enzyme in absence of substrates [17]; intramolecular autophosphorylation [2,3,4,5,6,7,16,26]; enzyme incorporates 1 mol of phosphate per mol of enzyme [22]; 4 phosphorylation sites on each heavy chain [4]; enzyme incorporates 20 mol phosphate per mol of kinase [16]; enzyme incorporates 2-3 mol phosphate per mol of 130 kDa subunit [6]; leads to activation of the phosphorylation activity towards myosin I [2, 3, 4, 5, 6, 17]; enzyme incorporates 6-8 mol phosphate per mol of enzyme [5]; the enzyme contains a cluster of 23 serine and threonine residues at the carboxyterminal end that might be the autophosphorylation domain [16]; increased activity during chemotaxis [16]) (Reversibility: ?) [2, 3, 4, 5, 6, 7, 16, 17, 20, 22, 26] P ADP + myosin heavy chain kinase phosphate [2, 3, 4, 5, 6, 7, 16, 17, 20, 26] S ATP + myosin regulatory light chain ( catalytic domain [25]) (Reversibility: ?) [25] P ADP + myosin regulatory light chain phosphate S ATP + peptide LMM58 of heavy chain ( 4 mol phosphate per mol, Thr-residues are phosphorylated [4]) (Reversibility: ?) [4] P ? S ATP + phosvitin ( no activity [7]; low activity [2]) (Reversibility: ?) [2, 7, 13] P ADP + phosvitin phosphate S ATP + smooth muscle myosin light chain (Reversibility: ?) [7] P ADP + smooth muscle myosin light chain phosphate S ATP + synthetic peptides ( variations in length, and number, location and kind of basic residues of the basic sequence of peptide GRGRSSVYS, overview [8]; peptide MH-3, corresponding to myosin II heavy chain from residues 2020 to 2035 except that Ser-2026 and Thr2031 have been replaced by alanine [3]) (Reversibility: ?) [3, 6, 8] P ? 190

2.7.11.7

Myosin-heavy-chain kinase

S ATP + troponin T (Reversibility: ?) [13] P ADP + troponin T phosphate S Additional information ( substrate specificity study [8]; enzyme contains several SH3-binding domains [23]; the Mg2+ -ATPase activity of the substrate myosin I is increased by its phosphorylation and the binding of F-actin [5,8,20]; enzyme possesses binding domains for the Ras-related GTP-binding proteins Cdc42 and Rac [19,23]; the Mg2+ -ATPase activity of the substrate myosin II is inhibited in vitro by its phosphorylation [9,15]; localization of autophosphorylation sites [20,25]; biochemical mechanism for the spatial regulation of myosin II filament disassembly acts via MHCK A activation by actin [28]; C-terminal phosphorylation of myosin heavy chain is required for regulation of myosin II filament assembly, regulation mechanism [27]; C-terminal phosphorylation of myosin heavy chain is required for regulation of myosin II filament assembly, the 3 isozymes MHCK A, MHCK B, and MHCK C show differential localization patterns in living cells [29]; MHCK A performs autophosphorylation [28]) (Reversibility: ?) [5, 8, 9, 15, 19, 20, 23, 25, 27, 28, 29] P ? Inhibitors Ca2+ /calmodulin ( weak [9]; no inhibition [2, 7, 13]; calmodulin-binding site: amino acid residues 51-80 [24]; competes with phospholipids [24]; binds to the enzyme and inhibits its activation by acidic phospholipids but not by guanosine 5-3-O-(thio)triphosphate-Rac1 [22]) [2, 7, 9, 13, 22, 24] EGTA ( no inhibition [2]) [2, 11, 12, 24] Heparin [13] Histone [3] KCl ( 80% inhibition at 90 mM [4]; strong, 90% inhibition at 60 mM, complete inhibition at 100 mM [2]; 60% inhibition at 0.1 M [9]) [2, 4, 9] Myosin I ( inhibits autophosphorylation [8]) [8] NaCl ( above 0.1 M [11,12]) [11, 12] Positively charged polypeptides ( strong inhibition, e.g. poly-(dLys), poly-(L-Lys), poly-(L-Arg) of different molecular weights [3]) [3] Additional information ( no inhibition by Ca2+ [2]; autoinhibitory domains, overview, autoinhibition is reversed by Rac [24]; strong inhibition of phosphorylation activity by increasing ionic strength [6,7]; ionic strength has no effect on autophosphorylation activity [6]; no inhibition by cAMP or cGMP [2,9]) [2, 6, 7, 9, 24] Cofactors/prosthetic groups ATP [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28]

191

Myosin-heavy-chain kinase

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Activating compounds acidic phospholipids ( functions cooperatively with acidic phospholipids to associate the enzyme with membranes [22]; enhance the activation of the enzyme by autophosphorylation [17,20,22]) [17, 20, 22] DNA ( autophosphorylation activity is increased 5-10fold in a Ca2+ independent manner [3]) [3] F-actin ( about 40fold activation of myosin heavy chain kinase A, structure and mechanism, stimulation via enzyme autophosphorylation [28]; i.e. filamentous actin, from rabbit muscle, about 40fold activation, coiled-coil domain binding structure and mechanism, overview, the enzymes coiled-coil structure mediates its oligomerization, cellular localization, and actin-binding activity of the MHCK A [27]) [27, 28] G-actin ( slight activation of MHCK A, activation is abolished by latrunculin A, which depolymerizes the actin filament [28]) [28] heparin ( autophosphorylation activity is increased 5-10fold in a Ca2+ -independent manner [3]) [3] phosphatidylinositol ( 10fold activation of autophosphorylation and kinase activity [22]) [8, 22, 23] phosphatidylinositol 4,5-bisphosphate ( 10fold activation of autophosphorylation and kinase activity [22]) [22] phosphatidylserine ( 10fold activation of autophosphorylation and kinase activity [22]; stimulation only of the autophosphorylated enzyme [8]; stimulates the enzymes autophosphorylation activity [5]) [5, 8, 17, 22, 23, 24] phospholipid vesicles ( autophosphorylation activity is increased 5-10fold in a Ca2+ -independent manner [3,8]; composed of phosphatidylserine or phosphatidylinositol, not phosphatidylcholine [3,8]; composed of phosphatidylethanolamine [8]) [3, 8] Rac ( reverses autoinhibition of the enzyme [24]; linoleic acid supports activation by Rac [23]; activation of autophosphorylation and kinase activity only in presence of phosphatidylserine, activates only the fully phosphorylated enzyme [23]) [23, 24] guanosine 5’-3-O-(thio)triphosphate-Rac1 ( functions cooperatively with acidic phospholipids to associate the enzyme with membranes [22]; i.e. GTPgS-Rac1 [22]; 10fold activation of autophosphorylation and kinase activity [22]) [22] Additional information ( no activation by cGMP [2,4,13]; no activation by phosphatidylcholine and phosphatidylethanolamine [23]; enzyme is stimulated by its autophosphorylation [5,6,17,20,22]; no activation by EGTA [2]; no activation by calmodulin, cAMP [2,4,7,13]; no activation by phosphatidylcholine and sphingosine [22]) [2, 3, 4, 5, 6, 7, 13, 17, 20, 21, 22, 23] Metals, ions Ca2+ ( not required [2, 3, 4, 7, 9, 10, 13]; slightly activates unphosphorlyated enzyme [24]; requirement, Ca2+ /calmodulin-dependent isozyme [11,12]) [2, 3, 4, 7, 9, 10, 11, 12, 13, 24, 28]

192

2.7.11.7

Myosin-heavy-chain kinase

KCl ( activates [28]) [28] Mg2+ ( requirement [2,3,4,6,7,9,10,11,12]; absolutely requires 1-2 mM Mg2+ [2,6]; cannot be substituted by Ca2+ or Mn2+ [2,9]; optimal activation at 6 mM [11]; maximal activity at 6-8 mM [4]) [2, 3, 4, 6, 7, 9, 10, 11, 12, 15, 17, 20, 23, 24, 25, 27, 28] Additional information ( no activation by Mn2+ [2,9]) [2, 9] Turnover number (min–1) 0.01 (RKKFGESEKTKTKEFL-amide, pH 7.0, 25 C [25]) [25] 0.3 (GRSARVSTYA, pH 7.0 [22]) [22] 0.55 (RKKFGESEKTKTKEFL-amide, pH 7.0, 25 C [25]) [25] 0.79 (RKKFGESEKTKTKEFL-amide, pH 7.0, 25 C [25]) [25] 4.6 (GRSARVSTYA, in presence of guanosine 5-3-O-(thio)triphosphate-Rac1, pH 7.0 [22]) [22] 4.7 (GRSARVSTYA, in presence of phosphatidylserine, pH 7.0 [22]) [22] 5 (AKRVSMMR, in presence of guanosine 5-3-O-(thio)triphosphate-Rac1, pH 7.0 [22]) [22] 5.4 (AKRVSMMR, in presence of phosphatidylserine, pH 7.0 [22]) [22] 14 (YAYDTRYRR, pH 7.0, 25 C [25]) [25] 71 (GRGRSSVYS, recombinant wild-type catalytic domain, pH 7.0, 30 C [21]) [21] 105 (GRGRSSVYS, recombinant T632A mutant catalytic domain, pH 7.0, 30 C [21]) [21] Additional information ( catalytic domain, several substrates [25]; catalytic domain mutants [21]) [21, 25] Specific activity (U/mg) 0.00032 ( partially purified enzyme [11]) [11] 0.00063 ( purified enzyme [12]) [12] 0.04 [5] 0.98 ( purified enzyme [2,6]; substrate myosin II heavy chain [6]) [2, 6] 2.1 ( purified enzyme [4]) [4] 3.03 ( purified enzyme, substrate histone 2A, 0.067 mM [7]) [7] 4.6 ( purified enzyme, substrate myosin IB, 0.0024 mM [7]) [7] Additional information [24] Km-Value (mM) 0.0002 (myosin IB, soluble, unphosphorylated enzyme, pH 7.0, 30 C [17]) [17] 0.0003 (myosin IA, soluble, unphosphorylated enzyme, pH 7.0, 30 C [17]) [17] 0.0005 (myosin IB, phospholipid-bound, unphosphorylated enzyme, pH 7.0, 30 C [17]) [17] 0.0007 (myosin IA, phospholipid-bound, unphosphorylated enzyme, pH 7.0, 30 C [17]) [17]

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0.0023 (myosin IA, soluble, phosphorylated enzyme, pH 7.0, 30 C [17]) [17] 0.0055 (myosin IB, soluble, phosphorylated enzyme, pH 7.0, 30 C [17]) [17] 0.015 (myosin heavy chain, pH 7.5, 22 C [4]) [4] 0.015 (protein LMM58 heavy chain, pH 7.5, 22 C [4]) [4] 0.016 (GRSARVSTYA, pH 7.0 [22]) [22] 0.017 (GRSARVSTYA, in presence of guanosine 5-3-O-(thio)triphosphate-Rac1, pH 7.0 [22]) [22] 0.019 (GRSARVSTYA, in presence of phosphatidylserine, pH 7.0 [22]) [22] 0.043 (ATP, pH 7.5, 30 C [7]) [7] 0.05 (GRGRSSVYS, pH 7.5, 30 C [5]; pH 7.0, 30 C [8]) [5, 8] 0.064 (GRGRSSVYS, phospholipid-bound, unphosphorylated enzyme, pH 7.0, 30 C [17]; soluble, phosphorylated enzyme, pH 7.0, 30 C [17]) [17] 0.098 (AKRVSMMR, in presence of guanosine 5-3-O-(thio)triphosphate-Rac1, pH 7.0 [22]) [22] 0.1 (RKKFGESEKTKTKEFL-amide, pH 7.5, 25 C [6]) [6] 0.11 (GRGRSSVYS, soluble, unphosphorylated enzyme, pH 7.0, 30 C [17]) [17] 0.116 (AKRVSMMR, in presence of phosphatidylserine, pH 7.0 [22]) [22] 0.15 (GRGRSSVYS, recombinant wild-type catalytic domain, pH 7.0, 30 C [21]) [21] 0.16 (ATP, recombinant wild-type catalytic domain, pH 7.0, 30 C [21]) [21] 0.2 (GRGRSSVYS, recombinant T632A mutant catalytic domain, pH 7.0, 30 C [21]) [21] 0.22 (RKKFGESEKTKTKEFL, recombinant MHCK B, pH 7.0, 22 C [15]) [15] 0.28 (RKKFGESEKTKTKEFL-amide, pH 7.0, 25 C [25]) [25] 0.3 (ATP, recombinant T632A mutant catalytic domain, pH 7.0, 30 C [21]) [21] 0.55 (YAYDTRYRR, pH 7.0, 25 C [25]) [25] Additional information ( catalytic domain mutants [21]; Km values for derivatives of peptide GRGRSSVYS [8]; F-actin binding kinetics [27]) [8, 21, 27] Ki-Value (mM) Additional information ( inhibition by positively charged polypeptides [3]) [3] pH-Optimum 7 ( assay at [15, 17, 22, 24, 25]) [15, 17, 22, 24, 25] 7-7.5 [2, 6, 7, 9, 11] 7.5 ( assay at [3,5,20,23]) [3, 4, 5, 20, 23] 194

2.7.11.7

Myosin-heavy-chain kinase

8 ( assay at [28]; actin binding assay at [27]) [12, 27, 28] pH-Range 6-8.5 ( about half-maximal activity at pH 6.0 [2,7]; about 50% activity at pH 6.0 [2]; about 80% activity at pH 6.0 [7]) [2, 7] 6-9 ( about half-maximal activity at pH 6.0, about 90% of maximal activity at pH 7.0 and pH 9.0 [12]) [12] Temperature optimum ( C) 20 ( assay at [24]; actin binding assay at [27]) [24, 27] 22 ( assay at [4,15]) [4, 15] 25 ( assay at [2, 3, 6, 25, 28]) [2, 3, 6, 25, 28] 30 ( assay at [5, 7, 8, 9, 11, 17, 20, 23]) [5, 7, 8, 9, 11, 17, 20, 23]

4 Enzyme Structure Molecular weight 107000 ( gel filtration [7]) [7] 160000 ( gel filtration [13]) [13] 240000 ( gel filtration [4]) [4] 490000 ( gel filtration, sucrose density gradient centrifugation [12]) [12] 700000 ( above, gel filtration [2,6]) [2, 6] Subunits ? ( x * 130000, SDS-PAGE [2,6]; x * 84000, SDS-PAGE [4]; x * 79300, determined from amino acid sequence, difference to MW of 97 kDa determined by SDS-PAGE is due to high proline content [23]; x * 86000, DNA sequence determination [14]; x * 94000, phosphorylated enzyme, SDS-PAGE [4]) [2, 4, 6, 14, 23] decamer ( 10 * 50000, SDS-PAGE, asymmetric complex with an axial ratio calculated for prolate ellipsoid of 6.1 [12]) [12] monomer ( 1 * 97000, unphosphorylated enzyme, SDS-PAGE [5]; 1 * 107000, phosphorylated enzyme, SDS-PAGE [5,7]) [5, 7] Additional information ( MHCK domain organization [29]; the enzymes coiled-coil structure mediates its oligomerization, cellular localization, and actin-binding activity of the MHCK A [27]) [27, 29] Posttranslational modification phosphoprotein ( MHCK A performs autophosphorylation, facilitated by F-actin requiring the coiled coil domain of the enzyme [28]) [28]

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Myosin-heavy-chain kinase

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5 Isolation/Preparation/Mutation/Application Source/tissue amoeba [6, 7, 9, 23, 26] brain [13] cell culture ( growth-phase cells contain enzyme form MHCK A [3]; starved-developing cells contain enzyme form MHCK B [3]; starved, developing cells [4]; growth-phase cells [2]) [2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18] cell suspension culture ( overexpresssing cells show slower growth and increased cell size [18]) [18] epithelium ( of the intestine, brush border cells [11,12]) [11, 12] small intestine ( epithelium [11,12]) [11, 12] Additional information ( enzyme expression only during development [16]) [16] Localization cell cortex [14, 26] cytoskeleton [29] cytosol [2, 3, 7, 9, 11, 26] lamellipodium [26] membrane ( bound [16]; associated [4]; association of the enzyme to membranes enhances the activity [22]; binding to the plasma membrane enhances the activity of unphosphorylated enzyme 20fold, substrate myosin I [17]) [4, 16, 17, 22] Additional information ( translocation of the enzyme within the cell responding to outer signals [26]; MHCK A is localized to actin-rich regions in mitotic cells [14]; the 3 isozymes MHCK A, MHCK B, and MHCK C show differential localization patterns in living cells, overview [29]; the enzymes coiled-coil structure mediates its oligomerization, cellular localization, and actin-binding activity of the MHCK A [27]) [14, 26, 27, 29] Purification (Ca2+ /calmodulin-dependent heavy chain kinase, 84fold) [12] (separation of the Ca2+ /calmodulin-dependent and -independent isozymes) [11] (enzyme copurifies with casein kinase II and Ca2+ -independent myosin kinase) [13] (14000fold to near homogeneity) [2] (isolation of highly phosphorylated and unphosphorylated myosin II heavy chain kinase A) [6] (recombinant His-tagged full-length isozyme MHCK A from Dictyostelium discoideum by affinity chromatography, recombinant GST-fusion truncation mutant Dcoil-MHCK A from Escherichia coli by affinity chromatography, the GST-tag is cleaved off by thrombin treatment) [27] (solubilized by high-salt extraction, affinity chromatography, 4600fold, to homogeneity) [4]

196

2.7.11.7

Myosin-heavy-chain kinase

(affinity chromatography using histone-resin) [5, 7] (partial) [9, 10] (recombinant His-tagged catalytic domains, wild-type and mutants, from Sf9 insect cells) [21] (to near homogeneity) [7] (recombinant His-tagged catalytic domain of MHCK A, expressed in Escherichia coli) [25] (recombinant His-tagged full-length isozyme MHCK A and truncation mutant Dcoil-MHCK A from Dictyostelium discoideum to homogeneity by affinity chromatography, recombinant GST-fusion coiled-coil domain and catalytic domain from Escherichia coli to near homogeneity by affinity chromatography) [28] (recombinant His-tagged or GFP-fusion proteins from Dictyostelium discoideum) [26] (MHCK B, recombinant from Dictyostelium discoideum cells as FLAGtagged protein) [15] (recombinant GST-fusion catalytic domain of MHCK B, expressed in Escherichia coli) [25] (recombinant peptides of the enzymes N-terminus from Escherichia coli, His- or FLAG-tagged, and recombinant enzyme from Sf9 insect cells) [24] (recombinant His-tagged catalytic domain of MHCK C, expressed in Escherichia coli) [25] Cloning (expression of GFP-tagged isozyme MHCK A in a Dictyostelium discoideum strain lacking endogenous MHCK A, expression of a truncation mutant Dcoil-MHCK A comprising residues 1-498 in Escherichia coli as GST-fused protein) [27] (expression of the 3 isozymes MHCK A, MHCK B, and MHCK C as GFPfusion proteins) [29] (gene mhck A, construction of overexpressing cell line by transfection of an expression plasmid into the deficient mhck A- cell line) [18] (cloning of wild-type and mutants of the catalytic domain of myosin I heavy chain kinase as His-tagged proteins and expression in Spodoptera frugiperda Sf9 cells via baculovirus infection system) [21] (construction of His-tagged or GFP fusion proteins with the full length enzyme or enzyme fragments, expression in Dictyostelium discoideum cells) [26] (expression of the His-tagged catalytic domain of MHCK A in Escherichia coli BL21(DE3)) [25] (overexpression of His-tagged full-length isozyme MHCK A and truncation mutant Dcoil-MHCK A comprising residues 1-498 in Dictyostelium discoideum, overexpression of isolated coiled-coil domain and of the catalytic domain in Escherichia coli as GST-fused proteins) [28] (MHCK B, overexpression as FLAG-tagged protein in AX2 cells or 3xALA cells of Dictyostelium discoideum) [15]

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(expression of the catalytic domain of MHCK B as a GST-fusion protein in Escherichia coli BL21(DE3)) [25] (DNA sequence determination and analysis, gene MHCK possesses all of the domains characteristic of members of the protein kinase C family) [16] (DNA sequence determination and analysis) [19] (DNA sequence determination and analysis) [22] (DNA and amino acid sequence determination and analysis) [23] (construction of His-tagged or FLAG-tagged N-terminal enzyme peptides by site directed mutagenesis for introduction of start and stop codons, expression in Escherichia coli BL21(DE3)) [24] (subcloning and expression in Escherichia coli, expression in Spodoptera frugiperda Sf9 cells via baculovirus infection system) [24] (expression of the His-tagged catalytic domain of MHCK C in Escherichia coli BL21(DE3)) [25] (DNA sequence determination and analysis) [14] Engineering C800A ( catalytic domain mutation, no remaining activity when expressed as full length enzyme or as catalytic domain only in a deficient Dictyostelium discoideum cell line [26]) [26] S627A ( catalytic domain mutant, mutation of potential phosphorylation site, no phosphorylation of the mutant, reduced activity, increased Km values for the substrates, but higher activity than the unphosphorylated wildtype enzyme [21]) [21] S627D ( catalytic domain mutant, mutation of potential phosphorylation site, no phosphorylation of the mutant, reduced activity, increased Km values for the substrates, the acidic residue cannot substitute for phospho-Ser [21]) [21] S627E ( catalytic domain mutant, mutation of potential phosphorylation site, no phosphorylation of the mutant, reduced activity, increased Km values for the substrates, the acidic residue cannot substitute for phospho-Ser [21]) [21] T631A ( catalytic domain mutant, mutation of a conserved Thr residue, full phosphorylation of the mutant, reduced activity, increased Km values for the substrates [21]) [21] T631D ( catalytic domain mutant, mutation of a conserved Thr residue, 95% phosphorylation of the mutant, highly reduced activity, increased Km values for the substrates [21]) [21] T631E ( catalytic domain mutant, mutation of a conserved Thr residue, no phosphorylation of the mutant, highly reduced activity, highly increased Km values for the substrates [21]) [21] T632A ( catalytic domain mutant, mutation of a nonconserved Thr residue, full phosphorylation of the mutant, increased activity, only slightly increased Km values for the substrates [21]) [21] T632D ( catalytic domain mutant, mutation of a nonconserved Thr residue, 80% phosphorylation of the mutant, reduced activity, increased Km values for the substrates [21]) [21]

198

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Myosin-heavy-chain kinase

T632E ( catalytic domain mutant, mutation of a nonconserved Thr residue, full phosphorylation of the mutant, reduced activity, increased Km values for the substrates [21]) [21] Additional information ( construction of single, double, and triple MHCK knockout cell lines concerning the 3 isozymes MHCK A, MHCK B, and MHCK C, the mutant cells defects in cytokinesis and myosin II overassembly increasing with the number of mutations, overview [29]; the truncation mutant Dcoil-MHCK A comprising residues 1-498 is not affected in its autophosphorylation rate by F-actin [27,28]) [27, 28, 29]

6 Stability pH-Stability 8 ( and above, unstable [2]) [2] Temperature stability 25 ( 1-2 h, stable [6]) [6] General stability information , freeze-thawing inactivates [11, 12] , loses about 25% of activity for every freeze-thaw cycle [6] , phosphorylated enzyme is somewhat less stable than unphosphorylated enzyme [6] , very unstable if exposed to low salt, i.e. 50 mM KCl or less, in the absence of sucrose [2] Storage stability , 0 C, at least 1 month [12] , 0 C, further purified Ca2+ /calmodulin-dependent isozyme, complete loss of activity within 1 day [11] , 0 C, partly purified Ca2+ /calmodulin-dependent isozyme, at least 2 weeks [11] , -20 C, in 25 mM HEPES buffer, pH 7.5, 1 mM DTT, 1 mM EDTA, 50 mM NaCl, 10% glycerol, or 20% sucrose, stable for 3 months [4] , -20 C, in 50% sucrose or glycerol, t1=2 : 2 weeks [4] , 0 C, highly purified preparation of unphosphorylated enzyme, 25% loss of activity per day [6] , 0 C, in 10 mM imidazole, pH 7, 100 mM KCl, 2 mM DTT, 60% sucrose, several weeks [2] , 0 C, partially purified preparation of unphosphorylated enzyme, several days [6] , indefinitely stable upon storage in liquid nitrogen, recovery of 60-70% activity after thawing [6] , -20 C, in 50% glycerol, 3 months stable and less than 20% loss of activity within 6 months [7] , -20 C, purified, stable up to 1 year [5] , 4 C, several months [9]

199

Myosin-heavy-chain kinase

2.7.11.7

References [1] Boeckmann, B.; Bairoch, A.; Apweiler, R.; Blatter, M.C.; Estreicher, A.; Gasteiger, E.; Martin M.J.; Michoud, K.; O’Donovan, C.; Phan, I.; Pilbout, S.; Schneider, M.: The SWISS-PROT protein knowledgebase and its supplement TrEMBL. Nucleic Acids Res., 31, 365-370 (2003) [2] Cote, G.P.; Bukiejko, U.: Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum. J. Biol. Chem., 262, 10651072 (1987) [3] Medley, Q.W.; Bagshaw, W.L.; Truong, T.; Cote, G.P.: Dictyostelium myosin II heavy-chain kinase A is activated by heparin, DNA and acidic phospholipids and inhibited by polylysine, polyarginine and histones. Biochim. Biophys. Acta, 1175, 7-12 (1992) [4] Ravid, S.; Spudich, J.A.: Myosin heavy chain kinase from developed Dictyostelium cells. Purification and characterization. J. Biol. Chem., 264, 15144-15150 (1989) [5] Lynch, T.J.; Brzeska, H.; Baines, I.C.; Korn, E.D.: Purification of myosin I and myosin I heavy chain kinase from Acanthamoeba castellanii. Methods Enzymol., 196, 12-23 (1991) [6] Medley, Q.W.; Lee, S.-F.; Cote, G.P.: Purification and characterization of myosin II heavy chain kinase A from Dictyostelium. Methods Enzymol., 196, 23-34 (1991) [7] Hammer, J.A.; Albanesi, J.P.; Korn, E.D.: Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii. J. Biol. Chem., 258, 10168-10175 (1983) [8] Brzeska, H.; Lynch, T.J.; Martin, B.; Corigliano-Murphy, A.; Korn, E.D.: Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides. J. Biol. Chem., 265, 16138-16144 (1990) [9] Cote, G.P.; Collins, J.H.; Korn, E.D.: Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II. J. Biol. Chem., 256, 12811-12816 (1981) [10] Maruta, H.; Korn, E.D.: Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+ -ATPase activity of Acanthamoeba myosin I. J. Biol. Chem., 252, 8329-8332 (1977) [11] Rieker, J.P.; Swanljung-Collins, H.; Montibeller, J.; Collins, J.H.: Isolation and characterization of calmodulin-dependent myosin heavy chain kinase from intestinal brush border. Methods Enzymol., 139, 105-114 (1987) [12] Rieker, J.P.; Swanljung-Collins, H.; Montibeller, J.; Collins, J.H.: Purification and characterization of a calmodulin-dependent myosin heavy chain kinase from intestinal brush border. J. Biol. Chem., 262, 15262-15268 (1987) [13] Murakami, N.; Matsumura, S.; Kumon, A.: Purification and identification of myosin heavy chain kinase from bovine brain. J. Biochem., 95, 651-660 (1984) [14] Nagasaki, A.; Itoh, G.; Yumura, S.; Uyeda, T.Q.: Novel myosin heavy chain kinase involved in disassembly of myosin II filaments and efficient cleavage in mitotic dictyostelium cells. Mol. Biol. Cell, 13, 4333-4342 (2002)

200

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[15] Rico, M.; Egelhoff, T.T.: Myosin heavy chain kinase B participates in the regulation of myosin assembly into the cytoskeleton. J. Cell. Biochem., 88, 521-532 (2003) [16] Ravid, S.; Spudich, J.A.: Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family. Proc. Natl. Acad. Sci. USA, 89, 5877-5881 (1992) [17] Wang, Z.Y.; Brzeska, H.; Baines, I.C.; Korn, E.D.: Properties of Acanthamoeba myosin I heavy chain kinase bound to phospholipid vesicles. J. Biol. Chem., 270, 27969-27976 (1995) [18] Kolman, M.F.; Futey, L.M.; Egelhoff, T.T.: Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development. J. Cell. Biol., 132, 101-109 (1996) [19] Lee, S.F.; Egelhoff, T.T.; Mahasneh, A.; Cote, G.P.: Cloning and characterization of a Dictyostelium myosin I heavy chain kinase activated by Cdc42 and Rac. J. Biol. Chem., 271, 27044-27048 (1996) [20] Brzeska, H.; Martin, B.M.; Korn, E.D.: The catalytic domain of Acanthamoeba myosin I heavy chain kinase. I. Identification and characterization following tryptic cleavage of the native enzyme. J. Biol. Chem., 271, 2704927055 (1996) [21] Szczepanowska, J.; Ramachandran, U.; Herring, C.J.; Gruschus, J.M.; Qin, J.; Korn, E.D.; Brzeska, H.: Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of Acanthamoeba myosin I heavy chain kinase. Proc. Natl. Acad. Sci. USA, 95, 4146-4151 (1998) [22] Lee, S.F.; Mahasneh, A.; de la Roche, M.; Cote, G.P.: Regulation of the p21activated kinase-related Dictyostelium myosin I heavy chain kinase by autophosphorylation, acidic phospholipids, and Ca2+ -calmodulin. J. Biol. Chem., 273, 27911-27917 (1998) [23] Brzeska, H.; Young, R.; Knaus, U.; Korn, E.D.: Myosin I heavy chain kinase: cloning of the full-length gene and acidic lipid-dependent activation by Rac and Cdc42. Proc. Natl. Acad. Sci. USA, 96, 394-399 (1999) [24] Brzeska, H.; Young, R.; Tan, C.; Szczepanowska, J.; Korn, E.D.: Calmodulinbinding and autoinhibitory domains of Acanthamoeba myosin I heavy chain kinase, a p21-activated kinase (PAK). J. Biol. Chem., 276, 4746847473 (2001) [25] Luo, X.; Crawley, S.W.; Steimle, P.A.; Egelhoff, T.T.; Cote, G.P.: Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family. J. Biol. Chem., 276, 17836-17843 (2001) [26] Steimle, P.A.; Licate, L.; Cote, G.P.; Egelhoff, T.T.: Lamellipodial localization of Dictyostelium myosin heavy chain kinase A is mediated via F-actin binding by the coiled-coil domain. FEBS Lett., 516, 58-62 (2002) [27] Russ, M.; Croft, D.; Ali, O.; Martinez, R.; Steimle, P.A.: Myosin heavy chain kinase A from Dictyostelium possesses a novel actin binding domain that cross-links actin filaments. Biochem. J., 395, 373-383 (2005) [28] Egelhoff, T.T.; Croft, D.; Steimle, P.A.: Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial 201

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regulation of myosin II filament disassembly. J. Biol. Chem., 280, 2879-2887 (2005) [29] Yumura, S.; Yoshida, M.; bpudi, V.; Licate, L.S.; Iwadate, Y.; Nagasaki, A.; Uyeda, T.Q.; Egelhoff, T.T.: Multiple myosin II heavy chain kinases: roles in filament assembly control and proper cytokinesis in Dictyostelium. Mol. Biol. Cell, 16, 4256-4266 (2005)

202

Fas-activated serine/threonine kinase

2.7.11.8

1 Nomenclature EC number 2.7.11.8 Systematic name ATP:[Fas-activated serine/threonine protein] phosphotransferas Recommended name Fas-activated serine/threonine kinase Synonyms FAST [1, 2, 3] Fas-activated serine/threonine phosphoprotein [3] CAS registry number 170347-50-9

2 Source Organism Homo sapiens (UNIPROT accession number: Q14296) [1] Homo sapiens (UNIPROT accession number: Q9JIX9) [2, 3]

3 Reaction and Specificity Catalyzed reaction ATP + [Fas-activated serine/threonine protein] = ADP + [Fas-activated serine/threonine phosphoprotein] Reaction type phospho group transfer Natural substrates and products S ATP + TIA-1 ( rapidly activated during Fas-mediated apoptosis. Phosphorylation of TIA-1 precedes the onset of DNA fragmentation, suggesting a role in signaling downstream events in the apoptotic program [1]) (Reversibility: ?) [1] P ADP + ?

203

Fas-activated serine/threonine kinase

2.7.11.8

S ATP + TIA-1 ( FAST serves as a sensor for mitochondrial stress modulating a TIA-1 regulated posttranscriptional stress response program, FAST might prevent TIA-1 mediated silencing of mRNA encoding inhibitors of of apoptosis [3]) (Reversibility: ?) [3] P ADP + phosphorylated TIA-1 S ATP + [Fas-activated serine/threonine protein] (Reversibility: ?) [2, 3] P ADP + [Fas-activated serine/threonine phosphoprotein] S Additional information ( FAST serves as a sensor for mitochondrial stress modulating a TIA-1 regulated posttranscriptional stress response program, FAST is a survival protein, modulating the NF-kB-dependent survival pathway, and has antiapoptotic effects inhibiting Fas-and UV-induced apoptosis, involving activation of caspase-3, its antiapoptotic effects are inhibited by TIA-1, FAST might prevent TIA-1 mediated silencing of mRNA encoding inhibitors of of apoptosis [3]; the interaction of the enzyme with protein BCL-XL is involved in regulation of mitochondrial metabolism during Fas-induced apoptosis [2]) (Reversibility: ?) [2, 3] P ? Substrates and products S ATP + TIA-1 ( rapidly activated during Fas-mediated apoptosis. Phosphorylation of TIA-1 precedes the onset of DNA fragmentation, suggesting a role in signaling downstream events in the apoptotic program [1]) (Reversibility: ?) [1] P ADP + ? S ATP + TIA-1 ( FAST serves as a sensor for mitochondrial stress modulating a TIA-1 regulated posttranscriptional stress response program, FAST might prevent TIA-1 mediated silencing of mRNA encoding inhibitors of of apoptosis [3]) (Reversibility: ?) [3] P ADP + phosphorylated TIA-1 S ATP + [Fas-activated serine/threonine protein] (Reversibility: ?) [2, 3] P ADP + [Fas-activated serine/threonine phosphoprotein] S Additional information ( FAST serves as a sensor for mitochondrial stress modulating a TIA-1 regulated posttranscriptional stress response program, FAST is a survival protein, modulating the NF-kB-dependent survival pathway, and has antiapoptotic effects inhibiting Fasand UV-induced apoptosis, involving activation of caspase-3, its antiapoptotic effects are inhibited by TIA-1, FAST might prevent TIA-1 mediated silencing of mRNA encoding inhibitors of of apoptosis [3]; the interaction of the enzyme with protein BCL-XL is involved in regulation of mitochondrial metabolism during Fas-induced apoptosis [2]; the enzyme interacts with BCL-XL at the outer mitochondrial membrane [3]; the enzyme interacts with BCL-XL requiring the

204

2.7.11.8

Fas-activated serine/threonine kinase

BCL-2-homology-3 (BH3)-related domain and the MTD domain of BCLXL [2]) (Reversibility: ?) [2, 3] P ? Cofactors/prosthetic groups ATP [2,3] Activating compounds Additional information ( rapidly activated during Fas-mediated apoptosis [1]) [1]

4 Enzyme Structure Posttranslational modification phosphoprotein ( enzyme is phosphorylated on serine and threonine residues. In response to Fas ligation, it is rapidly dephosphorylated and concomitantly activated to phosphorylate TIA-1 [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue HeLa cell [2, 3] JURKAT cell [1] Localization mitochondrial outer membrane ( interaction with BCL-XL [3]) [3] mitochondrion ( enzyme has a mitochondrial targeting domain [2,3]) [2, 3] Purification (partially, subcellular fractionation) [2] Cloning (overexpression of HA-tagged enzyme in HeLa cells and in COS-7 cells, expression of GFP-tagged enzyme in COS-7 cells) [2] (overexpression of HA-tagged enzyme in HeLa cells and in COS-7 cells, recombinant FAST promotes the expression of co-transfected reporter proteins, e.g. b-galactosidase, via its TIA-1 binding domain, and increases the expression of endogenous cIAP-1 and XIAP, but not GAPDH, in HeLa cells) [3]

References [1] Tian, Q.; Taupin, J.; Elledge, S.; Robertson, M.; Anderson, P.: Fas-activated serine/threonine kinase (FAST) phosphorylates TIA-1 during Fas-mediated apoptosis. J. Exp. Med., 182, 865-874 (1995)

205

Fas-activated serine/threonine kinase

2.7.11.8

[2] Li, W.; Kedersha, N.; Chen, S.; Gilks, N.; Lee, G.; Anderson, P.: FAST is a BCLXL-associated mitochondrial protein. Biochem. Biophys. Res. Commun., 318, 95-102 (2004) [3] Li, W.; Simarro, M.; Kedersha, N.; Anderson, P.: FAST is a survival protein that senses mitochondrial stress and modulates TIA-1-regulated changes in protein expression. Mol. Cell. Biol., 24, 10178-10732 (2004)

206

Goodpasture-antigen-binding protein kinase

2.7.11.9

1 Nomenclature EC number 2.7.11.9 Systematic name ATP:[Goodpasture antigen-binding protein] phosphotransferase Recommended name Goodpasture-antigen-binding protein kinase Synonyms collagen type IV a3 binding protein GPBP [2, 3, 4] START domain-containing protein 11 StAR-related lipid transfer protein 11 StARD11 CAS registry number 230316-19-5

2 Source Organism Mus musculus (UNIPROT accession number: Q9EQG9) [1, 2] Bos taurus (UNIPROT accession number: Q9GKI7) [2] Homo sapiens (UNIPROT accession number: Q9Y5P4) [2, 3, 4]

3 Reaction and Specificity Catalyzed reaction ATP + [Goodpasture antigen-binding protein] = ADP + [Goodpasture antigen-binding phosphoprotein] Reaction type phospho group transfer Natural substrates and products S ATP + [Goodpasture antigen-binding protein] ( autophosphorylation via the enzymes serine/threonine kinase activity that specifically targets a motif in the NC1 domain of the a3 chain of type IV collagen,

207

Goodpasture-antigen-binding protein kinase

2.7.11.9

the Good pasture autoantigen, the enzyme is involved in autoimmune processes and disease [4]) (Reversibility: ?) [4] P ADP + [Goodpasture antigen-binding phosphoprotein] S ATP + goodpasture antigen ( enzyme is involved in autoimmune pathogenesis. Enzyme expression is up-regulated in the striated muscle of a Goodpasture patient and in other autoimmune conditions including cutaneous lupus erythematosus, pemphigoid, and lichen planus [2]) (Reversibility: ?) [2] P ADP + phosphorylated goodpasture antigen [2] Substrates and products S ATP + [Goodpasture antigen-binding protein] ( autophosphorylation via the enzymes serine/threonine kinase activity that specifically targets a motif in the NC1 domain of the a3 chain of type IV collagen, the Good pasture autoantigen, the enzyme is involved in autoimmune processes and disease [4]; autophosphorylation via the enzymes serine/threonine kinase activity that specifically targets a motif in the NC1 domain of the a3 chain of type IV collagen, the Good pasture autoantigen [4]) (Reversibility: ?) [4] P ADP + [Goodpasture antigen-binding phosphoprotein] S ATP + goodpasture antigen ( phosphorylation in the N-terminal region of the goodpasture antigen [2,3]; enzyme is involved in autoimmune pathogenesis. Enzyme expression is up-regulated in the striated muscle of a Goodpasture patient and in other autoimmune conditions including cutaneous lupus erythematosus, pemphigoid, and lichen planus [2]) (Reversibility: ?) [2, 3] P ADP + phosphorylated goodpasture antigen [2] Cofactors/prosthetic groups ATP [4] Activating compounds Additional information ( expression of the Goodpasture antigenbinding protein is induced by tumor necrosis factor [4]) [4]

5 Isolation/Preparation/Mutation/Application Source/tissue HEK-293 cell [4] cell culture ( HeLa cells [2]) [2] Additional information ( enzyme is preferentially expressed in small vessels and histological structures targeted by natural autoimmune responses including alveolar and glomerular basement membranes [2]; preferential expression in the histological structures that are targets of common autoimmune responses [2]) [2]

208

2.7.11.9

Goodpasture-antigen-binding protein kinase

Cloning [2, 3] (DNA sequence determination and analysis, a TNF-responsive transcription unit that locates the enzyme in the signalling cascade of TNF, regulation of transcription, e.g. by tumor necrosis factor, via cooperation in a bidirectional transcription complex with the gene encoding the DNA polymerase kappa, NFkB, and a 140-bp promoter with a Sp1 site and a TATA-like element, promoter analysis, overview) [4]

References [1] Kawai, J.; Shinagawa, A.; Shibata, K.; Yoshino, M.; Itoh, M.; et al.: Functional annotation of a full-length mouse cDNA collection. Nature, 409, 685-690 (2001) [2] Raya, A.; Revert-Ros, F.; Martinez-Martinez, P.; Navarro, S.; Rosello, E.; et al.: Goodpasture antigen-binding protein, the kinase that phosphorylates the goodpasture antigen, is an alternatively spliced variant implicated in autoimmune pathogenesis. J. Biol. Chem., 275, 40392-40399 (2000) [3] Raya, A.; Revert, F.; Navarro, S.; Saus, J.: Characterization of a novel type of serine/threonine kinase that specifically phosphorylates the human goodpasture antigen. J. Biol. Chem., 274, 12642-12649 (1999) [4] Granero, F.; Revert, F.; Revert-Ros, F.; Lainez, S.; Martinez-Martinez, P.; Saus, J.: A human-specific TNF-responsive promoter for Goodpasture antigenbinding protein. FEBS J., 272, 5291-5305 (2005)

209

IkB kinase

2.7.11.10

1 Nomenclature EC number 2.7.11.10 Systematic name ATP:[IkB protein] phosphotransferase Recommended name IkB kinase Synonyms CHUK [21] IKK [23, 24, 25, 26, 27, 28, 29, 36, 37, 38, 39, 40, 42, 43, 44, 45, 48, 49, 50, 51, 52, 53, 54, 55, 61, 64] IKK complex [24, 25, 32, 37, 41, 46, 51] IKK-a [67] IKK-b [66] IKK-related kinase [72] IKK2 [56] IKKa [21, 58, 60, 62, 63, 68, 70] IKKb [30, 33, 35, 62, 63, 69, 71] IKKe [47, 57, 72] IKKi [72] IkB kinase [71] IkB kinase a [21] IkB kinase b [59] IkB kinase complex [31] IkB kinase-2 [56] I [58, 67] IkB kinase-b [66, 69] IkB-kinase [54] IkBa [10] IkBa kinase [34, 63] IkBb kinase [34] Ikka [67] TANK-binding kinase 1 [72] TBK1 [72] inhibitor of kB kinase [53] inhibitor of nuclear factor kB kinase-related kinase [72] inhibitor of nuclear factor k B kinase b subunit [3, 5, 15, 16]

210

2.7.11.10

IkB kinase

inhibitor of nuclear factor k B kinase b subunit inhibitor of nuclear factor k-B kinase a subunit [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14] inhibitor of nuclear factor k-B kinase a subunit [1, 3, 5, 9, 10, 16, 21, 22] inhibitor of nuclear factor k-B kinase e subunit [18, 19, 20] non-canonical IkBkinase e [57] serine/threonine-protein kinase pkn1 [17] CAS registry number 159606-08-3

2 Source Organism Mus musculus (no sequence specified) [28, 33, 37, 38, 39, 41, 42, 44, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 65, 67, 70, 72] Homo sapiens (no sequence specified) [23, 24, 25, 27, 29, 30, 31, 32, 34, 35, 36, 38, 40, 43, 45, 46, 47, 50, 53, 55, 62, 63, 64, 65, 66, 69, 71] Rattus norvegicus (no sequence specified) [68] Toxoplasma gondii (no sequence specified) [26, 37] Homo sapiens (UNIPROT accession number: O14920) [2, 3, 4, 5, 6, 7, 8] Homo sapiens (UNIPROT accession number: O15111) [2, 3, 4, 5, 9, 10, 11, 12, 13, 14] Mus musculus (UNIPROT accession number: O88351) [3, 5, 15, 16] Myxococcus xanthus (UNIPROT accession number: P33973) [17] Homo sapiens (UNIPROT accession number: Q14164) [18, 19, 20] Mus musculus (UNIPROT accession number: Q60680) [1, 3, 5, 9, 10, 16, 21, 22] Rattus norvegicus (UNIPROT accession number: Q9QY78) [3, 5] Mus musculus (UNIPROT accession number: Q9R0T8) [20]

3 Reaction and Specificity Catalyzed reaction ATP + [IkB protein] = ADP + [IkB phosphoprotein] ATP + [IkB protein] = ADP + [IkB phosphoprotein] ( activation and reaction mechanism [29]) Natural substrates and products S ATP + Bcl ( phosphorylation at the C-terminus of Bcl by IKKb disrupts Bcl10/Malt1 association and Bcl10-mediated signaling [65]) (Reversibility: ?) [65] P ADP + phosphorylated Bcl S ATP + Bcl | ( negative regulatory activity of the IKK complex in Bcl10 degradation, which is part of the regulatory mechanisms that precisely control the response to antigens, overview [71]) (Reversibility: ?) [71] 211

IkB kinase

2.7.11.10

P ADP + phosphorylated Bcl10 S ATP + IkB protein ( the enzyme targets the inhibitory IkB protein tightly bound to the transcription factor NF-kB for proteasomal degradation and allows the freed NF-kB to enter the nucleus where it can be transactivate its target gene, IKKa is involved in inflammation in macrophages [54]) (Reversibility: ?) [54] P ADP + phosphorylated IkB S ATP + IkBa ( a step in NF-kB activation, the IKK complex, composed of IKKa, IKKb, and NEMO/IKKg, is the convergence point for many diverse NFkB-activating stimuli including TNFa, LPS, and IL-1, overview, IKKb is the primary positive regulator of NFkB activity in inflammatory processes, is the molecular link between inflammation and cancer [69]; degradation of IkBa [71]) (Reversibility: ?) [63, 69, 71] P ADP + phosphorylated IkBa S ATP + [GST-IkB protein] (Reversibility: ?) [31] P ADP + [GST-IkB phosphoprotein] S ATP + [GST-IkBa protein] (Reversibility: ?) [32] P ADP + [GST-IkBa phosphoprotein] S ATP + [IkB protein] ( inhibition and degradation of IkB, an inhibitor of NF-kB retaining it in the cytoplasm, phosphorylation of IkB marks the protein for ubiquitination followed by degradation, activated NF-kB is translocated to the nucleus initiating signalling pathways, regulation mechanism, overview [44]; inhibitor substrate is bound to NF-kB [52]; parasite IKKa, localized in parasitophorous vacuole membrane, activates mouse intracellular NF-kB in early infection stage resulting in NF-kB nuclear translocation and subsequent gene expression independently from the host IKK complex [37]; phosphorylation of IkB results in its proteolytic degradation [49]; required for activation of NF-kB resulting in activation of signalling pathways [37]; signalling step of IKK bound to NFkB for subsequent ubiquitination of IkB and proteolytic degradation [27]) (Reversibility: ?) [25, 27, 28, 30, 37, 38, 42, 43, 44, 46, 49, 50, 52, 53] P ADP + [IkB phosphoprotein] S ATP + [IkBa protein] ( enzyme is involved in activation of pro-inflammation signalling [24]; parasite IKKa, localized in parasitophorous vacuole membrane, activates mouse intracellular NF-kB through phosphorylation of host IkBa at Ser32 and Ser36 in early infection stage resulting in NF-kB nuclear translocation and subsequent gene expression independently from the host IKK complex [26]) (Reversibility: ?) [24, 26, 34] P ADP + [IkBa phosphoprotein] S ATP + [IkBb protein] (Reversibility: ?) [35] P ADP + [IkBb phosphoprotein] S ATP + [NFkB subunit p56] (Reversibility: ?) [30] P ADP + [NFkB subunit p56]phosphate S ATP + [RelA/p65 protein] (Reversibility: ?) [42] P ADP + [RelA/p65 phosphoprotein] 212

2.7.11.10

IkB kinase

S ATP + [acetylated histone H3 protein] ( IKKa is required for histone function regulation in the nucleus [53]) (Reversibility: ?) [53] P ADP + [acetylated histone H3 phosphoprotein] S ATP + [histone H3 protein] ( histone H3 phosphorylation by IKK-a is critical for cytokine-induced gene expression [50]) (Reversibility: ?) [32, 5] P ADP + [histone H3 phosphoprotein] S ATP + a protein ( CHUK associates with the NF-kB inhibitory protein, IkB-a, in mammalian cells. CHUK specifically phosphorylates IkB-a on both Ser32 and Ser36, modifications that are required for targeted degradation of IkB-a via the ubiquitin-proteasome pathway [14]; the I k B/NF-k B system is a key determinant of mucosal inflammation and protection [3]; the expression of pkn1 is developmentally regulated to start immediately before spore formation. The enzyme plays an important role in the onset of proper differentiation [17]; phosphorylation of IkBs marks them out for destruction, thereby relieving their inhibitory effect on NF-kB [11]; phosphorylates IkB inhibitory proteins, causing their degradation and activation of transcription factor NF-kB, a master activator of inflammatory responses [10]) (Reversibility: ?) [3, 10, 11, 14, 17] P ADP + a phosphoprotein S ATP + protein p100 ( interaction with the NF-kB complex [57]) (Reversibility: ?) [57] P ADP + phosphorylated protein p100 S ATP + protein p165 ( p65 is part of the IKKe complex with p25, interaction with the NF-kB complex [57]) (Reversibility: ?) [57] P ADP + phosphorylated protein p165 S Additional information ( activation of heterodimeric nuclear transcription factor NFkB is an essential step in inflammation, e.g. resulting in osteoarthritis, ulcerative colitis, asthma, and Crohns disease, signalling step for phosphorylation, subsequent ubiquitination and proteolytic degradation of IkB, NFkB remains free after the reaction and is translocated to the nucleus, NFkB activation is also involved in development of diseases like cancer, gut ischemia-reperfusion, diabetes, or in transplant rejections, overview [27]; activation of NFkB by the catalytic subunit IKKb is required for signaling via the NFkB pathway in acute and systemic inflammation and for tissue protection [48]; defective ubiquination of the NF-kB essential modifier/IkB kinase-g complex leads to impaired cellular NFkB signalling and hypohidrotic ectodermal dysplasia with immunodeficiency HED-ID [31]; enzyme is responsible for NFkB activation, specific inhibition of IkB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in male Sprague-Dawley rats [43]; IkB kinases are essential key regulators of the NFkB pathways in the tooth development acting as stimulators, overview [28]; IKK activates and regulates NFkB important in intracellular signalling, overview [30]; IKK activates NF-kB through action of TNFa playing an important role in subsequent signaling pathways in213

IkB kinase

2.7.11.10

volved in e.g. apoptosis/cell survival, cell proliferation, and inflammation [34]; IKK activates NF-kB which initiates signaling pathways that play critical roles in a variety of physiological and pathological processes, e.g. promotion of cell survival inducing production of apoptosis inhibitors in normal and cancer cells, pathways overview, IKK/NF-kB links inflammation to cancer, regulation of IKK, overview [38]; IKK activates TNFa-dependent signaling pathways inducing 5-fluoro-2-deoxyuridine drug resistance in different cell lines, overview [36]; IKK is involved in NF-kB activation, IkB kinase IKKb, but not IKKa, is a critical mediator of NF-kB-dependent osteoclast survival preventing TNFa-induced cell death, and is required for formation of fully functional boneresorbing osteoclasts and for inflammation-induced bone loss [39]; IKK is required for activation of NF-kB and subsequent signalling pathways [49]; IKK is responsible for activation of NFkB, herpesvirus HSV-1 potently induces IkB kinase IKK causing persistent induction of NFkB resulting in transactivation of HIV-1-LTR-regulated genes and induction of HIV-1 replication in infected T-cells [23]; IKK is responsible for NF-kB activation by inactivating its inhibitor IkB, different inflammation stimuli induce distinct IKK activity profiles, molecular mechanism, overview [52]; IKK marks cytoplasmic NFkB inhibitors for proteolytic destruction playing a role in regulation of genetic cell cycle programs, IKK regulates nuclear translocation of transcription factor NFkB [29]; IKKa, not IKKb, is required for epidermal regeneration, IkB kinases are essential key regulators of the canonical and noncanonical NFkB pathways important for the expression of a wide variety of genes that are involved in the control of immune and inflammatory response, and in the regulation of cellular proliferation and survival, mechanism, overview [53]; IKKb is required for activation of NFkB, IKKb induces expression of epithelial sodium channel abg-ENaC in cell surfaces [33]; IKKb is required for regulation of NFkB activity and peripheral B cell survival and proliferation [41]; IKKb regulates the translocation of NF-kB from cytoplasm to nucleus earmarking the transcription factor for polyubiquitination and proteasome-mediated degradation, the cytokine TNFa-induced T-loop-phosphorylated IKKb becomes monoubiquitinated at Lyk163 proximal to the T-loop, mechanism of post-translational crosstalk, overview [35]; infection and genome insertion of human cytomegalovirus induces expression of the catalytic subunit IKK2 in the host cell which is required for viral induction of NF-kB activation and involved in viral replication and lytic cycle [45]; parasite Toxoplasma gondii IKKa, localized in parasitophorous vacuole membrane, activates the host intracellular NF-kB in early infection stage resulting in NF-kB nuclear translocation and subsequent gene expression independently from the host IKK complex [37]; subunit IKKb controls the activation of NF-kB, important in inflammation, IKKa plays a role in lyphoid organogenesis and suppresses NF-kB activity by accelerating both the turnover of the NFkB subunits RelA and cRel, and their removal from pro-inflammatory gene promoters, inactiva214

2.7.11.10

IkB kinase

tion of IKKa enhances inflammation and bacterial clearance in mice, overview [51]; the IKK complex is responsible for NFkB regulation [32]; the noncanonical IkB kinase homologue IKKe, beneath TANKbinding kinase-1 TBK-1, is required for activation of transcription factor NFkB and of interferon regulatory factor 3 IRF3, regulation and interaction, overview [47]; Vav-1 and IKKa subunit of IkB kinase functionally associate to induce NFkB activation in response to CD28 engagement [40]; activated IKK2 is responsible for induction of leucocyte infiltration in pancreatic acini, the mutant ICC2CA in pancreatic acinar cells increases tissue damage of secretagogue induced experimental pancreatitits, the enzyme is involved in the proinflammatory IKK/NF-kB pathway, overview [56]; hypoxia alters the cellular pool of IKKa and IKKb, and activates NFkB through a pathway involving activation of IkB kinase-b, IKKb, leading to phosphorylation-dependent degradation of IkBa and liberation of NFkB, overview, hypoxia-induced activation of the NFkB pathway is independent of HIF-1a, prolyl hydroxylase-1 negatively regulates IKKb [69]; IkB kinase b plays a critical role in metallothionein-1 expression and protection against arsenic toxicity, two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract reactive oxygen species accumulation, and second, when this pathway fails, excessive reactive oxygen species leads to activation of the MKK4-JNK pathway, resulting in apoptosis [59]; IkB kinase b plays an essential role in remodeling Carma1Bcl10-Malt1 complexes upon T cell activation, T cell receptor signaling to IkB kinase/NF-kB is controlled by PKCq-dependent activation of the Carma1, Bcl10, and Malt1 CBM complex, IKKb triggers the CBM complex formation and phosphorylation of Bcl by PMA/ionomycin or CD3/CD28, regulation, overview [65]; IkB kinase-a is critical for interferon-a production induced by Toll-like receptors 7 and 9, but IKK-a is dispensable for a cytoplasmic RNA helicase RIG-I-dependent cytosolic pathwayinduced production of IFN-a in MEF cells, overview [67]; IKK is responsible for activation of NF-kB by initiating the degradation of the NF-kB inhibitor IkB, subunits IKKa and IKKg/NEMO, not IKKb, are required for reovirus-induced NF-kB activation and apoptosis, overview [64]; IKK-b inhibition in vivo leads to reduction of rhinovirus-induced expression of CXCL8, CCL5, and IL-6, the enzyme is important in regulation of the NF-kB signaling pathway, overview [66]; IKK-related kinases tank-binding kinase 1 TBK1/IKKi and cullin-based ubiquitin ligases are involved in IFN regulatory factor-3, IRF-3, phosphorylation, activation, and degradation, IRF-3 activation is induced by viral infection, e.g. by HCMV, molecular mechanisms, detailed overview [61]; IKKa and IKKb are distinctly involved in ERK1-dependent, but IkBa-P65- and p100-p52-independent, upregulation of MUC5AC mucin transcription in case of infection by Streptococcus pneumoniae, MUC5AC mucin induction also requires pneumolysin and TLR4-dependent MyD88-IRAK1-TRAF6 signaling, molecular mechanism, overview [62]; 215

IkB kinase

2.7.11.10

IKKa enhances p73-mediated transactivation and pro-apoptotic functions in p53-deficient H1299 cells, stabilization of p73, but not of the antagonist p53, by nuclear IKKa mediates cisplatin-induced apoptosis, DNA damage-induced accumulation of both p53 and p73a is associated with the up-regulation of IKK-a and IKK-g, a functional interaction might exist between them in DNA damage-mediated apoptotic pathways [58]; IKKa is involved in the noncanonical NF-kB activation pathway, and plays an essential role in thymic organogenesis required for the establishment of self-tolerance, overview [60]; IKKa is not only a regulator of mammary epithelial proliferation, but is also an important contributor to ErbB2-induced oncogenesis, providing signals that maintain mammary tumor-initiating cells, IKKa activity is required for cyclin D1 induction and proliferation of lobuloalveolar epithelial cells, and is required for self-renewal of ErbB2/Her2-transformed mammary tumor-initiating cells, overview [70]; IKKb subunit of IKK complex is essential for the activation of NF-kB in response to various proinflammatory signals, Cys179 of IkB kinase b plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines, overview [55]; IKKe is important in the regulation of the alternative NF-kB activation pathway involving p52 and p65, IKKe interacts with p52 and promotes transactivation via p65 [57]; IKKe, i.e. IKKi, is implicated in virus induction of interferon-b, IFNb, and development of immunity, IKKe functions in a redundant role to its ubiquitous counterpart, TBK1, in the activation of IRF3 and IRF7 ex vivo, IKKe determines ISGF3 binding specificity, regulaiton, overview [72]; mechanisms/pathways of activation and derepression of the IKK complex, regulation, detailed overview [54]; the IkB kinase regulates chromatin structure during reconsolidation of conditioned fear memories, IKKa is involved in the regulation of histone H3 phosphorylation and acetylation at specific gene promoters in hippocampus in the NF-kB pathway, inhibition of IKKa regulation results in impairments in fear memory reconsolidation, mechanism, overview [68]) (Reversibility: ?) [23, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 45, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 72] P ? Substrates and products S ATP + Bcl ( phosphorylation at the C-terminus of Bcl by IKKb disrupts Bcl10/Malt1 association and Bcl10-mediated signaling [65]; phosphorylation at the C-terminus of Bcl by IKKb, inactive with a C-terminal 93 amino acid-deletion mutant of Bcl [65]) (Reversibility: ?) [65] P ADP + phosphorylated Bcl S ATP + Bcl ( Bcl10 is phosphorylated by the NEMO/IKK complex, recombinant substrate GST-Bcl10 expressed in HEK-293T cells, degradation of Bcl [71]) (Reversibility: ?) [71] P ADP + phosphorylated Bcl10

216

2.7.11.10

IkB kinase

S ATP + Bcl | ( negative regulatory activity of the IKK complex in Bcl10 degradation, which is part of the regulatory mechanisms that precisely control the response to antigens, overview [71]) (Reversibility: ?) [71] P ADP + phosphorylated Bcl10 S ATP + IkB protein ( the enzyme targets the inhibitory IkB protein tightly bound to the transcription factor NF-kB for proteasomal degradation and allows the freed NF-kB to enter the nucleus where it can be transactivate its target gene, IKKa is involved in inflammation in macrophages [54]) (Reversibility: ?) [54] P ADP + phosphorylated IkB S ATP + IkBa ( a step in NF-kB activation, the IKK complex, composed of IKKa, IKKb, and NEMO/IKKg, is the convergence point for many diverse NFkB-activating stimuli including TNFa, LPS, and IL-1, overview, IKKb is the primary positive regulator of NFkB activity in inflammatory processes, is the molecular link between inflammation and cancer [69]; degradation of IkBa [71]; phosphorylation of IkBa at Ser32 and Ser36 [54]; recombinant GST-fusion substrate [63]) (Reversibility: ?) [54, 63, 69, 71] P ADP + phosphorylated IkBa S ATP + IkBb ( phosphorylation of IkBa at Ser19 and Ser23 [54]) (Reversibility: ?) [54] P ADP + phosphorylated IkBb S ATP + [GST-IkB protein] (Reversibility: ?) [31] P ADP + [GST-IkB phosphoprotein] S ATP + [GST-IkBa protein] (Reversibility: ?) [24, 29, 32] P ADP + [GST-IkBa phosphoprotein] S ATP + [GST-IkBa1 -54 protein] ( recombinant substrate derived from mouse IkBa [26]) (Reversibility: ?) [26, 35, 36, 52] P ADP + [GST-IkBa1 -54 phosphoprotein] S ATP + [GST-IkBb protein] (Reversibility: ?) [44] P ADP + [GST-IkBb phosphoprotein] S ATP + [IkB protein] ( inhibition and degradation of IkB, an inhibitor of NF-kB retaining it in the cytoplasm, phosphorylation of IkB marks the protein for ubiquitination followed by degradation, activated NF-kB is translocated to the nucleus initiating signalling pathways, regulation mechanism, overview [44]; inhibitor substrate is bound to NF-kB [52]; parasite IKKa, localized in parasitophorous vacuole membrane, activates mouse intracellular NF-kB in early infection stage resulting in NF-kB nuclear translocation and subsequent gene expression independently from the host IKK complex [37]; phosphorylation of IkB results in its proteolytic degradation [49]; required for activation of NF-kB resulting in activation of signalling pathways [37]; signalling step of IKK bound to NFkB for subsequent ubiquitination of IkB and proteolytic degradation [27]; the enzyme is also active with a peptide derived from IkB protein spanning Ser32 and Ser36 which are phosphory-

217

IkB kinase

P S

P S P S

P S

P S

P S

P S

218

2.7.11.10

lated [43]) (Reversibility: ?) [25, 26, 27, 28, 30, 37, 38, 42, 43, 44, 46, 49, 50, 52, 53] ADP + [IkB phosphoprotein] ATP + [IkBa protein] ( enzyme is involved in activation of pro-inflammation signalling [24]; parasite IKKa, localized in parasitophorous vacuole membrane, activates mouse intracellular NF-kB through phosphorylation of host IkBa at Ser32 and Ser36 in early infection stage resulting in NF-kB nuclear translocation and subsequent gene expression independently from the host IKK complex [26]; IKKb-dependent phosphorylation and subsequent ubiquitin-dependent proteolytic degradation of IKKa, the 2 subunits have opposing function in NF-kB metabolism, overview [51]; phosphorylation at Ser32 and Ser36 [53]; phosphorylation on Ser32 and Ser36 [42]) (Reversibility: ?) [24, 26, 34, 42, 51, 53] ADP + [IkBa phosphoprotein] ATP + [IkBb protein] ( phosphorylation at Ser19 and Ser23 [53]) (Reversibility: ?) [35, 53] ADP + [IkBb phosphoprotein] ATP + [NFkB subunit p56] ( phosphorylation at serine residues, especially at S536, in the cytoplasm prior to NFkB p56 translocation to the nucleus [30]) (Reversibility: ?) [30] ADP + [NFkB subunit p56]phosphate ATP + [RelA/p65 protein] ( phosphorylation on Ser536 of the transactivation domain, dependent on lipopolysaccharide-induction, but not on Akt or p65 [42]) (Reversibility: ?) [42] ADP + [RelA/p65 phosphoprotein] ATP + [acetylated histone H3 protein] ( IKKa is required for histone function regulation in the nucleus [53]; phosphorylation at Ser10 [53]) (Reversibility: ?) [53] ADP + [acetylated histone H3 phosphoprotein] ATP + [histone H3 protein] ( histone H3 phosphorylation by IKK-a is critical for cytokine-induced gene expression [50]; phosphorylation at Ser10 by IKK-a [50]) (Reversibility: ?) [32, 5] ADP + [histone H3 phosphoprotein] ATP + a protein ( autophosphorylation at both Ser and Thr [17]; CHUK associates with the NF-kB inhibitory protein, IkB-a, in mammalian cells. CHUK specifically phosphorylates IkB-a on both Ser32 and Ser36, modifications that are required for targeted degradation of IkB-a via the ubiquitin-proteasome pathway [14]; the I k B/NF-k B system is a key determinant of mucosal inflammation and protection [3]; the expression of pkn1 is developmentally regulated to start immediately before spore formation. The enzyme plays an important role in the onset of proper differentiation [17]; phosphorylation of IkBs marks them out for destruction, thereby relieving their inhibitory effect on NF-kB [11]; phosphorylates IkB inhibitory proteins, causing their degradation and activation of transcription factor NF-kB, a

2.7.11.10

P S

P S

P S

IkB kinase

master activator of inflammatory responses [10]) (Reversibility: ?) [3, 10, 11, 14, 17] ADP + a phosphoprotein ATP + protein p100 ( interaction with the NF-kB complex [57]; required for the interaction with the NF-kB complex [57]) (Reversibility: ?) [57] ADP + phosphorylated protein p100 ATP + protein p165 ( p65 is part of the IKKe complex with p25, interaction with the NF-kB complex [57]; required for the interaction with the NF-kB complex [57]) (Reversibility: ?) [57] ADP + phosphorylated protein p165 Additional information ( activation of heterodimeric nuclear transcription factor NFkB is an essential step in inflammation, e.g. resulting in osteoarthritis, ulcerative colitis, asthma, and Crohns disease, signalling step for phosphorylation, subsequent ubiquitination and proteolytic degradation of IkB, NFkB remains free after the reaction and is translocated to the nucleus, NFkB activation is also involved in development of diseases like cancer, gut ischemia-reperfusion, diabetes, or in transplant rejections, overview [27]; activation of NFkB by the catalytic subunit IKKb is required for signaling via the NFkB pathway in acute and systemic inflammation and for tissue protection [48]; defective ubiquination of the NF-kB essential modifier/IkB kinase-g complex leads to impaired cellular NFkB signalling and hypohidrotic ectodermal dysplasia with immunodeficiency HED-ID [31]; enzyme is responsible for NFkB activation, specific inhibition of IkB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in male Sprague-Dawley rats [43]; IkB kinases are essential key regulators of the NFkB pathways in the tooth development acting as stimulators, overview [28]; IKK activates and regulates NFkB important in intracellular signalling, overview [30]; IKK activates NF-kB through action of TNFa playing an important role in subsequent signaling pathways involved in e.g. apoptosis/cell survival, cell proliferation, and inflammation [34]; IKK activates NF-kB which initiates signaling pathways that play critical roles in a variety of physiological and pathological processes, e.g. promotion of cell survival inducing production of apoptosis inhibitors in normal and cancer cells, pathways overview, IKK/NF-kB links inflammation to cancer, regulation of IKK, overview [38]; IKK activates TNFa-dependent signaling pathways inducing 5-fluoro-2-deoxyuridine drug resistance in different cell lines, overview [36]; IKK is involved in NF-kB activation, IkB kinase IKKb, but not IKKa, is a critical mediator of NF-kB-dependent osteoclast survival preventing TNFa-induced cell death, and is required for formation of fully functional bone-resorbing osteoclasts and for inflammation-induced bone loss [39]; IKK is required for activation of NF-kB and subsequent signalling pathways [49]; IKK is responsible for activation of NFkB, herpesvirus HSV-1 potently induces IkB kinase IKK causing persistent induction of NFkB resulting in transactivation of HIV-1219

IkB kinase

2.7.11.10

LTR-regulated genes and induction of HIV-1 replication in infected Tcells [23]; IKK is responsible for NF-kB activation by inactivating its inhibitor IkB, different inflammation stimuli induce distinct IKK activity profiles, molecular mechanism, overview [52]; IKK marks cytoplasmic NFkB inhibitors for proteolytic destruction playing a role in regulation of genetic cell cycle programs, IKK regulates nuclear translocation of transcription factor NFkB [29]; IKKa, not IKKb, is required for epidermal regeneration, IkB kinases are essential key regulators of the canonical and noncanonical NFkB pathways important for the expression of a wide variety of genes that are involved in the control of immune and inflammatory response, and in the regulation of cellular proliferation and survival, mechanism, overview [53]; IKKb is required for activation of NFkB, IKKb induces expression of epithelial sodium channel abg-ENaC in cell surfaces [33]; IKKb is required for regulation of NFkB activity and peripheral B cell survival and proliferation [41]; IKKb regulates the translocation of NF-kB from cytoplasm to nucleus earmarking the transcription factor for polyubiquitination and proteasome-mediated degradation, the cytokine TNFa-induced T-loop-phosphorylated IKKb becomes monoubiquitinated at Lyk163 proximal to the T-loop, mechanism of post-translational crosstalk, overview [35]; infection and genome insertion of human cytomegalovirus induces expression of the catalytic subunit IKK2 in the host cell which is required for viral induction of NF-kB activation and involved in viral replication and lytic cycle [45]; parasite Toxoplasma gondii IKKa, localized in parasitophorous vacuole membrane, activates the host intracellular NF-kB in early infection stage resulting in NF-kB nuclear translocation and subsequent gene expression independently from the host IKK complex [37]; subunit IKKb controls the activation of NF-kB, important in inflammation, IKKa plays a role in lyphoid organogenesis and suppresses NF-kB activity by accelerating both the turnover of the NFkB subunits RelA and c-Rel, and their removal from pro-inflammatory gene promoters, inactivation of IKKa enhances inflammation and bacterial clearance in mice, overview [51]; the IKK complex is responsible for NFkB regulation [32]; the noncanonical IkB kinase homologue IKKe, beneath TANK-binding kinase-1 TBK-1, is required for activation of transcription factor NFkB and of interferon regulatory factor 3 IRF3, regulation and interaction, overview [47]; Vav-1 and IKKa subunit of IkB kinase functionally associate to induce NFkB activation in response to CD28 engagement [40]; TNF-R1-activated IKKb phosphorylates IKKg and IKKa, autophosphorylation patterns involving K163 of IKKb, regulation, overview [35]; activated IKK2 is responsible for induction of leucocyte infiltration in pancreatic acini, the mutant ICC2CA in pancreatic acinar cells increases tissue damage of secretagogue induced experimental pancreatitits, the enzyme is involved in the proinflammatory IKK/NF-kB pathway, overview [56]; hypoxia alters the cellular pool of IKKa and IKKb, and activates NFkB through a pathway involving activation 220

2.7.11.10

IkB kinase

of IkB kinase-b, IKKb, leading to phosphorylation-dependent degradation of IkBa and liberation of NFkB, overview, hypoxia-induced activation of the NFkB pathway is independent of HIF-1a, prolyl hydroxylase1 negatively regulates IKKb [69]; IkB kinase b plays a critical role in metallothionein-1 expression and protection against arsenic toxicity, two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract reactive oxygen species accumulation, and second, when this pathway fails, excessive reactive oxygen species leads to activation of the MKK4-JNK pathway, resulting in apoptosis [59]; IkB kinase b plays an essential role in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation, T cell receptor signaling to IkB kinase/NF-kB is controlled by PKCq-dependent activation of the Carma1, Bcl10, and Malt1 CBM complex, IKKb triggers the CBM complex formation and phosphorylation of Bcl by PMA/ionomycin or CD3/CD28, regulation, overview [65]; IkB kinase-a is critical for interferon-a production induced by Toll-like receptors 7 and 9, but IKK-a is dispensable for a cytoplasmic RNA helicase RIG-I-dependent cytosolic pathway-induced production of IFN-a in MEF cells, overview [67]; IKK is responsible for activation of NF-kB by initiating the degradation of the NF-kB inhibitor IkB, subunits IKKa and IKKg/ NEMO, not IKKb, are required for reovirus-induced NF-kB activation and apoptosis, overview [64]; IKK-b inhibition in vivo leads to reduction of rhinovirus-induced expression of CXCL8, CCL5, and IL-6, the enzyme is important in regulation of the NF-kB signaling pathway, overview [66]; IKK-related kinases tank-binding kinase 1 TBK1/ IKKi and cullin-based ubiquitin ligases are involved in IFN regulatory factor-3, IRF-3, phosphorylation, activation, and degradation, IRF-3 activation is induced by viral infection, e.g. by HCMV, molecular mechanisms, detailed overview [61]; IKKa and IKKb are distinctly involved in ERK1-dependent, but IkBa-P65- and p100-p52-independent, upregulation of MUC5AC mucin transcription in case of infection by Streptococcus pneumoniae, MUC5AC mucin induction also requires pneumolysin and TLR4-dependent MyD88-IRAK1-TRAF6 signaling, molecular mechanism, overview [62]; IKKa enhances p73-mediated transactivation and pro-apoptotic functions in p53-deficient H1299 cells, stabilization of p73, but not of the antagonist p53, by nuclear IKKa mediates cisplatin-induced apoptosis, DNA damage-induced accumulation of both p53 and p73a is associated with the up-regulation of IKK-a and IKK-g, a functional interaction might exist between them in DNA damage-mediated apoptotic pathways [58]; IKKa is involved in the noncanonical NF-kB activation pathway, and plays an essential role in thymic organogenesis required for the establishment of self-tolerance, overview [60]; IKKa is not only a regulator of mammary epithelial proliferation, but is also an important contributor to ErbB2induced oncogenesis, providing signals that maintain mammary tumorinitiating cells, IKKa activity is required for cyclin D1 induction and 221

IkB kinase

2.7.11.10

proliferation of lobuloalveolar epithelial cells, and is required for selfrenewal of ErbB2/Her2-transformed mammary tumor-initiating cells, overview [70]; IKKb subunit of IKK complex is essential for the activation of NF-kB in response to various proinflammatory signals, Cys179 of IkB kinase b plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines, overview [55]; IKKe is important in the regulation of the alternative NF-kB activation pathway involving p52 and p65, IKKe interacts with p52 and promotes transactivation via p65 [57]; IKKe, i.e. IKKi, is implicated in virus induction of interferon-b, IFNb, and development of immunity, IKKe functions in a redundant role to its ubiquitous counterpart, TBK1, in the activation of IRF3 and IRF7 ex vivo, IKKe determines ISGF3 binding specificity, regulation, overview [72]; mechanisms/ pathways of activation and derepression of the IKK complex, regulation, detailed overview [54]; the IkB kinase regulates chromatin structure during reconsolidation of conditioned fear memories, IKKa is involved in the regulation of histone H3 phosphorylation and acetylation at specific gene promoters in hippocampus in the NF-kB pathway, inhibition of IKKa regulation results in impairments in fear memory reconsolidation, mechanism, overview [68]; IKKa interacts with p73 [58]; interaction analysis of IKKe with NF-kB complex components/p25/p65 by immunoprecipitation and mass spectrometric analysis, overview [57]; no activity with Rip2 [71]; the enzyme activity is included in a complex formed of the scaffold protein NF-kB essential modulator, i.e. NEMO or IKKg, and the IKKa and IKKb kinases, overview [54]) (Reversibility: ?) [23, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 45, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72] P ? Inhibitors (4-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)phenyl)acetic acid ( IC50 for the IKKb is 0.0022 mM [25]) [25] 15d-prostaglandin J2 ( strongly inhibits IKK [23]) [23] 2-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)benzoic acid ( IC50 for the IKK complex is above 0.1 mM [25]) [25] 2-benzamido-pyrimidines ( diverse derivatives, synthesis and inhibitory potential determination, overview [25]) [25] 3-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)benzoic acid ( IC50 for the IKKb is 0.001 mM [25]) [25] 4,8-dimethoxy-1-vinyl-b-carboline ( i.e. b-carboline alkaloid C-1, isolated from Melia azedarach var. japonica, inhibits IKK activity by reduction of IKK phosphorylation and degradation, and activation and nuclear translocation of NF-kB and subsequent signalling pathways [44]) [44] 4-(1-benzothiophen-2-yl)-N-[3-chloro-4-([4-pyrrolidin-1-yl-piperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 250 nM [25]) [25]

222

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IkB kinase

4-(1-benzothiophen-2-yl)-N-[3-methoxy-4-([4-pyrrolidin-1-yl-piperidin-1yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 150 nM [25]) [25] 4-(1-benzothiophen-2-yl)-N-[4-([4-(dimethyl-amino)piperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 80 nM, cellular profile [25]) [25] 4-(1-benzothiophen-2-yl)-N-[4-([4-pyrrolidin-1-yl-piperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 70 nM, cellular profile [25]) [25] 4-(1-benzothiophen-2-yl)-N-[4-(pyrrolidin-1-ylcarbonyl)phenyl]pyrimidin2-amine ( IC50 for the IKK complex is 0.0085 mM, cellular profile [25]) [25] 4-(5-(3-acetamino-3-methylbutyl)thiophen-2-yl)-N-[4-([4-pyrrolidin-1-yl-piperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 800 nM [25]) [25] 4-(5-(3-amino-3-ethylpentyl)thiophen-2-yl)-N-[4-([4-pyrrolidin-1-yl-piperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 30 nM [25]) [25] 4-(5-(3-amino-3-methyl-but-1-ynyl)thiophen-2-yl)-N-[4-([4-pyrrolidin-1-ylpiperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 200 nM [25]) [25] 4-(5-(3-amino-3-methyl-butyl)thiophen-2-yl)-N-[4-([4-pyrrolidin-1-yl-piperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 40 nM [25]) [25] 4-(5-(3-hydroxy-3-methylbutyl)thiophen-2-yl)-N-[4-([4-pyrrolidin-1-yl-piperidin-1-yl]carbonyl)phenyl]pyrimidin-2-amin ( IC50 for the IKK complex is 50 nM, cellular profile [25]) [25] 4-(5-(3-methoxypropyl)thiophen-2-yl)-N-[4-([4-pyrrolidin-1-yl-piperidin-1yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 100 nM, cellular profile [25]) [25] 4-(5-(4-hydroxybutyl)thiophen-2-yl)-N-[4-([4-pyrrolidin-1-yl-piperidin-1yl]carbonyl)phenyl]pyrimidin-2-amine ( IC50 for the IKK complex is 40 nM, cellular profile [25]) [25] 4-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)benzoic acid ( IC50 for the IKK complex is 0.018 mM, cellular profile [25]) [25] 5,6-dibromo-b-carboline ( inhibits IKK with IC50 of 600 nM [24]) [24] 5-aminosalicylate ( weak inhibition of IKK-2 [27]) [27] 5-bromo-6-chloro-b-carboline ( inhibits IKK with IC50 of 600 nM [24]) [24] 5-bromo-6-cyano-b-carboline ( inhibits IKK with IC50 of 0.0011 mM [24]) [24] 5-bromo-6-fluoro-b-carboline ( inhibits IKK with IC50 of 0.002 mM [24]) [24] 5-bromo-6-methoxy-b-carboline ( nonspecific inhibitor of IKK, inhibits IKK with IC50 of 0.004 mM [24]) [24]

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5-bromo-6-trifluoromethyl-b-carboline ( inhibits IKK with IC50 of 0.0011 mM [24]) [24] 5-bromo-b-carboline ( inhibits IKK with IC50 of 0.015 mM [24]) [24] 6,8-dichloro-b-carboline ( inhibits IKK with IC50 of 200 nM [24]) [24] 7-(OC(O)morpholine)-6,8-dichloro-b-carboline ( inhibits IKK with IC50 of 0.0032 mM [24]) [24] 7-(OCH2 (C6 H11 ))-6,8-dichloro-b-carboline ( inhibits IKK with IC50 of 0.003 mM [24]) [24] 7-(OCH2 CH(CH2 CH2 ))-6,8-dichloro-b-carboline ( inhibits IKK with IC50 of 80 nM [24]) [24] 7-ethoxy-6,8-dichloro-b-carboline ( inhibits IKK with IC50 of 140 nM [24]) [24] 7-hydroxy-6,8-dichloro-b-carboline ( inhibits IKK with IC50 of 0.011 mM [24]) [24] 7-methoxy-6,8-dichloro-b-carboline ( inhibits IKK with IC50 of 170 nM [24]) [24] 8-(NHC(O)(CH2 )3OH)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.02 mM [24]) [24] 8-(NHC(O)-2’-anisole)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.02 mM [24]) [24] 8-(NHC(O)-2’-pyridyl)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.001 mM [24]) [24] 8-(NHC(O)-3’-anisole)-6-chloro-b-carboline ( inhibits IKK with IC50 of 600 nM [24]) [24] 8-(NHC(O)-4’-anisole)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.0013 mM [24]) [24] 8-(NHC(O)-4’-pyridyl)-6-chloro-b-carboline ( inhibits IKK with IC50 of 300 nM [24]) [24] 8-(NHC(O)-phenyl)-6-chloro-b-carboline ( inhibits IKK with IC50 of 700 nM [24]) [24] 8-(NHC(O)CH2 -2’-pyridyl)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.003 mM [24]) [24] 8-(NHC(O)CH3 )-6-chloro-b-carboline ( inhibits IKK with IC50 of 600 nM [24]) [24] 8-(NHC(O)NHCH3 )-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.02 mM [24]) [24] 8-(NHC(O)OCH3 )-6-chloro-b-carboline ( inhibits IKK with IC50 of 700 nM [24]) [24] 8-(NHC(O)morpholine)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.02 mM [24]) [24] 8-(NHCH2 -phenyl)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.02 mM [24]) [24] 8-(NHS(O)2-phenyl)-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.02 mM [24]) [24]

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8-(NHS(O)2CH3 )-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.0083 mM [24]) [24] 8-amino-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.0013 mM [24]) [24] 8-amino-dimethyl-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.0018 mM [24]) [24] 8-amino-methyl-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.0018 mM [24]) [24] 8-nitro-6-chloro-b-carboline ( inhibits IKK with IC50 of 0.004 mM [24]) [24] AS602868 ( IKKb inhibitor [38]; inhibitor of IKK-b [66]) [38, 45, 66] acetylsalicylate ( weak inhibition of IKK-2 [27]) [27] acetylsalicylic acid ( nonspecific for IKK, anti-inflammatory [38]) [38] arsenite ( inhibition of IKKb [55]) [55] BMS-345541 ( specific for IKK-2, binds at an allosteric site [27]) [27] Bay 11-7082 [30] DDTC [68] N-(2-aminoethyl)-4-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)benzamide ( IC50 for the IKK complex is 0.0044 mM [25]) [25] N-(2-dimethyl-aminoethyl)-4-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)benzamide ( IC50 for the IKK complex is 300 nM, cellular profile [25]) [25] N-(2-pyrrolidin-1-yl-ethyl)-4-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)benzamide ( IC50 for the IKK complex is 500 nM [25]) [25] N-(3-methyl-butyl)-4-([4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino)benzamide ( IC50 for the IKKb is above 0.1 mM [25]) [25] PS-1145 ( i.e. 8-(NHC(O)-3-pyridyl)-6-chloro-b-carboline, inhibits IKK with IC50 of 100 nM in vitro, blocks phosphorylation of IkBa and subsequent activation of NFkB in vivo [24]) [24] PS1145 ( inhibits IKK, blocks TNFa activation by IKK in vivo [36]) [36] prostaglandin A1 ( strongly inhibits IKK [23]) [23] prostaglandin A2 ( strongly inhibits IKK [23]) [23] prostaglandin E1 ( inhibits IKK [23]) [23] prostaglandin E2 ( inhibits IKK [23]) [23] S1627 ( specific for the IKK complex, inhibits purified IKK in vitro with IC50 of 0.02 mM, inhibits IKK and NFkB nuclear translocation in vivo in umbilical vein endothelial cells and in rats, overview [43]) [43] salicylate ( weak inhibition of IKK-2 [27]) [27] antirheumatic gold compound ( inhibition of IKKb [55]) [55] arsenic trioxide ( nonspecific for IKK, acts on a cystein ersidue in the activation loop [38]) [38] curcumin ( weak inhibition of IKK-2 [27]) [27] cyclopentenone prostaglandins ( nonspecific for IKK, anti-inflammatory [38]; inhibition of IKKb [55]) [38, 55] 225

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cyclopentenone prostanoids ( inhibition of IKK and thus inhibition of NFkB-mediated HSV-1-induced HIV-1 replication [23]) [23] cyclopentone prostaglandines ( inhibition of IKK-2 [27]) [27] evodiamine ( alkaloid extracted from Evodia rutaecarpa fruits exhibiting antiproliferative, antimetastatic, and apoptotic activities, inhibits IKKa activity, suppresses IKKa phosphorylation and degradation, and specifically blocks NF-kB activation by IKK and other agents, translocation, and activity, overview [34]) [34] prostaglandin 2a ( inhibits IKK [23]) [23] sulfasalazine ( nonspecific for IKK, anti-inflammatory [38]; weak inhibition of IKK-2 [27]) [27, 38] sulindac sulfide ( nonspecific for IKK, anti-inflammatory [38]) [38] trans-resveratrol ( nonspecific for IKK, anti-inflammatory [38]) [38] Additional information ( CREB-binding protein-bound NEMO/IKKg inhibits IKKa and p65 transcriptional activities [32]; evodiamine analogue rutaecarpine does not inhibit IKK and NF-kB activation [34]; prolyl hydroxylase-1 negatively regulates IKKb, hydroxylation represses IKKb activity by altering protein expression or by preventing its activation through phosphorylation on S177/S181 [69]; simvastatin inhibits the TNFa-induced activation of IkB kinase and activation of NF-kB in vitro and in vivo, simvastatin highly affects the whole NF-kB-dependent pathway of signaling, overview [63]) [32, 34, 63, 69] Cofactors/prosthetic groups ATP ( binding involves activation loop Cys179 [55]; the binding involves Lys44 [58]) [24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 42, 43, 44, 46, 50, 52, 53, 54, 55, 57, 58, 60, 62, 63, 64, 65, 69, 71] Activating compounds CD28 ( activates Vav-1/IKKa and subsequently NFkB, meditated by Vav-1 [40]) [40] HSP70 ( the chaperone protein associates with the IKK complex and is required for its TNFa-induced activation [54]) [54] Hsp90 ( the chaperone protein associates with the IKK complex and is required for its TNFa-induced activation [54]) [54] NEMO ( i.e. NF-kB essential modifier, complexing with IkB kinase-g [35]; i.e. NF-kB essential modifier, complexing with IkB kinaseg and ubiquination, involving the zinc finger of NEMO and c-IAP1, are required for activation of the IkB kinase complex by inflammatory stimuli such as tumor necrosis factor TNFa [29,31]; i.e. NF-kB essential modifier, complexing with IkB kinase-g and ubiquitination, involving the zinc finger of NEMO and c-IAP1, are required for activation of the IkB kinase complex by inflammatory stimuli such as tumor necrosis factor TNFa [53]; i.e. NF-kB essential modifier, complexing with IkB kinase-g, regulates cytokineinduced IKK complex activity, NEMO/IKKg is shuttling between cytoplasm

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and nucleus, and binds CBP, being ininvolved in NFkB regulation [32]) [27, 29, 31, 32, 35, 53] TNFa ( activates histone H3 phosphorylation activity, TNFa-induced activation of IKK-b but not IKK-a leads to rapid NFkB activation and subsequent degradation of IkB [50]; and other cytokines, activation of IKK activating NF-kB [38]; induces IKK activity, activation is negatively feedback regulated [52]) [38, 50, 52] Tax ( viral oncoprotein, activates by direct binding, involved in ubiquitination and phosphorylation [35]) [35] Vav-1 ( cytoplasmic proto-oncogene, constitutively associates with IKKa in Jurkat and primary CD4+ cells [40]) [40] cdc37 ( the chaperone protein associates with the IKK complex and is required for its TNFa-induced activation [54]) [54] tumor necrosis factor TNFa ( cytokines activate the IKK complex, mechanism, overview [53]) [53] Additional information ( aldosterone regulates IKKb activity by enzyme induction [33]; cytokine-induced activation of IKK, e.g. by TNFa, required for NFkB activation, leads to rapid degradation of IkB [50]; herpesvirus HSV-1 potently induces IkB kinase IKK causing persistent induction of NFkB resulting in transactivation of HIV-1-LTR-regulated genes and induction of HIV-1 replication in infected T-cells [23]; IKK induction by cytokines, transcriptional activation by CREB-binding protein, i.e. CBP, after N-terminally binding to NEMO, which competes with IKKa and p65 [32]; IKK is induced by TNFa [24,31]; IKKg phosphorylation by IKKb is induced by the cytokine TNF-R1, IKKb is recruited to the cytoplasmic tail of TNF-R1 by adaptor proteins, such as RIP [35]; IKKg phosphorylation by IKKb is induced by the cytokine TNFa and the viral oncoprotein Tax [29]; infection and genome insertion of human cytomegalovirus induces expression of the catalytic subunit IKK2 in the host cell which is required for viral induction of NF-kB activation and involved in viral replication and lytic cycle [45]; lipopolysaccharides from Escherichia coli induce enzyme activity versus RelA/p65 and NFkB activation in vivo, induction is not inhibited by LY294002, overview [42]; lipopolysaccharides stimulate cytokine-mediated IKK activity being positively feedback regulated [52]; TNF-a induced association of IKK-a with p65 and CBP [50]; TNFa, TGF-b-activated kinase 1 TAK1, IKKa subunit and IKKb subunit induce IKK phosphorylation activity, overview [30]; type 1 TNFa induces the enzyme activity [48]; Bcl10 degradation depends on NEMO and CARMA1 [71]; hypoxia activates NFkB through a pathway involving activation of IkB kinase-b, IKKb [69]; IKKb is activated by phosphorylation at Ser171 and Ser181 of the activation loop [55]; no activation by arsenic nor H2 O2 [59]; the IkB complex gets activated by phosphorylation, the enzyme activity is induced by proinflammatory cytokines and by oxidative and genotoxic stress, mechanisms/pathways of activation and derepression of the IKK complex, overview [54]; TNFa induces activation of IkB kinase, which is inhibited by simvastatin [63]; virus-inducible ex-

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pression of IKKe, IKKe is activated by IFNb [72]) [23, 24, 29, 30, 31, 32, 33, 35, 42, 45, 48, 50, 52, 54, 55, 59, 63, 69, 71, 72] Metals, ions Mg2+ [27, 30, 31, 34, 36, 43, 44, 46, 54, 57, 60, 63, 64, 65, 71] Mn2+ [64] Specific activity (U/mg) Additional information ( development of real-time imaging for continous enzyme detection and kinetics in intact cells and living mice utilizing a recombinant IkBa-firefly luciferase reporter construct [49]) [49, 61] Km-Value (mM) Additional information ( kinetics of IkB phosphorylation by parasite IKK, host NF-kB activation reveals a biphasic, hierarchical, and temporally regulated response [37]) [37] pH-Optimum 7 ( assay at [58]) [58] 7.4 ( assay at [34,63,64]) [34, 63, 64] 7.5 ( assay at [31,36,43,46,57]) [31, 36, 43, 46, 57] 7.6 ( assay at [30]) [30] 8 ( assay at [44]) [44] Temperature optimum ( C) 25 ( assay at [43]) [43] 30 ( assay at [30, 31, 34, 36, 44, 58, 63, 64]) [30, 31, 34, 36, 44, 58, 63, 64] 37 ( assay at [46,57]) [46, 57]

4 Enzyme Structure Subunits oligomer ( the IKK complex is built by subunits IKKa, IKKb, and IKKg/NEMO [64]) [64] Additional information ( 2-step tetrameric oligomerization of IKKg, mediated by C-terminal coiled-coil domains, is obligatory for IKK complex phosphorylation activity and NF-kB activation in vivo, the IKK complex consists of 3 subunits: catalytic IKKa and IKKb, and noncatalytic IKKg, which bind to each other independently of IKKg oligomerization status, overview [46]; IKK complex is composed of catalytic IKK1 and IKK2, and regulatory IKKg, i.e. NEMO, subunits [25]; IKKb contains a leucine zipper, a T-loop, and a HLH domain important for phosphorylation and ubiquitination, the IKK complex consists of 3 subunits: catalytic IKKa and IKKb, and noncatalytic, regulatory NEMO/IKKg [35]; subunits IKK1 and IKK2 [45]; the IKK complex consists of 3 subunits: catalytic IKKa and IKKb, and noncatalytic NEMO/IKKg [27,29,51,53]; the IKK complex consists of 3 subunits: catalytic IKKa and IKKb, and noncatalytic

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NEMO/IKKg, complexes of IKKa and IKKg or IKKa homodimers are not stable [39]; the IKK complex consists of 3 subunits: catalytic IKKa and IKKb, and noncatalytic, regulatory NEMO/IKKg [32,38,53]; the IKK complex consists of 3 subunits: catalytic IKKa and IKKb, and regulatory IKKg [41]; the enzyme activity is included in a complex formed of the scaffold protein NF-kB essential modulator, i.e. NEMO or IKKg, and the IKKa and IKKb kinases, overview, the complex contains a zinc finger-like domain, N- and C-terminal coiled-coil domains, two a-helices and a leucine zipper [54]; within its activation loop, IKKb and IKKa contain an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases, while p65, p50, IkBa, and NEMO/IKKg are without this sequence [69]) [25, 27, 29, 32, 35, 38, 39, 41, 45, 46, 51, 53, 54, 69] Posttranslational modification phosphoprotein ( autophosphorylation at both Ser and Thr [17]; IKKb is phosphorylated at the T loop residues Ser171 and Ser181, chronic phosphorylation leads to attachment of one ubiquitin molecule at nearby Lys163, which in turn modulates the phosphorylation status at select C-terminal serines [35]; subunit IKKg is activated by phosphorylation via IKKb subunit at Ser31, Ser43, and Ser376, IKKg phosphorylation requires its zinc finger sequence at the C-terminus, phosphopeptide mapping [29]; IKKb is activated by phosphorylation at Ser171 and Ser181 of the activation loop [55]; the IkB complex gets activated by phosphorylation and then autophosphorylates IkBa at Ser32 and Ser36 [54]) [17, 29, 35, 54, 55] Additional information ( within its activation loop, IKKb contains an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases [69]) [69]

5 Isolation/Preparation/Mutation/Application Source/tissue 293T cell [31] ACH-2 cell ( chronically infected with HIV-1 [23]) [23] B-cell ( peripheral [41]) [41] B-lymphocyte ( peripheral [41]) [41] BEAS-2B cell ( bronchial epithelial cell line [66]) [66] Colo-201 cell ( colon cancer cell line [36]) [36] Colo-320 cell ( colon cancer cell line [36]) [36] H9 cell ( chronically infected with HIV-1 [23]) [23] HEK-293 cell ( embryonic kidney cell line [34]) [30, 34] HEK-293T cell [71] HeLa [30] HeLa cell ( induced by TNFa [24]) [24, 25, 31, 32, 46, 50, 53, 64] HeLa-S3 cell [43] JURKAT cell ( T-cell lymphoma cell line [34]) [23, 30, 34, 38, 40, 65] KBM-5 cell ( myeloid leukemia cell line [34]) [34, 63]

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MEF cell ( embryonic fibroblast cell line [52]; embryonic fibroblast cell line, from wild-type and IKKa- mice [51]; wild-type and IKKaor IKKb-deficient embryonic fibroblast cell line infected with parasite Toxoplasma gondii, RH strain [37]) [37, 42, 51, 52, 53, 54] NCI-H1299 cell ( lung adenocarcinoma cell line [34]) [34] RAW 264.7 cell ( macrophage cell line [54]; monocyte/ macrophage cell line [44]) [30, 42, 44, 54] T-cell [23, 40] T-lymphoblastoid cell line [23] T-lymphocyte [40] U-266 cell ( multiple myeloma cell line [34]) [34] bone [39] brain [68] breast [70] breast cancer cell [70] bronchus [66] cell culture ( cell line KG-1 [18]) [18] dendritic cell ( partly derived from bone marrow [67]) [67] embryo [28, 42, 50, 53] endothelial cell ( from umbilical vein [43]) [43] enterocyte [48] epidermis [38, 53] epithelial cell [62, 66] epithelium ( thymus [60]) [33, 60, 70] fibroblast ( embryonic [50]; adult and embryonic [53]; mouse embryonic fibrblasts MEF [61]; mouse embryonic fibroblasts MEF [59]; mouse embryonic fibroblasts, MEF [67,72]) [42, 50, 53, 54, 59, 61, 67, 72] hematopoietic cell [39] hepatocyte [38] hepatoma cell [38] hippocampus [68] intestinal mucosa [48] keratinocyte ( epidermal [38]) [38] liver [54] lung ( respiratory tract mucosa [62]) [62] lymphocyte ( primary [65]) [65] macrophage ( alveolar and peritoneal [51]) [42, 51, 54] mammary gland ( lobuloalveolar epithelial cell [70]) [70] monocyte [42] mpkCCDc14 cell ( immortal mpkCCDc14 cell line, derived from transgenic mice [33]) [33] neuron [68] osteoblast [39] osteoclast [39] pancreas [56] pancreatic acinar cell [56] 230

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peripheral blood mononuclear cell ( CD4+ [40]) [40, 65] squamous cell carcinoma [38] thymocyte [40, 60] thymus ( stroma, embryonic [60]) [60] tooth ( IKKa and IKKb expression patterns in different developmental stages during gestation and in different tooth regions, overview [28]) [28] umbilical vein [43] Localization cytoplasm ( predominantly [53]; IKK complex, NEMO/ IKKg is shuttling between cytoplasm and nucleus [32]; IKK-b and IKK-a [50]; IKKb and IKKa are almost entirely cytoplasmic, IKK complex component scaffold protein NF-kB essential modulator, i.e. NEMO or IKKg, shuttles between the cytoplasm and the nucleus in a CRM-dependent manner [54]) [27, 29, 30, 32, 34, 35, 37, 38, 39, 42, 44, 50, 51, 53, 54, 64] cytosol ( IKKa shuttles between the nucleus and cytoplasm in a CRM1-dependent fashion [58]) [43, 58, 69] membrane ( in T-cells [40]) [40] nucleus ( IKK-a [50]; NEMO/IKKg is shuttling between cytoplasm and nucleus [32]; IKK complex component scaffold protein NF-kB essential modulator, i.e. NEMO or IKKg, shuttles between the cytoplasm and the nucleus in a CRM-dependent manner [54]; nuclear accumulation of IKKa occurs in response to CDDP, IKKa shuttles between the nucleus and cytoplasm in a CRM1-dependent fashion [58]) [32, 43, 50, 53, 54, 58] vacuolar membrane ( parasitophorous, PVM [26,37]) [26, 37] Purification (native IkBa kinase from HeLa S3 cells by ammonium sulfate fractionation, ultrafiltration, and gel filtration) [43] (recombinant GST-fusion IKKg protein from Escherichia coli strain BL21 by glutathione affinity and anion exchange chromatography, and gel filtration, recombinant His-tagged IKKg protein from Escherichia coli strain BL21 by nickel affinity chromatography) [46] (recombinant His-tagged IKKb from Sf21 insect cells by nickel affinity chromatography) [30] Cloning (IKKe expression analysis) [72] (adenoviral-mediated transient IKKb expression in enzyme-deficient cells significantly reduces apoptosis of the cells in response to arsenic) [59] (expression of FLAG-tagged wild-type and mutant enzymes in HEK-293 and HeLa cells, expression of the C-terminal fragment, comprising amino acids 541-716, of IKKe with NF-kB2/p100/p52 in yeast two-hybrid cells) [57] (expression of IKKa in COS-7 cells, co-expression with p73a and Histagged ubiquitin, analysis of ubiquitinated compounds) [58]

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(expression of IKKb in the yeast two-hybrid system, interaction with epithelial sodium channel abg-ENaC, coexpression of IKKb and ab-FLAGg-ENaC in Xenopus laevis oocytes increases amilorid-sensitive current) [33] (expression of Myc-tagged IKK-a or IKK-b in embryonic fibroblasts MEF cell line) [50] (overexpression of a gain-of-function mutant of IKK2, IKK2-EE or IKK2CA, in transgenic mice pancreas using an inducible genetic tet system, the mice additionally overexpress CMV-rtTA protein, overview) [56] (coexpression of FLAG-atgged wild-type and mutant IKKb proteins with T7-tagged IKKg and HA-tagged ubiquitin in 293T cells) [35] (coexpression of FLAG-tagged IKKb, T7-tagged IKKg, and Tax in 293T cells and in murine embryonic fibroblasts devoid of the enzyme) [29] (coexpression of HA-tagged IKKb and GST-fusion IKKg in 293 cells, expression of GST-fusion or His-tagged IKKg protein in Escherichia coli strain BL21) [46] (coexpression of Myc-tagged NEMO/IKKg, FLAG-tagged p65, and HAtagged or GST-tagged CREB-binding protein in 293T cells or in mouse embryonic fibroblasts MEF cell line) [32] (expression of FLAG- or HA-tagged wild-type and mutant IKKb in HEK293 and HeLa cells) [55] (expression of Myc-tagged IKK-a or IKK-b in mouse embryonic fibroblasts MEF cell line) [50] (expression of mutant IKKb and IKKa in HeLa cells and in HEK293 cells, expression of His-tagged IKKb in Spodoptera frugiperda Sf21 cells via baculovirus infection) [30] (transient coexpression of IKKe, IRF3, and TBK1 or IKKb in 293T cells, transient coexpression of IFN-b or ISRE reporter genes and IKKe or TBK1 in HEK293 cells with or without FLAG or GFP tag) [47] (transient expression of HA-tagged IKKa, IKKb, and IKKa mutant L605R/F606P in Jurkat cells, transient coexpression of myc-tagged Vav-1 and IKKa in Jurkat cells) [40] (transient expression of wild-type and mutant FLAG-tagged IKK2 in HELF cells) [45] (transient expression of wild-type and mutant IKKb in HeLa cells) [69] (overexpression in Escherichia coli) [17] Engineering C179A ( site-directed mutagenesis of IKKb at the activation loop, the mutant shows reduced activation and activity compared to the wild-type enzyme, which is not restorable by TNF stimulation, activity of the mutant is partially recovered when its phosphorylation is enforced by coexpression with mitogen-activated protein kinase kinase kinases such as NF-kB inducing kinase, NIK, and MAPK/extracellular signal-regulated kinase kinase kinase 1, MEKK1, or when the serine residues are replaced with phospho-mimetic glutamate, the mutant is normal in dimer formation, while its activity abnormally responds to the change in the concentration of substrate ATP, overview [55]) [55]

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K106R ( site-directed mutagenesis, ubiquitination and phosphorylation of the mutant is unaltered compared to the wild-type IKKb [35]) [35] K147R ( site-directed mutagenesis, reduced monoubiquitination of IKKb mutant [35]) [35] K163R ( site-directed mutagenesis, monoubiquitination-defective mutant of IKKb retaining kinase activity in Tax-expressing cells [35]) [35] K171R ( site-directed mutagenesis, ubiquitination and phosphorylation of the mutant is unaltered compared to the wild-type IKKb [35]) [35] K18R ( site-directed mutagenesis, ubiquitination and phosphorylation of the mutant is unaltered compared to the wild-type IKKb [35]) [35] K198R ( site-directed mutagenesis, ubiquitination and phosphorylation of the mutant is unaltered compared to the wild-type IKKb [35]) [35] K234R ( site-directed mutagenesis, ubiquitination and phosphorylation of the mutant is unaltered compared to the wild-type IKKb [35]) [35] K238R ( site-directed mutagenesis, monoubiquitination-defective mutant of IKKb [35]) [35] K254R ( site-directed mutagenesis, ubiquitination and phosphorylation of the mutant is unaltered compared to the wild-type IKKb [35]) [35] K275R ( site-directed mutagenesis, ubiquitination and phosphorylation of the mutant is unaltered compared to the wild-type IKKb [35]) [35] K38A ( inactive IKKe mutant [57]) [57] K44A ( inactive IKKb mutant [33]; site-directed mutagenesis of subunit IKK2, catalytically inactive mutant and no NF-kB stimulation in infected cells in vivo [45]; site-directed mutagenesis, inactive ATP-binding site mutant, the kinase-deficient mutant IKK-a fails to stabilize p73 [58]) [33, 45, 58] K44M ( site-directed mutagenesis, inactive IKKa mutant [30]; site-directed mutagenesis, inactive IKKb mutant [30]) [30] K44R ( site-directed mutagenesis, monoubiquitination-defective mutant of IKKb [35]) [35] L605R/F606P ( IKKa mutant, defective in HLH domains, unable to bind wild-type Vav-1 [40]) [40] P191A ( inactive IKKb mutant [69]) [69] S177E/S181E ( site-directed mutagenesis, gain-of-function mutant of IKK2, IKK2-EE or IKK2CA, the mutant ICC2CA in pancreatic acinar cells increases tissue damage of secretagogue induced experimental pancreatitits, overview [56]) [56] Additional information ( construction of IKK knockout mice as a model system for drug development [38]; construction of IKKa-deficient and of IKKb-deficient mice and BM cell mutants, the mutants show defective osteoclastogenesis, wild-type osteoblasts can rescue osteoclastogenesis in IKKa- and IKKb-defective mutant BM cells, exogenic TNFa can rescue only IKKa-deficient mutants, TNFR1 can rescue IKKb-deficient osteoclast progenitors, but not prevent TNFa-induced apoptosis, overview [39]; construction of IKKg peptide fragements and sequence deletion mutants for determination of intermolecular interaction of IKKg required for 2-step tetramerization, overview [46]; disruption of the gene encoding IKKb leads 233

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to apoptotic tissue damage, e.g. in mucosa, and prevention of systemic inflammatory response, which results in the multiple organ dysfunction syndrome MODS [48]; IKKb-deficient B-cells are impaired in mitogenic responses to lipopolysaccharides, anti-CD40, and anti-IgM, and show high reduction of all peripheral B-cell subsets due to associated defects in cell survival [41]; IKKe and TBK1 interference RNA blocks IRF3 activation [47]; mutations of X-linked gene encoding IKKg can lead to immunodeficiency diseases interfering with NFkB-signaling [29]; disruption of Ikbke-/-, the gene encoding IKKe, results in a complete loss of the kinase in both mice and embryonic fibroblasts, generation of mice lacking IKKe, the mice produce normal amounts of IFNb, but are hypersusceptible to viral infection because of a defect in the IFN signaling pathway, phenotype, overview [72]; fibroblasts of ikkb-/- mice exhibit enhanced apoptosis in response to TNFa, NEMO/IKKg-deficient mice shows a phenotype with liver damage, but can be rescued by inactivation of the gene encoding the tumor necrosis factor-1, phenotypes, overview [54]; generation of IKKa-/- mice, an autoimmune disease phenotype is induced in athymic nude mice by grafting embryonic thymus from IKKa-deficient mice [60]; IKK knockout mice show downregulation of the CBM complex components Carma1 and Malt1, and impaired degradation of IkBa, overview [65]; IKK subunit-specific small interfering RNAs and cells deficient in individual IKK subunits show that IKKa subunit, not IKKb, is required for reovirus-induced NF-kB activation and apoptosis, overview, NF-kB activation and apoptosis are delayed in cells deficient in the IKK complex components IKKg/NEMO, overview [64]; IkkaAA/AA knockin mice, in which activation of IKKa is prevented by replacement of activation loop serines with alanines, exhibit delayed mammary gland growth during pregnancy, because IKKa activity is required for cyclin D1 induction and proliferation of lobuloalveolar epithelial cells, overview, retarded tumor development in response to either 7,12-dimethylbenzanthracene or the ErbB2/Her2 transgene but had no effect on MMTV-vHaras-induced cancer, overview [70]; IKKb-/- 3T3 fibroblasts show decreased expression of antioxidant genes, such as metallothionein 1, Mt1, IKKb null cells display a marked increase in arsenic-induced reactive oxygen species accumulation, which leads to activation of the MKK4-c-Jun NH2 -terminal kinase pathway, c-Jun phosphorylation, and apoptosis, overview [59]; small interfering RNA-mediated knockdown of endogenous IKK-a inhibits the CDDP-mediated accumulation of p73a, the kinase-deficient mutant form of IKK-a interacts with p73a, but fails to stabilize it, CDDP-mediated accumulation of endogenous p73a is not detected in mouse embryonic fibroblasts prepared from IKK-a-deficient mice, and CDDP sensitivity is significantly decreased compared to wild-type MEFs, overview [58]; TLR7/9-induced IFN-a production is severely impaired in constructed IKK-a-deficient plasmacytoid dendritic cells, whereas inflammatory cytokine induction is decreased but still occurrs, kinase-deficient IKK-a inhibits the ability of MyD88 to activate the Ifna promoter in synergy with IRF-7, expression of kinase-deficient IKK-a does not affect IRF-7-mediated promoter activation, but inhibits the enhancing effects ofMyD88 [67]; transfection of 234

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siRNA duplexes directed against IKKi and TBK1 downregulates the expression levels of both kinase isoforms by about 70% [61]) [29, 38, 39, 41, 46, 47, 48, 54, 58, 59, 60, 61, 64, 65, 67, 70, 72] Application analysis ( development of real-time imaging for continous enzyme detection and kinetics in intact cells and living mice utilizing a recombinant IkBa-firefly luciferase reporter construct, system can be used for determination of kinetics/pharmacodynamics of potential selective inhibitors, and for investigations of NF-kB signalling pathway activation [49]) [49] medicine ( IKK activates TNFa-dependent signaling pathways inducing drug resistance, e.g. increasing cell survival in anti-cancer treatment with 5-fluoro-2-deoxyuridine, overview [36]; inhibition of IKK-driven NF-kB activation offers a strategy for treatment of different malignancies and can convert inflammation-induced tumor growth to inflammation-induced tumor regression [38]; IKKa may represent a specific target for treatment of ErbB2-positive breast cancer [70]) [36, 38, 70] molecular biology ( IKK-a is a potential target for manipulating TLR-induced IFN-a production [67]) [67] pharmacology ( enzyme is a target for development of inhibitors of HIV-1 replication, overview [23]; IKK ia a good target for development of anti-rheumatic and anti-inflammatory drugs [25]; IKK is a target for development of therapeutics for treatment of diseases resulting from nuclear transcription factor NFkB pathogenesis [27]; IKKb/NF-kB inhibitors can be useful adjuvants for conventional chemotherapeutic drugs, ionizing radiation, or tumoricidal cytokines, e.g. IFNs or TRAIL [38]) [23, 25, 27, 38]

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[35] Carter, R.S.; Pennington, K.N.; Arrate, P.; Oltz, E.M.; Ballard, D.W.: Site-specific monoubiquitination of IkB kinase IKKb regulates its phosphorylation and persistent activation. J. Biol. Chem., 280, 43272-43279 (2005) [36] Wang, L.C.; Okitsu, C.Y.; Zandi, E.: Tumor necrosis factor a-dependent drug resistance to purine and pyrimidine analogues in human colon tumor cells mediated through IKK. J. Biol. Chem., 280, 7634-7644 (2005) [37] Molestina, R.E.; Sinai, A.P.: Host and parasite-derived IKK activities direct distinct temporal phases of NF-kB activation and target gene expression following Toxoplasma gondii infection. J. Cell Sci., 118, 5785-5796 (2005) [38] Luo, J.L.; Kamata, H.; Karin, M.: IKK/NF-kB signaling: balancing life and death - a new approach to cancer therapy. J. Clin. Invest., 115, 2625-2632 (2005) [39] Ruocco, M.G.; Maeda, S.; Park, J.M.; Lawrence, T.; Hsu, L.C.; Cao, Y.; Schett, G.; Wagner, E.F.; Karin, M.: IkB kinase IKKb, but not IKKa, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss. J. Exp. Med., 201, 1677-1687 (2005) [40] Piccolella, E.; Spadaro, F.; Ramoni, C.; Marinari, B.; Costanzo, A.; Levrero, M.; Thomson, L.; Abraham, R.T.; Tuosto, L.: Vav-1 and the IKKa subunit of IkB kinase functionally associate to induce NF-kB activation in response to CD28 engagement. J. Immunol., 170, 2895-2903 (2003) [41] Li, Z.W.; Omori, S.A.; Labuda, T.; Karin, M.; Rickert, R.C.: IKKb is required for peripheral B cell survival and proliferation. J. Immunol., 170, 4630-4637 (2003) [42] Yang, F.; Tang, E.; Guan, K.; Wang, C.Y.: IKKb plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J. Immunol., 170, 5630-5635 (2003) [43] Tegeder, I.; Niederberger, E.; Schmidt, R.; Kunz, S.; Guehring, H.; Ritzeler, O.; Michaelis, M.; Geisslinger, G.: Specific inhibition of IkB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in rats. J. Neurosci., 24, 1637-1645 (2004) [44] Yoon, J.W.; Kang, J.K.; Lee, K.R.; Lee, H.W.; Han, J.W.; Seo, D.W.; Kim, Y.K.: b-Carboline alkaloid suppresses NF-kB transcriptional activity through inhibition of IKK signaling pathway. J. Toxicol. Environ. Health, 68, 20052017 (2005) [45] Caposio, P.; Dreano, M.; Garotta, G.; Gribaudo, G.; Landolfo, S.: Human cytomegalovirus stimulates cellular IKK2 activity and requires the enzyme for productive replication. J. Virol., 78, 3190-3195 (2004) [46] Tegethoff, S.; Behlke, J.; Scheidereit, C.: Tetrameric oligomerization of IkB kinase g (IKKg) is obligatory for IKK complex activity and NF-kB activation. Mol. Cell. Biol., 23, 2029-2041 (2003) [47] Fitzgerald, K.A.; McWhirter, S.M.; Faia, K.L.; Rowe, D.C.; Latz, E.; Golenbock, D.T.; Coyle, A.J.; Liao, S.M.; Maniatis, T.: IKKe and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol., 4, 491-496 (2003) [48] Chen, L.W.; Egan, L.; Li, Z.W.; Greten, F.R.; Kagnoff, M.F.; Karin, M.: The two faces of IKK and NF-kB inhibition: prevention of systemic inflamma-

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[49] [50] [51] [52] [53] [54] [55] [56] [57] [58]

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[61]

[62]

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tion but increased local injury following intestinal ischemia-reperfusion. Nat. Med., 9, 575-581 (2003) Gross, S.; Piwnica-Worms, D.: Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat. Methods, 2, 607-614 (2005) Yamamoto, Y.; Verma, U.N.; Prajapati, S.; Kwak, Y.T.; Gaynor, R.B.: Histone H3 phosphorylation by IKK-a is critical for cytokine-induced gene expression. Nature, 423, 655-659 (2003) Lawrence, T.; Bebien, M.; Liu, G.Y.; Nizet, V.; Karin, M.: IKKa limits macrophage NF-kB activation and contributes to the resolution of inflammation. Nature, 434, 1138-1143 (2005) Werner, S.L.; Barken, D.; Hoffmann, A.: Stimulus specificity of gene expression programs determined by temporal control of IKK activity. Science, 309, 1857-1861 (2005) Yamamoto, Y.; Gaynor, R.B.: IkB kinases: key regulators of the NF-kB pathway. Trends Biochem. Sci., 29, 72-79 (2004) Gloire, G.; Dejardin, E.; Piette, J.: Extending the nuclear roles of IkB kinase subunits. Biochem. Pharmacol., 72, 1081-1089 (2006) Byun, M.S.; Choi, J.; Jue, D.M.: Cysteine-179 of IkB kinase b plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines. Exp. Mol. Med., 38, 546-552 (2006) Aleksic, T.; Baumann, B.; Wagner, M.; Adler, G.; Wirth, T.; Weber, C.K.: Cellular immune reaction in the pancreas is induced by constitutively active IkB kinase-2. Gut, 56, 227-236 (2007) Wietek, C.; Cleaver, C.S.; Ludbrook, V.; Wilde, J.; White, J.; Bell, D.J.; Lee, M.; Dickson, M.; Ray, K.P.; ONeill, L.A.: IkB kinase e interacts with p52 and promotes transactivation via p65. J. Biol. Chem., 281, 34973-34981 (2006) Furuya, K.; Ozaki, T.; Hanamoto, T.; Hosoda, M.; Hayashi, S.; Barker, P.A.; Takano, K.; Matsumoto, M.; Nakagawara, A.: Stabilization of p73 by nuclear IkB kinase-a mediates cisplatin-induced apoptosis. J. Biol. Chem., 282, 18365-18378 (2007) Peng, Z.; Peng, L.; Fan, Y.; Zandi, E.; Shertzer, H.G.; Xia, Y.: A critical role for IkB kinase b in metallothionein-1 expression and protection against arsenic toxicity. J. Biol. Chem., 282, 21487-21496 (2007) Kinoshita, D.; Hirota, F.; Kaisho, T.; Kasai, M.; Izumi, K.; Bando, Y.; Mouri, Y.; Matsushima, A.; Niki, S.; Han, H.; Oshikawa, K.; Kuroda, N.; Maegawa, M.; Irahara, M.; Takeda, K.; Akira, S.; Matsumoto, M.: Essential role of IkB kinase a in thymic organogenesis required for the establishment of selftolerance. J. Immunol., 176, 3995-4002 (2006) Bibeau-Poirier, A.; Gravel, S.P.; Clement, J.F.; Rolland, S.; Rodier, G.; Coulombe, P.; Hiscott, J.; Grandvaux, N.; Meloche, S.; Servant, M.J.: Involvement of the IkB kinase (IKK)-related kinases tank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation. J. Immunol., 177, 5059-5067 (2006) Ha, U.; Lim, J.H.; Jono, H.; Koga, T.; Srivastava, A.; Malley, R.; Pages, G.; Pouyssegur, J.; Li, J.D.: A novel role for IkB kinase (IKK) a and IKKb in ERK-dependent up-regulation of MUC5AC mucin transcription by Streptococcus pneumoniae. J. Immunol., 178, 1736-1747 (2007) 239

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[63] Ahn, K.S.; Sethi, G.; Aggarwal, B.B.: Simvastatin potentiates TNF-a-induced apoptosis through the down-regulation of NF-kB-dependent antiapoptotic gene products: role of IkBa kinase and TGF-b-activated kinase-1. J. Immunol., 178, 2507-2516 (2007) [64] Hansberger, M.W.; Campbell, J.A.; Danthi, P.; Arrate, P.; Pennington, K.N.; Marcu, K.B.; Ballard, D.W.; Dermody, T.S.: IkB kinase subunits a and g are required for activation of NF-kB and induction of apoptosis by mammalian reovirus. J. Virol., 81, 1360-1371 (2007) [65] Wegener, E.; Oeckinghaus, A.; Papadopoulou, N.; Lavitas, L.; Schmidt-Supprian, M.; Ferch, U.; Mak, T.W.; Ruland, J.; Heissmeyer, V.; Krappmann, D.: Essential role for IkB kinase b in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation. Mol. Cell, 23, 13-23 (2006) [66] Edwards, M.R.; Hewson, C.A.; Laza-Stanca, V.; Lau, H.T.; Mukaida, N.; Hershenson, M.B.; Johnston, S.L.: Protein kinase R, IkB kinase-b and NF-kB are required for human rhinovirus induced pro-inflammatory cytokine production in bronchial epithelial cells. Mol. Immunol., 44, 1587-1597 (2007) [67] Hoshino, K.; Sugiyama, T.; Matsumoto, M.; Tanaka, T.; Saito, M.; Hemmi, H.; Ohara, O.; Akira, S.; Kaisho, T.: IkB kinase-a is critical for interferon-a production induced by Toll-like receptors 7 and 9. Nature, 440, 949-953 (2006) [68] Lubin, F.D.; Sweatt, J.D.: The IkB kinase regulates chromatin structure during reconsolidation of conditioned fear memories. Neuron, 55, 942-957 (2007) [69] Cummins, E.P.; Berra, E.; Comerford, K.M.; Ginouves, A.; Fitzgerald, K.T.; Seeballuck, F.; Godson, C.; Nielsen, J.E.; Moynagh, P.; Pouyssegur, J.; Taylor, C.T.: Prolyl hydroxylase-1 negatively regulates IkB kinase-b, giving insight into hypoxia-induced NFkB activity. Proc. Natl. Acad. Sci. USA, 103, 1815418159 (2006) [70] Cao, Y.; Luo, J.L.; Karin, M.: IkB kinase a kinase activity is required for selfrenewal of ErbB2/Her2-transformed mammary tumor-initiating cells. Proc. Natl. Acad. Sci. USA, 104, 15852-15857 (2007) [71] Lobry, C.; Lopez, T.; Israel, A.; Weil, R.: Negative feedback loop in T cell activation through IkB kinase-induced phosphorylation and degradation of Bcl10. Proc. Natl. Acad. Sci. USA, 104, 908-913 (2007) [72] Tenoever, B.R.; Ng, S.L.; Chua, M.A.; McWhirter, S.M.; Garcia-Sastre, A.; Maniatis, T.: Multiple functions of the IKK-related kinase IKKe in interferon-mediated antiviral immunity. Science, 315, 1274-1278 (2007)

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cAMP-dependent protein kinase

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1 Nomenclature EC number 2.7.11.11 Systematic name ATP:protein phosphotransferase (cAMP-dependent) Recommended name cAMP-dependent protein kinase Synonyms PK-25 PK-A [97, 106] PKA [4, 5, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 67, 70, 71, 72, 73, 74, 75, 77, 78, 81, 82, 83, 84, 85, 86, 87, 88, 89, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 107, 108, 109, 110, 111] PKA C-a PKA C-b PKA C-g PKA Ca [76] PKAII [57] Tpk1 [100] cAMP-dependent protein kinase [4, 6, 46] cAMP-dependent protein kinase A [67, 78, 93, 104] cAMP-dependent protein kinase catalytic subunit [2, 24, 25, 29, 33, 34, 35, 36, 37, 38] cAMP-dependent protein kinase type 1 [19, 21, 22] cAMP-dependent protein kinase type 2 [19, 23, 39] cAMP-dependent protein kinase type 3 [18, 19, 20] cAMP-dependent protein kinase type I [66] cAMP-dependent protein kinase, a-catalytic subunit [1, 7, 8, 9, 10, 11, 15, 26, 27, 28, 32] cAMP-dependent protein kinase, b-1 catalytic subunit [13] cAMP-dependent protein kinase, b-2-catalytic subunit [12] cAMP-dependent protein kinase, b-catalytic subunit [14, 15, 16, 17, 30] cAMP-dependent protein kinase, g-catalytic subunit [30, 31] cAMP/protein kinase A [107] cAPK [3, 33, 68] cyclic AMP dependent protein kinase [40]

241

cAMP-dependent protein kinase

2.7.11.11

cyclic AMP-dependent kinase [3] cyclic AMP-dependent protein kinase [55, 56, 64, 65] cyclic AMP-dependent protein kinase A [83] cyclic AMP-protein kinase A [80] protein kinase A [40, 48, 49, 51, 54, 60, 61, 64, 71, 79, 82, 84, 97, 106, 110] protein kinase A type II [57] protein kinase-A [78] type I PKA [49, 66, 90] type I protein kinase A [69, 90] type II PKA [49] type II protein kinase A [69] Additional information ( see also EC 2.7.11.1 and EC 2.7.11.26 [67]; see also EC 2.7.11.26 [78]) [67, 78] CAS registry number 142008-29-5 142008-29-5 (cAMP-dependent protein kinase)

2 Source Organism













242

Cricetulus griseus (no sequence specified) [58] Cavia porcellus (no sequence specified) [54] mammalia (no sequence specified) [3] eukaryota (no sequence specified) [4, 5] Mus musculus (no sequence specified) [74, 76, 77, 82, 88, 89, 91, 95, 101, 105, 108, 111] Homo sapiens (no sequence specified) [46, 47, 52, 56, 60, 62, 63, 64, 65, 69, 72, 73, 78, 83, 85, 87, 88, 96, 102, 107, 109] Rattus norvegicus (no sequence specified) [41, 48, 53, 66, 67, 68, 71, 75, 79, 84, 87, 93, 94, 99, 103] Saccharomyces cerevisiae (no sequence specified) [86,100] Bos taurus (no sequence specified) ( There are four published structures of the Bacillus subtilis wild-type chroismate mutase (CM) with Protein Data Bank (PDB) codes 1COM, 2CHS, 2CHT, and 1DBF [45]) [45, 49, 50, 69, 73, 85, 90, 93, 110] Oryctolagus cuniculus (no sequence specified) [42, 61, 81] Zea mays (no sequence specified) [51] Pichia pastoris (no sequence specified) [40] Xenopus laevis (no sequence specified) [70, 104] Caenorhabditis elegans (no sequence specified) [97] Manduca sexta (no sequence specified) [59] Strongylocentrotus purpuratus (no sequence specified) [55] Mytilus galloprovincialis (no sequence specified) [43, 44, 92] Danio rerio (no sequence specified) [98] Caenorhabditis briggsae (no sequence specified) [97]

2.7.11.11

















cAMP-dependent protein kinase

Paramecium primaurelia (UNIPROT accession number: O00843) [6] Bos taurus (UNIPROT accession number: P00517) [7, 8, 9, 10, 11] Bos taurus (UNIPROT accession number: P24256) [12] Bos taurus (UNIPROT accession number: P05131) [13] Mus musculus (UNIPROT accession number: P68181) [14, 15, 16, 17] Cricetulus griseus (UNIPROT accession number: P68180) [15] Saccharomyces cerevisiae (UNIPROT accession number: P05986) [18, 19, 20] Saccharomyces cerevisiae (UNIPROT accession number: P06244) [19, 21, 22] Saccharomyces cerevisiae (UNIPROT accession number: P06245) [19, 23, 39] Drosophila melanogaster (UNIPROT accession number: P12370) [2, 24, 25] Homo sapiens (UNIPROT accession number: P17612) [26, 27, 28] Caenorhabditis elegans (UNIPROT accession number: P21137) [29] Homo sapiens (UNIPROT accession number: P22612) [30, 31] Homo sapiens (UNIPROT accession number: P22694) [30] Cricetulus griseus (UNIPROT accession number: P25321) [15] Rattus norvegicus (UNIPROT accession number: P27791) [32] Dictyostelium discoideum (UNIPROT accession number: P34099) [33, 34, 35] Schizosaccharomyces pombe (UNIPROT accession number: P40376) [36, 37] Ascaris suum (UNIPROT accession number: P49673) [38] Mus musculus (UNIPROT accession number: P05132) [1] Heteropneustes fossilis (no sequence specified) [80] Apis mellifera (UNIPROT accession number: Q6ZXJ1) [57] Amblyomma hebraeum (no sequence specified) [106]

3 Reaction and Specificity Catalyzed reaction ATP + a protein = ADP + a phosphoprotein ( catalytic mechanism [52]; activation mechanism [49,90]; activation involves the activation loop, a polypeptide region outside the active site cleft, which is reversibly phosphorylated at Thr197, phosphorylation leads to enzyme inhibition, activation mechanism, overview [4]; active site residues and structure, mechanism of ligand binding and ligand-induced conformational changes [76]; catalytic mechanism, active site structure, Tyr204 is required and involved in the hydrophobic network, substrate-induced interaction of Tyr204 and Asp166, overview [77]; catalytic site structure, Asp166 is the catalytic base invariant in all kinases, activation mechanism, overview [3]; random kinetic mechanism, active site structure with key residues E91, K72, N171, K168, the general base catalyst D166, and essential catalytic D184, overview, reaction mechanism [5]; substrate binding site structure invol243

cAMP-dependent protein kinase

2.7.11.11

ving E230, the electrostatic surface is important for substrate binding and catalysis, and also for the mechanism of closing the active site cleft of subunit C, conformation and mechanism, overview, the catalytic loop consists of residues Arg165-Asn171 [89]; the substrate binds to the regulatory subunit which is released from the catalytic subunit for enzyme activity, mechanism and structure-function relationship [48]; conserved residues K336 and H338 are important for catalytic activity, R324 is important in substrate binding [100]; phosphoryltransfer mechanism, substrate-binding site structure, the phosphorylated Thr197 in the catalytic subunit of cAMP-dependent protein is essential for activity, overview [111]) Natural substrates and products S ATP + A-kinase anchor protein ( phosphorylation at Ser1928 [94]) (Reversibility: ?) [94] P ADP + phosphorylated A-kinase anchor protein S ATP + BKCa channel ZERO ( recombinant murine HA-tagged tetrameric protein expressed in HEK-293 cells, activity with wild-type and mutant Y334V channel tetramers, but no activity with S899A mutant, phosphorylation at Ser899 activates the channels, overview [85]) (Reversibility: ?) [85] P ADP + phosphorylated BKCa channel ZERO S ATP + CRE-binding protein ( i.e. CREB, a transcription factor, activition by type I PKA [66]) (Reversibility: ?) [66] P ADP + CRE-binding phosphoprotein S ATP + CREB-binding protein ( i.e. CBP, activition by type II PKA [66]) (Reversibility: ?) [66] P ADP + CREB-binding phosphoprotein S ATP + Cav1.2 ( in anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons plays a critical role involving phosphorylation by the enzyme, PKA increases the activity of the L-type Ca2+ channel Cav1.2 in response to b-adrenergic stimulation in heart and brain [94]) (Reversibility: ?) [94] P ADP + phosphorylated Cav1.2 S ATP + Dot6 ( a protein implicated in telomere function [100]) (Reversibility: ?) [100] P ADP + phosphorylated Dot6 S ATP + EPS8 protein ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + EPS8 phosphoprotein S ATP + G protein-coupled receptor GRK1 ( recombinant FLAGtagged GRK1 expressed in HEK-293 cells, enzymatic inactivation of the receptor activity inhibiting rhodopsin phosphorylation [73]) (Reversibility: ?) [73] P ADP + phosphorylated G protein-coupled receptor GRK1 S ATP + G protein-coupled receptor GRK7 ( recombinant FLAGtagged GRK1 expressed in HEK-293 cells, enzymatic inactivation of the

244

2.7.11.11

P S

P S P S P S

P S

P S

P S P S P S P S

P S

P S

cAMP-dependent protein kinase

receptor activity inhibiting rhodopsin phosphorylation [73]) (Reversibility: ?) [73] ADP + phosphorylated G protein-coupled receptor GRK7 ATP + IP3R-2 ( phosphoregulation of the inositol 1,4,5-trisphosphate receptor subtype 2, PKA enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells, regulation, overview [103]) (Reversibility: ?) [103] ADP + phosphorylated IP3R-2 ATP + PDE11A protein ( sperm protein substrate [55]) (Reversibility: ?) [55] ADP + PDE11A phosphoprotein ATP + PDE5A protein ( sperm protein substrate [55]) (Reversibility: ?) [55] ADP + PDE5A phosphoprotein ATP + RGS protein ( recombinant HA-tagged substrate expressed in B35 cells, phosphorylation of RGS14 by PKA potentiates its activity toward Gai-GDP [48]) (Reversibility: ?) [48] ADP + RGS protein phosphate ATP + [t-protein] ( abnormal hyperphosphorylation of t by PKA is associated with Alzheimers disease and other tauopathies leading to neuronal degeneration [67]) (Reversibility: ?) [67, 78] ADP + O-phospho-[t-protein] ATP + a protein ( regulation by reversible phosphorylation, overview [4]; regulation of the enzyme involves reversible phosphorylation at the activation loop, and associative or dissociative mechanisms [5]; the enzyme is a key player in cellular responses to the second messenger cAMP [3]) (Reversibility: ?) [3, 4, 5] ADP + a phosphoprotein ATP + actin ( low activity [92]; sperm protein substrate [55]) (Reversibility: ?) [55, 92] ADP + phosphorylated actin ATP + adenylate kinase 1 ( sperm protein substrate [55]) (Reversibility: ?) [55] ADP + phosphorylated adenylate kinase 1 ATP + adenylate kinase 5 ( sperm protein substrate [55]) (Reversibility: ?) [55] ADP + phosphorylated adenylate kinase 5 ATP + autophagy-related protein kinase Atg1 ( initiating the degradative pathway, PKA regulates the association of Atg1 with the preautophagosomal structure PAS, overview [86]) (Reversibility: ?) [86] ADP + phosphorylated autophagy-related protein kinase Atg1 ATP + b-adrenergic receptor ( PKA phosphorylation mediates b1 adrenergic receptor endocytosis via the caveolae pathway [60]) (Reversibility: ?) [60] ADP + phosphorylated b-adrenergic receptor ATP + b1 -adrenergic receptor ( recombinant b1 -adrenergic receptor expressed in HEK-293 cell membranes, phosphorylation at Ser312 245

cAMP-dependent protein kinase

P S

P S P S

P S

P S P S

P S

P S

P S

P

246

2.7.11.11

is essential for activation of endocytic recycling of the agonist-internalized b1 -adrenergic receptor, b1 -AR mutant S312A is not recycled, overview [65]) (Reversibility: ?) [65] ADP + phosphorylated b1 -adrenergic receptor ATP + cAMP-responsive element binding protein ( the enzyme as well as GABAB receptors are involved in induction of cAMP-responsive element binding protein phosphorylation in hippocampus by g-hydroxybutyrate, overview [108]) (Reversibility: ?) [108] ADP + phosphorylated cAMP-responsive element binding protein ATP + catchin ( low activity [92]) (Reversibility: ?) [92] ADP + phosphorylated catchin ATP + cellular nucleic acid binding protein ( CNBP performs a fine tune expression regulation of a group of target including c-myc, during vertebrate embryogenesis, different phosphorylation patterns at different development al stages, overview [98]) (Reversibility: ?) [98] ADP + phosphorylated ATP + claudin-3 ( high activity in ovarian cancer cells with recombinantly overexpressed claudin-3, phosphorylation of claudin-3 affecting the barrier function with extracellular Ca2+ , overview [72]) (Reversibility: ?) [72] ADP + phosphorylated claudin-3 ATP + creatine kinase ( sperm protein substrate [55]) (Reversibility: ?) [55] ADP + phosphorylated creatine kinase ATP + ezrin ( recombinant GST-tagged wild-type ezrin transiently expressed in primary gastric parietal cells from stomach, recombinant mutant S66A and S66D ezrin proteins are no substrates, phosphorylation of ezrin leads to activation of parietal cells required for dilation of apical vacuolar membrane and histamine-stimulated acid secretion in gastric mucosa, overview [61]) (Reversibility: ?) [61] ADP + phosphoezrin ATP + histone deacetylase 8 ( FLAG-tagged class 1 histone deacetylase HDAC8 expressed in HeLa cells via adenovirus infection, phosphorylation at Ser39 reduces the enzyme activity of HDAC8, hyperphosphorylation inhibits the enzyme [83]) (Reversibility: ?) [83] ADP + phosphorylated histone deacetylase 8 ATP + lactate dehydrogenase subunit A mRNA ( the enzyme stabilizes lactate dehydrogenase LDH-A mRNA and increases intracellular LDH-A mRNA levels by phosphorylation of a cAMP-stabilizing region CSR on the 3-untranslated region of the LDH-A mRNA, regulation, mechanism [71]) (Reversibility: ?) [71] ADP + 3’-UTR phosphorylated lactate dehydrogenase subunit A mRNA ATP + merlin ( recombinant human substrate protein expressed in HEK-293 cells, activity of the catalytic subunit C, phosphorylation at Ser518 induces N-terminal binding of merlin to ezrin [64]) (Reversibility: ?) [64] ADP + phosphorylated merlin

2.7.11.11

cAMP-dependent protein kinase

S ATP + mitogen-activated protein kinase phosphatase-1 ( the enzyme enhances steroid hydroxylase CYP17 transcription via mitogen-activated protein kinase phosphatase-1 MKP-1 activation in H295R adrenocortical cells [63]) (Reversibility: ?) [63] P ADP + phosphorylated mitogen-activated protein kinase phosphatase-1 S ATP + paramyosin ( low activity [92]) (Reversibility: ?) [92] P ADP + phosphorylated paramyosin S ATP + ribosomal S6 protein (Reversibility: ?) [84] P ADP + phosphorylated ribosomal S6 protein S ATP + twitchin (Reversibility: ?) [92] P ADP + phosphorylated twitchin S ATP + type III inositol 1,4,5-trisphosphate receptor ( i.e. IP3 receptor, recombinantly expressed in HEK cells, forms tetrameric Ca2+ channels in the endoplasmic reticulum [47]) (Reversibility: ?) [47] P ADP + phosphorylated type III inositol 1,4,5-trisphosphate receptor S ATP + tyrosine hydroxylase ( enzyme is involved in the signal transduction regulatory mechanism in the triiodothyronine T3-activation of forebrain tyrosine hydroxylase [80]) (Reversibility: ?) [80] P ADP + phosphorylated tyrosine hydroxylase S ATP + ubiquinol-cytochrome c reductase complex core protein 2 ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + ubiquinol-cytochrome c reductase complex core phosphoprotein 2 S Additional information ( enzyme plays an essential role during differentiation and fruit morphogenesis in Dictyostelium discoideum [33]; the enzyme plays a central role in the control of mammalian sperm capacitation and motility [26]; activated PKA catalytic subunit C with cAMP or 8-bromocAMP, stimulated by b1 -adrenoreceptor activity, inhibits store-operated channel current activity in vascular tissue [81]; cAMP concentration and PKA activity are increased and important in exocytotic acrosome reaction taking place in fertilization when sperm contacts the egg jelly layer EJ, EJ component fucose sulfate polymer triggers sperm PKA activation [55]; cAMP-dependent activation of BKCa channels in pulmonary arterial smooth muscle is not catalyzed by PKA but by cGMPdependent protein kinase PKG, thus PKA is not involved in cAMP-induced signaling in pulmonary vasodilation [41]; enzyme regulation, overview [94]; induced by the pituitary follicle-stimulating hormone FSH PKA also catalyzes the dephosphorylation of residues T421 and S424 of the autoinhibitory domain of p70S6K, resulting in activation of p70S6K important for differentiation of Sertoli cells in male reproduction, regulation overview [84]; inositol 1,4,5-trisphosphate receptor-I is no substrate of PKA in vivo [42]; merlin acts as an PKA-anchoring protein being linked to the cAMP/PKA signaling pathway [64]; no activity with claudin-4 [72]; oestrogen-dependent increase in cAMP increases Ca2+ -dependent exocytosis through protein kinase A-dependent pathway and through alternative cAMP-guanine nucleotide exchange factor GEF/ Epac-dependent pathway in secretory cells [82]; PKA regulatory and 247

cAMP-dependent protein kinase

2.7.11.11

catalytic subunits are bound in the CSR complex including the protein kinase A anchoring protein AKAP 95 and several CSR-binding proteins, the complex acts in stabilizing the LDH-A mRNA, overview [71]; PKA type I regulates ethanol-induced cAMP response element-mediated gene expression via activation of CREB-binding protein and inhibition of MAPK, regulation overview [66]; PKA works coordinatedly with GSK3b, EC 2.7.11.1, on t phosphorylation [78]; regulation of GRK1 and GRK7 by cAMP level during light and dark phases [73]; tau becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain, inhibition of tau hyperphosphorylation inhibits an associated loss in spatial memory [67]; the enzyme activates NS1619-induced flavoprotein oxidation modulating mitochondrial Ca2+ -activated K+ channels, mechanism of the cardioprotective effect and modulation by PKA, overview [54]; the enzyme interacts with the neuron cortex membrane protein gravin via Ca2+ -independent binding to the regulatory RII enzyme subunit, gravin provides a platform to localize kinases, besides PKA also PKCa and PKCbII, in an isozyme-specific and activation-dependent manner at specific sites in neurons, gravin is strongly upregulated in cells during differentiation, overview [62]; the enzyme is not required for adenosine-induced dilation of intracerebral arterioles and regulation of cerebral blood flow [75]; the enzyme plays a central role in the adipokinetic signaling controling the mobilization of stored lipids in the fat body [59]; the enzyme plays a paradoxical role in cell motility facilitating and inhibiting actin cytoskeletal dynamics and cell migration, overview, the enzyme is regulated in a subcellular space during cell migration, regulatory subunits RII are enriched in protrusive cellular structures and pseudopodia formed during chemotaxis, anchoring of PKA inhibits pseudopod formation and cell migration, regulation of the process overview [87]; the enzyme regulates the G2/M transition in oocytes of Xenopus laevis, it also blocks progesterone-induced germinal vesicle envelope breakdown GVBD, Cdc-25-dependent dephosphorylation of Tyr15 in Cdc2, and synthesis of MEKK Mos [70]; cystic fibrosis results from mutations in the cystic fibrosis conductance regulator protein, CFTR, a cAMP/protein kinase A, PKA, and ATP-regulated Cl- channel, the formation of the cAMP/protein kinase Adependent annexin 2 S100A10 complex with cystic fibrosis conductance regulator protein, CFTR, regulates CFTR channel function, overview, PKA regulates anx 2 and S100A10 cellular distribution in HNE cells, modulation of PKA activity alters localization and distribution of anx 2’100A10 in HNE cells, overview [107]; inhibition of caspase-dependent spontaneous apoptosis via a cAMP-protein kinase A dependent pathway in neutrophils from sickle cell disease patients, overview [96]; PKA plays a crucial role in the release of the catch state of molluskan muscles, mechanism, overview [92]; redox regulation of cAMP-dependent protein kinase signaling: kinase versus phosphatase inactivation, in HeLa cells PKA activity follows a biphasic response to thiol oxidation, overview [102]; the enzyme is involved in cyclic nucleotide signaling, 248

2.7.11.11

cAMP-dependent protein kinase

overview, individually phosphorylated PKA-R isozymes are differentially targeted to distinct cellular compartments by AKAP-isozymes, providing a multifaceted platform for the kinase [105]; the enzyme plays a central role in regulation of diverse aspects of cellular activity, especially in the controlling of maturation of spermatids [106]) (Reversibility: ?) [26, 33, 41, 42, 54, 55, 59, 62, 64, 66, 67, 70, 71, 72, 73, 75, 78, 81, 82, 84, 87, 92, 94, 96, 102, 105, 106, 107] P ? Substrates and products S ATP + 5HT7 receptor ( recombinant rat 5HT7 receptor reconstituted in frog oocytes, overview [104]) (Reversibility: ?) [104] P ADP + phosphorylated 5HT7 receptor S ATP + A-kinase anchor protein ( phosphorylation at Ser1928 [94]; i.e. AKAP, phosphorylation at Ser1928, the rat enzyme is active with different AKAP substrate variants from different sources, overview [94]) (Reversibility: ?) [94] P ADP + phosphorylated A-kinase anchor protein S ATP + BKCa channel ZERO ( recombinant murine HA-tagged tetrameric protein expressed in HEK-293 cells, activity with wild-type and mutant Y334V channel tetramers, but no activity with S899A mutant, phosphorylation at Ser899 activates the channels, overview [85]; recombinant murine HA-tagged tetrameric protein expressed in HEK-293 cells, activity with wild-type and mutant Y334V channel tetramers, but no activity with S899A mutant, phosphorylation at Ser899, overview [85]; recombinant murine HA-tagged tetrameric protein expressed in HEK-293 cells, phosphorylation at Ser899 activates the channel [85]) (Reversibility: ?) [85] P ADP + phosphorylated BKCa channel ZERO S ATP + CRE-binding protein ( i.e. CREB, a transcription factor, activition by type I PKA [66]) (Reversibility: ?) [66] P ADP + CRE-binding phosphoprotein S ATP + CREB-binding protein ( i.e. CBP, activition by type II PKA [66]) (Reversibility: ?) [66] P ADP + CREB-binding phosphoprotein S ATP + Cav1.2 ( in anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons plays a critical role involving phosphorylation by the enzyme, PKA increases the activity of the l-type Ca2+ channel Cav1.2 in response to b-adrenergic stimulation in heart and brain [94]; an L-type calcium channel Cav1.2 [94]) (Reversibility: ?) [94] P ADP + phosphorylated Cav1.2 S ATP + Cav3.2 T-type Ca2+ channel ( recombinant human Ca2+ channel reconstituted in frog oocytes, overview [104]) (Reversibility: ?) [104] P ADP + phosphorylated Cav3.2 T-type Ca2+ channel

249

cAMP-dependent protein kinase

2.7.11.11

S ATP + ChChd3 ( an endogenous mitochondrial protein [101]) (Reversibility: ?) [101] P ADP + phosphorylated ChChd3 S ATP + Dot6 ( a protein implicated in telomere function [100]; a protein implicated in telomere function, substrate of Tpk1 [100]) (Reversibility: ?) [100] P ADP + phosphorylated Dot6 S ATP + EPS8 protein ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + EPS8 phosphoprotein S ATP + G protein-coupled receptor GRK1 ( recombinant FLAGtagged GRK1 expressed in HEK-293 cells, enzymatic inactivation of the receptor activity inhibiting rhodopsin phosphorylation [73]; phosphorylation at Ser21, enzymatic inactivation of the receptor activity inhibiting rhodopsin phosphorylation, no activity with GRK1 mutant S21A [73]; recombinant FLAG-tagged GRK1, phosphorylation at Ser21 [73]) (Reversibility: ?) [73] P ADP + phosphorylated G protein-coupled receptor GRK1 S ATP + G protein-coupled receptor GRK7 ( recombinant FLAGtagged GRK1 expressed in HEK-293 cells, enzymatic inactivation of the receptor activity inhibiting rhodopsin phosphorylation [73]; phosphorylation at Ser23 and Ser36, enzymatic inactivation of the receptor activity inhibiting rhodopsin phosphorylation, no activity with GRK1 mutants S23A/S36A and S23E/S36E [73]; recombinant FLAG-tagged GRK7, phosphorylation at Ser23 and Ser36 [73]) (Reversibility: ?) [73] P ADP + phosphorylated G protein-coupled receptor GRK7 S ATP + IP3R-2 ( phosphoregulation of the inositol 1,4,5-trisphosphate receptor subtype 2, PKA enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells, regulation, overview [103]) (Reversibility: ?) [103] P ADP + phosphorylated IP3R-2 S ATP + Kemptide ( activity of catalytic PKA subunit [58,69]; LRRASLG peptides substrate [74]; peptide substrate, activity of catalytic PKA subunit [70]) (Reversibility: ?) [58, 67, 69, 70, 74, 78] P ADP + phosphorylated Kemptide ( LRRA-phosphoserine-LG [74]) S ATP + Kemptide ( activity of the catalytic subunit C [43, 44]; activity of the catalytic subunit Ca [88]; catalytic subunit C [49]; enzyme activity is preferably activated by cAMP, 80% lower activity with cGMP [40]; LRRASLG peptide substrate [77]; LRRASLG peptide substrate, activity of the catalytic subunit C [90]; PKA-derived substrate, activity of the catalytic subunit Cg [88]) (Reversibility: ?) [40, 43, 44, 49, 77, 85, 88, 90] P ADP + Kemptide phosphate S ATP + l-pyruvate kinase ( recombinant rat protein substrate expressed in Escherichia coli, phosphorylation at Ser12, activity of catalytic PKA subunit [51]) (Reversibility: ?) [51] 250

2.7.11.11

cAMP-dependent protein kinase

P ADP + phosphorylated l-pyruvate kinase S ATP + LRRASLG ( i.e. Kemptide [59]; i.e. Kemptide, recombinant PKA catalytic subunit [52]) (Reversibility: ?) [52, 59] P ADP + LRRA-phosphoserine-LG S ATP + Nav1.4 T-type Na+ channel ( recombinant rat Na+ channel reconstituted in frog oocytes, overview [104]) (Reversibility: ?) [104] P ADP + phosphorylated Nav1.4 T-type Na+ channel S ATP + PDE11A protein ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + PDE11A phosphoprotein S ATP + PDE5A protein ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + PDE5A phosphoprotein S ATP + PLARTLSVAGLPGKK ( syntide 2-derived peptide substrate [59]) (Reversibility: ?) [59] P ADP + PLARTL-phosphoserine-VAGLPGKK S ATP + RFARKGSLREKNV ( protein kinase C-derived peptide, activity of the catalytic subunit Ca [88]; protein kinase C-derived peptide, activity of the catalytic subunit Cg [88]) (Reversibility: ?) [88] P ADP + RFARKG-phosphoserine-LREKNV S ATP + RFARKGSLRQKNV ( PKC-derived peptide substrate [59]) (Reversibility: ?) [59] P ADP + RFARKG-phosphoserine-LRQKNV S ATP + RGS protein ( recombinant HA-tagged substrate expressed in B35 cells, phosphorylation of RGS14 by PKA potentiates its activity toward Gai-GDP [48]; i.e. regulator of G protein signaling protein, recombinant His-tagged substrate R14-RSG residues 1-205 and 299-544, phosphorylation of wild-type R14-RSG at Ser258 and Thr494 [48]) (Reversibility: ?) [48] P ADP + RGS protein phosphate S ATP + RKRSRAE ( cGPK-1-derived peptide, activity of the catalytic subunit Ca [88]; cGPK-1-derived peptide, activity of the catalytic subunit Cg [88]) (Reversibility: ?) [88] P ADP + RKR-phosphoserine-RAE S ATP + RKRSRKE ( cGPK-2-derived peptide, activity of the catalytic subunit Ca [88]; cGPK-2-derived peptide, activity of the catalytic subunit Cg [88]) (Reversibility: ?) [88] P ADP + RKR-phosphoserine-RKE S ATP + RRASVA ( pyruvate kinase-derived peptide substrate comprising the phosphorylation site around Ser12 of the protein, activity of catalytic PKA subunit [51]) (Reversibility: ?) [51] P ADP + RRA-phosphoserine-VA S ATP + RRLSSLRA ( S6 kinase-derived peptide, activity of the catalytic subunit Ca [88]; S6 kinase-derived peptide, activity of the catalytic subunit Cg [88]) (Reversibility: ?) [88] P ADP + RRL-phosphoserine-phosphoserine-LRA 251

cAMP-dependent protein kinase

2.7.11.11

S ATP + SP20 ( substrate peptide, the C subunit SP20-binding residues are E203, F129, E170, E230, D166, and K168 [76]) (Reversibility: ?) [76] P ADP + SP20 phosphate S ATP + VASP ( i.e. vasodilator-stimulated phosphoprotein [87]) (Reversibility: ?) [87] P ADP + phosphorylated VASP S ATP + [t-protein] ( abnormal hyperphosphorylation of tau by PKA is associated with Alzheimers disease and other tauopathies leading to neuronal degeneration [67]; phosphorylation by PKA at Ser214, PKA catalyzes the reaction of EC 2.7.11.26 in brain [67]) (Reversibility: ?) [67, 78] P ADP + O-phospho-[t-protein] S ATP + a protein ( regulation by reversible phosphorylation, overview [4]; regulation of the enzyme involves reversible phosphorylation at the activation loop, and associative or dissociative mechanisms [5]; the enzyme is a key player in cellular responses to the second messenger cAMP [3]; phosphorylates nuclear proteins important for gene replication [3]; the enzyme phosphorylates the peptide substrate LRRASLG [5]) (Reversibility: ?) [3, 4, 5, 89] P ADP + a phosphoprotein S ATP + actin ( low activity [92]; sperm protein substrate [55]) (Reversibility: ?) [55, 92] P ADP + phosphorylated actin S ATP + adenylate kinase 1 ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + phosphorylated adenylate kinase 1 S ATP + adenylate kinase 5 ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + phosphorylated adenylate kinase 5 S ATP + autophagy-related protein kinase Atg1 ( initiating the degradative pathway, PKA regulates the association of Atg1 with the preautophagosomal structure PAS, overview [86]; phosphorylation at Ser508 and Ser515 [86]) (Reversibility: ?) [86] P ADP + phosphorylated autophagy-related protein kinase Atg1 S ATP + b-adrenergic receptor ( PKA phosphorylation mediates b1 adrenergic receptor endocytosis via the caveolae pathway [60]; mouse wild-type receptor, cytosolic phosphorylation domain near the transmembrane domains of the substrate, overview [60]) (Reversibility: ?) [60] P ADP + phosphorylated b-adrenergic receptor S ATP + b1 -adrenergic receptor ( recombinant b1 -adrenergic receptor expressed in HEK-293 cell membranes, phosphorylation at Ser312 is essential for activation of endocytic recycling of the agonist-internalized b1 -adrenergic receptor, b1 -AR mutant S312A is not recycled, overview [65]; phosphorylation at Ser312 in the third intracellular loop

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P S

P S P S P S

P S

P S

P S P S

P S

cAMP-dependent protein kinase

of the receptor protein by the PKA catalytic subunit [65]) (Reversibility: ?) [65] ADP + phosphorylated b1 -adrenergic receptor ATP + cAMP-responsive element binding protein ( i.e. CREB [108]; the enzyme as well as GABAB receptors are involved in induction of cAMP-responsive element binding protein phosphorylation in hippocampus by g-hydroxybutyrate, overview [108]) (Reversibility: ?) [108] ADP + phosphorylated cAMP-responsive element binding protein ATP + casein ( enzyme activity is preferably activated by cAMP, 25% lower activity with cGMP [40]) (Reversibility: ?) [40] ADP + phosphorylated casein ATP + catchin ( low activity [92]) (Reversibility: ?) [92] ADP + phosphorylated catchin ATP + ( CNBP performs a fine tune expression regulation of a group of target including c-myc, during vertebrate embryogenesis, different phosphorylation patterns at different development al stages, overview [98]; i.e. CNBP, a zinc finger protein, phosphorylation at multiple sites, putative phosphorylation sites are residues Ser4, Thr56, Ser70, and Ser158, substrate is recombinant GST- or His6-tagged CNBP [98]) (Reversibility: ?) [98] ADP + phosphorylated cellular nucleic acid binding protein ATP + claudin-3 ( high activity in ovarian cancer cells with recombinantly overexpressed claudin-3, phosphorylation of claudin-3 affecting the barrier function with extracellular Ca2+ , overview [72]; recombinant GST-fusion claudin-3 expressed in Escherichia coli, phosphorylation at Thr192, activity of catalytic PKA subunit [72]) (Reversibility: ?) [72] ADP + phosphorylated claudin-3 ATP + claudin-3 S199A ( recombinant GST-fusion claudin-3 mutant S199A expressed in Escherichia coli, phosphorylation at Thr192, activity of catalytic PKA subunit is lower compared to wild-type claudin-3 [72]) (Reversibility: ?) [72] ADP + phosphorylated claudin-3 S199A ATP + creatine kinase ( sperm protein substrate [55]) (Reversibility: ?) [55] ADP + phosphorylated creatine kinase ATP + ezrin ( recombinant GST-tagged wild-type ezrin transiently expressed in primary gastric parietal cells from stomach, recombinant mutant S66A and S66D ezrin proteins are no substrates, phosphorylation of ezrin leads to activation of parietal cells required for dilation of apical vacuolar membrane and histamine-stimulated acid secretion in gastric mucosa, overview [61]; phosphorylation at Ser66, catalytic subunit of PKA [61]) (Reversibility: ?) [61] ADP + phosphoezrin ATP + fat body triglyceride lipase ( protein substrate is phosphorylated at Ser563, but not activated by the enzyme, Ser563 is the regulatory site of the enzyme substrate [59]) (Reversibility: ?) [59] 253

cAMP-dependent protein kinase

P S P S P S P S P S P S

P S

P S P S

P S

P S

P

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ADP + phosphorylated fat body triglyceride lipase ATP + histone (Reversibility: ?) [59] ADP + phosphorylated histone ATP + histone 3s ( activity of the catalytic subunit Cg [88]) (Reversibility: ?) [88] ADP + phosphorylated histone 3s ATP + histone H2A ( enzyme activity is preferably activated by cAMP, 40% lower activity with cGMP [40]) (Reversibility: ?) [40] ADP + phosphorylated histone H2A ATP + histone H3S ( enzyme activity is preferably activated by cAMP, 60% lower activity with cGMP [40]) (Reversibility: ?) [40] ADP + phosphorylated histone H3S ATP + histone IIIS ( activity of the catalytic subunit Cg [88]) (Reversibility: ?) [88] ADP + phosphorylated histone IIIS ATP + histone deacetylase 8 ( FLAG-tagged class 1 histone deacetylase HDAC8 expressed in HeLa cells via adenovirus infection, phosphorylation at Ser39 reduces the enzyme activity of HDAC8, hyperphosphorylation inhibits the enzyme [83]; class 1 histone deacetylase HDAC8, recombinantly expressed FLAG-tagged GST-fusion substrate protein, Ser39 is the major phosphorylation site, low activity with HDAC8 mutant S39A [83]) (Reversibility: ?) [83] ADP + phosphorylated histone deacetylase 8 ATP + inositol 1,4,5-trisphosphate receptor-I ( isolated catalytic subunit of PKA, selective phosphorylation at Ser1589 and Ser1755 [42]) (Reversibility: ?) [42] ADP + phosphorylated inositol 1,4,5-trisphosphate receptor-I ATP + kemptide (Reversibility: ?) [5, 92, 95, 101, 102] ADP + phospho-kemptide ATP + lactate dehydrogenase subunit A mRNA ( the enzyme stabilizes lactate dehydrogenase LDH-A mRNA and increases intracellular LDH-A mRNA levels by phosphorylation of a cAMP-stabilizing region CSR on the 3-untranslated region of the LDH-A mRNA, regulation, mechanism [71]) (Reversibility: ?) [71] ADP + 3’-UTR phosphorylated lactate dehydrogenase subunit A mRNA ATP + maltose binding protein ( enzyme activity is preferably activated by cAMP, 35% lower activity with cGMP [40]) (Reversibility: ?) [40] ADP + phosphorylated maltose binding protein ATP + merlin ( recombinant human substrate protein expressed in HEK-293 cells, activity of the catalytic subunit C, phosphorylation at Ser518 induces N-terminal binding of merlin to ezrin [64]; a membrane/cytoskeleton linker protein, activity of the catalytic subunit C, phosphorylation at the N- and the C-terminus, e.g. at Ser518 [64]) (Reversibility: ?) [64] ADP + phosphorylated merlin

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S ATP + mitogen-activated protein kinase phosphatase-1 ( the enzyme enhances steroid hydroxylase CYP17 transcription via mitogen-activated protein kinase phosphatase-1 MKP-1 activation in H295R adrenocortical cells [63]; i.e. MKP-1 [63]) (Reversibility: ?) [63] P ADP + phosphorylated mitogen-activated protein kinase phosphatase-1 S ATP + paramyosin ( low activity [92]) (Reversibility: ?) [92] P ADP + phosphorylated paramyosin S ATP + phosphatase inhibitor PKI (Reversibility: ?) [57] P ADP + phosphorylated phosphatase inhibitor PKI S ATP + phosvitin ( enzyme activity is preferably activated by cAMP, 33% lower activity with cGMP [40]) (Reversibility: ?) [40] P ADP + phosphorylated phosvitin S ATP + protamine sulfate ( enzyme activity is about equally activated by cAMP and cGMP, and derivatives of cAMP [40]) (Reversibility: ?) [40] P ADP + phosphoprotamine sulfate S ATP + protein tyrosine phosphatase ( i.e. PTP, contains a PEST motif [87]) (Reversibility: ?) [87] P ADP + phosphorylated protein tyrosine phosphatase S ATP + pyruvate kinase ( enzyme activity is about equally activated by cAMP and cGMP [40]) (Reversibility: ?) [40] P ADP + phosphorylated pyruvate kinase S ATP + ribosomal S6 protein (Reversibility: ?) [84] P ADP + phosphorylated ribosomal S6 protein S ATP + ribosomal protein S6 ( substrate from purified Saccharomyces cerevisiae ribosomes, enzyme activity is preferably activated by cAMP, lower activity with cGMP [40]) (Reversibility: ?) [40] P ADP + phosphorylated ribosomal protein S6 S ATP + twitchin ( a high molecular mass protein associated with thick filaments, high activity [92]) (Reversibility: ?) [92] P ADP + phosphorylated twitchin S ATP + type III inositol 1,4,5-trisphosphate receptor ( i.e. IP3 receptor, recombinantly expressed in HEK cells, forms tetrameric Ca2+ channels in the endoplasmic reticulum [47]; phosphorylation sites are Ser916, Ser934, and Ser1832, Ser934 is the most susceptible site [47]) (Reversibility: ?) [47] P ADP + phosphorylated type III inositol 1,4,5-trisphosphate receptor S ATP + tyrosine hydroxylase ( enzyme is involved in the signal transduction regulatory mechanism in the triiodotyronone T3-activation of forebrain tyrosine hydroxylase [80]; 4 isoforms of the human enzyme as substrate, phosphorylation at Ser40 of the regulatory subunit of the substrate results in release of bound inhibiting catecholamines, e.g. dopamine and dihydroxyphenylalanine, from the tyrosine hydroxylase [79]; activity of catalytic PKA subunit [69]) (Reversibility: ?) [69, 79, 80] P ADP + phosphorylated tyrosine hydroxylase

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S ATP + ubiquinol-cytochrome c reductase complex core protein 2 ( sperm protein substrate [55]) (Reversibility: ?) [55] P ADP + ubiquinol-cytochrome c reductase complex core phosphoprotein 2 S N6 -benzyl-ATP + ChChd3 ( cofactor of mutant M120G, poor activity with the wild-type catalytic subunit [101]) (Reversibility: ?) [101] P N6 -benzyl-ADP + phosphorylated ChChd3 S N6 -phenethyl-ATP + ChChd3 ( cofactor of mutant M120G, poor activity with the wild-type catalytic subunit [101]) (Reversibility: ?) [101] P N6 -phenethyl-ADP + phosphorylated ChChd3 S Additional information ( substrate specificity, overview [88]; enzyme plays an essential role during differentiation and fruit morphogenesis in Dictyostelium discoideum [33]; the enzyme plays a central role in the control of mammalian sperm capacitation and motility [26]; activated PKA catalytic subunit C with cAMP or 8-bromo-cAMP, stimulated by b1 adrenoreceptor activity, inhibits store-operated channel current activity in vascular tissue [81]; cAMP concentration and PKA activity are increased and important in exocytotic acrosome reaction taking place in fertilization when sperm contacts the egg jelly layer EJ, EJ component fucose sulfate polymer triggers sperm PKA activation [55]; cAMP-dependent activation of BKCa channels in pulmonary arterial smooth muscle is not catalyzed by PKA but by cGMP-dependent protein kinase PKG, thus PKA is not involved in cAMP-induced signaling in pulmonary vasodilation [41]; enzyme regulation, overview [94]; induced by the pituitary follicle-stimulating hormone FSH PKA also catalyzes the dephosphorylation of residues T421 and S424 of the autoinhibitory domain of p70S6K, resulting in activation of p70S6K important for differentiation of Sertoli cells in male reproduction, regulation overview [84]; inositol 1,4,5-trisphosphate receptor-I is no substrate of PKA in vivo [42]; merlin acts as an PKA-anchoring protein being linked to the cAMP/ PKA signaling pathway [64]; no activity with claudin-4 [72]; oestrogen-dependent increase in cAMP increases Ca2+ -dependent exocytosis through protein kinase A-dependent pathway and through alternative cAMP-guanine nucleotide exchange factor GEF/Epac-dependent pathway in secretory cells [82]; PKA regulatory and catalytic subunits are bound in the CSR complex including the protein kinase A anchoring protein AKAP 95 and several CSR-binding proteins, the complex acts in stabilizing the LDH-A mRNA, overview [71]; PKA type I regulates ethanol-induced cAMP response element-mediated gene expression via activation of CREB-binding protein and inhibition of MAPK, regulation overview [66]; PKA works coordinatedly with GSK3b, EC 2.7.11.1, on t phosphorylation [78]; regulation of GRK1 and GRK7 by cAMP level during light and dark phases [73]; t becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain, inhibition of t hyperphosphorylation inhibits an associated loss in spatial memory [67]; the enzyme activates NS1619-induced flavoprotein oxidation modulating mitochondrial Ca2+ -activated K+ channels, mechanism 256

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of the cardioprotective effect and modulation by PKA, overview [54]; the enzyme interacts with the neuron cortex membrane protein gravin via Ca2+ -independent binding to the regulatory RII enzyme subunit, gravin provides a platform to localize kinases, besides PKA also PKCa and PKCbII, in an isozyme-specific and activation-dependent manner at specific sites in neurons, gravin is strongly upregulated in cells during differentiation, overview [62]; the enzyme is not required for adenosineinduced dilation of intracerebral arterioles and regulation of cerebral blood flow [75]; the enzyme plays a central role in the adipokinetic signaling controling the mobilization of stored lipids in the fat body [59]; the enzyme plays a paradoxical role in cell motility facilitating and inhibiting actin cytoskeletal dynamics and cell migration, overview, the enzyme is regulated in a subcellular space during cell migration, regulatory subunits RII are enriched in protrusive cellular structures and pseudopodia formed during chemotaxis, anchoring of PKA inhibits pseudopod formation and cell migration, regulation of the process overview [87]; the enzyme regulates the G2/M transition in oocytes of Xenopus laevis, it also blocks progesterone-induced germinal vesicle envelope breakdown GVBD, Cdc-25-dependent dephosphorylation of Tyr15 in Cdc2, and synthesis of MEKK Mos [70]; cAMP binds to the helical subunit C binding regions relayed by the highly dynamic switch of the Chelix of subunit RIa, which is linked to cAMP by a salt bridge essential for activation [90]; no activity with claudin-4, poor activity with claudin-3 mutant T192A [72]; PKA catalyzes t phosphorylation in brain, but phosphorylation of other proteins, EC 2.7.11.11, in different tissues [78]; poor activity on free amino acids, consensus sequence of PKA is R-RXS/T hyd [5]; potential substrate identification by an evolutionary proteomics approach, overview [86]; recognition motif and phosphorylation sequences, substrate binding structure, peptide binding in an extended conformation, N-terminal and C-terminal extensions bind pseudosubstrates [46]; sperm protein substrate identification using peptide analysis via tandem mass spectrometry [55]; substrate specificity profile utilizing l-pyruvate kinase mutants and pyruvate kinasederived peptide substrate mutants, mutated to different amino acids at positions 9,10, and 13, as protein substrates, overview [51]; substrate specificity, no activity with peptides derived from Thr-kinase or Tyr-kinase, overview [59]; substrates have differential effects on type I and type II PKA holoenzyme dissociation, the isolated catalytic subunit is catalytically active [49]; the enzyme autophosphorylates at Ser10, Thr197, and Ser338 of PKA Ca, substrate-induced conformational changes [76]; the enzyme performs autophosphorylation at K72, S338, and T197 [74]; the enzyme performs autophosphorylation at Ser10, Ser139, Thr197, and Ser338 [58]; the enzyme performs autophosphorylation, substrate specificity depends on activating agents cAMP or cGMP, overview [40]; cystic fibrosis results from mutations in the cystic fibrosis conductance regulator protein, CFTR, a cAMP/protein kinase A, PKA, and ATP-regulated Cl- channel, the formation of the cAMP/ 257

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protein kinase A-dependent annexin 2 S100A10 complex with cystic fibrosis conductance regulator protein, CFTR, regulates CFTR channel function, overview, PKA regulates anx 2 and S100A10 cellular distribution in HNE cells, modulation of PKA activity alters localization and distribution of anx 2-S100A10 in HNE cells, overview [107]; inhibition of caspasedependent spontaneous apoptosis via a cAMP-protein kinase A dependent pathway in neutrophils from sickle cell disease patients, overview [96]; PKA plays a crucial role in the release of the catch state of molluskan muscles, mechanism, overview [92]; redox regulation of cAMPdependent protein kinase signaling: kinase versus phosphatase inactivation, in HeLa cells PKA activity follows a biphasic response to thiol oxidation, overview [102]; the enzyme is involved in cyclic nucleotide signaling, overview, individually phosphorylated PKA-R isozymes are differentially targeted to distinct cellular compartments by AKAP-isozymes, providing a multifaceted platform for the kinase [105]; the enzyme plays a central role in regulation of diverse aspects of cellular activity, especially in the controlling of maturation of spermatids [106]; no activity with myosin heavy chain and tropomyosin [92]; phosphorylated residue pThr197 not only facilitates the phosphoryl transfer reaction by stabilizing the transition state through electrostatic interactions but also strongly affects its essential protein dynamics as well as the active site conformation, overview, free energy difference is 1.4 kcal/mol, it is necessary that Asp166 is available as the catalytic base to accept the hydroxyl proton in the late stages of the phosphoryl transfer [109]; the catalytic subunit has a cluster of nonconserved acidic residues, Glu127, Glu170, Glu203, Glu230, and Asp241, that are crucial for substrate recognition and binding [95]; the localization of the structural region of the reconstituted channel protein substrates contribute to PKA-mediated stimulation [104]; the regulatory subunit of PKA inhibits its kinase activity by shielding the catalytic subunit from physiological substrates, Asp170 not only plays a pivotal role in controlling the local conformation of the phosphate binding cassette, where cAMP docks, but also significantly affects the long-range cAMP-dependent interaction network that extends from the phosphate binding cassette to the three major sites of C-recognition [110]) (Reversibility: ?) [5, 26, 33, 40, 41, 42, 46, 49, 51, 54, 55, 58, 59, 62, 64, 66, 67, 70, 71, 72, 73, 74, 75, 76, 78, 81, 82, 84, 86, 87, 88, 90, 92, 94, 95, 96, 102, 104, 105, 106, 107, 109, 110] P ? Inhibitors (Rp)-adenosine 3’,5’-cyclic monophosphothioate ( i.e. (Rp)-cAMPS, analogue of cAMP, competitive inhibition of PKA activation by cAMP, binding site structure [50]) [50] (Sp)-adenosine 3’,5’-cyclic monophosphothioate ( i.e. (Sp)-cAMPS, analogue of cAMP, competitive inhibition of PKA activation by cAMP, binding site structure [50]) [50] ADP ( noncompetitive inhibition with respect to ATP [5]) [5]

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adenosine 5’-(b,g-imino)triphosphate ( binding structure at the ATP binding site [3]) [3] acetylsalicylic acid ( a non-steroidal anti-inflammatory drug, decreases adrenaline- or dibutyryl cAMP-stimulated glycerol release in isolated adipocytes [93]) [93] AdcAhxArg6 ( i.e. adenosine 5-carboxylic acid-6-aminohexanoic acid-l-arginine, peptide-nucleoside conjugate inhibitor, inhibition of the catalytic subunit, binding mechanism [52]) [52] Ca2+ ( strong inhibition [59]) [59] genistein ( partial inhibition [59]) [59] H-85 ( unspecific kinase inhibitor, inhibits 23% of the acrosome reaction in sperm at 0.03 mM [55]) [55] H-89 ( strong inhibition [83]; specific inhibitor [65]; inhibits autophosphorylation of the catalytic subunit C, reversible [74]; PKA inhibitor [80]; PKA inhibitor, inhibits 87% of the acrosome reaction in sperm at 0.03 mM [55]; potent inhibitor of PKA [59]; selective strong inhibitor [81]; selective, but not specific inhibitor of PKA [64]; i.e. N-[2-((4-bromocinnamyl)amino)ethyl]5-isoquinolinesulfonamide, as hydrochloride [108]; i.e. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide [98]) [55, 59, 60, 64, 65, 72, 74, 80, 81, 83, 98, 108] H7 ( specific protein kinase inhibitor [53]) [53] H89 ( specific inhibition [48]; i.e. N-[2-(4-bromocinnamylamino) ethyl]-5-isoquinoline [104]) [48, 102, 104] HA1077 ( partial inhibition [59]) [59] IP20 ( inhibitory peptide TTYADFIASGRTGRRN, residues 5-24 of inhibitor PKI, inhibits the ATPase function of the enzyme [77]; iodinated inhibitor peptide substrate [76]; peptide RRNAI, derived from the inhibitory sequence of RIa comprising residues 94-98, inhibition mechanism [91]) [76, 77, 89, 91] KT5720 ( PKA inhibitor [41]; specific for PKA [81]) [41, 81, 96] Mn2+ ( strong inhibition [59]) [59] N6 -benzyl-ATP ( preferred co-substrate of catalytic subunit mutant M120G, but inhibitory for the mutant, not the wild-type enzyme, versus kemptide, overview [101]) [101] N6 -phenethyl-ATP ( preferred co-substrate of catalytic subunit mutant M120G, but inhibitory for the mutant, not the wild-type enzyme, versus kemptide, overview [101]) [101] Naproxen ( a non-steroidal anti-inflammatory drug, decreases adrenaline- or dibutyryl cAMP-stimulated glycerol release in isolated adipocytes [93]) [93] Ni2+ ( inhibits sperm protein phosphorylation by the enzyme [55]) [55] PKI ( catalytic subunit Ca [88]; pseudo-substrate inhibitor [46]; recombinant His-tagged rabbit PKA inhibitor protein PKI,

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binding involves Arg133 [70]; specific PKA inhibitor protein [59]) [46, 59, 70, 88] Rp-8-CPT-cAMPS [75] Rp-adenosine 3’,5’-cyclic monophosphorothionate triethyl ammonium salt ( inhibitor of PKA [67]) [67] STVHEILCKLSLEG ( an acetylated peptide Ac1-14, but not the nonacetylated equivalent N1-14, inhibits PKA-dependent outwardly rectifying Clchannels, ORCC, and CFTRmediated currents, the peptide sequence is equivalent to the S100A10 binding site on anx 2, it disrupts the anx S100A10/CTFR interaction [107]) [107] staurosporine ( partial inhibition [59]) [9, 59] alsterpaullone ( 21% inhibition of CDK2 at 0.01 mM [45]) [45] ethylmaleimide ( complete inhibition at 2 mM [59]) [59] guanethidine ( noncompetitive inhibiting serine peptide analogue with respect to ATP [5]) [5] indirubin-3’-monoxime ( 20% inhibition of CDK2 at 0.01 mM [45]) [45] inhibitor peptide PKI5-24 ( inhibits the catalytic subunit [92]) [92] myristoylated PKA peptide inhibitor ( 14-22 amide, blocks egg jelly layer induction of PKA [55]) [55] myristoylated PKI ( specific inhibitor [65]) [65] myristoylated PKI14-22 amide ( selective peptide inhibitor of PKA [42]) [42] nifedipine ( inhibits sperm protein phosphorylation by the enzyme [55]) [55] nimesulide ( a non-steroidal anti-inflammatory drug, decreases adrenaline- or dibutyryl cAMP-stimulated glycerol release in isolated adipocytes [93]) [93] peptide inhibitor PKI [5] piroxicam ( a non-steroidal anti-inflammatory drug, decreases adrenaline- or dibutyryl cAMP-stimulated glycerol release in isolated adipocytes [93]) [93] protein kinase A inhibitor peptide PKI [104] protein kinase inhibitor PKI ( binding structure, overview [95]) [95] pseudosubstrate peptides ( IC50 values for inhibition of he regulatory subunits of PKA by endogenous inhibitors, overview [88]; IC50 values for inhibition of the regulatory subunits of PKA by endogenous inhibitors, overview [88]) [88] purvalanol ( 18% inhibition of CDK2 at 0.01 mM [45]) [45] regulatory subunit 1a [101] roscovitine ( 7% inhibition of CDK2 at 0.01 mM [45]) [45] Additional information ( modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups [7]; caveolae inhibitors inhibit the internalization of the b-adrenergic 260

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receptor by PKA [60]; complete enzyme inhibition inhibits pseudopod formation and cell migration [87]; molecular mechanism of enzyme inhibition by binding of catalytic and regulatory subunits via extended surface of subunit C, overview [91]; no inhibition by KT5823 [41]; no inhibition of catalytic subunit Cg by PKI [88]; no inhibition of CDK2 by kenpaullone [45]; phosphorylation of the activation loop leads to enzyme inhibition, in which the phosphorylated activation loop acts as an autoinhibitory substrate blocking the nucleotide binding pocket, competition with ATP [4,5]; the regulatory subunits competitively inhibit the PKA kinase activity [69]; cyclo-oxygenase-independent inhibitory effect of non-steroidal anti-inflammatory drugs, mechanism, overview [93]; PKA is inhibited by oxidation via either glutathionylation of Cys199 in the activation loop, or the formation of an internal disulfide bond between Cys199 and Cys343 [102]; the two isoforms of the regulatory subunit inhibit the catalytic subunit [92]) [4, 5, 7, 41, 45, 60, 69, 87, 88, 91, 92, 93, 102] Cofactors/prosthetic groups ATP ( binding mechanism [52]; as MgATP2- [59]; as MgATP2-, binding site structure [76]; binding pocket and small lobe structure, binding involves D166, N171, D184, and K72 [89]; binding site structure: spans both lobes, the N-terminal b-sheet and C-terminal a-helix of the core scaffold, binding of ATP and release of ADP in the open enzyme conformation, residues Asp184, Lys72, and Asp166 are involved, schematic overview [3]; binds between two lobes, directing the g-phosphate outwards while the adenine ring lies deep in the cleft between the lobes [46]; dependent on, the binding site is a deep pocket lined by hydrophobic residues, enzyme affinity for ATP is increased 2fold by phosphorylation of the activation loop at Thr197, ATP competes with the phosphorylated activation loop, that acts as an autoinhibitory substrate [5]; optimal at 0.0025 mM, binding involves phosphorylation of Thr197 [53]; the binding site is a deep pocket lined by hydrophobic residues, enzyme affinity for ATP is increased 2fold by phosphorylation of the activation loop at Thr197, ATP competes with the phosphorylated activation loop, that acts as an autoinhibitory substrate [4]; PKA-Mg2+ -ATP-substrate complex formation and effects on enzyme activity and stability, overview [109]; preferred cofactor of the wild-type catalytic subunit [101]) [3, 4, 5, 40, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 55, 57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 100, 101, 102, 103, 104, 105, 107, 108, 109, 110, 111] N6 -benzyl-ATP ( preferred co-substrate of catalytic subunit mutant M120G, but inhibitory versus kemptide, overview [101]) [101] N6 -phenethyl-ATP ( preferred co-substrate of catalytic subunit mutant M120G, but inhibitory versus kemptide, overview [101]) [101] cAMP ( dependent on [2, 6, 7, 8, 9, 12, 13, 14, 16, 17,

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24, 25, 29, 30, 33, 34, 35, 36, 37, 38]) [2, 6, 7, 8, 9, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 25, 26, 29, 30, 32, 33, 34, 35, 36, 37, 38] Activating compounds 8-CPT-cAMP ( induces release of the active catalytic subunits from the PKA holoenzyme [56]) [56] 8-bromo-cAMP ( stimulates [102]; activates [47]; activates to same extent as cAMP [40]) [40, 47, 54, 75, 81, 102, 104] 8-chloro-cAMP ( activates to same extent as cAMP [40]) [40] calcineurin ( i.e. CaN, is important for the cAMP/PKA-dependent anx 2-S100A10 complex formation [107]) [107] dibutyryl-cAMP ( activates in vitro [73]; induces MKP-1 mRNA and protein expression in H295R cells [63]) [63, 73] isoproterenol ( stimulates [102]) [102] prostaglandin E1 ( activates [47]) [47] vasoactive intestinal peptide ( i.e. VIP [42]) [42] adipokinetic hormone ( rapid enzyme activation [59]) [59] cAMP ( dependent on [41, 43, 45, 51, 52, 55, 58, 60, 61, 62, 64, 65, 66, 70, 71, 72, 73, 74, 76, 80, 82, 83, 85, 88, 89]; activates isozymes PKAmyt1 and PKAmyt2 [44]; binds to the 2 catalytic subunits and activates via dissociation of the regulatory dimer [5]; dependent on, 450% activation [59]; dependent on, activates the enzyme by partly dissociating the regulatory and the catalytic subunits, determination and analysis of the mechanism, the substrate plays a differential role in activation of type I versus type II holoenzyme [49]; dependent on, acts synergistically with cGMP, binding site domain structure on the regulatory subunit RII [57]; dependent on, binding site [50]; dependent on, competitive to regulatory subunit RIa, at low concentration the activation of PKA type I, but not of PKA type II, by cAMP is accelerated by the peptide or protein substrate by increased dissociation of subunits Ca and RIa, cAMP has low effect on RII/ PKA type II, overview [69]; dependent on, degree of enzyme activation by cAMP or cGMP depends on the substrate [40]; dependent on, stimulates phosphorylation of RGS protein [48]; dependent on, structural basis of activation mechanism, binding induces conformational changes in the regulatory subunit RIa [91]; dynamics of the binding domain structure, molecular binding mechanism, overview [90]; each monomer of the tetrameric enzyme contains 2 bindig sites A and B for cAMP, binding involves Arg230 of domain A and Arg359 in domain B of regulatory subunit RIIb, cAMP binding releases the catalytic subunits [68]; induces release of the active catalytic subunits from the PKA holoenzyme [56]; intracellular amount is controlled by the stimulating steroid hormone adrenocorticotropin ACTH in the adrenal cortex increasing PKA enzyme activity, cAMP induces MKP-1 mRNA and protein expression [63]; required for activity, activation is mediated by binding of cAMP to the regulatory subunits, causing release of the catalytic subunits [3]; required for PKA activation [78];

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required for PKA activity [67]) [3, 4, 5, 40, 41, 42, 43, 44, 45, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88, 89, 90, 91, 94, 96, 98, 101, 102, 103, 105, 110] cBIMPS [42] cGMP ( activates isozymes PKAmyt1 and PKAmyt2 [44]; activates, acts synergistically with cAMP [57]; activates, degree of enzyme activation by cAMP or cGMP depends on the substrate [40]) [40, 44, 57] forskolin ( activates at concentration above 0.001 mM [42]; activates PKA [67]; forskolin specifically induces tau hyperphosphorylation by PKA at Ser214 resulting in increased phosphorylation at Ser199, Ser22, Ser396, and Ser404 in N2a/t441 cells [78]; stimulates the cAMP-dependent enzyme [102]) [42, 54, 67, 75, 78, 84, 102, 104] isoprotenol ( at concentration below 0.001 mM [42]) [42] Additional information ( activation involves the activation loop, a polypeptide region outside the active site cleft, which is reversibly phosphorylated at Thr197, dephosphorylation leads to enzyme 23fold activation [5]; activation involves the activation loop, a polypeptide region outside the active site cleft, which is reversibly phosphorylated at Thr197, dephosphorylation leads to enzyme activation by 2-3fold [4]; barrestin is not required for phosphorylation activity and receptor substrate endocytosis [60]; dephosphorylation activity induced by the pituitary follicle-stimulating hormone FSH [84]; egg jelly layer EJ component fucose sulfate polymer triggers sperm PKA activation [55]; enzyme stimulation via increase of cAMP level trough 1-methyl-3-isobutylxanthine or theophylline [80]; forskolin activates via stimulation of cAMP production [41]; forskolin induces enzyme activity [47]; forskolin induces PKA activity [72,83]; forskolin stimulates the adenylyl cyclase, which activates the enzyme by increased cAMP level [48]; isozyme PKAmyt1 shows 2- and 3.5fold higher affinity for cAMP and cGMP, respectively, than PKAmyt2 [44]; mechanism, transitory activation of tau phosphorylation by PKA in Alzheimers disease, effects of durative incubation with activators, overview [78]; no enzyme activation or induction by adenosine [75]; no stimulation by N6 ,2-O-dibutyryl-cAMP [40]; phosphate-binding cassettes PCB of domain A and B of the regulatory subunits [57]; phosphorylation of CBP by PKA type I is induced by forskolin and ethanol, inhibition of MAPK activates the PKA type I [66]; PKA is induced by oestrogen 17b-oestradiol and forskolin [82]; activation loop phosphorylation at Thr197 regulates the enzymes catalytic activity, molecular mechanism, classical molecular dynamics simulations and ab initio QM/MM calculations are carried out on the wild-type PKA-Mg2+ - ATP-substrate complex and its dephosphorylated mutant, T197A, overview [109]; b-adrenergic stimulation of Ser1928 phosphorylation, l-type calcium channel Cav1.2 constitutively associates with the b2 -AR and PKA to efficiently respond to b-adrenergic activation [94]; g-hydroxybutyrate induces the enzyme in the hippocampus, the phosphorylation activity is increased via GABAB receptors, 263

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but a desensitization of the signaling pathway occurs after repeated administration of g-hydroxybutyrate, overview [108]; under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced [102]) [4, 5, 40, 41, 44, 47, 48, 55, 57, 60, 66, 72, 75, 78, 80, 82, 83, 84, 94, 102, 108, 109] Metals, ions Ca2+ ( extracellular Ca2+ is required for enzyme activity in sperm [55]) [55] Cd2+ ( can partially substitue Mg2+ [5]) [5] Co2+ ( can partially substitue Mg2+ [5]) [5] Mg2+ ( absolutely required, as MgATP2-, best at 0.5 mM Mg2+ , inhibitory at higher Mg2+ concentrations, cannot be substituted by Mn2+ [59]; activates the enzyme, binds to b- and g-phosphate of ATP and to Asp184 [3]; as MgATP2-, binding site structure [76]; dependent on, chelates the b- and g-phosphate of ATP, Mg2+ is the physiologic metal ion, other divalent cations are able to support nucleotide binding, but only Mn2+ , Co2+ , and Cd2+ can substitute Mg2+ in supporting the catalytic activity [5]; the Mg-positioning loop consists of residues Asp184-Phe187 [89]; PKA-Mg2+ -ATP-substrate complex formation and effects on enzyme activity and stability, overview [109]) [3, 4, 5, 40, 42, 43, 44, 47, 48, 49, 51, 52, 53, 55, 57, 58, 59, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88, 89, 90, 91, 93, 94, 95, 98, 100, 101, 102, 103, 104, 105, 108, 109, 111] Mn2+ ( can partially substitue Mg2+ [5]) [5] Zn2+ [88] Additional information ( in HeLa cells PKA activity follows a biphasic response to thiol oxidation, overview [102]) [102] Turnover number (min–1) 0.12 (N6 -phenethyl-ATP, pH 7.0, 37 C, recombinant mutant M120G catalytic subunit [101]) [101] 0.5 (N6 -benzyl-ATP, pH 7.0, 37 C, recombinant mutant M120G catalytic subunit [101]) [101] 10.6 (ATP, pH 7.0, 37 C, recombinant mutant M120G catalytic subunit [101]) [101] 19.6 (ATP, pH 7.0, 37 C, recombinant wild-type catalytic subunit [101]) [101] Specific activity (U/mg) 0.266 ( purified catalytic subunit [59]) [59] 0.31 ( purified recombinant catalytic subunit Cg, substrate Kemptide [88]) [88] 0.81 ( purified recombinant catalytic subunit Cg, substrate histone [88]) [88] 5.3 ( purified recombinant catalytic subunit Ca [88]) [88] Additional information ( measurement of cytosolic Ca2+ concentrations [82]) [44, 60, 82, 101]

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Km-Value (mM) 0.0011 (N6 -benzyl-ATP, pH 7.0, 37 C, recombinant mutant M120G catalytic subunit [101]) [101] 0.0015 (N6 -phenethyl-ATP, pH 7.0, 37 C, recombinant mutant M120G catalytic subunit [101]) [101] 0.0125 (Kemptide, pH 7.0, 30 C, recombinant catalytic subunit Cg [88]) [88] 0.0174 (ATP, pH 7.0, 37 C, recombinant wild-type catalytic subunit [101]) [101] 0.021 (RRASVA, pH 7.5, 25 C, catalytic subunit [51]) [51] 0.0253 (RFARKGSLREKNV, pH 7.0, 30 C, recombinant catalytic subunit Cg [88]) [88] 0.0305 (ATP, pH 7.0, 37 C, recombinant mutant M120G catalytic subunit [101]) [101] 0.031 (Kemptide, pH 7.0, 22 C, catalytic subunit [59]) [59] 0.0333 (RKRSRAE, pH 7.0, 30 C, recombinant catalytic subunit Cg [88]) [88] 0.0333 (RKRSRKE, pH 7.0, 30 C, recombinant catalytic subunit Cg [88]) [88] 0.038 (Kemptide, pH 7.0, 30 C, recombinant catalytic subunit Ca [88]) [88] 0.039 (MgATP2-, pH 7.0, 22 C, catalytic subunit [59]) [59] 0.05 (RFARKGSLREKNV, pH 7.0, 30 C, recombinant catalytic subunit Ca [88]) [88] 0.0503 (RRLSSLRA, pH 7.0, 30 C, recombinant catalytic subunit Cg [88]) [88] 0.1 (N6 -benzyl-ATP, above, pH 7.0, 37 C, recombinant wild-type catalytic subunit [101]) [101] 0.293 (RKRSRAE, pH 7.0, 30 C, recombinant catalytic subunit Ca [88]) [88] 0.338 (RRLSSLRA, pH 7.0, 30 C, recombinant catalytic subunit Ca [88]) [88] 0.5 (RKRSRKE, pH 7.0, 30 C, recombinant catalytic subunit Ca [88]) [88] 0.73 (histone, pH 7.0, 22 C, catalytic subunit [59]) [59] Additional information ( kinetics for cAMP binding to wild-type and mutant regulatory subunits RIIb, overview [68]; kinetics for catalytic subunit Cg [88]; kinetics of activation and autoinhibition, modeling, pre-steady-state kinetic methods, wild-type and mutant enzymes [4]; kinetics, random kinetic mechanism with kemptide as substrate, reaction kinetic can be influenced by the sort of substrate, pre-steadystate kinetics, slow structural changes during reaction [5]; kinetics, thermodynamics, isozymes PKAmyt1 and PKAmyt2 [44]; steady-state kinetics, second order rate constants, for the catalytic subunit [52]; kinetics of enzyme activity and activation [104]; PKA associates with substrates with a relatively low affinity [100]) [4, 5, 44, 52, 68, 88, 100, 104]

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Ki-Value (mM) 0.000048 (H-89, pH 7.0, 22 C, catalytic subunit [59]) [59] 0.00013 (AdcAhxArg6, pH 7.5, 30 C, recombinant catalytic subunit [52]) [52] 0.0039 (H7, recombinant wild-type catalytic subunit, pH 7.5 [53]) [53] 0.0049 (H7, recombinant mutant S338A catalytic subunit, pH 7.5 [53]) [53] Additional information ( inhibition kinetics for the catalytic subunit [52]) [52] pH-Optimum 6.5 ( assay at [56]) [56] 7 ( assay at [43, 44, 66, 79, 88, 101]) [43, 44, 59, 66, 79, 88, 101] 7.2 ( assay at [81,102]) [81, 102] 7.4 ( assay at [42, 60, 64, 67, 70, 78, 85, 103]) [42, 60, 64, 67, 70, 78, 85, 103] 7.5 ( assay at [40, 45, 51, 52, 53, 57, 58, 61, 63, 72, 108]) [40, 45, 51, 52, 53, 57, 58, 61, 63, 72, 108] 7.6 ( assay at [73]) [73] 8 ( assay at [55]) [55] Temperature optimum ( C) 4 ( assay at [79]) [79] 15 ( in vivo assay at [55]) [55] 20 ( assay at [57]) [57] 21 ( assay at room temperature [45]) [45] 22 ( assay at room temperature [43, 58, 59, 61]) [43, 58, 59, 61] 25 ( assay at [51]) [51] 30 ( assay at [40, 47, 52, 63, 64, 66, 67, 70, 72, 73, 74, 78, 83, 85, 86, 88, 103]) [40, 47, 52, 63, 64, 66, 67, 70, 72, 73, 74, 78, 83, 85, 86, 88, 103] 31 ( in vivo phosphorylation assay at [42]) [42] 37 ( assay at [60, 81, 101]) [60, 81, 101] 40 [44] Temperature range ( C) 10-40 ( isozyme PKAmyt1 shows a linear Arrhenius plot, while PKAmyt2 shows a break in Arrhenius plot at 15 C being much more temperature-sensitive above 15 C compared to isozyme PKAmyt1 [44]) [44]

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4 Enzyme Structure Molecular weight 37700 ( catalytic subunit, gel filtration [92]) [92] Additional information ( amino acid sequence of the catalytic subunit [10]) [10] Subunits ? ( x * 40800, recombinant catalytic subunit PKA Ca, SDS-PAGE [76]; x * 50000, about, regulatory subunit RII, SDSPAGE [40]; x * 40600, isozyme C1, x * 48200, isozyme C2, x * 52500, isozyme C3 [106]; x * 41000-44000, SDS-PAGE [97]; x * 57000, C-subunit, SDS-PAGE, x * 48000, RII-subunit, SDS-PAGE, x * 121000, AKAP121, SDS-PAGE [99]) [40, 76, 97, 99, 106] dimer ( 2 * 54000, (Rmyt1)2, nonreducing SDS-PAGE [43]) [43] monomer ( 1 * 41000, isolated recombinant catalytic subunit Cg from Sf9 cells, SDS-PAGE [88]; 1 * 42000, isolated recombinant catalytic subunit Ca from Escherichia coli, SDS-PAGE [88]; 1 * 45100, isolated catalytic subunit, SDS-PAGE [59]; 1 * 54000, Rmyt2, reducing or nonreducing SDS-PAGE, 1 * 54000, Rmyt1, reducing SDS-PAGE [43]; 1 x 40000, activated catalytic subunit Ca1, 1 x 47000, activated catalytic subunit Cb2 [56]; 1 * 40000, catalytic subunit, SDS-PAGE [92]) [43, 56, 59, 88, 92] oligomer [62] tetramer ( 2 regulatory subunits in a dimer bound to 2 catalytic subunits, the latter are released during activation by cAMP [68,71]; a heterotetramer composed of a regulatory dimer and 2 catalytic subunits, the tetramer is inactive, dissociation of the tetramer occurs during activation and cAMP binding [5]; a2 b2 , the tetramer is inactive, binding of ATP causes release of the 2 catalytic subunits in active conformation [3]) [3, 5, 68, 71] Additional information ( MW of the catalytic subunit determined by amino acid sequence is 40580 Da [11]; three different genes encode the catalytic subunits of the cAMP-dependent protein kinase [19]; MW of the catalytic subunit C is 39000-41000 Da [29]; PkaC is a catalytic subunit of the Dictyostelium discoideum cAPK [33]; the product of pka1 is a catalytic subunit of protein kinase A [36]; 2 catalytic subunits and 2 regulatory subunits form a tetramer, the inactive holoenzyme, upon cAMP binding the catalytic subunits are released as monomers and become catalytically active [59]; 2 isoforms of regulatory subunit termed Rmyt1, type 1, and Rmyt2, one form of catalytic subunit C, peptide mapping of Rmyt1 [43]; apoenzyme structure analysis, hydrophobic core network, overview [76]; Arg133 is essential for binding of the catalytic subunit C to the regulatory subunit RII [70]; catalytic subunit residue Ser338 stabilizes the enzyme [53]; determination of cAMP binding and interaction structures of subunits C and RIa, overview [90]; determination of Stokes radius of purified recombinant catalytic subunit Ca [88]; determination of Stokes ra-

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dius of purified recombinant catalytic subunit Cg [88]; enzyme consists of 2 catalytic subunits bound to a regulatory subunit dimer of subunits RIa and RIIa, the catalytic subunits are released upon activation by cAMP, 2 isoforms of catalytic subunit C exist, subunits Ca1 and Cb2 , which can colocalize in 1 holoenzyme complex [56]; holoenzyme structure [68]; isozymes PKAmyt1 and PKAmyt2 contain regulatory subunits Rmyt1 and Rmyt2, respectively [44]; structure modeling, structural elements, overview [5]; structure of catalytic subunit C with ATP binding cleft and activation loop, overview [58]; structure of residues 1-91 of the regulatory subunit [50]; structure of subunit C, subunit R, the cAMP binding site, and the activation loop, interaction sites, overview [91]; structure, deduced from DNA and amino acid sequence, analysis and comparison [57]; substrates have differential effects on type I and type II PKA holoenzyme dissociation, X-ray scattering measurements, overview [49]; the enzyme consists of an N-terminal small lobe and a C-terminal large lobe giving the catalytic core a bean-like structure and stabilizing the enzyme [46]; the enzyme possesses a regulatory and a catalytic subunit, 2 lobes are comprised in the core scaffold, the N-terminal b-sheet and C-terminal a-helix lobe, which are joined by a polypeptide chain, the active is located at the interface of the 2 lobes [3]; the substrate binds to the regulatory subunit which is released from the catalytic subunit for enzyme activity [48]; identification of protein kinase A regulatory isoforms and of 11 different Akinase anchoring proteins, AKAPs, in ventricular tissue, overview [105]; PKA is composed of the components C-PKA, R-PKA, and AKAP121 [99]; structure of the ternary PKA-substrate complex, overview [109]; the catalytic subunit has a cluster of nonconserved acidic residues, Glu127, Glu170, Glu203, Glu230, and Asp241, that are crucial for substrate recognition and binding, protein dynamics of the catalytic C-subunit, overview [95]; the regulatory subunit of PKA inhibits its kinase activity by shielding the catalytic subunit from physiological substrates, interdependence between the Asp170 relay site and the regulatory-catalytic subunit interaction interface [110]) [3, 5, 11, 19, 29, 33, 36, 43, 44, 46, 48, 49, 50, 53, 56, 57, 58, 59, 68, 70, 76, 88, 90, 91, 95, 99, 105, 109, 110] Posttranslational modification lipoprotein ( isozyme C1 cotains a consensus sequence for N-myristoylation [106]) [106] phosphoprotein ( phosphate groups at Thr196 and Ser337 [11]; autophosphorylation at Ser10, Ser139, Thr197, and Ser338 [58]; autophosphorylation at Thr197 required for activation [3]; phosphorylation at T197 by exogenous kinase PDK-I, the enzyme performs autophosphorylation at K72, S338 and T197, the latter is phosphorylated first and required for activity, phosphorylation of the other 2 positions take place only if T197 is already phosphorylated, mechanism [74]; regulation by phosphorylation at Thr197 of the activation loop, enhances ATP binding and phosphotransfer, but only slightly the substrate binding [5]; the enzyme autophosphorylates at Ser10, Thr197, and Ser338 of catalytic subunit PKA Ca

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[76]; the enzyme can be phosphorylated at Thr197, Ser338, Ser10, and Ser139, the phosphorylation status of the enzyme varies, and is chronically reduced in enzyme mutant E230Q [89]; the enzyme is regulated by reversible phosphorylation of the activation loop at Thr197, a polypeptide region outside the active site cleft, the enzyme is inhibited by phosphorylation, which also increases the affinity for ATP by 2fold, and activated by dephosphorylation [4]; the enzyme performs autophosphorylation [40]; activation loop phosphorylation at Thr197 regulates the enzymes catalytic activity, molecular mechanism, classical molecular dynamics simulations and ab initio QM/MM calculations are carried out on the wild-type PKAMg2+ - ATP-substrate complex and its dephosphorylated mutant, T197A, overview [109]; analysis of in vivo phosphorylation sites on PKA-R reveals the presence of several differentially phosphorylated PKA-R isoforms, using semiquantitative mass spectrometry, overview [105]; the catalytically active subunit mutant M120G is phosphorylated on Thr197 and Ser338 [101]) [3, 4, 5, 11, 40, 58, 74, 76, 89, 101, 105, 109] Additional information ( unmyristylated Ca2 may be essential for fertility in the male [27]) [27]

5 Isolation/Preparation/Mutation/Application Source/tissue 2008 cell ( ovarian cancer cell line [72]) [72] A-2780 cell ( ovarian cancer cell line [72]) [72] AR42J cell ( a pancreatic acinar cell line [103]) [103] B35 cell ( neuroblastoma cell line [48]) [48] BG-1 cell ( ovarian cancer cell line [72]) [72] C6 cell ( glioma cell line [71]) [71] CAOV-3 cell ( ovarian cancer cell line [72]) [72] CHO cell [58] H9C2-Mp cell [99] HEK cell ( embryonic kidney cell [47]) [47] HEK-293 cell [60, 64, 65, 73] HEY cell ( ovarian cancer cell line [72]) [72] HOSE-B cell ( immortilized ovarian surface epithelial cell line [72]) [72] HeLa cell [83, 102] IGROV-1 cell ( ovarian cancer cell line [72]) [72] JURKAT cell [56] NCI-H295R cell ( adrenocortical cells [63]) [63] Ng-108-15 cell ( neuroblastoma x glioma hybrid cell [66]) [66] NT2-N cell ( precursor-like neuronal cells and differentiated NT2 cells, model neurons [62]) [62] NTERA-2 cell [56] Neuro-2A cell [78] OVCA-420 cell ( ovarian cancer cell line [72]) [72] 269

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OVCA-429 cell ( ovarian cancer cell line [72]) [72] OVCA-432 cell ( ovarian cancer cell line [72]) [72] OVCA-433 cell ( ovarian cancer cell line [72]) [72] OVCAR-2 cell ( ovarian cancer cell line [72]) [72] OVCAR-3 cell ( ovarian cancer cell line [72]) [72] OVCAR-4 cell ( ovarian cancer cell line [72]) [72] OVCAR-5 cell ( ovarian cancer cell line [72]) [72] REF-52 cell ( embryonic fibroblast cell line [87]) [87] Sertoli cell [84] T-cell [56] T-lymphocyte [56] UCI-101 cell ( ovarian cancer cell line [72]) [72] WI-38 cell ( fetal lung fibroblast-like cell line [87]) [87] adipocyte [93] adrenal cortex [63] arteriole ( cerebral, microvascular, isolated from cortical vessels [75]) [75] brain [57, 67, 78, 94, 101, 108] cardiac muscle [11] cardiomyocyte [99] cell culture ( ovary cell line [15]; myoblast L6 cell line [32]) [15, 32] commercial preparation ( purified bovine PKA catalytic subunit C [85]; heart enzyme [93]) [85, 93] egg [97] embryo ( enzyme activity at different developmental stages, overview [98]) [29, 98] epididymis [93] epithelium ( nasal [107]) [107] fat body [59] fat pad [93] forebrain [80] gastric gland [61] glioma cell [71] heart [7, 54, 69, 93, 94, 99, 105] heart ventricle ( isozyme distribution [105]) [105] hippocampus [67, 108] hypothalamus [80] larva [29] lung [41] malpighian tubule [106] mantle [44] melanotroph [82] midgut [106] mucosa ( gastric [61]) [61] myocyte ( ventricular [54]; portal vein smooth muscle myocyte [81]) [54, 81] 270

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neuron [94] neutrophil ( from sickle cell disease patients [96]) [96] oocyte [70, 104] pancreas [103] pancreatic acinar cell [103] pituitary gland [82] portal vein ( portal vein smooth muscle myocyte [81]) [81] posterior adductor muscle [44, 92] retina [73] retinal rod [73] salivary gland [106] semen [55] smooth muscle ( portal vein smooth muscle myocyte [81]; pulmonary arterial [41]) [41, 81] smooth muscle cell ( gastric, primary [42]) [42] sperm [26, 27, 55] spermatid ( immature [106]) [106] stomach [42, 61] telencephalon [80] testis [31, 106] Additional information ( C a mRNA is widespread and highly expressed in brain, heart, adrenal gland, testis, lung, kidney, spleen and liver, whereas the Cb mRNA is unevenly expressed in the brain and adrenal gland and in much lesser amounts in other tissues [16]; expressed at a low level in cytosolic and particulate compartments during embryogenesis. As the nematodes progress from late embryonic stages to the newly hatched, first larval stage, C subunit content increases 15fold. High levels of C subunits are observed in several subsequent larval and adult stages of development [29]; high PKA activity in ovarian cancer cells [72]; expression of the genes kin-1 and F47F2.1b encoding two PK-A-like catalytic subunits during development in mixed stage populations, overview [97]; ubiquitous expression of all three isozymes with isozyme C1 showing the highst expression levels in all tissues [106]) [16, 29, 72, 97, 106] Localization cytoplasm ( activated type I PKA is not translocated to the nucleus [66]; primarily, enzyme can migrate to the nucleus upon activation [3]) [3, 65, 66, 86, 110] cytosol ( subunit RIa localizes at the membrane/cytosolic area, subunit RIIa is concentrated around the Golgi-centrosomal area, subunits Ca1 and Cb2 in colocalized with RIa and RIIa [56]) [29, 56, 59, 60, 71, 82, 108] membrane ( cAMP/PKA-dependent anx 2-S100A10 complex [107]) [107] microtubule [67, 78] mitochondrion ( localization of cAMP-dependent protein kinase, composed of the components C-PKA, R-PKA, and AKAP121, associated

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with the A-kinase anchor protein in the inner mitochondrial compartment [99]) [99, 101, 102] mitoplast [99] nucleus ( activated type II PKA is translocated to the nucleus [66]; enzyme can migrate from the cytoplasm to the nucleus upon activation [3]) [3, 66] ribosome ( 54 S subunit of the yeast mitochondrial ribosome [18]) [18] Additional information ( substrate ezrin is localized at the cytoskeleton of the apical plasma membrane [61]; the enzyme binds to and colocalizes with the kinase-anchoring protein gravin in the neuronal cortex membrane which is enriched in the inner peripheral cortex in close proximity to the plasma membrane [62]; subcellular localization study, overview [99]; individually phosphorylated PKA-R isozymes are differentially targeted to distinct cellular compartments by AKAP-isozymes [105]) [61, 62, 99, 105] Purification (recombinant wild-type and mutant PKAs from Escherichia coli strain BL21(DE3)) [58] (recombinant His-tagged mutant catalytic subunit M120G from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [101] (recombinant catalytic subunit C mutant E230Q from Escherichia coli) [89] (recombinant catalytic subunit Ca from Escherichia coli by nickel affinity chromatography, recombinant catalytic subunit Ca from Sf9 insect cells 62fold by adsorption chromatography and gel filtration) [88] (recombinant catalytic subunit PKA Ca from Escherichia coli strain BL21 by P11 cellulose and ion exchange chromatography) [76] (recombinant wild-type and mutant Y204A from Escherichia coli by ion exchange chromatography and gel filtration) [77] (recombinant His-tagged catalytic subunit Cg from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, recombinant catalytic subunit Cg 273fold from Sf9 insect cells by adsorption chromatography and gel filtration) [88] (recombinant His-tagged catalytic subunit C, and His-tagged wild-type and mutant regulatory subunits RIIb, by nickel affinity chromatography and dialysis) [68] (regulatory subunit RII from C6 cells by affinity chromatography on an 8-(6-aminohexyl)-amino-cAMP-resin) [71] (recombinant regulatory subunit deletion mutant RIa by affinity chromatography, elution with cGMP, and dialysis) [50] (recombinant wild-type and mutant subunit RIa residues 91-244 construct dimers from Escherichia coli strain BL21(DE3) by cAMP affinity chromatography, dialysis, and gel filtration) [90]

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(by ammonium sulfate fractionation, dialysis, DEAE ion exchange and P11 cellulose chromatography, and hydroxyapatite chromatography, concentration steps on PEG20000) [40] (recombinant N-terminally His6-tagged wild-type and mutant catalytic subunits C from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [70] (enzyme catalytic subunit from fat body 1773fold to homogeneity by ultracentrifugation and 3 steps of ion exchange chromatography) [59] (native enzyme from posterior adductor muscle by anion exchange chromatography, ultrafiltration, and gel filtration) [92] (partial purification of 2 isozymes PKAmyt1 and PKAmyt2 from posterior aductor muscle and mantle tissue by 2 steps of ion exchange chromatography, ammonium sulfate precipitation, and immunoaffinity chromatography) [44] (purification of the type 1 regulatory subunit Rmyt1 from mussel by DEAE ion exchange and immunoaffinity chromatography, the latter based on antibodies and cGMP-agarose, and gel filtration) [43] (PKAII holoenzyme from bee brain by ion exchange, adsorption, and cAMP-affinity chromatography, thereby dissociation of regulatory and catalytic subunits, and reconstitution) [57] Renaturation (reversible unfolding and folding in 8.5 M urea of recombinant Histagged wild-type and mutant regulatory subunits RIIb, overview) [68] (recombinant catalytic subunit C, regulatory subunit RIa, and regulatory subunit RIIb from Escherichia coli strain 222 by cAMP-affinity chromatography, gel filtration, and dialysis) [49] (reconstitution of holoenzyme from purified subunits in 50 mM TrisHCl, pH 7.8, 1 mM EDTA, 1 mM EGTA, 10 mM 2-mercaptoethanol, and 0.1 M NaCl) [57] Crystallization (crystallization in active conformation) [3] (X-ray diffraction structure analysis) [4] (X-ray diffraction structure analysis of the catalytic subunits and the regulatory R2 dimer, enzyme cocrystallized with ATP and a peptide inhibitor) [5] (complex between the catalytic subunit and a deletion mutant regulatory subunit RIa comprising residues 91-244, structure determination and analysis) [91] (crystallization of the muatnt E230Q with MgATP2- and protein kinase inhibitor is only possible as apoenzyme, analysis of the mechanism preventing ternary complex formation by relaxed-complex method) [95] (modification and computational analysis of the crystal structure of catalytic subunit in complex with two Mg2+ and a phosphorylated substrate peptide, molecular dynamics simulation overview) [111] (purified recombinant catalytic apo-subunit apo-PKA Ca phosphorylated at Ser10, Thr197, and Ser338, hanging drop vapour diffusion method, 273

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4 C, 0.02 ml of 7.5 mg/ml protein in solution 0.1 mM bicine buffer, pH 8.0, 150 mM ammonium acetate, 10 mM 2-mercaptoethanol, and 3% 2-methyl2,4-pentanediol versus 1 ml reservoir solution containing 0.1 mM Tris-HCl, pH 7.5, and 10-20% v/v 2-methyl-2,4-pentanediol, temperature-sensitive crystals need 6 months to 1 year to grow, cryoprotection by a gradient of 2methyl-2,4-pentanediol, X-ray diffraction structure determination and analysis at 2.9 A resolution, molecular replacement) [76] (purified recombinant catalytic subunit C mutant E230Q in ternary complex with ATP, Mg2+ , and IP20, hanging drop vapour diffusion method, protein solution versus reservoir solution containing 0.1 M bicine, pH 8.0, 13% 2-methyl-2,4-pentanediol, and 11% methanol, 4 C, cryoprotection by 15% glycerol, X-ray diffraction structure determination and analysis at 2.8 A resolution) [89] (purified recombinant wild-type and mutant PKA catalytic subunits, 0.002 ml 5 mg/ml protein in 50 mM bicine, pH 8.0, 200 mM ammonium acetate, 2 mM DTT, MgCl2 , ATP, and IP20, is mixed with 0.001 ml well solution containing 8-12% v/v 2-methyl-2,4-pentanediol and 10 mM DTT, addition of 7% v/v methanol before sealing, X-ray diffraction structure determination and analysis at 1.26 A resolution, modeling) [77] (purified recombinant regulatory subunit deletion mutant RIa bound to cAMP analogue ligands (Rp)-cAMPS or (Sp)-cAMPS resulting in (Rp)-RIa and (Sp)-RIa, hanging drop vapour diffusion method, mixing of 0.002ml protein solution containing 12 mg/ml protein in 50 mM MES, pH 7.5, 0.2 M NaCl, 2 mM EDTA, 2 mM EGTA, and 10 mM DTT, with 0.002 ml reservoir solution containing 1.0 M ammonium sulfate, 12.5% glycerol, 10 mM DTT, 0.1 M sodium acetate, pH 5.5, 22.5 C, 1 week, X-ray diffraction structure determination and analysis at 2.3-2.4 A resolution) [50] (crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket) [9] (crystal structures of catalytic subunit of cAMP-dependent protein kinase in complex with isoquinolinesulfonyl protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) [8] Cloning (expression of wild-type and mutant PKAs in Escherichia coli strain BL21(DE3)) [58] (expression of His-tagged catalytic subunit Ca in Escherichia coli strain BL21(DE3), expression of catalytic subunit Ca in Spodoptera frugiperda Sf9 cells via baculovirus infection system) [88] (expression of PKA catalytic subunit Ca as His-tagged protein in Escherichia coli strain BL21(DE3) and of HA-tagged wild-type and mutant PKA Ca in COS cells) [74] (expression of catalytic subunit C mutant E230Q in Escherichia coli) [89] (expression of catalytic subunit PKA Ca in Escherichia coli strain BL21) [76]

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(expression of wild-type and mutant Y204A in Escherichia coli) [77] (functional expression of His-tagged mutant catalytic subunit M120G in Escherichia coli strain BL21(DE3) requiring co-expression with PDK1 for stabilization, overview) [101] (recombinant expression of paralemmin 2-AKAP2 fusion protein in ventricular tissue) [105] (expression of His-tagged catalytic subunit Cg in Escherichia coli strain BL21(DE3), stable expression of catalytic subunit Cg in cAMP-resistant mouse Y1 adrenal mutant cell line, expression of catalytic subunit Cg in Spodoptera frugiperda Sf9 cells via baculovirus infection system) [88] (expression of the catalytic subunit C in HEK-293 cells) [64] (expression of His-tagged catalytic subunit C, and His-tagged wild-type and mutant regulatory subunits RIIb) [68] (subcloning and expression of His-tagged wild-type and mutant PKA catalytic subunits in Escherichia coli strain BL21(DE3), introduction of a tobacco etch virus cleavage site to remove the tag, expression of deuterated catalytic subunit in Escherichia coli strain CT19) [53] (expression of HA-tagged PKA-Atg1345-559 fusion protein in Saccharomyces cerevisiae) [86] (distinct expression of catalytic subunit C, regulatory subunit RIa, and regulatory subunit RIIb in Escherichia coli strain 222) [49] (expression of regulatory subunit deletion mutant RIa comprising residues 1-91) [50] (expression of wild-type and mutant subunit RIa residues 91-244 constructs, containing the minimal motifs for cAMP-dependent tight binding of subunit C, in Escherichia coli strain BL21(DE3)) [90] (expression of N-terminally His6-tagged wild-type and mutant catalytic subunits C in Escherichia coli strain BL21(DE3)) [70] (genes kin-1 and F47F2.1b encode two PK-A-like catalytic subunits, DNA and amino acid sequence determination and analysis, expression profiles) [97] (genes kin-1 and F47F2.1b encode two PK-A-like catalytic subunits, DNA and amino acid sequence determination and analysis, expression profiles) [97] (isolation of a full-length cDNA clone coding for Cb2 isoform of the bovine C-subunit) [12] [13] (characterization of genomic clones coding for the Ca and Cb subunits) [14] (isolation of a full-length cDNA clone encoding the Cb catalytic subunit of cAMP-dependent protein kinase from a brain cDNA library) [16] [15] [24] [27] [29] [30, 31] [30] 275

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(isolation of a full length cDNA clone encoding the C a type catalytic subunit of cAMP-dependent protein kinase) [32] (expression of the catalytic subunit DdPK3 in Escherichia coli) [35] (isolation of cDNA) [34] [36, 37] [38] (regulatory subunit RII, DNA sequence determination and analysis, phylogenetic analysis) [57] (DNA and amino acid sequence determination of three isozymes resulting from alternative 5’-splicing) [106] Engineering D170A ( site-directed mutagenesis of the regulatory subunit RIa, the mutation selectively reduces the negative cooperativity between the cAMPand C-recognition sites, i.e. the KD for the regulatory-catalytic subunit complex in the presence of cAMP is reduced by more than 12fold, without significantly compromising the high affinity of the regulatory subunit for both binding partners, conformational shifts upon mutation, and physiological implications of the dual functionality of the D170A mutant, overview [110]) [110] D328A ( site-directed mutagenesis of the catalytic subunit, mutant shows decreased interaction with the regulatory RI subunit [70]) [70] D328A/K72H ( site-directed mutagenesis of the catalytic subunit, inactive mutant showing decreased interaction with the regulatory RII subunit [70]) [70] E230Q ( site-directed mutagenesis, mutation of an acidic residue from the cluster around the active site of the catalytic subunit C, E230 forms the salt bridge required for interaction with the substrate, mutant shows decreased substrate recognition, phosphorylation degree, and activity compared to the wild-type subunit C, mutant has an open conformation and does not bind ligands like MgATP2- or IP20 inhibitor [89]; site-directed mutagenesis of the catalytic subunit, the mutation affects the local structure of the the F-to-G helix loop and asp241, and the binding of protein kinase inhibitor PKI, the mutant protein can only be crystallizes as apoenzyme [95]) [89, 95] I204A ( site-directed mutagenesis, subunit RIa residue mutation, slightly impaired activation of subunit C, mutant show a slightly lower melting temperature compared to the wild-type subunit RIa [90]) [90] K336A/H338A ( i.e. Tpk1K336A/H338A, site-directed mutagenesis, a catalytically inactive PKA variant, that exhibits a stable binding to the substrate [100]) [100] K72A ( site-directed mutagenesis, inactive unphosphorylated mutant [74]) [74] K72H ( site-directed mutagenesis of the catalytic subunit, inactive mutant [70]; site-directed mutagenesis, inactive unphosphorylated mutant [74]) [70, 74]

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K72M ( site-directed mutagenesis, inactive unphosphorylated mutant [74]) [74] K72R ( site-directed mutagenesis of the catalytic subunit, inactive mutant [70]; site-directed mutagenesis, inactive unphosphorylated mutant [74]) [70, 74] L203A ( site-directed mutagenesis, subunit RIa residue mutation, slightly impaired activation of subunit C, mutant show a slightly lower melting temperature compared to the wild-type subunit RIa [90]) [90] M120G ( site-directed mutagenesis in the ATP-binding pocket of the catalytic subunit, engineering of the PKA catalytic subunit to accept bulky N6 -substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src, overview, N6 -benzyl-ATP and N6 -phenethyl-ATP are the prefrred cofactors of the mutant [101]) [101] R133A ( site-directed mutagenesis of the catalytic subunit, mutant shows decreased interaction with the regulatory RII subunit [70]) [70] R133A/K72H ( site-directed mutagenesis of the catalytic subunit, inactive mutant showing decreased interaction with the regulatory RII subunit [70]) [70] R165A ( site-directed mutagenesis, the mutant cannot be phosphorylated at Thr197 of the activation loop, molecular dynamics simulations, overview [109]) [109] R230K ( site-directed mutagenesis, mutation of an A domain residue of regulatory subunit RIIb, the mutant possesses one high-affinity cAMP binding site and one low active binding site resulting in reduced overall cAMP binding [68]) [68] R241A ( site-directed mutagenesis, subunit RIa residue mutation, slightly impaired activation of subunit C, mutant show a slightly lower melting temperature compared to the wild-type subunit RIa [90]) [90] R324A ( i.e. Tpk1R324A, site-directed mutagenesis, a catalytically inactive PKA variant, that exhibits a stable binding to the substrate with increased affinity through a conformational change [100]) [100] R359K ( site-directed mutagenesis, mutation of a B domain residue of regulatory subunit RIIb, the mutant possesses one high-affinity cAMP binding site and one low active binding site resulting in reduced overall cAMP binding [68]) [68] S10A/S139D/S338D ( site-directed mutagenesis, mutant shows activity and properties similar to the wild-type enzyme [58]) [58] S10A/S139D/T197D/S338D ( site-directed mutagenesis, mutant shows reduced expression level in Escherichia coli and impaired folding [58]) [58] S10A/S139D/T197E/S338D ( site-directed mutagenesis, mutant shows reduced expression level in Escherichia coli and impaired folding [58]) [58] S338A ( site-directed mutagenesis, mutant is phosphorylated at T197, reduced activity compared to the wild-type enzyme [74]; site-specific mutagenesis, mutation of a residue in the hydrophobic motif at the Cterminus of the catalytic subunit, catalytic activity is similar to the wild-type 277

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catalytic subunit, reduced protein stability compared to the wild-type enzyme [53]) [53, 74] T197A ( site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [4]; site-directed mutagenesis, mutant is not phosphorylated, inactive mutant [74]; site-specific mutagenesis, mutation of activation loop residue of the catalytic subunit, inactive mutant, conformational changes and reduced protein stability compared to the wild-type enzyme [53]; site-directed mutagenesis, the side-chain conformations of the P-site Ser in the R165A mutant are similar to that in the wildtype PKA, although the replacement of Arg165 with Ala disconnects the interactions between the pThr 197 and the active site, molecular dynamics simulations, overview [109]) [4, 53, 74, 109] T197A/S338A ( site-specific mutagenesis, highly unstable protein, tends to aggregation [53]) [53] T197D ( site-directed mutagenesis, reduced activity compared to the wild-type enzyme [4]) [4] Y204A ( site-directed mutagenesis, mutation in the catalytic subunit, reduced catalytic efficiency compared to the wild-type enzyme [77]) [77] Y229A ( site-directed mutagenesis, subunit RIa residue mutation, slightly impaired activation of subunit C, mutant show a slightly lower melting temperature compared to the wild-type subunit RIa [90]) [90] Additional information ( construction and expression of a mouse mutant b-adrenergic receptor lacking the PKA phosphorylation sites in HEK-293 cells, and coexpression of murine GFP-tagged b-arrestin [60]; construction and expression of enzyme with inactive cAMP binding sites at the regulatory subunits resulting in impaired b1 -adrenergic receptor recycling in recombinant HEK-293 cells, overview [65]; construction of a deletion mutant of regulatory subunit RIIb lacking the complete domain B [68]; microinjection of mutant subunits C in Xenopus oocytes, overview [70]; removal of residues S10, S139, and S338 plus Akt/PKB-like mutations of the ATP binding site render PKA to a Akt/PKB similar enzyme, overview [58]; deletion of the AKAP150 gene abrogates stimulation of a1 1.2 phosphorylation on Ser1928 in vivo, overview [94]; reconstitution of the PKA cascade in Xenopus laevis oocytes, overview [104]; TPK1 overexpression resulted in a severe growth defect [100]) [58, 60, 65, 68, 70, 94, 100, 104] Application drug development ( recombinant PKA can be mutated to resemble Akt/PKB, which cannot be easily recombinantly expressed, mutant PKA can be used as surrogate enzyme for Akt/PKB in drug design [58]) [58] medicine ( PKA is a target for the therpeutical treatement of Alzheimers disease and other tauopathies [67]) [67]

278

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6 Stability General stability information , N-terminal and C-terminal extensions stabilize the catalytic core [46] Storage stability , -70 C, purified recombinant catalytic subunit Cg, 0.5 M NaCl, 10 mM potassium phosphate, pH 6.9, 1 mM EDTA, 30% glycerol, on freeze-thaw cycle, stable up to one year [88]

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[90] Vigil, D.; Lin, J.-H.; Sotriffer, C.A.; Pennypacker, J.K.; McCammon, J.A.; Taylor, S.S.: A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP. Protein Sci., 15, 113-121 (2006) [91] Kim, C.; Xuong, N.H.; Taylor, S.S.: Crystal structure of a complex between the catalytic and regulatory (RIa) subunits of PKA. Science, 307, 690-696 (2005) [92] Bejar, P.; Villamarin, J.A.: Catalytic subunit of cAMP-dependent protein kinase from a catch muscle of the bivalve mollusk Mytilus galloprovincialis: purification, characterization, and phosphorylation of muscle proteins. Arch. Biochem. Biophys., 450, 133-140 (2006) [93] Zentella de Pina, M.; Vazquez-Meza, H.; Agundis, C.; Pereyra, M.A.; Pardo, J.P.; Villalobos-Molina, R.; Pina, E.: Inhibition of cAMP-dependent protein kinase A: a novel cyclo-oxygenase-independent effect of non-steroidal anti-inflammatory drugs in adipocytes. Auton. Autacoid. Pharmacol., 27, 85-92 (2007) [94] Hall, D.D.; Davare, M.A.; Shi, M.; Allen, M.L.; Weisenhaus, M.; McKnight, G.S.; Hell, J.W.: Critical role of cAMP-dependent protein kinase anchoring to the l-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons. Biochemistry, 46, 1635-1646 (2007) [95] Ung, M.; Lu, B.; McCammon, J.A.: E230Q mutation of the catalytic subunit of cAMP-dependent protein kinase affects local structure and the binding of peptide inhibitor. Biopolymers, 81, 428-439 (2006) [96] Conran, N.; Almeida, C.B.; Lanaro, C.; Ferreira, R.P.; Traina, F.; Saad, S.T.; Costa, F.F.: Inhibition of caspase-dependent spontaneous apoptosis via a cAMP-protein kinase A dependent pathway in neutrophils from sickle cell disease patients. Br. J. Haematol., 139, 148-158 (2007) [97] Bowen, L.C.; Bicknell, A.V.; Tabish, M.; Clegg, R.A.; Rees, H.H.; Fisher, M.J.: Expression of multiple isoforms of the cAMP-dependent protein kinase (PK-A) catalytic subunit in the nematode, Caenorhabditis elegans. Cell. Signal., 18, 2230-2237 (2006) [98] Lombardo, V.A.; Armas, P.; Weiner, A.M.; Calcaterra, N.B.: In vitro embryonic developmental phosphorylation of the cellular nucleic acid binding protein by cAMP-dependent protein kinase, and its relevance for biochemical activities. FEBS J., 274, 485-497 (2007) [99] Sardanelli, A.M.; Signorile, A.; Nuzzi, R.; Rasmo, D.D.; Technikova-Dobrova, Z.; Drahota, Z.; Occhiello, A.; Pica, A.; Papa, S.: Occurrence of A-kinase anchor protein and associated cAMP-dependent protein kinase in the inner compartment of mammalian mitochondria. FEBS Lett., 580, 5690-5696 (2006) [100] Deminoff, S.J.; Howard, S.C.; Hester, A.; Warner, S.; Herman, P.K.: Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae. Genetics, 173, 1909-1917 (2006) [101] Schauble, S.; King, C.C.; Darshi, M.; Koller, A.; Shah, K.; Taylor, S.S.: Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit. J. Biol. Chem., 282, 14952-14959 (2007) 286

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[102] Humphries, K.M.; Pennypacker, J.K.; Taylor, S.S.: Redox regulation of cAMP-dependent protein kinase signaling: kinase versus phosphatase inactivation. J. Biol. Chem., 282, 22072-22079 (2007) [103] Regimbald-Dumas, Y.; Arguin, G.; Fregeau, M.O.; Guillemette, G.: cAMPdependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells. J. Cell. Biochem., 101, 609-618 (2007) [104] Kim, J.A.; Park, J.Y.; Kang, H.W.; Huh, S.U.; Jeong, S.W.; Lee, J.H.: Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A. J. Pharmacol. Exp. Ther., 318, 230-237 (2006) [105] Scholten, A.; van Veen, T.A.; Vos, M.A.; Heck, A.J.: Diversity of cAMP-dependent protein kinase isoforms and their anchoring proteins in mouse ventricular tissue. J. Proteome Res., 6, 1705-1717 (2007) [106] Tabish, M.; Clegg, R.A.; Turner, P.C.; Jonczy, J.; Rees, H.H.; Fisher, M.J.: Molecular characterisation of cAMP-dependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum. Mol. Biochem. Parasitol., 150, 330-339 (2006) [107] Borthwick, L.A.; McGaw, J.; Conner, G.; Taylor, C.J.; Gerke, V.; Mehta, A.; Robson, L.; Muimo, R.: The formation of the cAMP/protein kinase A-dependent annexin 2 S100A10 complex with cystic fibrosis conductance regulator protein (CFTR) regulates CFTR channel function. Mol. Biol. Cell, 18, 3388-3397 (2007) [108] Ren, X.; Mody, I.: g-Hydroxybutyrate induces cyclic AMP-responsive element-binding protein phosphorylation in mouse hippocampus: An involvement of GABAB receptors and cAMP-dependent protein kinase activation. Neuroscience, 141, 269-275 (2006) [109] Cheng, Y.; Zhang, Y.; McCammon, J.A.: How does activation loop phosphorylation modulate catalytic activity in the cAMP-dependent protein kinase: a theoretical study. Protein Sci., 15, 672-683 (2006) [110] Abu-Abed, M.; Das, R.; Wang, L.; Melacini, G.: Definition of an electrostatic relay switch critical for the cAMP-dependent activation of protein kinase A as revealed by the D170A mutant of RIa. Proteins, 69, 112-124 (2007) [111] Jin, H.; Wu, T.; Jiang, Y.; Zou, J.; Zhuang, S.; Mao, X.; Yu, Q.: Role of phosphorylated Thr-197 in the catalytic subunit of cAMP-dependent protein kinase. Theochem, 805, 9-15 (2007)

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1 Nomenclature EC number 2.7.11.12 Systematic name ATP:protein phosphotransferase (cGMP-dependent) Recommended name cGMP-dependent protein kinase Synonyms CGK [16, 23, 31, 41, 56] CGK 1 a CGK 1 b CGKI- a DG1 protein kinase [12] DG2P1 [41] DG2P2 [41] Foraging protein PKG [20, 22, 25, 26, 27, 30, 32, 33, 36, 42, 44, 47, 48, 50, 57, 58, 59, 62, 63, 67, 68, 70, 72, 73] PKG I [43, 64] PKG II [38] PKG Ia [61] PKG Ib [39] PKG type I [72] PKG-1 [21] PKG-I [24, 53] PKGI [42, 44, 45, 55, 65, 71] PKGII [29, 44] PKGIa [66] PKGIb [66] Type II cGMP-dependent protein kinase [34] cGK I [34] cGK II [18, 34, 37] cGKI [28, 35, 49, 51, 52, 54, 60] cGKI-b cGKII [29] cGKIb [39] cGMP kinase [31]

288

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cGMP-dependent protein kinase cGMP-dependent protein kinase 1, a isozyme [2, 3, 4, 5, 6, 7, 8, 16] cGMP-dependent protein kinase 1, b isozyme [7, 8, 9, 10, 19] cGMP-dependent protein kinase 2 [13, 14, 15, 17, 18] cGMP-dependent protein kinase I [28, 34, 35, 45, 54, 55] cGMP-dependent protein kinase I a [61] cGMP-dependent protein kinase II [29, 34] cGMP-dependent protein kinase Ib [39] cGMP-dependent protein kinase type I [51, 65] cGMP-dependent protein kinase type II [37, 38] cGMP-dependent protein kinase, isozyme 1 [1, 11, 12] cGMP-dependent protein kinase, isozyme 2 forms cD5/T2 [11] cGMP-dependent protein kinase-1 [21] cGMP-dependent protein kinase-I [24] cyclic GMP-dependent kinase [32] cyclic GMP-dependent kinase I [49] cyclic GMP-dependent protein kinase [40, 48, 59, 62] cyclic GMP-dependent protein kinase-1 [60] cyclic guanosine-3’,5’-monophoshate-dependent protein kinase [31] cyclic-GMP dependent protein kinase [58] guanosine 3,5-cyclic monophosphate-dependent protein kinase [70] guanosine cyclic 3’,5’-phosphate dependent protein kinase [6] type 1 PKG [58] type 1a PKG [27] type I b isozyme of cGMP-dependent protein kinase [9] type I cGMP-dependent protein kinase [34] Additional information ( the enzyme belongs to the AGC kinase family [63]) [63] CAS registry number 141588-27-4

2 Source Organism





Drosophila melanogaster (no sequence specified) [41, 73] Mammalia (no sequence specified) [23, 34] Chlamydomonas reinhardtii (no sequence specified) [33] Mus musculus (no sequence specified) [25, 29, 31, 32, 35, 37, 42, 49, 51, 52, 65] Homo sapiens (no sequence specified) [25, 27, 30, 31, 33, 37, 39, 40, 42, 45, 46, 50, 53, 54, 55, 56, 57, 58, 60, 63, 66, 69, 70] Rattus norvegicus (no sequence specified) [20, 26, 28, 31, 37, 38, 44, 47, 64, 65, 67, 70] Sus scrofa (no sequence specified) [72] Bos taurus (no sequence specified) [24,32,33,59,61,71]

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Oryctolagus cuniculus (no sequence specified) [21,22] Aplysia californica (no sequence specified) [33] Caenorhabditis elegans (no sequence specified) [33, 62] Plasmodium falciparum (no sequence specified) [48] Eimeria tenella (no sequence specified) [36, 68] Tetrahymena sp. (no sequence specified) [33] Plasmodium yoelii (no sequence specified) [33] Toxoplasma gondii (no sequence specified) [36, 68] Oryctolagus cuniculus (UNIPROT accession number: O77676) [2] Bos taurus (UNIPROT accession number: P00516) [3, 4, 5, 6, 7] Homo sapiens (UNIPROT accession number: P14619) [8, 9] Bos taurus (UNIPROT accession number: P21136) [7, 10] Drosophila melanogaster (UNIPROT accession number: P32023) [11, 33] Drosophila melanogaster (UNIPROT accession number: Q03042) [1, 11, 12] Drosophila melanogaster (UNIPROT accession number: Q03043) [11] Homo sapiens (UNIPROT accession number: Q13237) [13, 14, 15] Homo sapiens (UNIPROT accession number: Q13976) [8, 16] Mus musculus (UNIPROT accession number: Q61410) [17] Rattus norvegicus (UNIPROT accession number: Q64595) [18] Mus musculus (UNIPROT accession number: Q9Z0Z0) [19] Plasmodium falsiparum (no sequence specified) [33] Paramecium tetraurelia (UNIPROT accession number: Q869J9) [33] Eimeria tenella (UNIPROT accession number: Q8MMZ8) [33] Cryptosporidium parvum (UNIPROT accession number: Q8MMZ6) [33] Toxoplasma gondii (UNIPROT accession number: Q8MMP4) [33] Eimeria maxima (UNIPROT accession number: Q8MMZ5) [33] Hydra oligactis (UNIPROT accession number: O17474) [33] Apis mellifera (UNIPROT accession number: Q8SSX4) [33] Paramecium tetraurelia (UNIPROT accession number: Q86pK0) [33] Oryzias latipes (UNIPROT accession number: Q75PY4) [43] Oryzias latipes (UNIPROT accession number: Q75PY3) [43]

3 Reaction and Specificity Catalyzed reaction ATP + a protein = ADP + a phosphoprotein Natural substrates and products S ATP + Akt (Reversibility: ?) [66] P ADP + phosphorylated Akt S ATP + CFTR ( phosphorylation leads to stimulation of chloride channels [31]) (Reversibility: ?) [31] P ADP + phosphorylated CFTR

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S ATP + FoxO1a ( interaction of enzyme with FocO1a is involved in regulation of myoblast cell fusion during myogenesis [49]) (Reversibility: ?) [49] P ADP + phosphorylated FoxO1a S ATP + IRAG ( i.e. inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, phosphorylation by isoyzme cGKIb, which is complexed with the substrate and the inositol 1,4,5-trisphosphate receptor in a ternary complex [23]; phosphorylation by cGKIb mediates the inhibition of inositol-1,4,5-trisphosphate-dependent Ca2+ -release, a mechanism leading to smooth muscle relaxation, involved in platelet aggregation [31]; phosphorylation by cGKIb mediates the inhibition of inositol-1,4,5-trisphosphate-dependent Ca2+ -release, one mechanism leading to smooth muscle relaxation, involved in platelet aggregation [31]) (Reversibility: ?) [23, 31] P ADP + phosphorylated IRAG S ATP + Na+ /K+ ATPase ( the enzyme has a positive regulatory function on the ion pump activity induced by No and glutamate via increased cGMP, overview [26]) (Reversibility: ?) [26] P ADP + phosphorylated S ATP + Rac1 ( the enzyme activates the recombinant Myc-tagged small GTPase Rac1 in HEK-293 cells leading to activation of PAK1, the enzyme induces phosphorylation of p38 which activates MAPK, regulation, overview [27]) (Reversibility: ?) [27] P ADP + phosphorylated Rac-1 S ATP + Rho ( phosphorylation leads to inhibition of Rho [31]) (Reversibility: ?) [31] P ADP + phosphorylated rho S ATP + RhoA ( phosphorylation at Ser188 by isoyzme cGKI leading to translocation of RhoA from the membrane to the cytosol and inactivates RhoA [23]) (Reversibility: ?) [23] P ADP + phosphorylated RhoA S ATP + TPIa ( involved in desensitization of TPIa signalling [31]) (Reversibility: ?) [31] P ADP + phosphorylated TPIa S ATP + TRIM39 (Reversibility: ?) [55] P ADP + phosphorylated TRIM39 S ATP + TRIM39R ( PKGI interacts with TRIM39R, a Rpp21 domain-containing TRIM protein, in the kinase phosphorylation domain [55]) (Reversibility: ?) [55] P ADP + phosphorylated TRIM39R S ATP + TRPC3 ( involved in inhibition of store operated Ca2+ influx [31]) (Reversibility: ?) [31] P ADP + phosphorylated TRPC3 S ATP + big potassium channel BKCa ( phosphorylation by cGKI leads to activation of the channel, a mechanism leading to Ca2+ dependent smooth muscle relaxation [31]) (Reversibility: ?) [31] P ADP + phosphorylated big potassium channel BKCa 291

cGMP-dependent protein kinase

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S ATP + cysteine-riche protein 2 ( i.e. CRP2, phosphorylation by cGKI and cGKII [31]) (Reversibility: ?) [31] P ADP + phosphorylated vasodilator-stimulated phosphoprotein S ATP + glycogen synthase kinase-3 ( phosphorylation at Ser9 by PKGI and PKGII inhibiting the glycogen synthase kinase-3 inducing dephosphorylation of C/EBPb, i.e. the CCAAT enhancer-binding protein b, important in regulation of gene expression during cell proliferation, differentiation, and apoptosis [44]) (Reversibility: ?) [44] P ADP + phosphorylated glycogen synthase kinase 3 S ATP + human serotonin transporter ( i.e. hSERT, the naturally occuring hSERT mutant I425V is associated with obsessive-compulsive disorder and other neuropsychiatric disorders [69]) (Reversibility: ?) [69] P ADP + phosphorylated human serotonin transporter S ATP + inositol 1,4,5-trisphosphate receptor-I ( the phosphorylation of the receptor inhibits inositol 1,4,5-trisphosphate-induced Ca2+ release [22]) (Reversibility: ?) [22] P ADP + phosphorylated inositol 1,4,5-trisphosphate receptor-I S ATP + maxiK channel ( phosphorylation by isoyzme cGKI enhances the channel activity [23]) (Reversibility: ?) [23] P ADP + phosphorylated maxiK channel S ATP + myosin phosphatase ( phosphorylation by isoyzme cGKIa leads to decreased level of phosphorylated MLC [23]) (Reversibility: ?) [23] P ADP + phosphorylated RhoA S ATP + p21-activated kinase ( PKG phosphorylation of p21-activated kinase, i.e. Pak, regulates HeLa cell and human umbilical vein endothelial cell morphology and cellular remodeling, time course of Pak1 activation in vivo, overview, PKG stimulates association of vasodilator-stimulated phosphoprotein VASP with Pak [63]) (Reversibility: ?) [63] P ADP + phosphorylated p21-activated kinase S ATP + phosphodiesterase 5 ( phosphorylation at Ser92 by isoenzyme cGKI leads to enhanced hydrolysis of cGMP, regulatory function [23]) (Reversibility: ?) [23] P ADP + phosphorylated phosphodiesterase 5 S ATP + phospholamban ( phosphorylation leads to stimulation of the sarcoendoplasmic reticulum pump Ca2+ -ATPase, i.e. SERCA [31]) (Reversibility: ?) [31] P ADP + phosphorylated phospholamban S ATP + phospholipase Cb3 ( potentially involved in inhibition of phospholipase C activity [31]) (Reversibility: ?) [31] P ADP + phosphorylated phospholipase Cb3 S ATP + septin 3 ( potentially involved in vesicle trafficking [31]) (Reversibility: ?) [31] P ADP + phosphorylated septin 3 S ATP + target membrane SNARe complex ( the enzyme is involved in regulation of degranulation of leukocytes by phosphorylation

292

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P S P S

P S P S

cGMP-dependent protein kinase

of target membrane SNARe complex together with the phosphoinositide 3-kinase [67]) (Reversibility: ?) [67] ADP + phosphorylated target membrane SNARe complex ATP + troponin T ( phosphorylation by isoyzme cGKI in cardiomyocytes [23]) (Reversibility: ?) [23] ADP + phosphorylated troponin T ATP + vasodilator stimulated phosphoprotein ( phosphorylation of vasodilator stimulated phosphoprotein, i.e. VASP, induces detachment of VASP and other cytoskeletal proteins from focal adhesion, phosphorylated VASP may prevent dendritic cells from migrating towards CCl19 [56]) (Reversibility: ?) [56, 58] ADP + phosphorylated vasodilator stimulated phosphoprotein ATP + vasodilator-stimulated phosphoprotein (Reversibility: ?) [31] ADP + phosphorylated vasodilator-stimulated phosphoprotein Additional information ( enzyme plays a crucial role in the relaxation of vascular smooth muscle by lowering the intracellular level of calcium [8]; the enzyme is thought to be involved in the regulation of intestinal ion transport and fluid secretion [14]; enzyme is involved in inhibition of platelet aggregation, relaxation of smooth muscle cells, and control of cardiocyte contractility. Pathophysiological implication of the type I cGK in cardiovascular diseases, including hypertension and atherosclerosis [16]; plays a pivotal role in the regulation of intestinal fluid balance in man [15]; amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase [3]; alternative splicing of PKGI in angiotensin-hypertension, mechanism for nitrate tolerance in vascular smooth muscle [28]; cGK is a key enzyme in the nitric oxide-cGMP and natriuretic signalling cascades, it modulates smooth muscle relaxation, platelet aggregation, renin release, intestinal secretion, learning amd memory, cGKII is involved in androsterone homeostasis, pleiotropic effets of the enzyme on diverse tissues, overview [31]; cGKI mediates NO signaling and plays a proatherogenic role in vascular smooth muscle cells, enzyme activation in primary smoothe muscle cells results in increased levels of proliferation and vascular cell adhesion molecule-1, peroxisome proliferato-activated receptor g, and phosphatidylinositol 3-kinase/Akt signaling, and in decreased plasminogen activator inhibitor 1 mRNA, overview [52]; cGMP-activated PKGIa inhibits thrombin receptor-mediated Ca2+ mobilization in vascular smooth muscle cells, cGMP-activated PKGIb causes inhibition in vitro but has nearly no effect in vivo, the thrombin receptor is activated by thrombin receptor activating peptide TRAP, or lysophosphatidic acid, or U4, overview [46]; cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein b functions through inhibition of glycogen synthase kinase-3 [44]; DG2P1 and DG2P2 are involved in behaviour and epithelial transport [41]; isozymes cGK I and cGK II are functionally distinct, isozyme cGK II is involved in regulation of electrolyte and water secretion by 293

cGMP-dependent protein kinase

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epithelial tissues in response to luminocrinic hormones guanylin and uroguanylin and in the secretory diarrhea provoked by heat-stable enterotoxins, isozyme cGK II also plays a role in the regulation of endochondral ossification by C-type natriuretic peptide, in renin secretion by the kidney, aldosterone secretion by the adrenal, and adjustment of the biological clock [34]; NO induces enzyme activity responsible for modulation/ inhibition of voltage-gated CaV1 and CaV2.2, i.e. L- and N-type, Ca2+ channels in neuroblastoma cells, in cardiovascular and smooth muscle cells Ca2+ channel activity is inhibited by direct nitrosylation of the channel proteins by NO, overview [50]; PKG-1 is involved in NO-induced cGMP-mediated increase in corpus cavernosum smooth muscle relaxation and penile erection, enzyme disfunction can cause erectile dysfunction, diabetes reduces corpus cavernosum smooth muscle relaxation [21]; PKGII modulates mPer1 and mPer2 gene induction, not by phosphorylation of CREB at Ser133, and influences phase shifts of the circadian clock, regulation model, overview [29]; PKGII regulates basal level of aldosterone production by zona glomerulosa cells without increasing expression of the steroidogenic acute regulatory protein gene [38]; sequential activation of p38 and ERK pathways by the enzyme leading to activation of the platelet integrin aIIb b3 [25]; the enzyme activates diverse signal transduction pathways, isozyme cGKI is responsible for smooth muscle relaxation by lowering the cytosolic calcium level and/or by desensitization of the contractile elements, smooth muscle contraction and enzyme-mediated smooth muscle relaxation mechanisms, detailed overview, cGKI affects the Ca2+ efflux from the endoplasmic reticulum and the cytosolic Ca2+ concentration [23]; the enzyme is involved in age-dependent behavioural change from nurse to a foraging way of life by increased gene amfor expression [33]; the enzyme is involved in cGMP-dependent male gametogenesis, i.e. exflagellation, together with Ca2+ release [33]; the enzyme is involved in coccidian parasite motility and invasion and is important in host-parasite interaction [36]; the enzyme is involved in gliding motility and cell invasion [33]; the enzyme is involved in locomotion behaviour of the larvae in presence of food, so-called rover and sitter gene ecoding an isozyme termed forager, overview [33]; the enzyme is required for adenosine-induced dilation of intracerebral arterioles and thus in regulation of cerebral blood flow, mechanism [47]; the enzyme mediates an exocytosis pathway via glycoprotein Ib-IX, and an aggregation-dependent platelet secretion pathway important for amplifying platelet activation, in stabilizing thrombin, and in arteriosclerosis and vascular remodeling, signaling mechanism activating the enzyme, overview [42]; the enzyme mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo [35]; the enzyme negatively regulates gene expression of thrombospondin 1, which activates the transforming growth factor-b, at both transcriptional and posttranscriptional level inhibiting the fibrogenic potential fo high d-glucose levels, overview [30]; the enzyme performs autophosphorylation at serines 110, 114, 117, 126, and 445 294

2.7.11.12

cGMP-dependent protein kinase

and at Thr109 in vitro and in vivo [37]; the NO/cGMP pathway including inhibits Ca2+ -dependent and turbulence-induced Rap 1 activation in human platelets mediated by cGKI in platelets and megakaryocytes [54]; the phorbol myristate acetate-stimulated PKC isozyme activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase [20]; through its c-Myc-, AP-1- and PEA3binding motifs the enzyme regulates the expression of thioredoxin and thioredoxin peroxidase-1 during hormesis in response to oxidative stress-induced apoptosis, the enzyme is involved in cell protection against apoptosis and lipid oxidation [40]; cGMP-activated PKGI inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes and protects them from ischemia and reperfusion injury, cGMP-activated PKG I does not inhibit MKK3- and MKK6-p38 MAPK signaling, overview [65]; guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the postsynaptic differentiation of developing neuromuscular junctions, molecular mechanism [59]; NO and cGMP protein kinase regulate dendritic-cell migration toward the lymph-node-directing chemokine CCL19, cGMP/cGK pathway, overview [56]; PKG has an instructive signaling role to control many aspects of animal physiology [62]; PKG I plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, regulation of PKG I expression by Rho and Kruppel-like transcription factor-4, RhoA is involved in decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury, overview [64]; PKG Ia attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte, overview [66]; PKG inhibits p38 MAPK activation in platelets [57]; PKG is involved in the regulation of basal tension and plays a primary role in relaxation induced by nitrovasodilators, whereas PKA may play a minor role [72]; PKGI, in part, mediates the regulation of pulmonary cell proliferation and phenotype caused by NO and cGMP, in cells that lack PKGI such as highly passaged vascular smooth muscle cells, NO and cGMP do not regulate cell proliferation and phenotype [55]; PKGIa is a major branch point in the nitric oxide and natriuretic peptide-induced cGMP-signaling pathway. PKG plays a pivotal role in several important biological processes such as the regulation of smooth muscle relaxation, and synaptic plasticity [61]; reduced enzyme activity leads to increased thermotolerance of synaptic transmission at the larval neuromuscular junction, PKG and the for gene polymorphism associated with the allelic variation in for may provide populations with natural variantion in heat stress tolerance, the for gene function in behaviour is conserved across most organisms, overview [73]; the enzyme is involved in development of anoikis, an essential process in which a loss of adhesion to the substratum alters intracellular signaling pathways that lead to apoptosis [58]; transcriptional regulation by the cgMP/PKG pathway affects the amount of IL-6 expression, cGMP-induced PKG activates the IL-6 promoter involving cis-acting DNA elements and transcription factors, 295

cGMP-dependent protein kinase

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the activation is prevented by actinomycin D, regulation system, overview [70]) (Reversibility: ?) [3, 8, 14, 15, 16, 20, 21, 23, 25, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 40, 41, 42, 44, 46, 47, 50, 52, 54, 55, 56, 57, 58, 59, 61, 62, 64, 65, 66, 70, 72, 73] P ? Substrates and products S ATP + Akt ( phosphorylation at Ser473 [66]) (Reversibility: ?) [66] P ADP + phosphorylated Akt S ATP + Arg-Lys-Arg-Ser-Arg-Ala-Glu ( i.e. Glasstide, a commercial peptide substrate [48]) (Reversibility: ?) [48] P ADP + Arg-Lys-Arg-O-phospho-Ser-Arg-Ala-Glu S ATP + CFTR ( phosphorylation leads to stimulation of chloride channels [31]; phosphorylation by cGKI and cGKII [31]) (Reversibility: ?) [31] P ADP + phosphorylated CFTR S ATP + EKRKRTYETF ( p65-derived synthetic peptide substrate [32]) (Reversibility: ?) [32] P ADP + phosphorylated EKRKRTYETF S ATP + FoxO1a ( interaction of enzyme with FocO1a is involved in regulation of myoblast cell fusion during myogenesis [49]; enzyme expression is activated by FoxO1a which is then phosphorylated by cGKI for feedback inhibition of FoxO1a DNA binding, phosphorylation of Ser152-155 and Ser184, and weakly of Ser266 and Ser268 [49]) (Reversibility: ?) [49] P ADP + phosphorylated FoxO1a S ATP + GKKRKRSRKES ( i.e. MCPD-4, a commercial peptide substrate [48]) (Reversibility: ?) [48] P ADP + GKKRKRS(P)RKES S ATP + GRTGRRNSI ( i.e. PKI, a commercial peptide substrate [48]) (Reversibility: ?) [48] P ADP + phosphorylated GRTGRRNSI S ATP + IRAG ( i.e. inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, phosphorylation by isoyzme cGKIb, which is complexed with the substrate and the inositol 1,4,5-trisphosphate receptor in a ternary complex [23]; phosphorylation by cGKIb mediates the inhibition of inositol-1,4,5-trisphosphate-dependent Ca2+ -release, a mechanism leading to smooth muscle relaxation, involved in platelet aggregation [31]; phosphorylation by cGKIb mediates the inhibition of inositol-1,4,5-trisphosphate-dependent Ca2+ -release, one mechanism leading to smooth muscle relaxation, involved in platelet aggregation [31]; i.e. inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, phosphorylation by isoyzme cGKIb [23]; i.e. inositol-1,4,5-trisphosphate-receptor-associated cGMP kinase substrate, phosphorylation by cGKIb [31]) (Reversibility: ?) [23, 31] P ADP + phosphorylated IRAG

296

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cGMP-dependent protein kinase

S ATP + Kemptide ( i.e. LRRASLG [32]; i.e. LRRASLG, a commercial peptide substrate [48]) (Reversibility: ?) [32, 48] P ADP + phosphorylated Kemptide ( i.e. LRRA-phosphoserine-LG [32]) S ATP + MQLRRPSDRE ( p65-derived synthetic peptide substrate [32]) (Reversibility: ?) [32] P ADP + phosphorylated MQLRRPSDRE S ATP + Maxi-K channel (Reversibility: ?) [28] P ADP + phosphorylated Maxi-K channel S ATP + NF-kB ( recombinant human NF-kB p49 and p50 expressed in 293T cells [32]) (Reversibility: ?) [32] P ADP + phosphorylated NF-kB S ATP + Na+ /K+ ATPase ( the enzyme has a positive regulatory function on the ion pump activity induced by No and glutamate via increased cGMP, overview [26]) (Reversibility: ?) [26] P ADP + phosphorylated Na+ /K+ ATPase S ATP + RKRSRAE ( cGK-specific peptide substrate [41]; commercial synthetic peptide substrate [39]) (Reversibility: ?) [39, 41] P ADP + RKRS(P)RAE S ATP + Rac1 ( the enzyme activates the recombinant Myc-tagged small GTPase Rac1 in HEK-293 cells leading to activation of PAK1, the enzyme induces phosphorylation of p38 which activates MAPK, regulation, overview [27]; no activity with Rac-1 mutant T17N [27]) (Reversibility: ?) [27] P ADP + phosphorylated Rac-1 S ATP + Rho ( phosphorylation leads to inhibition of Rho [31]; a small GTP-binding protein, cGKI [31]) (Reversibility: ?) [31] P ADP + phosphorylated rho S ATP + RhoA ( phosphorylation at Ser188 by isoyzme cGKI leading to translocation of RhoA from the membrane to the cytosol and inactivated RhoA [23]; phosphorylation at Ser188 by isoyzme cGKI [23]) (Reversibility: ?) [23] P ADP + phosphorylated RhoA S ATP + TPIa ( involved in desensitization of TPIa signalling [31]; phosphorylation by cGKIb [31]) (Reversibility: ?) [31] P ADP + phosphorylated TPIa S ATP + TRIM39 ( phosphorylation of the tripartite motif protein 39 in the conserved phosphorylation domain by PKGI, recombinantly expresses TRIM39 substrate [55]) (Reversibility: ?) [55] P ADP + phosphorylated TRIM39 S ATP + TRIM39R ( PKGI interacts with TRIM39R, a Rpp21 domain-containing TRIM protein, in the kinase phosphorylation domain [55]; phosphorylation of the endogenous putative PKGI-interactor and TRIM39 variant in the conserved phosphorylation domain by PKGI, recombinantly expresses TRIM39R substrate [55]) (Reversibility: ?) [55] P ADP + phosphorylated TRIM39R

297

cGMP-dependent protein kinase

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S ATP + TRPC3 ( involved in inhibition of store operated Ca2+ influx [31]; phosphorylation by cGKIb [31]) (Reversibility: ?) [31] P ADP + phosphorylated TRPC3 S ATP + VASP (Reversibility: ?) [54] P ADP + VAS(P)P S ATP + big potassium channel BKCa ( phosphorylation by cGKI leads to activation of the channel, a mechanism leading to Ca2+ dependent smooth muscle relaxation [31]; phosphorylation by cGKI [31]) (Reversibility: ?) [31] P ADP + phosphorylated big potassium channel BKCa S ATP + cysteine-riche protein 2 ( i.e. CRP2, phosphorylation by cGKI and cGKII [31]) (Reversibility: ?) [31] P ADP + phosphorylated vasodilator-stimulated phosphoprotein S ATP + glycogen synthase kinase-3 ( phosphorylation at Ser9 by PKGI and PKGII inhibiting the glycogen synthase kinase-3 inducing dephosphorylation of C/EBPb, i.e. the CCAAT enhancer-binding protein b, important in regulation of gene expression during cell proliferation, differentiation, and apoptosis [44]) (Reversibility: ?) [44] P ADP + phosphorylated glycogen synthase kinase 3 S ATP + glycogen synthase kinase3 ( phosphorylation at Ser9 by PKGI and PKGII inhibiting the glycogen synthase kinase-3, substrate phosphorylation site mapping [44]) (Reversibility: ?) [44] P ADP + phosphorylated glycogen synthase kinase 3 S ATP + histone ( DG1 [41]) (Reversibility: ?) [41] P ADP + phosphorylated histone S ATP + histone H2B (Reversibility: ?) [48] P ADP + phosphorylated histone H2B S ATP + human serotonin transporter ( i.e. hSERT, the naturally occuring hSERT mutant I425V is associated with obsessive-compulsive disorder and other neuropsychiatric disorders [69]; i.e. hSERT, no activity with hSERT mutants T276A or T276D, active with hSERT mutant I425V [69]) (Reversibility: ?) [69] P ADP + phosphorylated human serotonin transporter S ATP + inositol 1,4,5-trisphosphate receptor-I ( the phosphorylation of the receptor inhibits inositol 1,4,5-trisphosphate-induced Ca2+ release [22]; selective phosphorylation at Ser1755 [22]) (Reversibility: ?) [22] P ADP + phosphorylated inositol 1,4,5-trisphosphate receptor-I S ATP + maxiK channel ( phosphorylation by isoyzme cGKI enhances the channel activity [23]; phosphorylation of the PKC-phosphorylated substrate at Ser1072 by isoyzme cGKI [23]) (Reversibility: ?) [23] P ADP + phosphorylated maxiK channel S ATP + myosin phosphatase ( phosphorylation by isoyzme cGKIa leads to decreased level of phosphorylated MLC [23]; i.e. MP, phosphorylation of the 130 kDa myosin-binding subunit by isoyzme cGKIa [23]) (Reversibility: ?) [23] 298

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P ADP + phosphorylated RhoA S ATP + p21-activated kinase ( PKG phosphorylation of p21-activated kinase, i.e. Pak, regulates HeLa cell and human umbilical vein endothelial cell morphology and cellular remodeling, time course of Pak1 activation in vivo, overview, PKG stimulates association of vasodilator-stimulated phosphoprotein VASP with Pak [63]; activation of the wildtype p21-activated kinase, i.e. Pak1, PKG phosphorylates wild-type and mutant K299R Pak, PKG phosphorylates especially N-terminal amino acids 1-74, e.g. Ser21, and does not phosphorylate a fragment encompassing amino acids 67-149 and only weakly phosphorylated fragments from the central region of Pak1 encompassing amino acids 147-231 and the Cterminal catalytic region, amino acids 231-544, no activity with Pak1 mutant S21A, overview [63]) (Reversibility: ?) [63] P ADP + phosphorylated p21-activated kinase S ATP + p65 ( activity with recombinant wild-type p65 and mutants T305A and S276A [32]) (Reversibility: ?) [32] P ADP + phosphorylated p65 S ATP + peptide W15 ( peptide substrate sequence TQAKRKKSLAMA [61]) (Reversibility: ?) [61] P ADP + phosphorylated peptide W15 S ATP + phosphodiesterase 5 ( phosphorylation at Ser92 by isoenzyme cGKI leads to enhanced hydrolysis of cGMP, regulatory function [23]; phosphorylation at Ser92 by isoyzme cGKI [23]) (Reversibility: ?) [23] P ADP + phosphorylated phosphodiesterase 5 S ATP + phospholamban ( phosphorylation leads to stimulation of the sarcoendoplasmic reticulum pump Ca2+ -ATPase, i.e. SERCA [31]) (Reversibility: ?) [31] P ADP + phosphorylated phospholamban S ATP + phospholipase Cb3 ( potentially involved in inhibition of phospholipase C activity [31]; phosphorylation by cGKIb [31]) (Reversibility: ?) [31] P ADP + phosphorylated phospholipase Cb3 S ATP + protein ( autophosphorylation [4,19]) (Reversibility: ?) [4, 19] P ADP + phosphoprotein S ATP + septin 3 ( potentially involved in vesicle trafficking [31]; phosphorylation by cGKIb [31]; substrate from rat nerves: recombinant His-tagged septin 3 expressed in Escherichia coli or purified from brain and prepared in synaptosomes, phosphorylation at Ser91 and Ser92, no activity with septin 3 mutants S91A and S92A [24]) (Reversibility: ?) [24, 31] P ADP + phosphorylated septin 3 S ATP + septin 3 peptide 86-98 (Reversibility: ?) [24] P ADP + phosphorylated septin 3 peptide 86-98

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S ATP + steroidogenic acute regulatory protein ( i.e. StAR, recombinant GST-tagged substrate expressed in HEK-293 cells, phosphorylation in vitro at Ser55, Ser56, and Ser99 [38]) (Reversibility: ?) [38] P ADP + phosphorylated steroidogenic acute regulatory protein S ATP + target membrane SNARe complex ( the enzyme is involved in regulation of degranulation of leukocytes by phosphorylation of target membrane SNARe complex together with the phosphoinositide 3-kinase [67]) (Reversibility: ?) [67] P ADP + phosphorylated target membrane SNARe complex S ATP + troponin T ( phosphorylation by isoyzme cGKI in cardiomyocytes [23]; phosphorylation by isoyzme cGKI [23]) (Reversibility: ?) [23] P ADP + phosphorylated troponin T S ATP + vasodilator stimulated phosphoprotein ( phosphorylation of vasodilator stimulated phosphoprotein, i.e. VASP, induces detachment of VASP and other cytoskeletal proteins from focal adhesion, phosphorylated VASP may prevent dendritic cells from migrating towards CCl19 [56]; phosphorylation at Ser239 [56]) (Reversibility: ?) [56, 58] P ADP + phosphorylated vasodilator stimulated phosphoprotein S ATP + vasodilator-stimulated phosphoprotein ( i.e. VASP, phosphorylation at Ser239 by cGKI [31]) (Reversibility: ?) [31] P ADP + phosphorylated vasodilator-stimulated phosphoprotein S Additional information ( enzyme plays a crucial role in the relaxation of vascular smooth muscle by lowering the intracellular level of calcium [8]; the enzyme is thought to be involved in the regulation of intestinal ion transport and fluid secretion [14]; enzyme is involved in inhibition of platelet aggregation, relaxation of smooth muscle cells, and control of cardiocyte contractility. Pathophysiological implication of the type I cGK in cardiovascular diseases, including hypertension and atherosclerosis [16]; plays a pivotal role in the regulation of intestinal fluid balance in man [15]; amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase [3]; alternative splicing of PKGI in angiotensin-hypertension, mechanism for nitrate tolerance in vascular smooth muscle [28]; cGK is a key enzyme in the nitric oxide-cGMP and natriuretic signalling cascades, it modulates smooth muscle relaxation, platelet aggregation, renin release, intestinal secretion, learning amd memory, cGKII is involved in androsterone homeostasis, pleiotropic effets of the enzyme on diverse tissues, overview [31]; cGKI mediates NO signaling and plays a proatherogenic role in vascular smooth muscle cells, enzyme activation in primary smoothe muscle cells results in increased levels of proliferation and vascular cell adhesion molecule-1, peroxisome proliferato-activated receptor g, and phosphatidylinositol 3-kinase/Akt signaling, and in decreased plasminogen activator inhibitor 1 mRNA, overview [52]; cGMP-activated PKGIa inhibits thrombin receptor-mediated Ca2+ mobilization in vascular smooth muscle cells, cGMP-activated PKGIb causes inhibition in vitro but has nearly no effect 300

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cGMP-dependent protein kinase

in vivo, the thrombin receptor is activated by thrombin receptor activating peptide TRAP, or lysophosphatidic acid, or U4, overview [46]; cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein b functions through inhibition of glycogen synthase kinase-3 [44]; DG2P1 and DG2P2 are involved in behaviour and epithelial transport [41]; isozymes cGK I and cGK II are functionally distinct, isozyme cGK II is involved in regulation of electrolyte and water secretion by epithelial tissues in response to luminocrinic hormones guanylin and uroguanylin and in the secretory diarrhea provoked by heat-stable enterotoxins, isozyme cGK II also plays a role in the regulation of endochondral ossification by C-type natriuretic peptide, in renin secretion by the kidney, aldosterone secretion by the adrenal, and adjustment of the biological clock [34]; NO induces enzyme activity responsible for modulation/ inhibition of voltage-gated CaV1 and CaV2.2, i.e. L- and N-type, Ca2+ channels in neuroblastoma cells, in cardiovascular and smooth muscle cells Ca2+ channel activity is inhibited by direct nitrosylation of the channel proteins by NO, overview [50]; PKG-1 is involved in NO-induced cGMP-mediated increase in corpus cavernosum smooth muscle relaxation and penile erection, enzyme disfunction can cause erectile dysfunction, diabetes reduces corpus cavernosum smooth muscle relaxation [21]; PKGII modulates mPer1 and mPer2 gene induction, not by phosphorylation of CREB at Ser133, and influences phase shifts of the circadian clock, regulation model, overview [29]; PKGII regulates basal level of aldosterone production by zona glomerulosa cells without increasing expression of the steroidogenic acute regulatory protein gene [38]; sequential activation of p38 and ERK pathways by the enzyme leading to activation of the platelet integrin aIIb b3 [25]; the enzyme activates diverse signal transduction pathways, isozyme cGKI is responsible for smooth muscle relaxation by lowering the cytosolic calcium level and/or by desensitization of the contractile elements, smooth muscle contraction and enzyme-mediated smooth muscle relaxation mechanisms, detailed overview, cGKI affects the Ca2+ efflux from the endoplasmic reticulum and the cytosolic Ca2+ concentration [23]; the enzyme is involved in age-dependent behavioural change from nurse to a foraging way of life by increased gene amfor expression [33]; the enzyme is involved in cGMP-dependent male gametogenesis, i.e. exflagellation, together with Ca2+ release [33]; the enzyme is involved in coccidian parasite motility and invasion and is important in host-parasite interaction [36]; the enzyme is involved in gliding motility and cell invasion [33]; the enzyme is involved in locomotion behaviour of the larvae in presence of food, so-called rover and sitter gene ecoding an isozyme termed forager, overview [33]; the enzyme is required for adenosine-induced dilation of intracerebral arterioles and thus in regulation of cerebral blood flow, mechanism [47]; the enzyme mediates an exocytosis pathway via glycoprotein Ib-IX, and an aggregation-dependent platelet secretion pathway important for amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling, sig301

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naling mechanism activating the enzyme, overview [42]; the enzyme mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo [35]; the enzyme negatively regulates gene expression of thrombospondin 1, which activates the transforming growth factor-b, at both transcriptional and posttranscriptional level inhibiting the fibrogenic potential fo high d-glucose levels, overview [30]; the enzyme performs autophosphorylation at serines 110, 114, 117, 126, and 445 and at Thr109 in vitro and in vivo [37]; the NO/cGMP pathway including inhibits Ca2+ -dependent and turbulence-induced Rap 1 activation in human platelets mediated by cGKI in platelets and megakaryocytes [54]; the phorbol myristate acetate-stimulated PKC isozyme activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase [20]; through its c-Myc-, AP-1- and PEA3binding motifs the enzyme regulates the expression of thioredoxin and thioredoxin peroxidase-1 during hormesis in response to oxidative stress-induced apoptosis, the enzyme is involved in cell protection against apoptosis and lipid oxidation [40]; no activity with the cAMP-responsive element binding protein, i.e. CREB [29]; cGMP-activated PKGI inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes and protects them from ischemia and reperfusion injury, cGMP-activated PKG I does not inhibit MKK3- and MKK6p38 MAPK signaling, overview [65]; guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the postsynaptic differentiation of developing neuromuscular junctions, molecular mechanism [59]; NO and cGMP protein kinase regulate dendritic-cell migration toward the lymph-node-directing chemokine CCL19, cGMP/ cGK pathway, overview [56]; PKG has an instructive signaling role to control many aspects of animal physiology [62]; PKG I plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, regulation of PKG I expression by Rho and Kruppel-like transcription factor-4, RhoA is involved in decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury, overview [64]; PKG Ia attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte, overview [66]; PKG inhibits p38 MAPK activation in platelets [57]; PKG is involved in the regulation of basal tension and plays a primary role in relaxation induced by nitrovasodilators, whereas PKA may play a minor role [72]; PKGI, in part, mediates the regulation of pulmonary cell proliferation and phenotype caused by NO and cGMP, in cells that lack PKGI such as highly passaged vascular smooth muscle cells, NO and cGMP do not regulate cell proliferation and phenotype [55]; PKGIa is a major branch point in the nitric oxide and natriuretic peptideinduced cGMP-signaling pathway. PKG plays a pivotal role in several important biological processes such as the regulation of smooth muscle relaxation, and synaptic plasticity [61]; reduced enzyme activity leads to increased thermotolerance of synaptic transmission at the larval neuromuscular junction, PKG and the for gene polymorphism associated with 302

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the allelic variation in for may provide populationswith natural variantiion in heat stress tolerance, the for gene function in behaviour is conserved across most organisms, overview [73]; the enzyme is involved in development of anoikis, an essential process in which a loss of adhesion to the substratum alters intracellular signaling pathways that lead to apoptosis [58]; transcriptional regulation by the cgMP/PKG pathway affects the amount of IL-6 expression, cGMP-induced PKG activates the IL-6 promoter involving cis-acting DNA elements and transcription factors, the activation is prevented by actinomycin D, regulation system, overview [70]; structure-function relationship of PKG [62]) (Reversibility: ?) [3, 8, 14, 15, 16, 20, 21, 23, 25, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 40, 41, 42, 44, 46, 47, 50, 52, 54, 55, 56, 57, 58, 59, 61, 62, 64, 65, 66, 70, 72, 73] P ? Inhibitors 1H-[1,24]oxadiazolo-[4,3-a]quinoxalin-1-one [47] 4-[2-(4-fluorophenyl)-5-(1-methylpiperidin-4-yl)-1H-pyrrol-3-yl]pyridine ( a synthetic compound [68]) [68] 4-[2-(fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine ( inhibitor of apicomplexan parasite enzyme, IC50 is 1 nM for the enzyme of Eimeria tenella, mechanism [33]; inhibitor of apicomplexan parasite enzyme, mechanism [33]) [33] 4-methoxy-3,6-diphenylcyclohexa-3,5-diene-1,2-dione ( isolated from a microbial extract of a Phoma sp. [68]) [68] 6’-methoxy-1,1’:4’,1’’-terphenyl-2,2’,5’-triol ( isolated from a microbial extract of a Phoma sp. [68]) [68] 6’-methoxy-1,1’:4’,1’’-terphenyl-2,2’,5,5’-tetrol ( isolated from a microbial extract of a Phoma sp. [68]) [68] 6-anilino-5,8-quinolinedione [40] 7-nitroindazole [40] 8-(4-chlorophenylthio)guanosine 3’,5’-cyclic monophosphate [63] 8-(4-chlorophenylthio)guanosine 3’,5’-cyclic monophosphorothioate Rp-isomer [69] angiotensin ( causes a decrease in expression of isozyme cGKIa, and an increase in expression of isozyme cGKIb [28]) [28] d-glucose ( high levels reduce enzyme activity in vivo leading to increased thrombospondin 1-dependent activation of transforming growth factor-b, and increased TGF- b-dependent expression of fibronectin and collagene type IV [30]) [30] DT-2 peptide ( a HIV tat peptide sequence, blocks type 1 PKG [58]) [58] KT5823 ( i.e. (8R,9S,11S)-(-)-9-methoxy-carbamyl8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H-2,7b,11a-trizadizobenzo9(a,g)cycloocta(c,d,e)-trinden-1-one, selective PKG inhibitor [22]; PKG inhibitor [43]; specific antagonist [20]; specific for PKG [26]; specific PKG inhibitor [50]; staurosporine analogue, inhibits in vitro by selective blockage of ATP binding [31]; a specific PKG inhibitor,

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PKG inhibition induces rapid thermotolerance of neural circuitry [73]; in vivo inhibition in leukemia cells causes reduced agonist-stimulated b-hexosaminidase release and abolishes vesicular fusion with the plasma membrane [67]) [20, 22, 25, 26, 31, 42, 43, 50, 67, 73] KT5926 [33] N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide [47] Rp-8-Br-PET-cGMPS ( PKG-I inhibition leads to abolished induction of p38 by thrombin [25]; the PKG inhibitor has no effect in vivo on the basal tension of vessels treated with nitro-l-arginine, nitro-l-arginine plus indomethacin, or of vessels denuded of the endothelium, overview [72]) [25, 31, 42, 72] Rp-8-Br-cGMP ( in vivo inhibition in leukemia cells causes reduced agonist-stimulated b-hexosaminidase release and abolishes vesicular fusion with the plasma membrane [67]) [67] Rp-8-Br-pCPT-cGMPS ( i.e. Rp-8-bromo-(4-chlorophenylthio)guanosine 3,5-cyclic monophosphate [31]) [31] Rp-8-p-CPT-cGMP ( i.e. Rp-8-(4-chlorophenylthio)guanosine 3,5cyclic monophosphate [38]) [38] Rp-8-pCPT-cGMP ( i.e. 8-(4-chlorophenylthio)guanosine-3,5-cyclic monophosphorothionate Rp isomer, inhibits DG2P1 and DG2P2 at 0.01 and 0.1 mM, respectively [41]; i.e. Rp-8-(4-chlorophenylthio)guanosine 3,5-cyclic monophosphate, cGKI inhibitor, reverses the NO effects [54]; Rp-i.e. 8-(4-chlorophenylthio)guanosine 3,5-cyclic monophosphate [42]) [41, 42, 54] Rp-8-pCPT-cGMPS ( PKG inhibitor [40]) [32, 40, 59] Rp-8-pCTP-cGMPS ( selective for PKG [47]) [47] Rp-pCPT-cGMPS [25, 42] staurosporine [33] pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine ( inhibits native and recombinant enzyme, shows antiparasitic activity being active against Plasmodium falsiparum blood cell stages cultured in vitro, IC50 for the native enzyme is 8.35 nM, and for the recombinant FLAG-tagged enzyme 3.48 nM [48]) [48] pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine ( inhibits downstream of calcium-dependent events, inhibition is not reversible by calcium ionophores or ethanol, inhibits uracil uptake, blocks the attachment of the sporozoite to the host cells and inhibits parasite invasion and gliding motility, inhibits the secretion of micronemal adhesins MIC1 and MIC2 by the parasite, no inhibition of mutants T770M and T770Q [36]; inhibits downstream of calcium-dependent events, inhibition is not reversible by calcium ionophores or ethanol, inhibits uracil uptake, blocks the attachment of the tachyzoite to the host cells and inhibits parasite invasion and gliding motility, inhibits the secretion of micronemal adhesin MIC2 by the parasite, no inhibition of mutants T761M and T761Q [36]) [36]

304

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regulatory subunit of photoreceptor cGMP phosphodiesterase ( inhibits PKGIa, the N-terminal 61 amino acids are important containing a cationic region [71]) [71] terferol ( isolated from a microbial extract of a Phoma sp. [68]) [68] Additional information ( cGK II contains an autoinhibitory region [34]; enzyme activity is reduced in diabetic rabbits [21]; inhibition by DT-2 fusion peptide of in vitro peptide inhibitor W45 and the membrane translocation signal sequences from HIV tyrosine aminotransferase protein, and DT-3 fusion peptide of W45 and the Drosophila antennapedia homeodomain [31]; inhibition of PKG affects platelet secretion, platelet aggregation, and Ca2+ mobilization [42]; no inhibition by myristoylated PKI14-22 amide [22]; PKG-I inhibition leads to abolished induction of p38 by thrombin [25]; PKGI contains autoinhibitory subdomains in the regulatory domains and two allosteric cGMP-binding sites of isozymes a and b [45]; PKGII inhibition inhibits aldosterone production [38]; the enzyme is inactivated by expression of a dominant negative mutant type 1a PKG construct [27]; the enzyme possesses an N-terminal autoinhibitory sequence [33]; the enzyme possesses an N-terminal autoinhibitory sequence ERKVQKAIKQQE [33]; the enzyme possesses an N-terminal autoinhibitory sequence ERNKKKAIFSND [33]; no inhibition by the PKA-specific inhibitor myristoylated PKI [72]; Rho and Krueppel-like transcription factor-4 KLF4, a bound nuclear protein, suppress PKG I expression requiring Sp1 consensus sequences in the PKG I promoter, suppression in a in a cell density-dependent manner [64]; the enzyme is regulated via negative feedback [56]) [21, 22, 25, 27, 31, 33, 34, 38, 42, 45, 56, 64, 72] Cofactors/prosthetic groups 8-bromo-cGMP ( 50% activation at 0.00004 mM [12]) [12] 8-bromo-cAMP ( 50% activation at 0.00062 mM [12]) [12] ATP [21, 22, 23, 24, 26, 27, 31, 32, 37, 38, 39, 41, 42, 44, 45, 47, 48, 49, 53, 54, 55, 56, 58, 61, 62, 63, 65, 66, 67, 68, 69, 70, 72, 73] cAMP ( 50% activation at 0.0117 mM, activation is not cooperative [12]) [1,12] cGMP ( dependent on [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19]; cGMP, 0.010 mM, stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold [12]; 50% activation at 0.00019 mM, cooperative activation [12]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19] cIMP ( 50% activation at 0.0053 mM [12]) [12] Activating compounds 20 kDa heat shock protein ( i.e. Hsp20, involved in cGKI-induced smooth muscle relaxation, probably via the actin binding sequence [23]) [23] 305

cGMP-dependent protein kinase

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8-(4-chlorophenylthio)guanosine 3’,5’-cyclic monophosphate [57] 8-APT-cGMP ( cGKIa-selective activator [28]) [28] 8-Br-PET-cGMP ( activation of cGKIa and cGKII, specificity overview [31]) [31] 8-Br-cGMP ( activates [27]; activation of cGKIa and cGKII, specificity overview [31]; selective for PKG [47]) [27, 31, 40, 47, 50, 52, 57, 69] 8-bromo-cGMP ( PKG activator 8-bromo-cGMP abolishes thermoprotective effect of a prior heat shock [73]) [25, 26, 46, 58, 67, 70, 73] 8-NBD-cGMP ( i.e. 8-[[2-[(7-nitro-4-benzofurazanyl)amino]ethyl]thio]guanosine-3,5-cyclic monophosphate, poor, non-cooperative binding [33]) [33] 8-bromo-cyclic GMP [59] 8-pCPT-CGMP ( i.e. 8-(4-chlorophenylthio)guanosine 3,5-cyclic monophosphate [24, 31, 38]; i.e. 8-(4-chlorophenylthio)guanosine 3,5-cyclic monophosphate, 358% activation, inhibitor KT5823 abolishes the activating effect [22]; i.e. 8-(4-chlorophenylthio)guanosine 3,5-cyclic monophosphate, activation of cGKIa and cGKII, specificity overview [31]; i.e. i.e. 8-(4-chlorophenylthio)guanosine 3,5-cyclic monophosphate, cGMP analogue, membrane-permeable [54]) [22, 24, 31, 38, 54] 8-pCPT-GMP [25] adenosine ( adenosine-induced vasodilation in cerebral microvessesls involves cGMP and cGMP-dependent protein kinase [47]) [47] angiotensin ( causes an increase in expression of isozyme cGKIb, and a decrease in expression of isozyme cGKIa [28]) [28] Bay412272 ( YC1 analogue, stimulates the enzyme in a NO-dependent manner, inhibition of phosphodiesterase 5 [31]) [31] FoxO1a ( directly activates enzyme expression binding to DNA in skeletal muscle [49]) [49] NO ( NO stimulates PKGI in vascular smooth umscle cells contributing to cytoskelatl kinetics and phenotype, overview [55]) [55, 56] PET-cGMP ( activation of cGKIa and cGKII, specificity overview [31]) [31] Sp-8-pCTP-cGMPS ( cyclic guanosine monophosphate-dependent protein kinase activator, selective for PKG [47]) [47] Vasoactive intestinal peptide ( i.e. VIP [22]) [22] YC1 ( i.e. 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole, hemdependent activation of cGK, inhibition of phosphodiesterase 5 [31]) [31] cAMP ( crossactivation at high levels in vivo [22]) [22] cGMP ( dependent on [21, 27, 35, 40, 43, 46, 49, 50, 51, 54]; activates both PKGI and PKGII [44]; activates native and recombinant enzyme [37]; activates native and recombinant enzyme, the activation of recombinant cGKII is reduced because the enzyme already partially phosphorylated [37]; binding site analysis, positive cooperativity at low concentration [33]; cGK I and cGK II contain a high and a low affinity binding site for cGMP with opposite orientation in cGK II compared 306

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to cGK I [34]; cooperative kinetics of stimulation [48]; highly activating, binding site analysis [33]; highly activating, binding site analysis, cooperative binding [33]; highly dependent on, about 10fold activation, binding and activation mechanisms for wild-type and mutant PKG-Ib, modeling, the enzyme elongates upon cGMP binding [53]; level is increased by NO and glutamate [26]; required by the full length enzyme for activity, the activity of the recombinant catalytic domain is independent of cGMP [30]; selective for PKG [47]; stimulates DG2P1 and DG2P2, and DG1, the latter maximally at 20 mM [41]; two allosteric binding sites in the regulatory domains in PKGI isozymes, structure and mechanism, enzyme elongation by cGMP binding ehich induces conformational changes, overview [45]; molecular mechanism and thermodynamics [61]) [21, 22, 23, 24, 26, 27, 28, 30, 31, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 65, 67, 70, 72, 73] cgs-21680 [47] formyl-methionyl-leucyl-phenylalanine [67] forskolin ( crossactivation at high levels above 0.001 mM in vivo [22]) [22] isoprotenol ( crossactivation at high levels above 0.001 mM in vivo [22]) [22] lipopolysaccharides ( induces enzyme expression and activates the enzyme [56]) [56] sodium nitroprusside ( i.e. SNP, selective activator of PKG, 414% activation, inhibitor KT5823 abolishes the activating effect [22]; i.e. SNP, selective for PKG [47]) [22, 47] Additional information ( aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme [4]; cGKI activity is induced by NO via increased cGMP production [54]; cGKII is upregulated by aldosterone inducation e.g. via low salt diet, NO induces cGMP production and thus increases enzyme activity, cGK is also activates via increased cGMP level by inhibition of the phosphodiesterase 5 by sildenafil, vardenafil, and taladafil, or by inhibition of Ca2+ /calmodulin-specific cGMP-dependent phosphodiesterase 1 [31]; enzyme is induced by NO via cGMP [27]; glutamate induced NO, and increased NO release from sodium nitroprusside and S-nitroso-N-acetylpenicillamine increase cGMP level which increases PKG activity, overview [26]; high glucose levels induce NO-induced stimulation of cGMP production and increase PKG-1 activity [21]; isozymes cGK I and cGK II exhibit different affinity for cGMP analogues, overview [34]; NO activates the enzyme through elevation of cGMP concentration, reduced in hypertensive rats due to a shift in relation of cGKIa to cGKIb [28]; NO activates the enzyme via increased cGMP level leading to vascular relaxation [46]; NO induces cGMP production and thus increases enzyme activity [51]; NO induces cGMP production which activates the enzyme [23]; NO induces the enzyme via increased 307

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cGMP production [40]; NO, from NO donor sodium nitroprusside, induces cGMP production and thus increases enzyme activity [35,50]; the enzyme mediates NO antiatherogenic and proatherogenic effects [52]; mechanical stress also induces the enzyme, e.g. in lung epithelial cells, umbilical vein endothelial cells, and osteoblasts [70]; Rac1 stimulates PKG I expression [64]) [4, 21, 23, 26, 27, 28, 31, 34, 35, 40, 46, 50, 51, 52, 54, 64, 70] Metals, ions Ca2+ ( required for e.g. inhibition of inositol-1,4,5-trisphosphate-dependent Ca2+ -release [31]) [31] Mg2+ ( maximal activation at 4050 mM [12]; required by DG2P1 and DG2P2, and DG1 [41]) [12, 22, 24, 27, 31, 32, 37, 39, 41, 42, 45, 48, 55, 61, 62, 63, 65, 66, 68, 72, 73] Specific activity (U/mg) 0.000006 ( purified recombinant FLAG-tagged enzyme, substrate Glasstide [48]) [48] 0.000028 ( purified recombinant FLAG-tagged enzyme, substrate Kemptide [48]) [48] 0.000037 ( purified recombinant FLAG-tagged enzyme, substrate MCPD-4 [48]) [48] 0.000041 ( purified recombinant FLAG-tagged enzyme, substrate histone H2B [48]) [48] 0.0004 ( purified recombinant FLAG-tagged enzyme, substrate PKI [48]) [48] 0.005 ( recombinant catalytic domain in rat mesangial cell extract in absence of cGMP [30]) [30] 0.011 ( recombinant catalytic domain in rat mesangial cell extract in presence of cGMP [30]) [30] 0.02 ( recombinant tetracyclin-induced catalytic domain in rat mesangial cell extract in absence of cGMP [30]) [30] 0.026 ( recombinant tetracyclin-induced catalytic domain in rat mesangial cell extract in presence of cGMP [30]) [30] 2.72 ( purified monomeric PKG-Ib deletion mutant D1-52 [53]) [53] Additional information ( activity in vasodilation of arterioles measured in passive diameter [47]; the enzyme shows low basal activity [33]) [24, 33, 36, 47] Km-Value (mM) 0.00008 (hSERT T276D/I425V) [69] 0.00011 (hSERT T276D) [69] 0.00012 (hSERT T276A/I425V) [69] 0.00013 (hSERT T276E) [69] 0.00019 (hSERT T276A) [69] 0.00024 (hSERT) [69] 0.00026 (hSERT I425V) [69] 0.0104 (ATP, pH 7.4, 30 C, recombinant PKG Ib deletion mutant D152 [39]) [39]

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0.0133 (ATP, pH 7.4, 30 C, recombinant wild-type PKG Ib [39]) [39] 0.0139 (ATP, pH 7.4, 30 C, recombinant PKG Ib leucine zipper mutant [39]) [39] 0.0263 (RKRSRAE, pH 7.4, 30 C, recombinant PKG Ib deletion mutant D1-52 [39]) [39] 0.0304 (RKRSRAE, pH 7.4, 30 C, recombinant wild-type PKG Ib [39]) [39] 0.0313 (RKRSRAE, pH 7.4, 30 C, recombinant PKG Ib leucine zipper mutant [39]) [39] 0.062 (GRTGRRNSI, pH 7.4, 37 C, recombinant FLAG-tagged enzyme [48]) [48] 0.0865 (septin 3 peptide 86-98, pH 7.4, 30 C, PKG-I [24]) [24] Additional information ( kinetics [48,69]; cGMP binding kinetics for recombinant regulatory domains of PKGI isozymes [45]; dissociation constants for cGMP binding by wild-type and mutant PKG-Ib [53]; kinetics and dissociation constants of cGMP [39]; thermodynamics of PKGIa activation by cGMP, overview [61]) [39, 45, 48, 53, 61, 69] Ki-Value (mM) 0.035 (Rp-8-Br-PET-cGMPS, cGKI [31]) [31] 0.23 (KT5823, cGKIa [31]) [31] 0.23-0.5 (Rp-8-Br-pCPT-cGMPS, cGKII [31]) [31] 0.5 (Rp-8-Br-pCPT-cGMPS, cGKI [31]) [31] 0.9 (Rp-8-Br-PET-cGMPS, cGKII [31]) [31] Additional information [36] pH-Optimum 6.8-7.6 ( assay at, study of pH responsiveness of asteriole enzymes in vivo [47]) [47] 6.9 ( assay at, substrate Kemptide [32]) [32] 7.4 ( assay at [22, 24, 37, 39, 48, 55, 63, 67, 72]; assay at, in vivo [26]) [22, 24, 26, 37, 39, 48, 55, 63, 67, 72] 7.5 ( assay at [27,41]; assay at, substrate NF-kB [32]) [27, 32, 41] Temperature optimum ( C) 22 ( assay at room temperature [32]) [32] 30 ( assay at [24,37,39,63,72]) [24, 37, 39, 63, 72] 31 ( in vivo phosphorylation assay at [22]) [22] 34 ( assay at, in vivo [26]) [26] 37 ( assay at [48,67]; in vivo assay at [47]) [47, 48, 67]

4 Enzyme Structure Molecular weight 76000 ( recombinant deletion mutant D1-52, monomeric form, sedimentation equilibrium analysis [39]) [39]

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86000 ( recombinant PKG Ib mutant L17A/I24A, monomeric form at 0.95 nM concentration, sedimentation equilibrium analysis [39]) [39] 88000 ( recombinant PKG Ib mutant L31A/I38A, monomeric form at 0.95 nM concentration, sedimentation equilibrium analysis [39]) [39] 89000 ( recombinant PKG Ib mutant L3A/L10A/L45A/I52A, monomeric form, sedimentation equilibrium analysis [39]) [39] 91000 ( recombinant PKGIa D349-670, sucrose density gradient centrifugation [45]) [45] 92000 ( recombinant PKG Ib mutant L31A/I38A/L45A/I52A, monomeric form, sedimentation equilibrium analysis [39]) [39] 96000 ( recombinant PKG Ib mutant L3A/L10A/L17A/I24A, monomeric form, sedimentation equilibrium analysis [39]) [39] 98000 ( recombinant PKG Ib leucine zipper mutant, monomeric form, sedimentation equilibrium analysis [39]) [39] 99000 ( recombinant PKGIb D364-685, sucrose density gradient centrifugation [45]) [45] 113000-125000 ( gel filtration [33]) [33] 120000 ( recombinant PKG Ib mutant L17A/I24A, dimeric form at 51 nM concentration, sedimentation equilibrium analysis [39]) [39] 140000 ( recombinant PKG Ib mutant L31A/I38A, dimeric form at 51 nM concentration, sedimentation equilibrium analysis [39]) [39] 170000 ( recombinant PKG Ib mutants L3A/L10A, L17A, and I24A, dimeric forms, sedimentation equilibrium analysis [39]) [39] 180000 ( recombinant wild-type PKG Ib, dimeric form, sedimentation equilibrium analysis [39]) [39] Subunits ? ( x * 76331 [6]; x * 77803, calculation from nucleotide sequence [9]; x * 65000, recombinant DG1, SDS-PAGE, x * 84000, recombinant DG2P1 and DG2P2, SDS-PAGE [41]; x * 98000, native enzyme, SDS-PAGE [48]; x * 75000, PKG type I, SDS-PAGE [72]) [6, 9, 41, 48, 72] dimer ( 2 * 40000, about, recombinant PKGIa D349-670, sequence calculation, 2 * 41000, about, recombinant PKGIb D364-685, sequence calculation [45]; dimerization of PKG Ib via the N-terminal dimerization domain containing an extensive leucine zipper motif modifying enzyme activity [39]; 2 * 76000, wild-type enzyme, SDS-PAGE [61]) [39, 45, 61] monomer ( 1 * 113000-125000, functional enzyme, SDS-PAGE [33]; structure analysis of PKG-Ib deletion mutant D1-52, light scattering [53]) [33, 53] Additional information ( domain structure [33]; analysis of the leucine zipper motif within the N-terminal dimerization domain, overview [39]; dimerization of isozymes PKGIa and Ib, each subunit contains a regulatory R domain and a catalytic C domain [45]; domain structure of cGK II compared to the structure of cGK Ia and b, cGK II is composed of an N-terminal regulatory domain, a dimerization, an autoin-

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hibitory region, two cGMP-binding domains, and a C-terminal catalytic domain [34]; peptide fragment identification by mass spectrometry after limited proteolysis of wild-type PKG in the absence and presence of cGMP, overview [61]; structure-function relationship of PKG [62]) [33, 34, 39, 45, 61, 62] Posttranslational modification phosphoprotein ( autophosphorylation [4, 5, 33]; autophosphorylation by DG1 [41]; autophosphorylation of isozymes at a Thr in the catalytic domain is essential, phosphorylation sites, also for other kinases, overview [33]; autophosphorylation of isozymes, phosphorylation sites, also for other kinases, overview [33]; the enzyme performs autophosphorylation at serines 110, 114, 117, 126, and 445 and at Thr109, phosphorylation at Ser110 and Ser97 of the recombinant enzyme expressed in Sf9 insect cells, phosphorylation site mapping [37]) [4, 5, 33, 37, 41]

5 Isolation/Preparation/Mutation/Application Source/tissue CCD-841 cell ( colonic epithelial cell line [58]) [58] CS54 cell ( pulmonary artery smooth muscle cell line [64]) [64] Co-403 cell ( artery smooth muscle cell line [46]) [46] HEK-293 cell [27, 31] HEp-2 cell [55] HeLa cell [63] IEC-CF7 cell ( intestinal cell line [31]) [31] IMR-32 cell ( neuroblastoma cell line [50]) [50] MEG-01 cell ( megakaryocyte cell line [54]) [54] Purkinje cell [31] RBL-2H3 cell ( a basophilic leukemia cell line [67]) [67] SCHNEIDER-2 cell [41] SH-SY5Y cell ( neuroblastoma cell line [40]) [40] UMR-106 cell ( primary psteoblast cells [44]) [44, 70] adrenal gland ( cortex, PKGI [38]) [38] amygdala [51] aorta ( smooth muscle [31]; high expression of isozymes PKG-1a and PKG-1b [21]) [21, 31, 46, 64] arteriole ( cerebral, microvascular, isolated from cortical vessels [47]) [35, 47] artery [46] auditory vesicle ( embryonal, high expression of isozyme PKGIb [43]) [43] bladder [31] blood platelet [25, 31, 42, 54, 57]

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blood vessel ( PKGI [38]; pulmonary arterial, smooth msucle [20]) [20, 35, 38] brain ( isozyme cGK II [34]) [18, 31, 34, 51, 71] cardiac myocyte [31] cardiomyocyte [23, 65, 66] central nervous system [51] cerebellum ( Purkinje cells [31]; predominant expression of isozyme cGKIa [51]) [31, 51] cerebral cortex ( predominant expression of isozyme cGKIb [51]) [51] chondrocyte [31] colon [58] commercial preparation ( native purified from lung or recombinant [32]) [32, 71] coronary artery [72] corpus cavernosum ( corpus cavernosum smooth muscle, high expression of isozymes PKG-1a and PKG-1b, corpus cavernosum smooth muscle of diabetic rabbits sho reduced expression of isozyme PKG-1a [21]) [21] corpus striatum [26] dendritic cell ( derived from CD14+ monocytes [56]) [56] embryo ( expression of both PKGIa and PKGIb after late gastrula stage [43]) [43] endothelial cell ( HUVEC [63]) [63] epithelial cell [58, 70] epithelium ( intestinal [37]) [37, 41] eye ( similar expression of isozymes cGKIa and cGKIb [51]) [51, 71] fibroblast ( in normal diploid fibroblasts, the gene is constitutively expressed during cell-cycle and population doubling levels [13]) [13, 27] gastrointestinal tract [31] gill arch ( embryonal, high expression of isozyme PKGIa [43]) [43] head ( of the fly [41]) [41] heart ( expressed at a much higher level in newborn than in adult [2]; low expression of isozymes PKG-1a and PKG-1b [21]) [2, 21, 31, 60] hippocampus ( predominant expression of isozyme cGKIb [51]) [31, 51] hypothalamus ( predominant expression of isozyme cGKIb [51]) [29, 51] intestine ( epithelium [37]; intestinal mucosa [18]; isozyme cGK II [34]) [18, 31, 34, 37] kidney ( isozyme cGK II [34]) [18, 31, 32, 34] larva [73] leukemia cell [67] leukocyte [67] 312

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lung ( epithelium [70]) [5, 6, 20, 24, 32, 55, 70] malpighian tubule [41] mast cell [67] medulla oblongata ( predominant expression of isozyme cGKIa [51]) [51] megakaryocyte ( MEG-01 cell line [54]) [54] muscle [73] myoblast ( primary [49]) [49] myocyte [49] myotube [49] neuroblastoma cell [50] neuromuscular junction [59] neuron ( enteric [31]; photosensitive [71]) [31, 71] olfactory bulb ( predominant expression of isozyme cGKIb [51]) [51] osteoblast ( primary [70]) [44, 70] osteosarcoma cell [70] penis [31] pineal peduncle [51] pituitary gland ( similar expression of isozymes cGKIa and cGKIb [51]) [51] placenta [9, 39] prostate gland ( transition zone, distribution of cGKI [60]) [60] retina [51] skeletal muscle cell ( embryonic [59]) [49, 59] small intestine [14] smooth muscle ( corpus cavernosum smooth muscle, high expression of isozymes PKG-1a and PKG-1b, corpus cavernosum smooth muscle of diabetic rabbits show reduced expression of isozyme PKG-1a [21]; e.g. aortic [23]; gastric, primary [22]; pulmonary arterial [20]; high expression levels of cGKI in vascular and intestinal smooth muscle, both isozymes in cardiovascular muscle, fibromuscular stroma [60]; pulmonary artery [64]) [20, 21, 22, 23, 28, 31, 55, 60, 64] smooth muscle cell ( vascular [46]; vascular, aortic [52]) [46, 52] spinal cord ( embryonal, high expression of isozyme PKGIa [43]) [43] sporozoite [33, 36] stomach [22] subcommissural organ [51] suprachiasmatic nucleus ( of the hypothalamus [29]) [29] tachyzoite [33, 36] trachea ( trachea smooth muscle [7]) [7] umbilical vein ( HUVEC [63]) [63] umbilical vein endothelium [70] urinary bladder ( expression of isozymes PKG-1a and PKG-1b [21]) [21] 313

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vascular cell [64] zona glomerulosa cell ( adrenal, PKGI, expression of PKGII is restricted to adrenal zona glomerulosa cells [38]) [38] zona glomerulus ( expression of cGKII [31]) [31] Additional information ( distribution of cGKI isozymes in brain and eye, overview [51]; isozymes cGK I and cGK II show different tissue distribution and expression patterns [34]; a and b isozymes of cGKI show a different tissue distribution [60]; no expression of PKG in colon carcinoma cell lines SW480 and SW620 [58]) [34, 51, 58, 60] Localization cytoplasm ( PKG1, primarily in cGMP untreated cells [70]) [70] cytosol ( mainly [27]; DG1 [41]) [23, 27, 28, 41] extracellular ( dynamic membrane localization [27]) [27] membrane ( isozyme cGK II bound by a myristoyl moiety [34]) [18, 34] nucleus ( PKG1, primarily in cGMP or 8-bromo-cGMp treated cells [70]) [70] plasma membrane ( DG2P1 and DG2P2 [41]) [27, 41] Purification (recombinant His-tagged cGKII from Sf9 cells by nikel affinity chromatography) [37] (recombinant His-tagged cGKII from Sf9 cells by nikel affinity chromatography) [37] (recombinant PKGIa D349-670 and PKGIb D364-685 from Sf9 insect cells by 8-aminohexylamino-cAMP affinity chromatography and gel filtration) [45] (recombinant wild-type and mutant PKG Ib from Sf9 insect cells by 8amino-hexylamino-cAMP affinity chromatography and gel filtration) [39] (isozyme PKG-Ia) [22] (native enzyme is purified partially by cGMP affnity and ion exchange chromatography, recombinant FLAG-tagged enzyme from Toxoplasma gondii by anti-FLAG affinity chromatography) [48] Cloning (in vitro transcription of DG2P1 and DG2P2, and DG1 by RT-PCR of cDNAs from tubule cells, targeted expression of DG2P1 and DG2P2, and DG1 in malpigian tubule using the UAS/GAL4 system, differential localization of all 3 enzyme forms in transgenic flies) [41] (the enzyme is encoded by the foraging gene for which shows polymorphisms associated with the allelic variation in for) [73] (phylogenetic analysis) [33] (DNA sequence determination of isozyme PKGIa, functional co-expression of His-tagged enzyme in 293T cells with human NF-kB p49 and p50, NF-kB activates PKG, which increases 5fold transactivation activity of p65 from consensus sites but not from a nonconsensus NF-kB reporter) [32]

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(expression of His-tagged cGKII in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [37] (adenoviral transfection of rat adult cardiomyocytes with the human isozyme PKGIa and its catalytically inactive mutant PKGIaK390A) [66] (alternate splicing of the 5’ end of a pre-mRNA encoded by a single gene results in isozymes a and b PKGI, expression of the constitutively active catalytic region of PKGI from a lung cDNA library, and co-expression with TRIM39R in a yeast two hybrid system) [55] (expression of GFP-tagged dominant negative mutant type 1-a PKG in A-549 cells and of untagged mutant in HEK-293 cells) [27] (expression of His-tagged cGKII in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [37] (expression of PKGIa D349-670 and PKGIb D364-685 consisting of the regulatory domains in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [45] (expression of isozyme PKG1b in COS-7 or 293T cells) [63] (placental PKG Ib, expression of wild-type and mutant enzymes in Escherichia coli and in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [39] (stable expression of PKGIa and PKGIb in CHO-K1 cells, overexpression of PKGIa and PKGIb in Co403 cells by adenoviral infection, only expression of PKGIa after treatment with 8-bromo-cGMP leads to inhibition of thrombin receptor-mediated Ca2+ mobilization) [46] (stable tetracycline-inducible expression of the catalytic enzyme domain in Rattus norvegicus mesangial cells leading to suppression of glucose-induced expression of thrombospondin 1 and thus of transforming growth factor-b activaation and the TGF-b-dependent expression of fibronectin and collagene type IV) [30] (co-expression of FLAG-tagged p38a MAPK, and PKG Ia or PKG I-DN192 plasmid expression vectors, and stimulation with 8-pCPT-cGMP, in HEK293 cells) [65] (expression of PKGI and PKGII in UMR106 cells, PKGI expression enhances the glycosynthase kinase-3 phosphorylation to a greater extent than PKGII) [44] (expression of wild-type and mutant cGKIIs in COS-1 cells) [37] (expression of PKGIa in transgenic Xenopus laevis embryos) [59] (overexpression of wild-type and mutant enzymes in Spodoptera frugiperda Sf9 cells using the baculovirus infection method) [61] (phylogenetic analysis) [33] (gene egl-4, expression analysis of wild-type and mutant enzymes) [62] (phylogenetic analysis) [33] (DNA and amino acid sequence determination and analysis, transient expression of FLAG-tagged enzyme in Toxoplasma gondii parasites, comparison of properties of the recombinant enzyme to the native one) [48] (phylogenetic analysis) [33] (expression in COS cells) [2]

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(cDNA of the two isoforms of bovine cGMP-dependent protein kinase) [7] (characterization of the human gene encoding the type I a and type I b cGMP-dependent protein kinase) [8] [7] (phylogenetic analysis) [33] (expressed in Sf9 cells) [12] [13, 14] [8, 16] (expression in Cos-1 cells) [17] [18] (expression of tagged enzyme in Escherichia coli, mutational analaysis, phylogenetic analysis) [33] (expression of tagged enzyme in Escherichia coli, mutational analaysis, phylogenetic analysis) [33] (phylogenetic analysis, expression in Toxoplasma gondii) [33] (phylogenetic analysis) [33] (phylogenetic analysis) [33] (phylogenetic analysis) [33] (phylogenetic analysis) [33] (phylogenetic analysis) [33] (DNA sequence determination and analysis, expression in NG108-15 hybrid cells constructed of mouseneuroblastoma cell line N18TG-2 and rat glioma cell line C6BU-1) [43] (DNA sequence determination and analysis, expression in NG108-15 hybrid cells constructed of mouseneuroblastoma cell line N18TG-2 and rat glioma cell line C6BU-1) [43] Engineering I24A ( site-directed mutagenesis, unaltered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] K482A ( site-directed mutagenesis, transfection of the catalytically inactive PKGII mutant inhibits aldosterone production in zona glomerulosa cells [38]) [38] L17A ( site-directed mutagenesis, unaltered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] L17A/I24A ( site-directed mutagenesis, altered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] L17A/I24A/L31A/I38A/L45A/I52A ( site-directed mutagenesis, altered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] L31A/I38A ( site-directed mutagenesis, unaltered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] L31A/I38A/L45A/I52A ( site-directed mutagenesis, altered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] L3A/L10A ( site-directed mutagenesis, altered dimerization and molecular weight compared to the wild-type enzyme [39]) [39]

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L3A/L10A/L17A/I24A ( site-directed mutagenesis, altered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] L3A/L10A/L45A/I52A ( site-directed mutagenesis, altered dimerization and molecular weight compared to the wild-type enzyme [39]) [39] R77L ( site-directed mutagenesis, the PKGIa mutation not only stabilizes the N-terminus but extends a stabilizing effect on the remaining domains of the enzyme as well [61]) [61] S110A/S114A/S445A ( site-directed mutagenesis, the mutant shows 60% reduced autophosphorylation activity compared to the wild-type GKII [37]) [37] S79D ( replacement of an autophosphorylated Ser79 of cGKIb with an aspartic acid results in a mutant kinase with constitutive kinase activity in vitro and in vivo. The cGKIbS79D mutant localized to the cytoplasm and is only a weak activator of CRE-dependent gene transcription [19]) [19] T761M ( site-directed mutagenesis, mutant enzyme is not inhibited by pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol3-yl]pyridine [36]) [36] T761Q ( site-directed mutagenesis, mutant enzyme is not inhibited by pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol3-yl]pyridine [36]) [36] T770M ( site-directed mutagenesis, mutant enzyme is not inhibited by pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol3-yl]pyridine [36]) [36] T770Q ( site-directed mutagenesis, mutant enzyme is not inhibited by pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol3-yl]pyridine [36]) [36] Additional information ( constructed PKG-knockout mutant parasite strains are refractory to to inhibitor pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine with reduced sensitivity, complementation by expression of Eimeria tenella PKG [36]; constructed PKG-knockout mutant parasite strains are refractory to to inhibitor pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine4-yl)-1H-pyrrol-3-yl]pyridine with reduced sensitivity, complementation by expression of Toxoplasma gondii PKG [36]; construction of a PKG Ib leucine zipper mutant, and of the deletion mutant D1-52 lacking the N-terminal part, mutants show slightly altered properties, overview [39]; construction of a PKG-Ib deletion mutant D1-52 [53]; construction of cGKI knockout mice [52]; construction of PKGI knockout mutant mice, phenotype analysis, platelet secretion, platelet aggregation, and Ca2+ mobilization are affected [42]; construction of tissue-specific cGK-knockout mice causing several dysfunction phenotypes, overview [31]; construction of transgenic flies by targeted expression of DG2P1 and DG2P2, and DG1 in malpigian tubule using the UAS/GAL4 system, differential localization of all 3 enzyme forms in transgenic flies, phenotypes, overview [41]; expression of constitutively active enzyme prevents glucose stimulation of thrombospondin 1 expression and TGF-b activity [30]; injection of PKG-Iaspecific antisense or overexpression constructs into two-cell stage embryos 317

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causes severe abnormalities in the developing embryos, phenotypes, overview [43]; injection of PKG-Ib-specific antisense or overexpression constructs into two-cell stage embryos causes severe abnormalities in the developing embryos, phenotypes, overview [43]; PKG-I knockout mutant mice show no induction of p38 by thrombin [25]; PKGII-deficient mice are defective in renal functiona regulation, and in resetting the circadian clock because light induction of mPer2 gene in the hypothalamus is strongly reduced in the early peroid of the night, whereas mPer1 gene induction is elevated [29]; primary myoblast of enzyme-deficient mice mutants show excessive cell fusion [49]; the enzyme is inactivated by expression of a dominant negative mutant PKG construct [27]; constructing PKG I-DN192, a PKG I mutant lacking the N-terminal 92 amino acids [65]; construction of a D1-77 truncation mutant [61]; construction of the gainof-function mutant egl-4 of PKG, mutation causes increased normal gene activity although it is associated with a reduced EGL-4 protein level, multiple phenotypes of egl-4 dominant mutants, detailed analysis, overview, isolation of egl-4 loss-of-function mutations in cis to ad450sd and of extragenic suppressors of ad450sd, overview [62]; during ischemia and reperfusion in vivo, mice with a cardiac myocyte-restricted deletion of PKG I display a more pronounced interaction of TAB1 with p38MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared to wildtype littermates [65]; enzyme inhibition by a dominant negative PKG construct G1aR-GFP, enzyme inhibition blocks apoptosis in suspended CCD841 cells [58]; expression of wild-type and mutant human serotonin transporters in HeLa cells and effects on activation through phosphorylation by PKG and cell metabolism, overview [69]; overexpression in Xenopus laevis of either guanylate cyclase or PKG in embryos increases acetylcholine receptor aggregate area by 60-170%, whereas expression of a dominant negative form of guanylate cyclase inhibits endogenous aggregation by 50%, overview [59]; overexpression of active PKGIa, but not of the inactive mutant K390A, increases of Akt and inhibits necrosis and apoptosis in cardiomyocytes, overview [66]; siRNA-mediated down-regulation of RhoA derepresses PKG I expression [64]) [25, 27, 29, 30, 31, 36, 39, 41, 42, 43, 49, 52, 53, 58, 59, 61, 62, 64, 65, 66, 69] Application drug development ( enzyme is a target for drug development [33]; the enzyme is a target for antiparasitic drugs, e.g. pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine [48]; the enzyme is a target for drug development [31]) [31, 33, 48] medicine ( gene transfer of the catalytic domain of the enzyme might provide a new strategy for treatment of diabetic renal fibrosis [30]) [30] pharmacology ( enzyme inhibitors are useful in treatment of diverse physiological dysfunctions, overview [31]; the enzyme is a

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target for coccidiostat pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4yl)-1H-pyrrol-3-yl]pyridine [36]) [31, 36]

References [1] Adams, M.D.; Celniker, S.E.; Holt, R.A.; Evans, C.A.; Gocayne, J.D.; et al.: The genome sequence of Drosophila melanogaster. Science, 287, 2185-2195 (2000) [2] Kumar, R.; Joyner, R.W.; Komalavilas, P.; Lincoln, T.M.: Analysis of expression of cGMP-dependent protein kinase in rabbit heart cells. J. Pharmacol. Exp. Ther., 291, 967-975 (1999) [3] Hashimoto, E.; Takio, K.; Krebs, E.G.: Amino acid sequence at the ATPbinding site of cGMP-dependent protein kinase. J. Biol. Chem., 257, 727733 (1982) [4] Heil, W.G.; Landgraf, W.; Hofmann, F.: A catalytically active fragment of cGMP-dependent protein kinase. Occupation of its cGMP-binding sites does not affect its phosphotransferase activity. Eur. J. Biochem., 168, 117121 (1987) [5] Takio, K.; Smith, S.B.; Walsh, K.A.; Krebs, E.G.; Titani, K.: Amino acid sequence around a “hinge“ region and its “autophosphorylation“ site in bovine Lung cGMP-dependent protein kinase. J. Biol. Chem., 258, 5531-5536 (1983) [6] Takio, K.; Wade, R.D.; Smith, S.B.; Krebs, E.G.; Walsh, K.A.; Titani, K.: Guanosine cyclic 3’,5’-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families. Biochemistry, 23, 42074218 (1984) [7] Wernet, W.; Flockerzi, V.; Hofmann, F.: The cDNA of the two isoforms of bovine cGMP-dependent protein kinase. FEBS Lett., 251, 191-196 (1989) [8] Orstavik, S.; Natarajan, V.; Tasken, K.; Jahnsen, T.; Sandberg, M.: Characterization of the human gene encoding the type I a and type I b cGMP-dependent protein kinase (PRKG1). Genomics, 42, 311-318 (1997) [9] Sandberg, M.; Natarajan, V.; Ronander, I.; Kalderon, D.; Walter, U.; Lohmann, S.M.; Jahnsen, T.: Molecular cloning and predicted full-length amino acid sequence of the type I b isozyme of cGMP-dependent protein kinase from human placenta. Tissue distribution and developmental changes in rat. FEBS Lett., 255, 321-329 (1989) [10] Ruth, P.; Pfeifer, A.; Kamm, S.; Klatt, P.; Dostmann, W.R.; Hofmann, F.: Identification of the amino acid sequences responsible for high affinity activation of cGMP kinase Ia. J. Biol. Chem., 272, 10522-10528 (1997) [11] Kalderon, D.; Rubin, G.M.: cGMP-dependent protein kinase genes in Drosophila. J. Biol. Chem., 264, 10738-10748 (1989) [12] Foster, J.L.; Higgins, G.C.; Jackson, F.R.: Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase. J. Biol. Chem., 271, 23322-23328 (1996) [13] Fujii, M.; Ogata, T.; Takahashi, E.; Yamada, K.; Nakabayashi, K.; Oishi, M.; Ayusawa, D.: Expression of the human cGMP-dependent protein kinase II 319

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[28] Gerzanich, V.; Ivanov, A.; Ivanova, S.; Yang, J.B.; Zhou, H.; Dong, Y.; Simard, J.M.: Alternative splicing of cGMP-dependent protein kinase I in angiotensin-hypertension: novel mechanism for nitrate tolerance in vascular smooth muscle. Circ. Res., 93, 805-812 (2003) [29] Oster, H.; Werner, C.; Magnone, M.C.; Mayser, H.; Feil, R.; Seeliger, M.W.; Hofmann, F.; Albrecht, U.: cGMP-dependent protein kinase II modulates mPer1 and mPer2 gene induction and influences phase shifts of the circadian clock. Curr. Biol., 13, 725-733 (2003) [30] Wang, S.; Wu, X.; Lincoln, T.M.; Murphy-Ullrich, J.E.: Expression of constitutively active cGMP-dependent protein kinase prevents glucose stimulation of thrombospondin 1 expression and TGF- b activity. Diabetes, 52, 2144-2150 (2003) [31] Schlossmann, J.; Hofmann, F.: cGMP-dependent protein kinases in drug discovery. Drug Discov. Today, 10, 627-634 (2005) [32] He, B.; Weber, G.F.: Phosphorylation of NF-kB proteins by cyclic GMP-dependent kinase. A noncanonical pathway to NF-kB activation. Eur. J. Biochem., 270, 2174-2185 (2003) [33] Baker, D.A.; Deng, W.: Cyclic GMP-dependent protein kinases in protozoa. Front. Biosci., 10, 1229-1238 (2005) [34] Vaandrager, A.B.; Hogema, B.M.; de Jonge, H.R.: Molecular properties and biological functions of cGMP-dependent protein kinase II. Front. Biosci., 10, 2150-2164 (2005) [35] Koeppen, M.; Feil, R.; Siegl, D.; Feil, S.; Hofmann, F.; Pohl, U.; de Wit, C.: cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo. Hypertension, 44, 952-955 (2004) [36] Wiersma, H.I.; Galuska, S.E.; Tomley, F.M.; Sibley, L.D.; Liberator, P.A.; Donald, R.G.: A role for coccidian cGMP-dependent protein kinase in motility and invasion. Int. J. Parasitol., 34, 369-380 (2004) [37] Vaandrager, A.B.; Hogema, B.M.; Edixhoven, M.; van den Burg, C.M.; Bot, A.G.; Klatt, P.; Ruth, P.; Hofmann, F.; Van Damme, J.; Vandekerckhove, J.; de Jonge, H.R.: Autophosphorylation of cGMP-dependent protein kinase type II. J. Biol. Chem., 278, 28651-28658 (2003) [38] Gambaryan, S.; Butt, E.; Marcus, K.; Glazova, M.; Palmetshofer, A.; Guillon, G.; Smolenski, A.: cGMP-dependent protein kinase type II regulates basal level of aldosterone production by zona glomerulosa cells without increasing expression of the steroidogenic acute regulatory protein gene. J. Biol. Chem., 278, 29640-29648 (2003) [39] Richie-Jannetta, R.; Francis, S.H.; Corbin, J.D.: Dimerization of cGMP-dependent protein kinase Ib is mediated by an extensive amino-terminal leucine zipper motif, and dimerization modulates enzyme function. J. Biol. Chem., 278, 50070-50079 (2003) [40] Andoh, T.; Chiueh, C.C.; Chock, P.B.: Cyclic GMP-dependent protein kinase regulates the expression of thioredoxin and thioredoxin peroxidase-1 during hormesis in response to oxidative stress-induced apoptosis. J. Biol. Chem., 278, 885-890 (2003)

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[41] MacPherson, M.R.; Lohmann, S.M.; Davies, S.A.: Analysis of Drosophila cGMP-dependent protein kinases and assessment of their in vivo roles by targeted expression in a renal transporting epithelium. J. Biol. Chem., 279, 40026-40034 (2004) [42] Li, Z.; Zhang, G.; Marjanovic, J.A.; Ruan, C.; Du, X.: A platelet secretion pathway mediated by cGMP-dependent protein kinase. J. Biol. Chem., 279, 42469-42475 (2004) [43] Yamamoto, T.; Suzuki, N.: Expression and function of cGMP-dependent protein kinase type I during medaka fish embryogenesis. J. Biol. Chem., 280, 16979-16986 (2005) [44] Zhao, X.; Zhuang, S.; Chen, Y.; Boss, G.R.; Pilz, R.B.: Cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein b functions through inhibition of glycogen synthase kinase-3. J. Biol. Chem., 280, 32683-32692 (2005) [45] Richie-Jannetta, R.; Busch, J.L.; Higgins, K.A.; Corbin, J.D.; Francis, S.H.: Isolated regulatory domains of cGMP-dependent protein kinase Ia and Ib retain dimerization and native cGMP-binding properties, and undergo isoform-specific conformational changes. J. Biol. Chem., 281, 6977-6984 (2006) [46] Christensen, E.N.; Mendelsohn, M.E.: Cyclic GMP-dependent protein kinase Ia inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells. J. Biol. Chem., 281, 8409-8416 (2006) [47] West, G.A.; Meno, J.R.; Nguyen, T.S.; Ngai, A.C.; Simard, J.M.; Winn, H.R.: cGMP-dependent and not cAMP-dependent kinase is required for adenosine-induced dilation of intracerebral arterioles. J. Cardiovasc. Pharmacol., 41, 444-451 (2003) [48] Diaz, C.A.; Allocco, J.; Powles, M.A.; Yeung, L.; Donald, R.G.; Anderson, J.W.; Liberator, P.A.: Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): Antiparasitic activity of a PKG inhibitor. Mol. Biochem. Parasitol., 146, 78-88 (2006) [49] Bois, P.R.; Brochard, V.F.; Salin-Cantegrel, A.V.; Cleveland, J.L.; Grosveld, G.C.: FoxO1a-cyclic GMP-dependent kinase I interactions orchestrate myoblast fusion. Mol. Cell. Biol., 25, 7645-7656 (2005) [50] Grassi, C.; Dscenzo, M.; Azzena, G.B.: Modulation of CaV1 and CaV2.2 channels induced by nitric oxide via cGMP-dependent protein kinase. Neurochem. Int., 45, 885-893 (2004) [51] Feil, S.; Zimmermann, P.; Knorn, A.; Brummer, S.; Schlossmann, J.; Hofmann, F.; Feil, R.: Distribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina. Neuroscience, 135, 863868 (2005) [52] Wolfsgruber, W.; Feil, S.; Brummer, S.; Kuppinger, O.; Hofmann, F.; Feil, R.: A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells. Proc. Natl. Acad. Sci. USA, 100, 13519-13524 (2003) [53] Wall, M.E.; Francis, S.H.; Corbin, J.D.; Grimes, K.; Richie-Jannetta, R.; Kotera, J.; Macdonald, B.A.; Gibson, R.R.; Trewhella, J.: Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 100, 2380-2385 (2003)

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[54] Danielewski, O.; Schultess, J.; Smolenski, A.: The NO/cGMP pathway inhibits Rap 1 activation in human platelets via cGMP-dependent protein kinase I. Thromb. Haemost., 93, 319-325 (2005) [55] Roberts, J.D.; Chiche, J.D.; Kolpa, E.M.; Bloch, D.B.; Bloch, K.D.: cGMP-dependent protein kinase I interacts with TRIM39R, a novel Rpp21 domaincontaining TRIM protein. Am. J. Physiol. Lung Cell Mol. Physiol., 293, L903-L912 (2007) [56] Giordano, D.; Magaletti, D.M.; Clark, E.A.: Nitric oxide and cGMP protein kinase (cGK) regulate dendritic-cell migration toward the lymph-node-directing chemokine CCL19. Blood, 107, 1537-1545 (2006) [57] Begonja, A.J.; Geiger, J.; Rukoyatkina, N.; Rauchfuss, S.; Gambaryan, S.; Walter, U.: Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase. Blood, 109, 616-618 (2007) [58] Hou, Y.; Wong, E.; Martin, J.; Schoenlein, P.V.; Dostmann, W.R.; Browning, D.D.: A role for cyclic-GMP dependent protein kinase in anoikis. Cell. Signal., 18, 882-888 (2006) [59] Godfrey, E.W.; Longacher, M.; Neiswender, H.; Schwarte, R.C.; Browning, D.D.: Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction. Dev. Biol., 307, 195-201 (2007) [60] Waldkirch, E.S.; Uckert, S.; Langnaese, K.; Richter, K.; Jonas, U.; Wolf, G.; Andersson, K.E.; Stief, C.G.; Hedlund, P.: Immunohistochemical distribution of cyclic GMP-dependent protein kinase-1 in human prostate tissue. Eur. Urol., 52, 495-501 (2007) [61] Scholten, A.; Fuss, H.; Heck, A.J.; Dostmann, W.R.: The hinge region operates as a stability switch in cGMP-dependent protein kinase I a. FEBS J., 274, 2274-2286 (2007) [62] Raizen, D.M.; Cullison, K.M.; Pack, A.I.; Sundaram, M.V.: A novel gain-offunction mutant of the cyclic GMP-dependent protein kinase egl-4 affects multiple physiological processes in Caenorhabditis elegans. Genetics, 173, 177-187 (2006) [63] Fryer, B.H.; Wang, C.; Vedantam, S.; Zhou, G.L.; Jin, S.; Fletcher, L.; Simon, M.C.; Field, J.: cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association. J. Biol. Chem., 281, 11487-11495 (2006) [64] Zeng, Y.; Zhuang, S.; Gloddek, J.; Tseng, C.C.; Boss, G.R.; Pilz, R.B.: Regulation of cGMP-dependent protein kinase expression by Rho and Kruppellike transcription factor-4. J. Biol. Chem., 281, 16951-16961 (2006) [65] Fiedler, B.; Feil, R.; Hofmann, F.; Willenbockel, C.; Drexler, H.; Smolenski, A.; Lohmann, S.M.; Wollert, K.C.: cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes. J. Biol. Chem., 281, 32831-32840 (2006) [66] Das, A.; Smolenski, A.; Lohmann, S.M.; Kukreja, R.C.: Cyclic GMP-dependent protein kinase Ia attenuates necrosis and apoptosis following ische-

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[67]

[68]

[69]

[70]

[71] [72]

[73]

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mia/reoxygenation in adult cardiomyocyte. J. Biol. Chem., 281, 38644-38652 (2006) Nanamori, M.; Chen, J.; Du, X.; Ye, R.D.: Regulation of leukocyte degranulation by cGMP-dependent protein kinase and phosphoinositide 3-kinase: potential roles in phosphorylation of target membrane SNARE complex proteins in rat mast cells. J. Immunol., 178, 416-427 (2007) Zhang, C.; Ondeyka, J.G.; Herath, K.B.; Guan, Z.; Collado, J.; Pelaez, F.; Leavitt, P.S.; Gurnett, A.; Nare, B.; Liberator, P.; Singh, S.B.: Highly substituted terphenyls as inhibitors of parasite cGMP-dependent protein kinase activity. J. Nat. Prod., 69, 710-712 (2006) Zhang, Y.W.; Gesmonde, J.; Ramamoorthy, S.; Rudnick, G.: Serotonin transporter phosphorylation by cGMP-dependent protein kinase is altered by a mutation associated with obsessive compulsive disorder. J. Neurosci., 27, 10878-10886 (2007) Broderick, K.E.; Zhang, T.; Rangaswami, H.; Zeng, Y.; Zhao, X.; Boss, G.R.; Pilz, R.B.: Guanosine 3,5-cyclic monophosphate (cGMP)/cGMP-dependent protein kinase induce interleukin-6 transcription in osteoblasts. Mol. Endocrinol., 21, 1148-1162 (2007) Guo, L.W.; Ruoho, A.E.: Inhibition of cGMP-dependent protein kinase by the regulatory subunit of photoreceptor cGMP phosphodiesterase. Neurosci. Lett., 401, 252-255 (2006) Qin, X.; Zheng, X.; Qi, H.; Dou, D.; Raj, J.U.; Gao, Y.: cGMP-dependent protein kinase in regulation of basal tone and in nitroglycerin- and nitricoxide-induced relaxation in porcine coronary artery. Pfluegers Arch., 454, 913-923 (2007) Dawson-Scully, K.; Armstrong, G.A.; Kent, C.; Robertson, R.M.; Sokolowski, M.B.: Natural variation in the thermotolerance of neural function and behavior due to a cGMP-dependent protein kinase. PLoS ONE, 2, e773 (2007)

Protein kinase C

2.7.11.13

1 Nomenclature EC number 2.7.11.13 Systematic name ATP:protein phosphotransferase (diacylglycerol-dependent) Recommended name protein kinase C Synonyms calcium-dependent protein kinase C [90, 91, 92] Capkc1p [78] PAK-1 [96] PICK1 [60] PKC [1, 2, 3, 4, 98, 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, 114, 116, 117, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153] PKC Apl I [90] PKC Apl II [93] PKC bII [113] PKC dII [8] PKC dIII [46] PKC i [77] PKC l [95] PKC z [25] PKC-L [69] PKC-d [85] PKC-e [57] PKC-like kinase [126] PKC-z [32] PKC1B [72] PKCa [102, 115, 118] PKCd [47, 112] PKCn [26] PKD2 [7] a-PKC [61] calcium-independent protein kinase C [90, 91, 92, 93, 94] ePKC nPKC h [63]

325

Protein kinase C

2.7.11.13

protein kinase C [55] protein kinase C a [59] protein kinase C bII [113] protein kinase C d [8, 46, 112] protein kinase C, D2 type [7, 24] protein kinase C, a type [27, 28, 34, 35, 45, 58, 59, 60, 61, 62] protein kinase C, b type [5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 27, 29, 39, 40, 41, 42, 43, 44, 45] protein kinase C, brain isoenzyme [33] protein kinase C, d type [6, 8, 20, 21, 22, 23, 46, 47, 48, 49, 50, 51, 85, 86] protein kinase C, e type [50, 51, 54, 56, 57, 80] protein kinase C, h type [63, 67, 68, 69, 97] protein kinase C, g type [19, 29, 30, 31, 32, 34, 36, 37, 38, 53] protein kinase C, i type [76, 77, 95] protein kinase C, m type [87] protein kinase C, n type [26] protein kinase C, q type [82, 83] protein kinase C, z type [25, 32, 50, 51, 52, 81, 84] protein kinase C-e [80] protein kinase C-h [97] protein kinase C-like [79] protein kinase C-like 1 [64, 65, 66, 67, 70, 71, 73, 78, 88, 89, 96] protein kinase C-like 2 [67, 72, 73, 74, 75, 88] protein kinase Ca [115, 118] protein kinase Cd [6, 47] protein kinase D2 [7] protein kinase-C [138] Additional information ( PKC occurs in multiple isozymes, designated as classical, novel, and atypical isozymes, belonging to the PKC family [107]; PKCs form a family of related serine/threonine kinases that are part of the AGC-type kinase, kinase G/protein kinase C-family kinase, superfamily [130]; the enzyme belongs to the family of serine/threonine kinases [129]) [107, 129, 130] CAS registry number 141436-78-4 (calcium-dependent protein kinase C)

2 Source Organism



326

Cavia porcellus (no sequence specified) [109] Drosophila melanogaster (no sequence specified) [3] Mammalia (no sequence specified) [2] eukaryota (no sequence specified) [1, 3, 4]

2.7.11.13

Protein kinase C

Mus musculus (no sequence specified) [103, 107, 110, 119, 126, 130, 135, 141, 142, 143, 145, 147, 151] Homo sapiens (no sequence specified) [3, 101, 102, 110, 112, 115, 116, 117, 118, 124, 126, 128, 131, 132, 133, 134, 136, 140, 146, 148, 149, 151, 152, 153] Rattus norvegicus (no sequence specified) [98, 99, 100, 104, 105, 106, 108, 113, 114, 118, 120, 121, 122, 125, 127, 137, 138, 139, 143, 144, 147, 150, 151] Saccharomyces cerevisiae (no sequence specified) [3] Bos taurus (no sequence specified) [111] Xenopus laevis (no sequence specified) [131] Caenorhabditis elegans (no sequence specified) [126] Lycopersicon esculentum (no sequence specified) [126] Mus musculus (UNIPROT accession number: P04410) [5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19] Mus musculus (UNIPROT accession number: P28867) [6, 8, 20, 21, 22, 23] Homo sapiens (UNIPROT accession number: Q9BZL6) [7, 24] Oryctolagus cuniculus (UNIPROT accession number: O19111) [25] Homo sapiens (UNIPROT accession number: O94806) [26] Bos taurus (UNIPROT accession number: P04409) [27, 28] Bos taurus (UNIPROT accession number: P05126) [27, 29] Homo sapiens (UNIPROT accession number: P05129) [29, 30, 31, 32] Drosophila melanogaster (UNIPROT accession number: P05130) [33] Rattus norvegicus (UNIPROT accession number: P05696) [27, 34, 35] Mus musculus (UNIPROT accession number: P05697) [19, 34, 36, 37, 38] Homo sapiens (UNIPROT accession number: P05771) [39, 40, 41, 42, 43, 44] Oryctolagus cuniculus (UNIPROT accession number: P05772) [45] Rattus norvegicus (UNIPROT accession number: P09215) [46, 47, 48, 49, 50, 51] Rattus norvegicus (UNIPROT accession number: P09216) [50, 51] Rattus norvegicus (UNIPROT accession number: P09217) [50, 51, 52] Oryctolagus cuniculus (UNIPROT accession number: P10102) [27, 45] Oryctolagus cuniculus (UNIPROT accession number: P10829) [53] Oryctolagus cuniculus (UNIPROT accession number: P10830) [54] Drosophila melanogaster (UNIPROT accession number: P13678) [55] Mus musculus (UNIPROT accession number: P16054) [56, 57] Homo sapiens (UNIPROT accession number: P17252) [58, 59] Mus musculus (UNIPROT accession number: P20444) [60, 61, 62] Mus musculus (UNIPROT accession number: P23298) [63] Saccharomyces cerevisiae (UNIPROT accession number: P24583) [64, 65, 66] Homo sapiens (UNIPROT accession number: P24723) [67, 68, 69] Caenorhabditis elegans (UNIPROT accession number: P34722) [70, 71] Caenorhabditis elegans (UNIPROT accession number: P34885) [72] Schizosaccharomyces pombe (UNIPROT accession number: P36582) [73]

327

Protein kinase C

2.7.11.13

Schizosaccharomyces pombe (UNIPROT accession number: P36583) [73, 74, 75] Homo sapiens (UNIPROT accession number: P41743) [76, 77] Candida albicans (UNIPROT accession number: P43057) [78] Aspergillus niger (UNIPROT accession number: Q00078) [79] Homo sapiens (UNIPROT accession number: Q02156) [80] Mus musculus (UNIPROT accession number: Q02956) [81] Homo sapiens (UNIPROT accession number: Q04759) [82, 83] Homo sapiens (UNIPROT accession number: Q05513) [32, 84] Homo sapiens (UNIPROT accession number: Q05655) [85,86] Homo sapiens (UNIPROT accession number: Q15139) [87] Homo sapiens (UNIPROT accession number: Q16512) [67, 88, 89] Homo sapiens (UNIPROT accession number: Q16513) [67,88] Aplysia californica (UNIPROT accession number: Q16974) [90, 91, 92] Aplysia californica (UNIPROT accession number: Q16975) [90, 91, 92, 93, 94] Mus musculus (UNIPROT accession number: Q62074) [95] Rattus norvegicus (UNIPROT accession number: Q63433) [89,96] Rattus norvegicus (UNIPROT accession number: Q64617) [97] Trichoderma reesei (UNIPROT accession number: Q99014) [79] Heteropneustes fossilis (no sequence specified) [123] Giardia duodenalis (UNIPROT accession number: Q1H8W8) [129]

3 Reaction and Specificity Catalyzed reaction ATP + a protein = ADP + a phosphoprotein ( regulation of enzyme activity involves the activation loop, a polypeptide region outside the active site cleft, which is reversibly phosphorylated at a Thr residue [4]) Natural substrates and products S ATP + Akt ( phosphorylation by PKC isozymes at Ser473, regulation of the phosphorylation activity by PKCbII in mast cells stimulated by stem cell factor or interaleukin-3, and in serum-stimulated fibroblasts, and in antigen-receptor stimulated T- or B-lymphocytes, overview [119]) (Reversibility: ?) [119] P ADP + phosphorylated Akt S ATP + Btk ( specific phosphorylation leads to inhibition of Btk membrane translocation and activation and the downstream events that promote PKCb activation, mechanism, overview [107]) (Reversibility: ?) [107] P ADP + phosphorylated Btk S ATP + Na,K-ATPase (Reversibility: ?) [137] P ADP + phosphorylated Na,K-ATPase

328

2.7.11.13

Protein kinase C

S ATP + [low density lipoprotein receptor-related protein] ( involved in regulation of endocytosis and association with adaptor molecules, e.g. Shc, Dab1, or CED-6/GULP [118]) (Reversibility: ?) [118] P ADP + phosphorylated [low density lipoprotein receptor-related protein] S ATP + a protein (Reversibility: ?) [1, 2, 3, 4] P ADP + a phosphoprotein S ATP + calcium-independent phospholipase A2 ( PKC regulates membrane-associated, calcium-independent phospholipase A2 in coronary artery endothelial cells in competition to thrombin, overview [101]) (Reversibility: ?) [101] P ADP + phosphorylated calcium-independent phospholipase A2 S ATP + diacylglycerol kinase-z ( the enzyme, especially isozyme PKCa, inhibits binding of diacylglycerol kinase-zeta to the retinoblastoma protein [134]) (Reversibility: ?) [134] P ADP + phosphorylated diacylglycerol kinase-z S ATP + endothelial nitric oxide synthase ( the enzyme is responsible for the negative regulation of endothelial nitric oxide synthase, a key enzyme in nitric oxide-mediated signal transduction, phosphorylation reduces the affinity of nitric oxide synthase for calmodulin [111]) (Reversibility: ?) [111] P ADP + phosphorylated endothelial nitric oxide synthase S ATP + histone H1-IIIS ( isozyme PKCb [129]) (Reversibility: ?) [129] P ADP + phosphorylated histone H1-IIIS S ATP + inhibitory killer cell Ig-like receptor ( the enzyme regulates expression and function of inhibitory killer cell Ig-like receptors in NK cells, KIR negatively regulate NK cell cytotoxicity by activating Src homology 2 domain-containing protein tyrsine phospatase 1 and 2, overview [148]) (Reversibility: ?) [148] P ADP + phosphorylated inhibitory killer cell Ig-like receptor S ATP + insulin receptor substrate 1 ( phosphorylation of insulin receptor substrate proteins on serine residues is an important posttranslational modification that is linked to insulin resistance, overview [150]) (Reversibility: ?) [150] P ADP + phosphorylated insulin receptor substrate 1 S ATP + metabotropic glutamate receptor 5 ( isozyme-specific phosphorylation of metabotropic glutamate receptor 5 by PKCd blocks Ca2+ oscillation and oscillatory translocation of Ca2+ -dependent PKCg [116]) (Reversibility: ?) [116] P ADP + phosphorylated metabotropic glutamate receptor 5 S ATP + myristoylated alanine-rich C kinase substrate ( the substrate is involved in actincytoskeletal rearrangement in response to extracellular stimuli, phosphorylation of MARCKS is dramatically upregulated specifically in microglial cells after kainic acid-induced seizures, but not in other types of glial cells, overview [145]) (Reversibility: ?) [145] P ADP + phosphorylated myristoylated alanine-rich C kinase substrate

329

Protein kinase C

2.7.11.13

S ATP + phosphoinositide-dependent kinase ( phosphoinositide-dependent kinases are conserved substrates of PKC [126]) (Reversibility: ?) [126] P ADP + phosphorylated phosphoinositide-dependent kinase S ATP + serotonin N-acetyltransferase ( PKC regulates the activity and stability of serotonin N-acetyltransferase in vivo, recombinant enzyme expressed in COS-7 cells [98]) (Reversibility: ?) [98] P ADP + phosphorylated serotonin N-acetyltransferase S ATP + sodium channel ( PKC mediates inhibition of sodium channels and tetrodotoxin-sensitive transient sodium current by phosphorylation by topiramate, overview [138]) (Reversibility: ?) [138] P ADP + phosphorylated sodium channel S ATP + vasodilator-stimulated phosphoprotein ( phosphorylation at Ser157 [130]) (Reversibility: ?) [130] P ADP + phosphorylated vasodilator-stimulated phosphoprotein S Additional information ( constitutive enzyme [32]; protein kinase C b controls nuclear factor kB activation in B cells through selective regulation of the kB kinase a [5]; PKC d is involved in fundamental cellular functions regulated by diacylglycerols and mimicked by phorbol esters [22]; the enzyme is a phorbol ester receptor [28]; overproduction of protein kinase C causes disordered growth control in rat fibroblasts, activation of PKC may be of central importance in the process of multistage carcinogenesis [15]; PKC z exhibits a constitutive kinase [32]; PKC z action is involved in growth and differentiation of the collecting duct [25]; PKC dIII may show a dominant negative effect against PKC dI [46]; protein kinase Cd controls self-antigeninduced B-cell tolerance [6]; key regulatory role in a variety of cellular functions, including apoptosis, as well as cell growth and differentiation [8]; PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle [66]; plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors [60]; protein kinase C-e increases growth and cause malignant transformation when overexpressed in NIH3T3 cells the catalytic domain of PKC-e, in reciprocal PKC-d and PKC-e chimeras, is responsible for conferring tumorgenicity to NIH3T3 cells, whereas both regulatory and catalytic domains of PKC-e contribute to in vitro transformation [56]; in normal rat kidney cells, predominant phosphorylation of a 30000 Da protein at serine residues, constitutive low level expression in normal tissues, elevated expression levels in selected tumor cell lines, a role of PKC m in signal transduction pathways related to growth control [87]; PKC-d desensitizes the Pkc1-mediated pathway by regulating an aspect of G protein function [85]; enzyme may play a role in signal transduction and growth regulatory pathways unique to hematopoietic cells [83]; activation of metabotropic glutamate receptor 5 induces translocation of PKCg and 330

2.7.11.13

Protein kinase C

PKCd to plasma membrane and elicits cyclical translocations of myristoylated alanine-rich PKC substrate from plasma membrane to cytosol [116]; alterations in PKC isoenzyme a, d, and e expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy [125]; bidirectional regulation of renal cortical Na+ ,K+ -ATPase by PKC [99]; calcium-dependent protein kinase C activation occurs in acutely isolated neurons during oxygen and glucose deprivation [127]; Helicobacter pylori activates the enzyme in gastric epithelial cells limiting interleukin-8 production, induced by the pathogen, through suppression of extracellular signal-regulated kinase ERK activation, mechanism overview [124]; identification of isozymes being involved in calcium-independent phospholipase A2 activation in coronary artery endothelial cells,overview [101]; intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C bII mediated by phosphatidylinositol 3-kinase and mammalian target of rapamycin-dependent pathways, PKC bII regulation and degradation control, overview [113]; isozyme-specific and developmental stage-specific alterations in brain PKC following exposure to a polychlorinated biphenyl mixture [105]; PKC is involved in cellular processes like cell growth, cytoskeleton remodelling, and gene expression regulation, emerging and diverse roles of PKC isozymes in immune cell signalling, i.e macrophage activation, signal transduction pathway of PKC in B- and Tlymphocytes, overview, isozyme theta is associated to NFkappa-B activation in T-cells [103]; PKC is involved in regulation of acetylcholine release in cholinergic nervous activity in the central nervous system in hypertension [121]; PKC is involved in signal transduction in the triiodothyronine-activation of forebrain, i.e. telencephalon and hypothalamus [123]; PKC isozymes are involved in signal transduction, required for defense against fungal infection [126]; PKC isozymes are involved in signal transduction, required for host defense [126]; PKC mediates NF-kB activation, pathway overview, PKC isozymes have distinct roles in regulation of B-cell activation and function, e.g. signal transduction, isozyme PKCb is essential for B-cell antigen receptor-induced NF-kB activation and B-cell survival, isozyme PKCd is required for maintainance of self-reactive B-cell tolerance [107]; PKC regulates Na,K-ATPase isozyme a1 specifically via isozyme-specific regions, while it has no effect on isozyme a3, overview [104]; PKCg positively regulates P2X7 receptor-mediated calcium signalling in type-2 astrocytes, mechanism, overview [106]; PKCq is important in regulation of immune response in T-cells, overview, PKC is involved in NF-kB activation pathways in vivo, overview, PKC isozymes are involved in signal transduction, and thereby in cell adhesion and migration, isozyme PKCq is recruited to the plasma membrane by a cytoskeleton-dependent mechanism regulating the Rac-1 guanine nucelotide exchange protein Vav-1 [126]; PKCq is important in regulation of immune response in T-cells, overview, PKC is involved in NF-kB activation pathways in vivo, overview, PKCd acts as negative regulator by feedback inhibition of the CDg3 subunit important for immune 331

Protein kinase C

2.7.11.13

response in T lymphocytes, overview, PKC isozymes are involved in signal transduction, and thereby in cell adhesion and migration, isozyme PKCtheta is recruited to the plasma membrane by a cytoskeleton-dependent mechanism regulating the Rac-1 guanine nucelotide exchange protein Vav-1 [126]; regulation of amphetamine-stimulated dopamine efflux by PKCbII, overview [120]; some PKC isozymes inhibit BKCa channel activity in pulmonary arterial smooth muscle via inhibition of cAMP-induced activation of the BKCa channel [100]; the enzyme is essential for transduction of signals in a wide range of cell types, including neurons [109]; the enzyme is important in signal transduction from extracellular signals to intracellular responses involving the second messengers Ca2+ and diacylglycerol, processing mechanism and regulation overview [110]; the enzyme is important in signal transduction from extracellular signals to intracellular responses involving the second messengers Ca2+ and diacylglycerol, processing mechanism and regulation overview, PKC is important in diverse biological functions, e.g. in cell proliferation, cell differentiation, immune response, cancer, and memory [110]; activation-induced upregulation of inhibitory killer Ig-like receptors, KIR, is regulated by PKC at the posttranscriptional level via the cytoplasmic tail of KIR by stimulation of the maturation processes in the endoplasmic reticulum-Golgi and by promoting the recycling of surface KIR through sorting endosomes, isozyme PKCd plays a role in the exocytosis of KIR in secretory lysosomes, overview [146]; involvement of isozyme PKCa in the early action of angiotensin II type 2 effects on neurite outgrowth in NG108-15 cells, the angiotensin II type 2-receptor inhibits PKC a and p21ras activity, inhibition of PKCa is not directly involved in the Rap1-MEK-p42/p44mapk cascade, overview [143]; isozyme PKC-d inhibits colon cancer cell proliferation by selective changes in cell cycle, arresting cells in the G1 phase, and cell death regulators enhancing apoptosis using two different mechanisms, e.g. by downregulation expression of cyclin E and D1, and Bcl-2, inducing Bax expression, and altering levels of p27Kip1 and p21Waf1, PKC-d is thus an important tumor suppressor in colonic carcinogenesis, overview [152]; isozyme PKCb is involved in encystment [129]; isozyme PKCd inhibits the production of proteolytic enzymes in murine mammary cells, the PKCd effect is mediated by the MEK/ERK pathway, overview [142]; PKC isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from single mouse pancreatic islets, overview [135]; PKC signalling has been suggested to play a role in Ca2+ entry, granule secretion, aIIbb3 activation and outside-in signalling, PKC also is involved in receptor desensitization, extrusion of intracellular Ca2+ , secretion and actin-mediated filopodia formation, PKC negatively regulates platelet activation and the diverse processes in which active platelet are involved, detailed overview, PKC increases Ca2+ extrusion from the cytosol and desensitizes some G-protein coupled receptors, isozyme PKCd inhibits platelet aggregation by inhibiting VASP phosphorylation at Ser157, reducing filopodial extension, overview, PKCd is required for dense gran332

2.7.11.13

Protein kinase C

ule secretion following stimulation by thrombin, and plays a negative regulatory role in dense granule secretion when platelets are stimulated by convulxin, PKCq is also required for outside-in signalling and, as with PKCb, platelets deficient in PKCq do not fully spread on a fibrinogencoated surface [130]; regulation of PKC isozyme expression, overview, specific PKC isozymes act as transducers and modulators of insulin signaling, the activation of PKC isozymes by insulin is modified by several effectors, signaling cascade, overview, isozyme PKCa might play a role in insulin resistance, overview [151]; regulation of PKC isozyme expression, overview, specific PKC isozymes act as transducers and modulators of insulin signaling, the activation of PKC isozymes by insulin is modified by several effectors, signaling cascade, overview, isozyme PKCa might play a role in insulin resistance, overview, isozyme PKCe forms signaling complexes with Raf-1 and ERK, isozyme PKCq inactivation prevents fat-induced defects in insulin signaling and glucose transport in skeletal muscle [151]; the enzyme plays a key role in the mechanism of cerebral ischemic/hypoxic preconditioning, the isozymes are differently involved in neuroprotection, overview [140]; the enzyme plays a role in aldosterone-induced non-genomic inhibition of basolateral potassium IKCa channels in human colonic crypts [149]) (Reversibility: ?) [5, 6, 8, 15, 22, 25, 28, 32, 46, 56, 60, 66, 83, 85, 87, 99, 100, 101, 103, 104, 105, 106, 107, 109, 110, 113, 116, 120, 121, 123, 124, 125, 126, 127, 129, 130, 135, 140, 142, 143, 146, 149, 151, 152] P ? Substrates and products S ATP + Akt ( phosphorylation by PKC isozymes at Ser473, regulation of the phosphorylation activity by PKCbII in mast cells stimulated by stem cell factor or interaleukin-3, and in serum-stimulated fibroblasts, and in antigen-receptor stimulated T- or B-lymphocytes, overview [119]; Akt substrate is protein kinase B, phosphorylation by PKC isozymes at Ser473 involving phorbol esters [119]; phosphorylation on Ser473 by isozyme PKCbII [151]) (Reversibility: ?) [119, 15] P ADP + phosphorylated Akt S ATP + Bcl-xL ( recombinant human substrate, recombinant isozyme PKCd [147]; recombinant human substrate, recombinant isozymes PKCe and PKCzeta [147]) (Reversibility: ?) [147] P ADP + phosphorylated Bcl-xL S ATP + Btk ( specific phosphorylation leads to inhibition of Btk membrane translocation and activation and the downstream events that promote PKCb activation, mechanism, overview [107]; specific phosphorylation of the tyrosine protein kinase at Ser180 within the Techomology domain [107]) (Reversibility: ?) [107] P ADP + phosphorylated Btk S ATP + EF factor 1 ( i.e. eukaryotic translation elongation factor 1-a1, in vitro phosphorylation [133]) (Reversibility: ?) [133] P ADP + phosphorylated EF factor 1

333

Protein kinase C

2.7.11.13

S ATP + Fyn ( Fyn is a tyrosine protein kinase of the Src family, phosphorylation at a serine residue [112]) (Reversibility: ?) [112] P ADP + phosphorylated Fyn S ATP + IRS ( isozymes PKCa and PKCd, phosphorylation on Ser307 [151]) (Reversibility: ?) [151] P ADP + phosphorylated IRS S ATP + Na,K-ATPase ( substrate in membrane vesicles from the rectal gland of the spiny dog fish Squalus acanthias [137]) (Reversibility: ?) [137] P ADP + phosphorylated Na,K-ATPase S ATP + PKC-a-derived peptide ( in the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC a phosphorylates a PKC-a pseudosubstrate-derived peptide, an epidermal-growth-factorreceptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-a-derived peptide [32]) (Reversibility: ?) [32] P ADP + phosphorylated PKC-a-derived peptide S ATP + RFARKGSLRQKNV (Reversibility: ?) [136] P ADP + phosphorylated RFARKGSLRQKNV S ATP + RFARKGSLRQKNV ( a commercially available peptide substrate [132]) (Reversibility: ?) [132] P ADP + phoshorylated RFARKGSLRQKNV S ATP + RFARKGSLRQKNV ( synthetic peptide substrate [133]) (Reversibility: ?) [133] P ADP + RFARKGphosphoSLRQKNV S ATP + [low density lipoprotein receptor-related protein] ( involved in regulation of endocytosis and association with adaptor molecules, e.g. Shc, Dab1, or CED-6/GULP [118]; i.e. LPR, phosphorylation of cytoplasmic domain residues T63, T16, S3, S73, S76, and S79 [118]; i.e. LPR, phosphorylation of cytoplasmic domain serine and threonine residues [118]) (Reversibility: ?) [118] P ADP + phosphorylated [low density lipoprotein receptor-related protein] S ATP + a protein (Reversibility: ?) [1, 2, 3, 4] P ADP + a phosphoprotein S ATP + calcium-independent phospholipase A2 ( PKC regulates membrane-associated, calcium-independent phospholipase A2 in coronary artery endothelial cells in competition to thrombin, overview [101]) (Reversibility: ?) [101] P ADP + phosphorylated calcium-independent phospholipase A2 S ATP + calmodulin ( phosphorylation at Thr497 in the calmodulin-binding domain, binding is competitive to substrate endothelial nitric oxide synthase [111]) (Reversibility: ?) [111] P ADP + phosphorylated calmodulin S ATP + diacylglycerol kinase-z ( the enzyme, especially isozyme PKCa, inhibits binding of diacylglycerol kinase-z to the retinoblastoma protein [134]; isozyme PKCa [134]) (Reversibility: ?) [134] P ADP + phosphorylated diacylglycerol kinase-z 334

2.7.11.13

Protein kinase C

S ATP + endothelial nitric oxide synthase ( the enzyme is responsible for the negative regulation of endothelial nitric oxide synthase, a key enzyme in nitric oxide-mediated signal transduction, phosphorylation reduces the affinity of nitric oxide synthase for calmodulin [111]; phosphorylation at Thr497 in the calmodulin-binding domain, binding is competitive to substrate calmodulin [111]) (Reversibility: ?) [111] P ADP + phosphorylated endothelial nitric oxide synthase S ATP + hematopoietic-specific G-protein Ga15 ( recombinant substrate expressed in COS-7 cells, phosphorylation at Ser334, mutant Gprotein Ga15 S334A is inactive as substrate for PKC [122]) (Reversibility: ?) [122] P ADP + phosphorylated hematopoietic-specific G-protein Ga15 S ATP + hematopoietic-specific G-protein Ga15 peptide ( the PKC phosphorylation site peptide sequence RKGSR includes Ser334 [122]) (Reversibility: ?) [122] P ADP + phosphorylated hematopoietic-specific G-protein Ga15 peptide S ATP + hematopoietic-specific G-protein Ga16 ( substrate from HL-60 cells, phosphorylation at Ser336, mutant G-protein Ga16 S336A is inactive as substrate for PKC [122]) (Reversibility: ?) [122] P ADP + phosphorylated hematopoietic-specific G-protein Ga16 S ATP + hematopoietic-specific G-protein Ga16 peptide ( the PKC phosphorylation site peptide sequence KKGARSRR includes Ser336 [122]) (Reversibility: ?) [122] P ADP + phosphorylated hematopoietic-specific G-protein Ga16 peptide S ATP + histone ( in presence of absence of phosphatidylserinecontaining liposomes [137]) (Reversibility: ?) [137] P ADP + phosphorylated histone S ATP + histone H1 ( PKD2 activated by phorbol esters efficiently phosphorylate the exogenous substrate histone H1 [7]) (Reversibility: ?) [7] P ADP + phosphorylated histone H1 S ATP + histone H1-IIIS ( isozyme PKCb [129]) (Reversibility: ?) [129] P ADP + phosphorylated histone H1-IIIS S ATP + histone H2 ( in vitro phosphorylation [133]) (Reversibility: ?) [133] P ADP + phosphorylated histone H2 S ATP + histone H3 ( in vitro phosphorylation [133]) (Reversibility: ?) [133] P ADP + phosphorylated histone H3 S ATP + histone III-SS ( commercial substrate [114]) (Reversibility: ?) [114] P AP + phosphorylated histone III-SS S ATP + histone IIIS ( poor substrate [97]) (Reversibility: ?) [97] P ADP + phosphorylated histone IIIS

335

Protein kinase C

2.7.11.13

S ATP + inhibitory killer cell Ig-like receptor ( the enzyme regulates expression and function of inhibitory killer cell Ig-like receptors in NK cells, KIR negatively regulate NK cell cytotoxicity by activating Src homology 2 domain-containing protein tyrsine phospatase 1 and 2, overview [148]; phosphorylation of the cytoplasmic tail, mutational analysis of kinase phosphorylation sites, e.g. Ser394, activity with substrate mutants, overview [148]) (Reversibility: ?) [148] P ADP + phosphorylated inhibitory killer cell Ig-like receptor S ATP + insulin receptor ( isozyme PKCa [151]) (Reversibility: ?) [151] P ADP + phosphorylated insulin receptor S ATP + insulin receptor substrate 1 ( phosphorylation of insulin receptor substrate proteins on serine residues is an important posttranslational modification that is linked to insulin resistance, overview [150]; a bona fide substrate for conventional isozymes PKCa, PKCzeta, and PKCd, phosphorylation of IRS1 at Ser24 in the N-terminal pleckstrin homology domain of IRS1, additionally isozyme PKCa is indirectly involved in Ser307 and Ser612 phosphorylation [150]) (Reversibility: ?) [150] P ADP + phosphorylated insulin receptor substrate 1 S ATP + lamin A ( a nuclear membrane protein, isozyme PKCa [133]) (Reversibility: ?) [133] P ADP + phosphorylated lamin A S ATP + lamin C ( a nuclear membrane protein, isozyme PKCa, phosphorylation at Ser572 [133]) (Reversibility: ?) [133] P ADP + phosphorylated lamin C S ATP + metabotropic glutamate receptor 5 ( isozyme-specific phosphorylation of metabotropic glutamate receptor 5 by PKCd blocks Ca2+ oscillation and oscillatory translocation of Ca2+ -dependent PKCg [116]; phosphorylation at Thr840, PKCd is active with wild-type substrate and mutant T840A, but not with mutant T840D, no phosphorylation by PKCg [116]) (Reversibility: ?) [116] P ADP + phosphorylated metabotropic glutamate receptor 5 S ATP + myelin basic protein (Reversibility: ?) [77, 78] P ADP + phosphorylated myelin basic protein S ATP + myristoylated alanine-rich C kinase substrate ( the substrate is involved in actincytoskeletal rearrangement in response to extracellular stimuli, phosphorylation of MARCKS is dramatically upregulated specifically in microglial cells after kainic acid-induced seizures, but not in other types of glial cells, overview [145]) (Reversibility: ?) [145] P ADP + phosphorylated myristoylated alanine-rich C kinase substrate S ATP + phosphoinositide-dependent kinase ( phosphoinositide-dependent kinases are conserved substrates of PKC [126]; i.e. PDK, phosphorylation at Ser744 and Ser748 in the activation loop of the catalytic domain of PKD [126]; i.e. PDK, phosphorylation at Ser744 and Ser748 in the activation loop of the catalytic domain of PKD, reaction is regulated by diacylglycerol [126]) (Reversibility: ?) [126] 336

2.7.11.13

Protein kinase C

P ADP + phosphorylated phosphoinositide-dependent kinase S ATP + phospholipase D1 ( phosphorylation by PKC [115]) (Reversibility: ?) [115] P ADP + phosphorylated phospholipase D1 S ATP + phospholipase D2 ( phosphorylation by PKCa [115]) (Reversibility: ?) [115] P ADP + phosphorylated phospholipase D2 S ATP + protein ( autophosphorylation [87, 88, 95]) (Reversibility: ?) [87, 88, 95] P ADP + phosphoprotein S ATP + ribosomal protein S6-(229-239) peptide analogue (Reversibility: ?) [96] P ADP + phosphorylated ribosomal protein S6-(229-239) peptide analogue S ATP + serotonin N-acetyltransferase ( PKC regulates the activity and stability of serotonin N-acetyltransferase in vivo, recombinant enzyme expressed in COS-7 cells [98]; recombinant enzyme expressed in COS-7 cells, phosphorylation at Thr29 [98]) (Reversibility: ?) [98] P ADP + phosphorylated serotonin N-acetyltransferase S ATP + sodium channel ( PKC mediates inhibition of sodium channels and tetrodotoxin-sensitive transient sodium current by phosphorylation by topiramate, overview [138]) (Reversibility: ?) [138] P ADP + phosphorylated sodium channel S ATP + vasodilator-stimulated phosphoprotein ( phosphorylation at Ser157 [130]) (Reversibility: ?) [130] P ADP + phosphorylated vasodilator-stimulated phosphoprotein S N6 -phenyl-ATP + RFARKGSLRQKNV ( synthetic peptide substrate, recombinant isozyme PKCa mutant M417A [133]) (Reversibility: ?) [133] P N6 -phenyl-ADP + RFARKGphosphoSLRQKNV S Additional information ( substrate specificity [3]; preferably phosphorylates the Saccharomyces cerevisiae Pkc1p pseudosubstrate peptide and myelin basic protein, but not histones, protamine or dephosphorylated casein [78]; phorbol ester receptor/ protein kinase [63]; constitutive enzyme [32]; protein kinase Cb controls nuclear factor kB activation in B cells through selective regulation of the kB kinase a [5]; PKC d is involved in fundamental cellular functions regulated by diacylglycerols and mimicked by phorbol esters [22]; the enzyme is a phorbol ester receptor [28]; overproduction of protein kinase C causes disordered growth control in rat fibroblasts, activation of PKC may be of central importance in the process of multistage carcinogenesis [15]; PKC z exhibits a constitutive kinase [32]; PKC z action is involved in growth and differentiation of the collecting duct [25]; PKC dIII may show a dominant negative effect against PKC dI [46]; protein kinase Cd controls self-antigeninduced B-cell tolerance [6]; key regulatory role in a variety of cellular functions, including apoptosis, as well as cell growth and differentia337

Protein kinase C

2.7.11.13

tion [8]; PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle [66]; plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors [60]; protein kinase C-e increases growth and cause malignant transformation when overexpressed in NIH3T3 cells the catalytic domain of PKC-e, in reciprocal PKC-d and PKC-e chimeras, is responsible for conferring tumorgenicity to NIH3T3 cells, whereas both regulatory and catalytic domains of PKC-e contribute to in vitro transformation [56]; in normal rat kidney cells, predominant phosphorylation of a 30000 Da protein at serine residues, constitutive low level expression in normal tissues, elevated expression levels in selected tumor cell lines, a role of PKC m in signal transduction pathways related to growth control [87]; PKC-d desensitizes the Pkc1-mediated pathway by regulating an aspect of G protein function [85]; enzyme may play a role in signal transduction and growth regulatory pathways unique to hematopoietic cells [83]; activation of metabotropic glutamate receptor 5 induces translocation of PKCg and PKCd to plasma membrane and elicits cyclical translocations of myristoylated alanine-rich PKC substrate from plasma membrane to cytosol [116]; alterations in PKC isoenzyme a, d, and e expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy [125]; bidirectional regulation of renal cortical Na+ ,K+ -ATPase by PKC [99]; calcium-dependent protein kinase C activation occurs in acutely isolated neurons during oxygen and glucose deprivation [127]; Helicobacter pylori activates the enzyme in gastric epithelial cells limiting interleukin-8 production, induced by the pathogen, through suppression of extracellular signal-regulated kinase ERK activation, mechanism overview [124]; identification of isozymes being involved in calcium-independent phospholipase A2 activation in coronary artery endothelial cells,overview [101]; intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C bII mediated by phosphatidylinositol 3-kinase and mammalian target of rapamycin-dependent pathways, PKC bII regulation and degradation control, overview [113]; isozyme-specific and developmental stage-specific alterations in brain PKC following exposure to a polychlorinated biphenyl mixture [105]; PKC is involved in cellular processes like cell growth, cytoskeleton remodelling, and gene expression regulation, emerging and diverse roles of PKC isozymes in immune cell signalling, i.e macrophage activation, signal transduction pathway of PKC in B- and Tlymphocytes, overview, isozyme theta is associated to NFkappa-B activation in T-cells [103]; PKC is involved in regulation of acetylcholine release in cholinergic nervous activity in the central nervous system in hypertension [121]; PKC is involved in signal transduction in the triiodothyronine-activation of forebrain, i.e. telencephalon and hypothalamus [123]; PKC isozymes are involved in signal transduction, required for defense against fungal infection [126]; PKC isozymes are 338

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Protein kinase C

involved in signal transduction, required for host defense [126]; PKC mediates NF-kB activation, pathway overview, PKC isozymes have distinct roles in regulation of B-cell activation and function, e.g. signal transduction, isozyme PKCb is essential for B-cell antigen receptor-induced NF-kB activation and B-cell survival, isozyme PKCd is required for maintainance of self-reactive B-cell tolerance [107]; PKC regulates Na,K-ATPase isozyme a1 specifically via isozyme-specific regions, while it has no effect on isozyme a3, overview [104]; PKCg positively regulates P2X7 receptor-mediated calcium signalling in type-2 astrocytes, mechanism, overview [106]; PKCq is important in regulation of immune response in T-cells, overview, PKC is involved in NF-kB activation pathways in vivo, overview, PKC isozymes are involved in signal transduction, and thereby in cell adhesion and migration, isozyme PKCq is recruited to the plasma membrane by a cytoskeleton-dependent mechanism regulating the Rac-1 guanine nucelotide exchange protein Vav-1 [126]; PKCq is important in regulation of immune response in T-cells, overview, PKC is involved in NF-kB activation pathways in vivo, overview, PKCd acts as negative regulator by feedback inhibition of the CDg3 subunit important for immune response in T lymphocytes, overview, PKC isozymes are involved in signal transduction, and thereby in cell adhesion and migration, isozyme PKCq is recruited to the plasma membrane by a cytoskeleton-dependent mechanism regulating the Rac-1 guanine nucelotide exchange protein Vav-1 [126]; regulation of amphetamine-stimulated dopamine efflux by PKCbII, overview [120]; some PKC isozymes inhibit BKCa channel activity in pulmonary arterial smooth muscle via inhibition of cAMP-induced activation of the BKCa channel [100]; the enzyme is essential for transduction of signals in a wide range of cell types, including neurons [109]; the enzyme is important in signal transduction from extracellular signals to intracellular responses involving the second messengers Ca2+ and diacylglycerol, processing mechanism and regulation overview [110]; the enzyme is important in signal transduction from extracellular signals to intracellular responses involving the second messengers Ca2+ and diacylglycerol, processing mechanism and regulation overview, PKC is important in diverse biological functions, e.g. in cell proliferation, cell differentiation, immune response, cancer, and memory [110]; activation of phospholipase D2 by 4b-phorbol 12-myristate 13-acetate-induced PKCa does not require phosphorylation, overview [115]; activity with wild-type and mutant substrate low density lipoprotein receptor-related protein, overview [118]; interaction between PKCd and Fyn leads to phosphorylation on tyrosine and on serine, respectively, with Fyn kinase being activated by the snake venom alboaggregin-A [112]; isozymes a, d, and e perform autophosphorylation [125]; myristoylated alanine-rich PKC substrate is repetitively phosphorylated by oscillating gPKC on the plasma membrane [116]; phosphorylation by PKC is involved in regulation of hematopoietic-specific G-protein Ga15 and Ga16, deletion of the PKC phosphorylation site leads to inhibition of receptor-coupled phospholipase C activation [122]; PKC interacts 339

Protein kinase C

2.7.11.13

with CARMA-1, involved in signaling from PKC to NF-kB [107]; regulatory interactions of isozymes with other kinases, overview [126]; substrate specificity of PKC isozymes, PKC isozymes interacting proteins, overview [103]; the enzyme depends on basic residues for substrate recognition, autoregulation by a pseudosubstrate mechanism, overview [2]; activation-induced upregulation of inhibitory killer Ig-like receptors, KIR, is regulated by PKC at the posttranscriptional level via the cytoplasmic tail of KIR by stimulation of the maturation processes in the endoplasmic reticulum-Golgi and by promoting the recycling of surface KIR through sorting endosomes, isozyme PKCd plays a role in the exocytosis of KIR in secretory lysosomes, overview [146]; involvement of isozyme PKCa in the early action of angiotensin II type 2 effects on neurite outgrowth in NG108-15 cells, the angiotensin II type 2-receptor inhibits PKC a and p21ras activity, inhibition of PKCa is not directly involved in the Rap1-MEK-p42/p44mapk cascade, overview [143]; isozyme PKC-d inhibits colon cancer cell proliferation by selective changes in cell cycle, arresting cells in the G1 phase, and cell death regulators enhancing apoptosis using two different mechanisms, e.g. by downregulation expression of cyclin E and D1, and Bcl-2, inducing Bax expression, and altering levels of p27Kip1 and p21Waf1, PKC-d is thus an important tumor suppressor in colonic carcinogenesis, overview [152]; isozyme PKCb is involved in encystment [129]; isozyme PKCd inhibits the production of proteolytic enzymes in murine mammary cells, the PKCd effect is mediated by the MEK/ERK pathway, overview [142]; PKC isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from single mouse pancreatic islets, overview [135]; PKC signalling has been suggested to play a role in Ca2+ entry, granule secretion, aIIbb3 activation and outside-in signalling, PKC also is involved in receptor desensitization, extrusion of intracellular Ca2+ , secretion and actin-mediated filopodia formation, PKC negatively regulates platelet activation and the diverse processes in which active platelet are involved, detailed overview, PKC increases Ca2+ extrusion from the cytosol and desensitizes some G-protein coupled receptors, isozyme PKCd inhibits platelet aggregation by inhibiting VASP phosphorylation at Ser157, reducing filopodial extension, overview, PKCd is required for dense granule secretion following stimulation by thrombin, and plays a negative regulatory role in dense granule secretion when platelets are stimulated by convulxin, PKCq is also required for outside-in signalling and, as with PKCb, platelets deficient in PKCq do not fully spread on a fibrinogencoated surface [130]; regulation of PKC isozyme expression, overview, specific PKC isozymes act as transducers and modulators of insulin signaling, the activation of PKC isozymes by insulin is modified by several effectors, signaling cascade, overview, isozyme PKCa might play a role in insulin resistance, overview [151]; regulation of PKC isozyme expression, overview, specific PKC isozymes act as transducers and modulators of insulin signaling, the activation of PKC isozymes by insulin is modified by several effectors, signaling cascade, overview, isozyme PKCa 340

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Protein kinase C

might play a role in insulin resistance, overview, isozyme PKCe forms signaling complexes with Raf-1 and ERK, isozyme PKCq inactivation prevents fat-induced defects in insulin signaling and glucose transport in skeletal muscle [151]; the enzyme plays a key role in the mechanism of cerebral ischemic/hypoxic preconditioning, the isozymes are differently involved in neuroprotection, overview [140]; the enzyme plays a role in aldosterone-induced non-genomic inhibition of basolateral potassium IKCa channels in human colonic crypts [149]; PKCbII specifically binds to b-actin [151]) (Reversibility: ?) [2, 3, 5, 6, 8, 15, 22, 25, 28, 32, 46, 56, 60, 63, 66, 78, 83, 85, 87, 99, 100, 101, 103, 104, 105, 106, 107, 109, 110, 112, 113, 115, 116, 118, 120, 121, 122, 123, 124, 125, 126, 127, 129, 130, 135, 140, 142, 143, 146, 149, 151, 152] P ? Inhibitors 1,1’-(1,10-decanediyl)bis[1-amino isoquinolinium] diiodide [136] 1,1’-(1,10-decanediyl)bis[2-amino-1-methylbenzimidazolium] diiodide [136] 1,1’-(1,10-decanediyl)bis[2-methylbenzothiazolium] diiodide [136] 1,1’-(1,10-decanediyl)bis[2-methylbenzoxazolium] diiodide [136] 1,1’-(1,10-decanediyl)bis[2-methylquinolinium] diiodide [136] 1,1’-(1,10-decanediyl)bis[4-N,N,dimethylaminoquinolinium] diiodide [136] 1,1’-(1,10-decanediyl)bis[4-amino-2-methyl quinolinium] diiodide [136] 1,1’-(1,10-decanediyl)bis[4-aminoquinolinium] diiodide [136] 1,1’-(1,10-decanediyl)bis[quinolinium] diiodide [136] 1,1’-decane-1,10-diylbis(4-aminopyridinium) diiodide [136] 1,1’-decane-1,10-diylbis[4-(dimethylamino)pyridinium] [136] 1,6-bis[N-(1-methylquinolinium-2-methyl)amino]-hexane diiodide [136] 1-(5-isoquinolinesulfonyl)-2-methylpiperazine [8] 3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (2-methyl-1H-indol-3-yl-BIM-1) ( a bisindolylmaleimide analogue of BIM-1 inhibitor [132]) [132] 4-amino-1,2-dimethylquinolinium [136] 4-amino-1-decyl-2-methylquinolinium iodide [136] AVGPRPQT [131] BIM-I ( inhibits encystment in vivo [129]) [129] CG53353 ( inhibitor of isozyme PKCbII [151]) [151] CRLVLASC ( targets isozyme PKCg, blocks formalin-induced pain response [131]) [131] chelerythrine chloride ( interacts with the catalytic domain of PKC, inhibits encystment in vivo [129]) [129] EAVSLKPT ( targets isozyme PKCe, reverses psi eRACK-mediated protection, and decreases formalin-induced pain response [131]) [131]

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ethanol ( 0.03%, inhibition of PKCg oscillation at 0.1 mM [116]) [116] FLLDPY [131] FNGLLKIKI ( protects from cardiac ischaemic injury [131]) [131] FTRKRQRAMRRVHQ ( autoregulatory pseudosubstrate sequence, residues 24-40, lung enzyme [3]) [3] GF109203X ( inhibits classical and novel, but not atypical PKC isozymes [99]; PKC-specific inhibitor [124]; specific inhibition of PKC [98]) [98, 99, 124] Go6976 [130] Gç-6976 ( IC50 of 2.3-7.9 nM [120]; inhibits classical, but not novel and atypical PKC isozymes [99]; selective inhibition of isozymes PKCa, PKCb, and PKC-my [100]) [99, 100, 120] Gç-6976 ( an inhibitor of isozymes cPKCa, -b, and -my [150]; inhibits classical isozymes [143]) [143, 149, 150] Gç-6983 ( an inhibitor of isozyme PKCb, but not of PKCmy [150]) [150] H-7 ( specific PKC inhibitor [121]) [121] HDAPIGYD ( protects from cardiac ischaemic injury, from graft coronary artery disease, and activates potassium current, inhibits sodium current [131]) [131] K-252a ( strong inhibition [118]) [118] KGDYEKILVALCGGN ( blocks Xenopus oocyte maturation [131]; targets isozyme PKCb [131]) [131] KLFIMNL ( targets isozyme PKCbI, inhibits cardiomyocyte hypertrophy [131]) [131] KQKTKTIK T ( blocks Xenopus oocyte maturation [131]; targets isozyme PKCb [131]) [131] LEPEGK [131] LY333531 ( specific inhibition of PKCb [99]) [99] LY379196 ( highly specific for PKCb, inhibits amphetamine-stimulated dopamine efflux in striatum in vivo [120]) [120] MDPNGLSDPYVKL ( targets isozyme PKCb, blocks Ca2+ current [131]) [131] MRAAEDPM ( increased injury from cardiac ischaemia [131]) [131] N,N,N,N’,N’,N’-hexaethyldecane-1,10-diaminium [136] N-cyclohexa-1,3-dien-1-yl-N,N,N’,N’,N’-pentaphenyldecane-1,10-diaminium [136] N6 -Dimethylaminopurine [1] NGRKI [131] NKMKSRLRKGALKKNV ( autoregulatory pseudosubstrate sequence, residues 24-40 [3]) [3] PAWHD [131] PYIALNVD [131] QEVIRNN ( targets isozyme PKCbII, inhibits cardiomyocyte hypertrophy and activates potassium channels [131]) [131]

342

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Protein kinase C

QLMGGLHRHGAIINRKE ( autoregulatory pseudosubstrate sequence, residues 391-407 [3]) [3] RACK1 peptide I homologue [131] Ro-31-8220 ( strong inhibition of PKCa [115]) [115] Ro-32-0432 ( strong inhibition [118]) [118] SFNSYELGSL ( targets isozyme PKCd, protects from cardiac ischaemic injury, from cerebral injury, from graft coronary artery disease, and increases fibroblast proliferation [131]) [131] SIKIWD ( targets isozyme PKCb [131]) [131] SIYRRGARRWRKLYRAN ( targets isozyme PKCzeta, which leads to inhibition of fibroblast proliferation [131]) [131] SLNPEWNE ( targets isozyme PKCb [131]) [131] SRIGQ [131] staurosporine ( strong inhibition [118]; inhibits all PKC isozymes [99]; PKC inhibitory alkaloid isolated from the bacterium Lentzea albida, formerly Streptomyces staurosporeus [126]; poorly selective for, unspecific inhibitor of a broad range of protein kinases [1]) [1, 73, 99, 118, 126] Tween 80 ( inhibits the enzyme at high concentration [3]) [3] alsterpaullone ( 7% inhibition of PKCa at 0.01 mM [102]) [102] angiotensin II type 2-receptor ( inhibits isozyme PKCa [143]) [143] arachidonoyl fluoroamethylketone ( inhibition of PKCg oscillation at 0.1 mM [116]) [116] bisindolylmaleimide ( relatively specific for protein kinase C [1]; strong inhibition of PKCa [115]) [1, 115] bisindolylmaleimide derivatives [126] bromoerol lactone ( slight inhibition of PKCg oscillation at 0.1 mM [116]) [116] calphostin C ( PKC-specific inhibitor [124]; interacts with the regulatory domain of PKC, inhibits encystment in vivo [129]) [124, 129] calphostin-C ( inhibition of triiodothyronine-activation in forebrain, overview, can be partially reversed by triiodothyronone, i.e. T3 [123]) [123] compound 48/80 ( complete inhibition of PKCg oscillation at 0.1 mM [116]) [116] curcumin ( inhibits or activates PKC dependent on Ca2+ and the presence of membranes or phosphatidylserine, respectively, in presence of phosphatidylserine curcumin activates PKC, in presence of membranes with phosphatidylserine the enzyme is inhibited, Ca2+ competes with curcumin at the regulatory domainbinding site, overview, effects on different isozymes, overview [137]) [137] cytochalasine D ( inhibition of PKCg oscillation at 0.01 mM [116]) [116] indirubin-3’-monoxime ( 32% inhibition of PKCa at 0.01 mM [102]) [102] 343

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methylphenylethynyl pyridine ( complete inhibition of PKCg oscillation at 0.05 mM [116]) [116] phorbol 12,13-dibutyrate ( activates Na+ ,K+ -ATPase activity at low concentration, but inhibits it at high concentration, the inhibitory effect can be reversed by preincubation with ethoxyresorufin, 17-octadecynoic acid, wortmannin, LY294002, cytochlasin D, or latrunculin B [99]) [99] propidiumiodide [136] rottlerin ( inhibits PKCd [126]; inhibits PKCd, but does not block its translocation [112]; selective inhibition of isozyme PKCd [100]; slight inhibition of PKCa [115]; specific for PKCd [120]; a selective inhibitor of nPKCd and PKCtheta [150]; specific inhibitor of isozyme PKCd [130]) [100, 112, 115, 120, 126, 130, 150] safranine O [136] thapsigargin ( complete inhibition of PKCg oscillation at 0.005 mM [116]) [116] thymeleatoxin ( activates Na+ ,K+ -ATPase activity at low concentration, but inhibits it at high concentration [99]) [99] Additional information ( insensitive to caspase-3 [8]; insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain [32]; F-actin, latrunculin B, and phalloidin do not modulate the initial steps of enzyme activation process in living nerve cells [108]; no inhibition by genistein and H-89 [118]; no inhibition of PKCa by roscovitine, and purvalanol [102]; no inhibition of PKCd by bisindolylmaleimide I [112]; no inhibition of PKCg oscillation by EGTA, aristolochic acid, and colchicine [116]; PKC isozymes interacting proteins, overview [103]; rapamycin and wortmannin inhibit PKC bII turnover [113]; synthesis of peptides behaving as pseudosubstrates, determination of inhibitory potential [3]; synthesis of peptides behaving as pseudosubstrates, determination of inhibitory potential, sequences of the different enzyme forms, required sequence properties, overview [3]; the enzyme is inhibited by its regulatory subunit masking the active site, autoregulation by a pseudosubstrate mechanism, overview [2]; inhibition of PKC by dequalinium analogues of bis-quaternary dequalinium salts, structure-activity studies on head group variations, overview, Mulliken charges on N1 of the model compounds used for correlation, overview [136]; peptides derived from the PKC-cognate proteins are useful competitive inhibitors of PKC signalling, inhibitory peptide inhibit enzyme activity and/or enzyme translocation, physiological effects, overview [131]) [2, 3, 8, 32, 102, 103, 108, 112, 113, 116, 118, 131, 136] Cofactors/prosthetic groups ATP ( preferred by the wild-type PKCa [133]) [1, 2, 3, 4, 98, 101, 102, 108, 111, 112, 113, 114, 115, 116, 118, 119, 122, 123, 125, 126, 129, 130, 132, 133, 134, 136, 137, 138, 143, 145, 147, 148, 149, 150, 151]

344

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Protein kinase C

N6 -phenyl-ATP ( preferred by PKCa mutant M417A to phosphorylate peptide and protein substrates, no activity with the wild-type PKCa [133]) [133] Activating compounds 1,2-diacyl-sn-glycerol [148] 1,2-diolein ( activates, required [129]) [129] 1,2-dioleoyl-sn-glycerol ( activates [132]) [132] 1-oleoyl-2-acetyl-sn-glycerol [138] 12-O-tetradecanoylphorbol 13-acetate ( activates [132]; activates isozyme PKCe [141]; activates PKC [137]; activates PKC isozymes [152]; activates PKCa isozyme about 1.6fold [134]; modeling of complex formation with PKCg, competes with hypericin for binding to the regulatory domain of PKC [153]) [132, 134, 137, 141, 146, 148, 152, 153] 12-O-tetradecanoylphorbol-13-acetate [120] 4b-phorbol 12-myristate 13-acetate ( activates PKCa-mediated activation of phospholipase D [115]) [115] anionic phospholipids ( isozymes a, bI, bII, and g require anionic phospholipids for activity, isozymes d, e, h, and q do not [109]) [109] arachidonic acid ( alone or a combination of g-linolenic acid and phosphatidylserine slightly enhances PKC z activity [32]; slightly enhances PKC z activity [32]) [32] cardiolipin ( activates [96]) [96] diacylglycerols ( activate PKC isozymes [130]) [130] epidermal growth factor ( EGF, activates isozyme PKCa in the fetal lung during signaling involved in lung maturation, overview [144]) [144] fatty acids ( activation mechanism [151]; activation mechanism, isozyme PKCd is involved in fatty acid-induced hepatic insulin resistance [151]) [151] insulin ( activates PKC isozymes a, bII, d, and zeta in several cell types, activation mechanism [151]; activates PKC isozymes a, bII, d, and z in several cell types, activation mechanism, isozyme PKCd is involved in fatty acid-induced hepatic insulin resistance [151]) [151] kainic acid ( phosphorylation of MARCKS by PKC isozymes is dramatically upregulated specifically in microglial cells after kainic acid-induced seizures, but not in other types of glial cells, overview, upregulation of isozymes PKCa, PKCbI, PKCbII, and PKCd [145]) [145] palmitate ( activates isozyme PKCq, but not isozymes PKCa and PKCe [151]) [151] phorbol 12-myristate 13-acetate ( activates PKC 4fold [98]; binds to the C1 domains of isozymes PKCa and PKCg, differential roles of C1A and C1B, overview [114]; i.e. PMA [117]; mediates isozyme translocation [106]; i.e. PMA, inhibits IKCa channels [149]; stimulates IRS1 Ser24 phosphorylation in vivo [150]) [98, 99, 100, 101, 104, 106, 111, 114, 117, 124, 126, 142, 149, 150] phorbol esters ( stimulate [79]; bind to and stimulate the kinase activity of PKC-L [69]) [69, 79]

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phosphatidylserine ( stimulates [90]; activation mechanism [151]; plus diacylglycerol, activates [32]; a combination of g-linolenic acid and phosphatidylserine slightly enhances PKC z activity [32]; dependent upon phosphatidylserine or diacylglycerol for maximal activation [86]; PKC Apl I requires much less phosphatidylserine for activation than does purified PKC Apl II [90,93]; activates, required [129]) [32, 86, 90, 93, 111, 129, 148, 151] phospholipid ( stimulates [79]; dependent on [97]; activity is dependent on [50]; strict dependence on the presence of phospholipids [23]; significantly dependent on phospholipid when assayed with calf thymus H1 histone as a phosphate acceptor protein [52]) [23, 50, 52, 79, 97] phospholipids ( activate [129]; regulatory function for isozymes a, bI, bII, g, d, e, h, and q [126]) [126, 129] curcumin ( inhibits or activates PKC dependent on Ca2+ and the presence of membranes or phosphatidylserine, respectively, in presence of phosphatidylserine curcumin activates PKC, in presence of membranes with phosphatidylserine the enzyme is inhibited, Ca2+ competes with curcumin at the regulatory domainbinding site, overview, effects on different isozymes, overview [137]) [137] diacylglycerol ( activates [36, 129]; activity is dependent on [50]; plus phosphatidylserine, activates [32]; independent of the presence of Ca2+ or diacylglycerol, when assayed with calf thymus H1 histone as a phosphate acceptor protein [52]; dependent upon phosphatidylserine or diacylglycerol for maximal activation [86]; binds to C1 domain of PKC, regulatory function for isozymes a, bI, bII, g, d, e, h, and q, overview [126]; binds to the C1 domains of isozymes PKCa and PKCg, differential roles of C1A and C1B, overview [114]; isozymes a, bI, bII, and g require diacylglycerol for activity, isozymes d, e, h, and q do not [109]; required, second messenger involved in PKC regulation [110]) [32, 36, 50, 52, 86, 99, 109, 110, 114, 126, 129, 150] dioleoylglycerol [111] g-linolenic acid ( a combination of g-linolenic acid and phosphatidylserine slightly enhances PKC z activity [32]) [32] gastrin ( physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor [7]) [7] hypericin ( competes with 12-O-tetradecanoylphorbol 13-acetate for binding to the regulatory domain of PKC, localization of PKC isozymes a, d, and g, high affinty binding and interaction with the C1B domain of PKC, molecular modeling [153]) [153] kenpaullone ( 20% activation of PKCa at 0.01 mM [102]) [102] mezerein ( stimulates classical, but not novel PKC isozymes [99]) [99] phorbol 12,13-dibutyrate ( activates PKC, activates Na+ ,K+ ATPase activity at low concentration, but inhibits it at high concentration [99]) [99, 126] 346

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Protein kinase C

phorbol dibutyrate ( activates [36]) [36] phosphatidylserine-diolein [122] thymeleatoxin ( activates Na+ ,K+ -ATPase activity at low concentration, but inhibits it at high concentration [99]; activates Ca2+ -dependent PKC isozymes, reversal by chronic thymeleatoxin pretreatment of the effects of carbachol on glucose-induced Ca2+ oscillations and pulsatile 5-HT release, overview [135]) [99, 100, 135] transforming growth factor a ( TGFa, activates isozyme PKCa in the fetal lung during signaling involved in lung maturation, overview [144]) [144] Additional information ( cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme [32]; activation mechanism of isozymes PKCa and PKCg [114]; activation mechanism of isozymes, overview [126]; autoregulation by a pseudosubstrate mechanism, overview [2]; F-actin, latrunculin B, and phalloidin do not modulate the initial steps of enzyme activation process in living nerve cells [108]; Helicobacter pylori activates the enzyme in gastric epithelial cells [124]; phorbol 12-myristate 13-acetate and sugars activate PKC bII activation and degradation via dephosphorylation and ubiquitinylation pathway [113]; PKC activation is influenced by intracellular Ca2+ concentration and activity of the metabotropic glutamate receptor 5 [116]; PKC isozymes interacting proteins, overview [103]; the isozymes show varying activation requirements [109]; no activation by 4a-phorbol 12-myristate 13-acetate [149]; no activation by 4a-phorbol-12,13-didecanoate [134]; no activation by C2 ceramide or chronic insulin treatment [150]) [2, 32, 103, 108, 109, 113, 114, 116, 124, 126, 134, 149, 150] Metals, ions Ca2+ ( activates [91, 148]; no activation [23]; Ca2+ -mediated interactions between the two domains could contribute to enzyme activation as well as to the creation of a positively charged phosphatidylserine-binding site [9]; activity is independent of Ca2+ [32]; enzyme is dependent on Ca2+ [90]; activates, required [129,130]; binds to C2 domain of PKC, regulatory fucntion for isozymes a, bI, bII, and g, overview [126]; binds to C2 domain of PKC, regulatory function for isozymes a, bI, bII, and g, overview [126]; calcium-dependent PKC activation in acutely isolated neurons during oxygen and glucose deprivation, involved in PKC activity regulation during ischemia, overview [127]; dependent on, depends on the isozyme, e.g. isozyme bII and g are dependent on Ca2+ , while isozyme d is not [116]; isozymes a, bI, bII, and g require Ca2+ for activity, isozymes d, e, h, and q do not [109]; PKC isozymes are divided into groups of calcium-independent and of calcium-dependent isozymes [120]; required, second messenger involved in PKC regulation, some isozymes are Ca2+ -independent [110]; activates Ca2+ -dependent PKC isozymes [135]; activation mechanism [151]; PKCa is a Ca2+ -dependent isozyme [144]; stimulates the en-

347

Protein kinase C

2.7.11.13

zyme, binds to the regulatory domain of PKC, competes with effector curcumin, overview, no effect by cysteine [137]) [9, 23, 32, 90, 91, 98, 108, 109, 110, 111, 114, 116, 120, 122, 126, 127, 129, 130, 132, 133, 135, 136, 137, 143, 144, 148, 151] Mg2+ [1, 2, 3, 4, 98, 111, 112, 114, 122, 132, 133, 134, 136, 138, 143, 145, 147, 149, 150] Mn2+ [112] Additional information ( presence of only one cysteine-rich, zinc finger-like domain, absence of an apparent Ca(2+)-binding region [77]) [77] Specific activity (U/mg) Additional information [101, 152] Km-Value (mM) 0.0036 (S6-(229-239) peptide, enzyme activated by cardiolipin [96]) [96] 0.0093 (ATP, pH 7.4, 37 C, recombinant wild-type isozyme PKCa [133]) [133] 0.0124 (N6 -phenyl-ATP, pH 7.4, 37 C, recombinant isozyme PKCa mutant M417A [133]) [133] 0.0828 (ATP, pH 7.4, 37 C, recombinant isozyme PKCa mutant M417A [133]) [133] Additional information ( kinetics [132]; binding kinetics of phorbol 12-myristate 13-acetate and diacylglycerol [114]; binding kinetics of PKC isozymes to hypericin and phorbol esters, overview [153]) [114, 132, 153] Ki-Value (mM) Additional information ( Ki values of the pseudosubstrates in nano- to micromolar range [3]) [3] pH-Optimum 6.2 ( assay at [123]) [123] 7.2 ( assay at [112]) [112] 7.4 ( assay at [98, 111, 114, 127, 133, 136, 143]) [98, 111, 114, 127, 133, 136, 143] 7.5 ( assay at [102, 122, 124, 129, 132, 137]) [102, 122, 124, 129, 132, 137] Temperature optimum ( C) 21 ( assay at room temperature [102]) [102] 22 ( assay at room temperature [112,114]) [112, 114] 24 ( assay at [137]) [137] 25 ( assay at [111]) [111] 30 ( assay at [98, 122, 123, 129, 132, 148]) [98, 122, 123, 129, 132, 148] 36 ( assay at [127]) [127] 37 ( assay at [117, 118, 133, 143]; assay at, in vivo [124]) [117, 118, 124, 133, 143]

348

2.7.11.13

Protein kinase C

4 Enzyme Structure Subunits ? ( x * 78000, SDS-PAGE [142]; x * 120000, SDS-PAGE [89]; x * 105000, SDS-PAGE [7]; x * 64000, SDS-PAGE [52]; x * 65000 [77]; x * 116000 [96]; x * 103925, calculation from nucleotide sequence [89]; x * 126000 [79]; x * 78000-80000, PKC bII, SDS-PAGE [113]; x * 82000, isozyme PKCa, SDS-PAGE [143]) [7, 52, 77, 79, 89, 96, 113, 142, 143] Additional information ( PKC domain composition [110]; PKC domain structure, overview [103]) [103, 110] Posttranslational modification phosphoprotein ( phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulates the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulate autophosphorylation of PKD2 in intact cells, C-terminal Ser876 is an in vivo phosphorylation site within PKD2 that is correlated with the activation status of the kinase [7]; phosphorylation of Thr642 is an early event in the processing of newly synthesized protein kinase C b 1 and is essential for its activation [11]; activation loop phosphorylation of PKCd in response to serum stimulation of cells is PI 3-kinase-dependent and is enhanced by PDK1 coexpression [20]; COOH-terminal autophosphorylation sites are critical for enzyme function and possibly subcellular localization in COS cells [13]; processing by protein kinase C cannot occur until the enzyme is first phosphorylated by a protein kinase C kinase [12]; protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop T500 renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus T641 maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus S660 regulates the enzyme’s subcellular localization [10]; isozymes a, d, and e require trans- and autophosphorylation for activity [125]; phosphorylation of PKCny leads to its activation in response to B-cell receptor engagement in subcellular compartment redistribution [107]; phosphorylation regulates the enzyme activity, the enzyme is phosphorylated by PDK-1 at the activation loop [4]; phosphorylation of isozyme PKCe at Ser279 is required for translocation to the nucleus during which the enzyme looses the phsophate group at Ser279, overview [141]) [4, 7, 10, 11, 12, 13, 20, 107, 125, 141] Additional information ( two types of complementary DNA clones for rat brain protein kinase C, these clones encode 671 and 673 amino acid sequences, which differ from each other only in the carboxyl-terminal regions of approximately 50 amino acid residues. This difference seems to result from alternative splicing [18]) [18]

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Protein kinase C

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5 Isolation/Preparation/Mutation/Application Source/tissue 3T3 cell [62] AGS cell ( AGS-B cell [7]) [7] B-cell ( PKC isozyme expression patterns [107]) [107] B-cell lymphoma cell [128] B-lymphocyte [103, 107, 119, 126] Burkitt lymphoma cell ( high expression of isozyme PKCa in most cell lines, overview [128]) [128] CACO-2 cell [152] DAUDI cell ( Burkitt lymphoma cell line [128]) [128] FL-18 cell ( follicular lymphoma cell line [128]) [128] FL-218 cell ( follicular lymphoma cell line [128]) [128] H4IIE cell [151] HBL-1 cell ( diffuse large B-cell lymphoma cell line [128]) [128] HBL-10 cell ( Burkitt lymphoma cell line [128]) [128] HBL-11 cell ( myeloma cell line [128]) [128] HBL-2 cell ( diffuse large B-cell lymphoma cell line [128]) [128] HBL-3 cell ( precursor T-lymphoblastic leukemia cell line [128]) [128] HBL-4 cell ( Burkitt lymphoma cell line [128]) [128] HBL-5 cell ( Burkitt lymphoma cell line [128]) [128] HBL-6 cell ( diffuse large B-cell lymphoma cell line [128]) [128] HBL-7 cell ( Burkitt lymphoma cell line [128]) [128] HBL-8 cell ( Burkitt lymphoma cell line [128]) [128] HBL-9 cell ( Burkitt lymphoma cell line [128]) [128] HEK-293 cell [116] HL-60 cell [7] HeLa cell [110] JURKAT cell ( isozyme PKCa [128]) [128, 146] Kobayashi cell ( Burkitt lymphoma cell line [128]) [128] MKN-45 cell [124] Ng-108-15 cell ( a hybridomal cell line of a rat neuroblastoma and a mouse glioma, expression of isozymes PKCa, PKCe, PKCi, and PKCz, no expression of isozymes PKCb, PKCd, PKCg, PKCm, and PKCq [143]) [143] NIH-3T3 cell [151] NK-92 cell [148] PC-12 cell ( PKCa [108]) [108] RAJI cell ( Burkitt lymphoma cell line [128]) [128] RAW 264.7 cell ( virus-induced macrophage cell line [103]) [103] RBA-2 cell ( cultured type-2 astrocyte cell line [106]) [106] SH-SY5Y cell [140] T-cell ( isozyme PKCa [128]) [128]

350

2.7.11.13

Protein kinase C

T-lymphocyte ( peripheral blood CD8+ T cell [146]) [103, 119, 126, 146] U-87MG cell [153] WI-38 cell ( fibroblast cell line [118]) [118] adipocyte [151] adipose tissue [151] aorta [111] artery ( coronary [101]; pulmonary [100]) [100, 101, 131] astrocyte ( primary, cortical [106]) [106] blood platelet ( isozyme PKCd [112]) [82, 112, 130] blood vessel ( arterial pulmonary [100]) [100] bone marrow [119] brain ( in adult brain, the relative activities of a-, b I-, b II-, and g-subspecies are roughly 16%, 8%, 55%, and 21% [35]; isozyme-specific and developmental stage-specific alterations in brain PKC following exposure to a polychlorinated biphenyl mixture [105]) [8, 17, 18, 19, 22, 28, 34, 35, 45, 50, 52, 54, 77, 81, 82, 95, 105, 111, 122, 138, 139, 140, 143, 151] capillary ( isozyme g [109]) [109] cardiac myocyte ( expression of several isozymes [125]) [125] cell culture ( primary [151]; PR17 cells and wild-type HL-60 cells [58]; fibrosarcoma cell line [61]; epidermoid carcinoma line A431 [69]; insulin-secreting cell line RINm5F [77]; Swiss 3T3 fibroblasts [62]; undifferentiated mouse embryonal carcinoma cell line P19, NIH 3T3 cells [95]; several hemopoietic tumor lines [81]; NIH/hIR/rIRS1cells [150]) [58, 61, 62, 69, 77, 81, 95, 104, 150, 151] central nervous system [121, 145] cerebellum ( granule cells [108]; isozyme PKCg [128]; ontogenetic isozyme profiling [105]; isozymes a, bI, bII, g, d, e, l, h, q, and z [139]) [105, 108, 128, 139] colon [149] commercial preparation ( purified PKC from brain [122]; recombinant isozymes [119]; recombinant PKCa [115,118]) [115, 118, 119, 122] corpus striatum [120, 121] crypt cell ( from normal sigmoid colon [149]) [149] duodenum ( isozymes g, and e [109]) [109] encysting cell [129] endothelial cell ( from coronary artery [101]; isozyme g [109]) [101, 109] endothelium ( aortic [111]) [111] enteric nervous system ( several isozymes [109]) [109] epithelium [124, 140, 144] fibroblast ( isozyme b [109]; primary from fetal lung [144]) [15, 109, 118, 119, 131, 141, 144, 151] fibrosarcoma cell line [61] 351

Protein kinase C

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forebrain [123] frontal cortex [84] gastric cell ( epithelial cell [124]) [124] glial cell ( glial network [109]) [109, 145] glioma cell [153] granule cell ( primary [108]; primary cerebellar, cultured [139]) [108, 139] heart [68, 69, 125, 131] hematopoietic cell ( hematopoietic stemcell and hemopoietic progenitor cells [24]; enzyme is predominantly expressed in [83]) [23, 24, 83, 122] hepatocyte ( from fetal liver [119]) [119] hepatoma cell ( constitutively active isozyme PKCd [151]) [151] hippocampus ( ontogenetic isozyme profiling [105]; cellular distribution and phosphorylation of MARCKS by PKC isozymes in the hippocampus following kainic acid-induced seizures, immunohistochemic detection, overview [145]) [89, 105, 127, 145] hypothalamus [123] ileum ( myenteric neurons, isozymes g, lambda, and e [109]) [109] intestine [113] jejunum [113] kidney ( cortex [25]; low expression in vascular elements and high expression in tubule epithelium, highly expressed in proximal tubule, thick limb, and collecting duct [25]) [8, 25, 87, 95, 99] larva ( abundant at the earliest larval stage, but their relative concentrations decrease coordinately in late larvae [72]) [72] left ventricle [125] leukocyte [41] liver [96, 151] lung ( fetal, isozymes PKCa, PKCe, PKCm, and PKCz, isozyme PKCa is localized in the cuboidal epithelium and in mesenchyme [144]) [3, 63, 68, 69, 77, 81, 82, 100, 118, 144] lymph vessel ( isozyme g [109]) [109] lymphoma cell [128] mammary gland [142] mast cell ( derived from bone marrow [119]) [119] megakaryoblast [82] microglia [145] myeloid cell ( ABPL-3 myeloid tumor [23]) [23] myenteron ( myenteric neuron, isozyme b in submucosal ganglia of neurons, isozymes g, h, and q are present in intrinsic primary afferent neurons, but not in other myenteric neurons, low expression of isozyme a, isozyme e [109]) [109] myotube ( isozyme PKCbII [151]) [151] nerve [121] nervous system [91] neuroblastoma cell [140] 352

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neuron ( acutely dissociated CA1 neurons in non-excitotoxic ischemia [127]) [127, 138, 140] ovary ( highly expressed [8]) [8, 95] pancreatic islet [77, 135] pineal gland [98] plant [126] renal cortex [99] sensory cell [92] skeletal muscle ( isozyme q [103]; isozyme PKCq is decreased in obese patients skeletal muscle [151]) [82, 103, 151] skin [63, 68, 69] small intestine ( myenteric neurons [109]) [109] smooth muscle ( no isozyme b, high expression of isozyme d [109]; pulmonary arterial [100]) [100, 109] smooth muscle cell ( pulmonary cell line [118]) [118] telencephalon [123] testis ( highly expressed [8]) [8, 81, 95] thymocyte [8] trophozoite [129] white muscle ( increased expresison of isozyme PKCtheta in insulinresistant rats [151]) [151] Additional information ( expression analysis of isozymes a, bII, g, and d in reactive lymphoid tissues, B-cell lymphoma and lymphoma cell lines, overview [128]; PKC isozymes are expressed in a wide range of tissues, some isozymes are tissue-specific [103]; tissue- and cell-typespecific expression of isozymes, overview, involved in regulation of organ, e.g. intestinal, function [109]; developmental expression patterns of isozymes PKCa, PKCe, PKCm, and PKCz [144]; tissue distribution of PKC isozymes, overview [140]) [103, 109, 128, 140, 144] Localization Golgi apparatus ( isozyme PKCe in a perinuclear site is associated with the Golgi apparatus, phosphorylation of isozyme PKCe at Ser279 is required for translocation to the nucleus during which the enzyme looses the phsophate group at Ser279, overview [141]) [141, 146] cytoplasm ( isozyme PKCd [128]; isozymes b, g, d, e [109]; non-activated PKCbII and PKCbI [117]) [46, 96, 108, 109, 112, 117, 128] cytoskeleton ( associated with [72]) [72] cytosol ( stimulation by phorbol ester causes weak translocation of dIII-GFP from the cytosol to the plasma membrane [46]; isozyme e, g, and a [105]; translocation of PKCg from cytosol to membrane [116]; PKC is commonly associated with a rapid redistribution of the kinase from the cytosol to membranes [129]) [46, 103, 105, 116, 129, 130, 139, 140, 144] endoplasmic reticulum [146]

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endosome ( activated PKCbII and PKCbI are translocated from cytosol, juxtanuclear location, isozyme-specific translocation of PKCbII and not PKCbI to a juxtanuclear subset of recycling endosomes, involvement of phospholipase D [117]) [117] intracellular ( intracellular localization pattern during cyst formation [129]; localization of PKC isozymes a, d, and g, is influenced by hypericin, high affinity binding and interaction with the C1B domain of PKC, molecular modeling [153]) [129, 153] membrane ( associated with [72]; bound to via second messengers and phorbol esters, the ligand-binding domains are required for membrane targeting [110]; cellular membrane translocation mechanism of isozyme PKCa and PKCg [114]; isozyme e, g, and a [105]; jejunal brush border membrane [113]; translocation [115]; PKC is commonly associated with a rapid redistribution of the kinase from the cytosol to membranes [129]) [61, 72, 103, 105, 108, 110, 113, 114, 115, 120, 129, 140, 144] microtubule ( isozymes PKC-b and PKC-g are recruited to microtubules by LFA-1 signalling, i.e. lymphocyte-function-associated antigen-1 signalling [103]) [103] particle-bound [139] perinuclear space ( isozyme PKCe in a perinuclear site is associated with the Golgi apparatus, phosphorylation of isozyme PKCe at Ser279 is required for translocation to the nucleus during which the enzyme looses the phsophate group at Ser279, overview [141]) [60, 140, 141] plasma membrane ( stimulation by phorbol ester causes weak translocation of dIII-GFP from the cytosol to the plasma membrane [46]; activated PKCbII and PKCbI are translocated from cytosol [117]; PKC is recruited by other kinases [126]; translocation of PKCg from cytosol to membrane, PKCg oscillates on the membrane induced by glutamate and activated metabotropic glutamate receptor 5, overview [116]) [46, 104, 106, 108, 112, 116, 117, 126] soluble [143] Additional information ( isozyme PKCtheta is recruited to the plasma membrane by a cytoskeleton-dependent mechanism regulating the Rac-1 guanine nucelotide exchange protein Vav-1 [126]; no isozyme d in the nucleus [109]; phorbol 12-myristate 13-acetate-mediated translocation of isozymes a, bI, bII, and g [106]; PKC is translocated from cytoplasm to membrane upon stimulation, latrunculin B does not influence enzyme localization [108]; PKCd is translocated from cytosol to plasma membrane in platelets activated by the snake venom alboaggregin-A [112]; subcellular distribution of isozymes in brain regions during development [105]; subcellular distribution of isozymes PKCbII and PKCbI [117]; analysis of subcellular distribution of isozymes in rat cerebellum, overview [139]; cell passage induces rapid translocation of isozyme PKCe to the periphery where it appears to colocalize with F-actin, which renders the cells proliferative again while PKCe return to the perinuclear site [141]; quantitative determination of membrane translocation of iso354

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zymes under hypoxic conditions in neuroblastoma SH-SY5Y cells, overview [140]; subcellular distribution of isozymes PKCa, PKCe, PKCmy, and PKCzeta [144]; subcellular localization study, during encystment PKCb redistributs to plasma membrane from cytosol [129]) [105, 106, 108, 109, 112, 117, 126, 129, 139, 140, 141, 144] Purification (recombinant full-length enzyme by nickel affinity chromatography, proteolytic clevage of the recombinant full-length enzyme to obtain the catalytic domain, followed by anion exchange chromatography and gel filtration) [132] (isozyme PKC bII from jejunal loops) [113] (isozymes partially from primary cerebellar granule cells, Tris buffer leads to degradation of the proteins) [139] (recombinant full-length isozymes PKCa and PKCg from Sf9 cells by ion exchange chromatography, recombinant His-tagged isolated isozymes PKCa and PKCg domains C1 from Escherichia coli strain BL21(DE3) inclusion bodies by nickel affinity chromatography) [114] [23] [28] (partial purification of the PKC-zeta isoenzyme) [32] (partial) [50] (partial) [50] (partial) [32] [96] (partial) [79] (native isozyme partially by anion exchange chromatography and gel filtration) [129] Crystallization (purified recombinant isozyme PKCbII catalytic domain, residues 321673, hanging drop vapour diffusion method, 8 mg/ml protein in 0.1 M acetamidoiminodiacetic acid, pH 6.5, and 1.7-2.3 M sodium acetate, 19 C, X-ray diffraction structure determination and analysis at 3.2 A resolution, molecular replacement, modeling) [132] (crystal structure of the cys2 activator-binding domain of protein kinase C d in complex with phorbol ester) [21] (crystal structure of PKC-d C2 domain. Structural elements unique to this C2 domain include a helix and a protruding b hairpin which may contribute basic sequences to a membrane-interaction site) [48] Cloning (co-expression of PKC isozymes and insulin in HEK-293 or CHO cells, interaction analysis, overview) [151] (expression of GFP-tagged PKC) [110] (expression of isozymes PKCe and PKCzeta in Saccharomyces cerevisiae, subcloning in Escherichia coli) [147]

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(expression of wild-type and mutant isozyme PKCe in NIH3T3 fibroblasts as GFP-tagged proteins) [141] (overexpression PKCd in NMuMG cells, a normal immortalized mammary cell line derived from NAMRU mice) [142] (co-expression of PKC isozymes and insulin in HEK-293 or CHO cells, interaction analysis, overview) [151] (expression of FLAG-tagged wild-type and mutant isozyme PLCa in MCF- 10A human breast epithelial cells) [133] (expression of GFP-tagged PKC) [110] (expression of GFP-tagged wild-type PKC isozymes in HEK-293 cells, and of GFP-tagged PKCg wild-type and mutants lacking the C1 or C2 domain in HEK-293 cells) [116] (expression of full-length His-tagged isoform PKCbII in Spodoptera frugiperda Sf21 cells) [132] (expression of wild-type and mutant isoyzem PKCa in COS-7 cells, coexpression of PKCa with diacylglycerol kinase-zeta and retinoblastoma protein in COS-7 cells) [134] (isozymes PKCbII and PKCbI are products of alternative splicing of gene PKCb, expression of GFP- or HA-tagged isozymes PKCbII, wild-type and mutant, and PKCbI in HEK-293 or HeLa cells) [117] (co-expression of PKC isozymes and insulin in HEK-293 or CHO cells, interaction analysis, overview) [151] (expression of full-length isozymes PKCa and PKCg in Spodoptera frugiperda Sf9 cells using the baculovirus infection system, expression of Histagged isolated isozymes PKCa and PKCg domains C1 in Escherichia coli strain BL21(DE3)) [114] (expression of isozyme PKCbI in HEK-293 cells, and co-expression with dopamine transporter significantly enhancing the amphetamine-stimulated dopamine efflux) [120] (expression of isozyme PKCd in Saccharomyces cerevisiae, subcloning in Escherichia coli) [147] [17] (expression in COS cells) [19] (expression of mutant enzymes in COS cells) [12] (isolation of cDNA clones encoding protein kinase C) [16] (isolation of cloned mouse protein kinase C b-II cDNA) [14] (wild-type and mutant enzymes overexpressed in COS cells) [13] (PKCdII expressed in COS-1 cells) [8] [7, 24] [25] (expression in recombinant baculovirus-infected insect cells) [32] (structure and nucleotide sequence of a Drosophila melanogaster protein kinase C gene) [33] (cDNA sequence encoding mouse PKC-g isolated from a C57BL/6 brain cDNA library) [37] (expression in COS cells) [19] [40] 356

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(the 5’ segment of the gene for protein kinase C b is cloned from a human leukocyte genomic library in EMBL3 bacteriophage) [41] [51] (cDNA cloning of an alternative splicing variant of protein kinase C d, expression of truncated form of PKC dIII fused to green fluorescent protein in CHO-K1 cells) [46] (expressed in COS 7 cells) [50] [79] (protein kinase C-e E1 and E2, expression in Sf9 cells, the recombinant protein displays protein kinase C activity and phorbol ester binding activity) [80] (expressed in insect cells via a baculovirus expression vector, a 75000 Da protein is synthesized which, unlike other PKC isoforms, does not bind phorbol ester, even at very high concentrations) [81] [83] (expression in COS cells) [82] (expression in recombinant baculovirus-infected insect cells, overexpression in NIH 3T3 cells or insect cells) [32] (isolation of cDNA) [84] (expression in the baculovirus insect-cell expression system) [86] (expression in COS1 cells) [88] (expression in COS7 cells) [89] (expression in COS1 cells) [88] [92] [92] (COS cells transfected with the PKC l expression plasmid) [95] (expression in COS cells) [97] [79] (gene pkcb, isozyme PKCb, DNA and amino acid sequence determination and analysis, expression of the catalytic domain from PKCb) [129] Engineering D116A ( site-directed mutagenesis, isozyme PKCg domain C1B mutant shows unaltered activity [114]) [114] D55A ( site-directed mutagenesis, isozyme PKCa and PKCg mutants show unaltered activity and ligand binding, isozyme PKCa shows enhanced activation by phospholipids [114]) [114] K368A ( a dominant-negative mutant of PKCa [143]) [143] K368D ( site-directed mutagenesis of isozyme PKCa ATP-binding site, a dominant-negative mutant [134]) [134] K371R ( site-directed mutagenesis, inactive isozyme PKCbII mutant [117]) [117] M417A ( site-directed mutagenesis, utilizes the alternate cofactor N6 -phenyl-ATP [133]) [133] S279A ( site-directed mutagenesis of the isozyme PKCe phosphorylation site results in altered subcellular localization not in the perinulcear/ Golgi site [141]) [141]

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S279E ( site-directed mutagenesis of the isozyme PKCe phosphorylation site results in altered subcellular localization not in the perinulcear/ Golgi site [141]) [141] S279T ( site-directed mutagenesis of the isozyme PKCe phosphorylation site results in altered subcellular localization not in the perinulcear/ Golgi site [141]) [141] T50 0E ( expression as a catalytically active protein kinase C in COS cells [12]) [12] T50 0V ( expression as a catalytically inactive protein kinase C in COS cells [12]) [12] W58A ( site-directed mutagenesis, isozyme PKCg domain C1A mutant shows impaired binding of phorbol 12-myristate 13-acetate and diacylglycerol [114]) [114] Y123A ( site-directed mutagenesis, isozyme PKCg domain C1B mutant shows impaired binding of phorbol 12-myristate 13-acetate and diacylglycerol [114]) [114] Additional information ( construction of isozyme PKCq T-cell knockout mice, which show slightly impaired immune system and are defective in NF-kB activation, overview, isozyme PKCd knockout mice show a deregulated immune system, overview [126]; construction of isozymeknockout mice, immunological phenotypes of isozyme a, b, d, e, q, and z knockout mice, overview [103]; construction of PKC isozyme, e.g. PKCz-deficient or PKCd-deficient, knockout mice, construction of PKCb-deficient B-cells [107]; construction of PKC isozyme-deletion mutant mice leading to important metabolic deficiencies in the mutant mice [110]; downregulation of PKCd by short interfering RNA, deletion of domain C2 of PKCg leads to complete loss of activity, while deletion of C1 domain shows no effect on PKCg activity [116]; depletion of PKCd using RNAi leads to a marked increase in both urokinase-type plasminogen activator and matrix metalloproteinase-9 secretion, while PKCd overexpression significantly decreases urokinase-type plasminogen activator and matrix metalloproteinase9 production, two proteases associated with migratory and invasive capacities [142]; siRNA knockdown of isozyme PKC-d in Caco-2 cells leads to increased cell proliferation and apoptosis [152]; specific modulation of apoptosis and Bcl-xL phosphorylation in recombinant yeast by isozymes PKCe and PKCzeta [147]) [103, 107, 110, 116, 126, 142, 147, 152]

6 Stability General stability information , native PKCb is very sensitive to protease degradation [129]

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References [1] MacKintosh, C.; MacKintosh, R.W.: Inhibitors of protein kinases and phosphatases. Trends Biochem. Sci., 19, 444-448 (1994) [2] Kemp, B.E.; Parker, M.W.; Hu, S.; Tiganis, T.; House, C.: Substrate and pseudosubstrate interactions with protein kinases: determinants of specificity. Trends Biochem. Sci., 19, 440-444 (1994) [3] Kemp, B.E.; Pearson, R.B.; House, M.: Pseudosubstrate-based peptide inhibitors. Methods Enzymol., 201, 287-304 (1991) [4] Adams Joseph, A.: Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model?. Biochemistry, 42, 601-607 (2003) [5] Saijo, K.; Mecklenbrauker, I.; Santana, A.; et al.: Protein kinase C b controls nuclear factor kB activation in B cells through selective regulation of the IkB kinase a. J. Exp. Med., 195, 1647-1652 (2002) [6] Mecklenbrauker, I.; Saijo, K.; Zheng, N.Y.; Leitges, M.; Tarakhovsky, A.: Protein kinase C d controls self-antigen-induced B-cell tolerance. Nature, 416, 860-865 (2002) [7] Sturany, S.; Van Lint, J.; Muller, F.; Wilda, M.; Hameister, H.; Hocker, M.; Brey, A.; Gern, U.; Vandenheede, J.; Gress, T.; Adler, G.; Seufferlein, T.: Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases. J. Biol. Chem., 276, 3310-3318 (2001) [8] Sakurai, Y.; Onishi, Y.; Tanimoto, Y.; Kizaki, H.: Novel protein kinase C d isoform insensitive to caspase-3. Biol. Pharm. Bull., 24, 973-977 (2001) [9] Sutton, R.B.; Sprang, S.R.: Structure of the protein kinase Cb phospholipid-binding C2 domain complexed with Ca2+ . Structure, 6, 1395-1405 (1998) [10] Keranen, L.M.; Dutil, E.M.; Newton, A.C.: Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr. Biol., 5, 13941403 (1995) [11] Zhang, J.; Wang, L.; Schwartz, J.; Bond, R.W.; Bishop, W.R.: Phosphorylation of Thr642 is an early event in the processing of newly synthesized protein kinase Cb1 and is essential for its activation. J. Biol. Chem., 269, 19578-19584 (1994) [12] Orr, J.W.; Newton, A.C.: Requirement for negative charge on “activation loop“ of protein kinase C. J. Biol. Chem., 269, 27715-27718 (1994) [13] Zhang, J.; Wang, L.; Petrin, J.; Bishop, W.R.; Bond, R.W.: Characterization of site-specific mutants altered at protein kinase Cb1 isozyme autophosphorylation sites. Proc. Natl. Acad. Sci. USA, 90, 6130-6134 (1993) [14] Tang, Y.M.; Ashendel, C.L.: Isolation of cloned mouse protein kinase C bII cDNA and its sequence. Nucleic Acids Res., 18, 5310 (1990) [15] Housey, G.M.; Johnson, M.D.; Hsiao, W.L.; O’Brian, C.A.; Murphy, J.P.; Kirschmeier, P.; Weinstein, I.B.: Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell, 52, 343-354 (1988)

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[30] Dryja, T.P.; McEvoy, J.; McGee, T.L.; Berson, E.L.: No mutations in the coding region of the PRKCG gene in three families with retinitis pigmentosa linked to the RP11 locus on chromosome 19q. Am. J. Hum. Genet., 65, 926-928 (1999) [31] Al-Maghtheh, M.; Vithana, E.N.; Inglehearn, C.F.; Moore, T.; Bird, A.C.; Bhattacharya, S.S.: Segregation of a PRKCG mutation in two RP11 families. Am. J. Hum. Genet., 62, 1248-1252 (1998) [32] Kochs, G.; Hummel, R.; Meyer, D.; Hug, H.; Marme, D.; Sarre, T.F.: Activation and substrate specificity of the human protein kinase C a and z isoenzymes. Eur. J. Biochem., 216, 597-606 (1993) [33] Rosenthal, A.; Rhee, L.; Yadegari, R.; Paro, R.; Ullrich, A.; Goeddel, D.V.: Structure and nucleotide sequence of a Drosophila melanogaster protein kinase C gene. EMBO J., 6, 433-441 (1987) [34] Ono, Y.; Fujii, T.; Igarashi, K.; Kikkawa, U.; Ogita, K.; Nishizuka, Y.: Nucleotide sequences of cDNAs for a and g subspecies of rat brain protein kinase C. Nucleic Acids Res., 16, 5199-5200 (1988) [35] Kikkawa, U.; Ogita, K.; Ono, Y.; Asaoka, Y.; Shearman, M.S.; Fujii, T.; Ase, K.; Sekiguchi, K.; Igarashi, K.; Nishizuka, Y.: The common structure and activities of four subspecies of rat brain protein kinase C family. FEBS Lett., 223, 212-216 (1987) [36] Xu, R.X.; Pawelczyk, T.; Xia, T.H.; Brown, S.C.: NMR structure of a protein kinase C-g phorbol-binding domain and study of protein-lipid micelle interactions. Biochemistry, 36, 10709-10717 (1997) [37] Bowers, B.J.; Parham, C.L.; Sikela, J.M.; Wehner, J.M.: Isolation and sequence of a mouse brain cDNA coding for protein kinase C-g isozyme. Gene, 123, 263-265 (1993) [38] Chen, K.H.; Widen, S.G.; Wilson, S.H.; Huang, K.P.: Characterization of the 5’-flanking region of the rat protein kinase C g gene. J. Biol. Chem., 265, 19961-19965 (1990) [39] Loftus, B.J.; Kim, U.J.; Sneddon, V.P.; Kalush, F.; Brandon, R.; Fuhrmann, J.; Mason, T.; Crosby, M.L.; Barnstead, M.; Cronin, L.; Deslattes Mays, A.; Cao, Y.; Xu, R.X.; Kang, H.L.; Mitchell, S.; Eichler, E.E.; Harris, P.C.; Venter, J.C.; Adams, M.D.: Genome duplications and other features in 12 Mb of DNA sequence from human chromosome 16p and 16q. Genomics, 60, 295-308 (1999) [40] Mahajna, J.; King, P.; Parker, P.; Haley, J.: Autoregulation of cloned human protein kinase C b and g gene promoters in U937 cells. DNA Cell Biol., 14, 213-222 (1995) [41] Obeid, L.M.; Blobe, G.C.; Karolak, L.A.; Hannun, Y.A.: Cloning and characterization of the major promoter of the human protein kinase C b gene. Regulation by phorbol esters. J. Biol. Chem., 267, 20804-20810 (1992) [42] Kubo, K.; Ohno, S.; Suzuki, K.: Nucleotide sequence of the 3’ portion of a human gene for protein kinase C b I/b II. Nucleic Acids Res., 15, 71797180 (1987) [43] Kubo, K.; Ohno, S.; Suzuki, K.: Primary structures of human protein kinase C b I and b II differ only in their C-terminal sequences. FEBS Lett., 223, 138-142 (1987) 361

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[44] Coussens, L.; Rhee, L.; Parker, P.J.; Ullrich, A.: Alternative splicing increases the diversity of the human protein kinase C family. DNA, 6, 389394 (1987) [45] Ohno, S.; Kawasaki, H.; Imajoh, S.; Suzuki, K.; Inagaki, M.; Yokokura, H.; Sakoh, T.; Hidaka, H.: Tissue-specific expression of three distinct types of rabbit protein kinase C. Nature, 325, 161-166 (1987) [46] Ueyama, T.; Ren, Y.; Ohmori, S.; Sakai, K.; Tamaki, N.; Saito, N.: cDNA cloning of an alternative splicing variant of protein kinase C d (PKC dIII), a new truncated form of PKCd, in rats. Biochem. Biophys. Res. Commun., 269, 557-563 (2000) [47] Kurkinen, K.M.; Keinanen, R.A.; Karhu, R.; Koistinaho, J.: Genomic structure and chromosomal localization of the rat protein kinase Cd-gene. Gene, 242, 115-123 (2000) [48] Pappa, H.; Murray-Rust, J.; Dekker, L.V.; Parker, P.J.; McDonald, N.Q.: Crystal structure of the C2 domain from protein kinase C-d. Structure, 6, 885-894 (1998) [49] Garcia-Paramio, P.; Cabrerizo, Y.; Bornancin, F.; Parker, P.J.: The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation. Biochem. J., 333, 631-636 (1998) [50] Ono, Y.; Fujii, T.; Ogita, K.; Kikkawa, U.; Igarashi, K.; Nishizuka, Y.: The structure, expression, and properties of additional members of the protein kinase C family. J. Biol. Chem., 263, 6927-6932 (1988) [51] Ono, Y.; Fujii, T.; Ogita, K.; Kikkawa, U.; Igarashi, K.; Nishizuka, Y.: Identification of three additional members of rat protein kinase C family: d-, e- and z-subspecies. FEBS Lett., 226, 125-128 (1987) [52] Ono, Y.; Fujii, T.; Ogita, K.; Kikkawa, U.; Igarashi, K.; Nishizuka, Y.: Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties. Proc. Natl. Acad. Sci. USA, 86, 3099-3103 (1989) [53] Ohno, S.; Kawasaki, H.; Konno, Y.; Inagaki, M.; Hidaka, H.; Suzuki, K.: A fourth type of rabbit protein kinase C. Biochemistry, 27, 2083-2087 (1988) [54] Ohno, S.; Akita, Y.; Konno, Y.; Imajoh, S.; Suzuki, K.: A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family. Cell, 53, 731-741 (1988) [55] Schaeffer, E.; Smith, D.; Mardon, G.; Quinn, W.; Zuker, C.: Isolation and characterization of two new Drosophila protein kinase C genes, including one specifically expressed in photoreceptor cells. Cell, 57, 403-412 (1989) [56] Wang, Q.J.; Acs, P.; Goodnight, J.; Blumberg, P.M.; Mischak, H.; Mushinski, J.F.: The catalytic domain of PKC-e, in reciprocal PKC-d and -e chimeras, is responsible for conferring tumorgenicity to NIH3T3 cells, whereas both regulatory and catalytic domains of PKC-e contribute to in vitro transformation. Oncogene, 16, 53-60 (1998) [57] Schaap, D.; Parker, P.J.; Bristol, A.; Kriz, R.; Knopf, J.: Unique substrate specificity and regulatory properties of PKC-e: a rationale for diversity. FEBS Lett., 243, 351-357 (1989) [58] McSwine-Kennick, R.L.; McKeegan, E.M.; Johnson, M.D.; Morin, M.J.: Phorbol diester-induced alterations in the expression of protein kinase C

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[59] [60] [61] [62] [63]

[64] [65] [66] [67] [68]

[69]

[70]

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Rhodopsin kinase

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1 Nomenclature EC number 2.7.11.14 Systematic name ATP:rhodopsin phosphotransferase Recommended name rhodopsin kinase Synonyms G protein-coupled receptor kinase [57] G protein-coupled receptor kinase 1 [10] G protein-coupled receptor kinase GRK7 [9] G-protein coupled protein kinase 7a [51] G-protein coupled protein kinase 7b [51] G-protein coupled receptor kinase [44, 52] GPCR kinase 1 [50] GPRK1 [52] GRK1 [44, 50, 51, 52, 57, 59] GRK7 [9, 44, 52, 57] RK [45, 47, 52] SQRK [56] cone-specific kinase GRK7 [51] kinase (phosphorylating), opsin kinase (phosphorylating), rhodopsin opsin kinase rhodopsin kinase [2, 3, 4, 5, 6, 7, 8] Additional information ( the enzyme belongs to the G-protein coupled receptor kinase family [49]; the enzyme belongs to the G protein-coupled receptor kinase family [56]) [49, 56] CAS registry number 54004-64-7

2 Source Organism Drosophila melanogaster (no sequence specified) [52] Mammalia (no sequence specified) [1] Mus musculus (no sequence specified) [27, 30, 43, 45, 49, 50, 59]

370

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Homo sapiens (no sequence specified) [25, 27, 30, 31, 36, 43, 44, 53, 58] Rattus norvegicus (no sequence specified) [30, 31, 43, 47] Bos taurus (no sequence specified) [2, 3, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 29, 30, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 46, 48, 54, 55] Rana pipiens (no sequence specified) [12] Oryzias latipes (no sequence specified) [43] Musca domestica (no sequence specified) [23] Loligo pealei (no sequence specified) [28, 56] Danio rerio (no sequence specified) [51] Ovis sp. (no sequence specified) [32] Bos taurus (UNIPROT accession number: P28327) [2, 3] Homo sapiens (UNIPROT accession number: Q15835) ( S-adenosylmethionine decarboxylase proenzyme 2 [6]) [4, 5, 6, 43] Rattus norvegicus (UNIPROT accession number: Q63651) [6, 7] Mus musculus (UNIPROT accession number: Q9WVl4) [8] Homo sapiens (UNIPROT accession number: Q8WTQ7) [9] Drosophila melanogaster (UNIPROT accession number: P32865) [10] Gallus gallus (UNIPROT accession number: O73685) [34, 36, 43] Octopus dofleini (UNIPROT accession number: O97020) [38, 43] Loligo pealei (UNIPROT accession number: Q9N2R0) [43] Danio rerio (UNIPROT accession number: Q49HM9) [51] Danio rerio (UNIPROT accession number: Q49HM8) [51] Danio rerio (UNIPROT accession number: Q1XHM0) [57] Danio rerio (UNIPROT accession number: Q49HN0) [57] Danio rerio (UNIPROT accession number: Q1XHL8) [57] Danio rerio (UNIPROT accession number: Q1XHL7) [57]

3 Reaction and Specificity Catalyzed reaction ATP + rhodopsin = ADP + phosphorhodopsin ( mechanism [16, 17, 19, 21, 22]; molecular mechanism of trans-phosporylation [49]) Reaction type phospho group transfer Natural substrates and products S ATP + M opsin ( M opsin binds to cone arrestin during cone phototransduction in the retina [50]) (Reversibility: ?) [50] P ADP + phosphorylated m opsin S ATP + S opsin ( S opsin binds to cone arrestin during cone phototransduction in the retina [50]) (Reversibility: ?) [50] P ADP + phosphorylated S opsin S ATP + a protein (Reversibility: ?) [1] P ADP + a phosphoprotein

371

Rhodopsin kinase

2.7.11.14

S ATP + arrestin ( phosphorylation of arrestin in the presence of Ca2+ , occurs only after photoactivation in vivo, dual role of RK in the inactivation of the squid visual system [28]) (Reversibility: ?) [28] P ADP + phosphoarrestin S ATP + arrestin ( the enzyme interacts with the squid visual arrestin, arrestin binding to photoactivated rhodopsin is a key mechanism of desensitization, overview [56]) (Reversibility: ?) [56] P ADP + phospho-arrestin S ATP + cone opsin ( light-dependent phosporylation [51]) (Reversibility: ?) [51] P ADP + phosphorylated cone opsin S ATP + opsin ( the enzyme is involved in the opsin deactivation process, GRK subtypes play a role in phosphorylating non-visual opsins in the particular extraretinal tissues [57]) (Reversibility: ?) [57] P ADP + phosphoopsin S ATP + rhodopsin (Reversibility: ?) [56] P ADP + phospho-rhodopsin S ATP + rhodopsin ( light-dependent deactivation of rhodopsin involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase [2]; enzyme is required for normal rhodopsin deactivation. Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase [8]; null mutations in the rhodopsin kinase gene are a cause of Oguchi disease and extend the known genetic heterogeneity in congenital stationary night blindness [5]) (Reversibility: ?) [2, 4, 5, 6, 8] P ADP + phosphorylated rhodopsin S ATP + rhodopsin ( phosphorylation of enzyme may represent one of the control mechanisms for rhodopsin phosphorylation [20]; specific and Ca2+ -dependent recoverin/RK interaction may play an important role in photoreceptor light adaptation [39]; RK partially terminates the biochemical events that follow photon absorption [40]; deactivation of photoexcited rhodopsin by its phosphorylation by RK, in vivo, since ATP is present, RK exists in an autophosphorylated state [30]; natural substrate is photoactivated rhodopsin [25]; important enzyme of phototransduction [31,34,36]; major regulatory mechanism for the control of photorhodopsin transduction pathway [22]; the deactivation of photoexcited rhodopsin requires multiple phosphorylations [30]; light-dependent initiating of deactivation of rhodopsin [2,34,36]; initiation of deactivation of photoexcited visual pigments in rod and cone photoreceptors, recoverin is a Ca2+ -dependent negative regulator of RK in vertebrate phototransduction [27]; involved in a mechanism for quenching or terminating the visual signal involving the interaction of metarhodopsin II with RK and arrestin, phosphorylation of light-activated rhodopsin by RK is the key step in the signal-termination reaction [24,31]; second messenger-independent protein kinase, involved in the deac372

2.7.11.14

P

S P S

P

Rhodopsin kinase

tivation of photolyzed rhodopsin [3]; involved in quenching of the excitational pathway of phototransduction [17]; dual role of RK in the inactivation of the squid visual system [28]; enzyme in vivo is probably inactive in the dark, but is almost fully activated in the light [12]; phosphorylation of rhodopsin may control passive permeability to certain ions in rod outer segments, so mediating the responsiveness to a light impulse [11]; involved in the inactivation of light-sensitive opsins in pineal, which contains a functional photoreceptive system [6]; localization of enzyme enables it to quench immediately the activated form of the photopigment [23]; activity is increased in diabetes due to upregulation of the enzyme and downregulation of inhibitory recoverin and transducin [47]; differential spatial and temporal phosphorylation of the visual receptor rhodopsin at the primary phosphorylation sites Ser334 and Ser338 in mice exposed to light, phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin [45]; GRK1 and GRK7 [44]; the enzyme phosphorylates activated rhodopsin, light causes phosphorylation of nonactivated rhodopsin in intact rod photoreceptor cells [49]) (Reversibility: ir) [2, 3, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 54] ADP + phosphorhodopsin [2, 3, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43] ATP + rhodopsin 1 (Reversibility: ?) [52] ADP + phosphorhodopsin 1 Additional information ( kinase plays a role in human visual signaling [9]; RK phosphorylates other color opsins in vivo [2]; analysis of the deactivation of the cone phototransduction cascade in mouse retina and the role of GRK1, GRK1-dependent phosphorylation of cone opsins and binding to cone arrestin leads to association of cone arrestins to the membrane in a light-dependent manner [50]; cone-specific kinase GRK7 is essential for cone vision [51]; dark-adapted retina rhodopsin is also phosphorylated in transgenic photoreceptor cells overexpressing the human S opsin mutant K296E [49]; for cone vision GRK1 is not essential [51]; absence of PrBP/d, a ubiquitous prenyl binding protein, in Pde6d knockout mice retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy, overview [59]) (Reversibility: ?) [2, 9, 49, 50, 51, 59] ?

Substrates and products S ATP + 338-SKTETSQVAPA-348 ( peptide containing the last 11 amino acids of the C-terminal of bovine rhodopsin [29,32]; phosphorylated at Ser-343, about 11% of the rate with rhodopsin, photo-

373

Rhodopsin kinase

P S P S P S P S P S P S P S

P S

P S

P S P S P

374

2.7.11.14

activated rhodopsin-dependent, soluble active kinase catalyzes photoactivated rhodopsin-independent peptide phosphorylation [29]; only in the presence of photoactivated rhodopsin, which activates RK for peptide phosphorylation, also activated by metarhodopsin III, but not by opsin, up to 60% of the rate with photoactivated rhodopsin, light-dependent phosphorylation [32]) (Reversibility: ?) [29, 32] ? ATP + Ac-RRRAAAAASAAA-NH2 ( synthetic peptide substrate [23]) (Reversibility: ?) [23] ? ATP + DDEASTTVSKTETSQVARRR ( synthetic peptide C, very poor substrate [16]) (Reversibility: ?) [16] ? ATP + M cone opsin ( light-dependent, multi-site phosporylation [50]) (Reversibility: ?) [50] ADP + phosphorylated M cone opsin ATP + M opsin ( M opsin binds to cone arrestin during cone phototransduction in the retina [50]) (Reversibility: ?) [50] ADP + phosphorylated m opsin ATP + RRREEEEESAAA ( synthetic peptide substrate [25]) (Reversibility: ?) [25] ADP + RRREEEEE-(P)SAAA ATP + S cone opsin ( light-dependent, multi-site phosporylation [50]) (Reversibility: ?) [50] ADP + phosphorylated S cone opsin ATP + S opsin ( S opsin binds to cone arrestin during cone phototransduction in the retina [50]; trans-phosporylation is induced by activated rhodopsin [49]) (Reversibility: ?) [49, 5] ADP + phosphorylated S opsin ATP + arrestin ( phosphorylation of arrestin in the presence of Ca2+ , Ca2+ may facilitate arrestin-binding to RK [28]; phosphorylation of arrestin in the presence of Ca2+ , occurs only after photoactivation in vivo, dual role of RK in the inactivation of the squid visual system [28]) (Reversibility: ?) [28] ADP + phosphoarrestin ATP + arrestin ( the enzyme interacts with the squid visual arrestin, arrestin binding to photoactivated rhodopsin is a key mechanism of desensitization, overview [56]) (Reversibility: ?) [56] ADP + phospho-arrestin ATP + b-adrenergic receptor ( phosphorylates rhodopsin better than bAR [14]) (Reversibility: ?) [14] ADP + phospho-b-adrenergic receptor ATP + cone opsin ( light-dependent phosporylation [51]) (Reversibility: ?) [51] ADP + phosphorylated cone opsin

2.7.11.14

Rhodopsin kinase

S ATP + opsin ( the enzyme is involved in the opsin deactivation process, GRK subtypes play a role in phosphorylating non-visual opsins in the particular extraretinal tissues [57]) (Reversibility: ?) [57] P ADP + phosphoopsin S ATP + peptide ( corresponding to the C-terminus and loop 5-6 of opsin, poor substrates, phosphorylates serine and threonine residues in each peptide [22]; acid-rich peptides, RK prefers acid residues localized to the C-terminal side of the serine [24,31]; acidic peptides, stimulated by photolyzed rhodopsin, K-491 of RK participates in substrate binding [40]; containing sites phosphorylated in rhodopsin [19,31]; monophosphorylated [17]; lower amount of phosphoryl group incorporation than of rhodopsin [19]; low catalytic efficiency of RK toward a peptide containing its major autophosphorylation site [3]) (Reversibility: ?) [3, 17, 19, 22, 24, 30, 31, 40, 43] P ADP + phosphopeptide S ATP + protein ( autophosphorylation [3]) (Reversibility: ?) [3] P ADP + phosphoprotein S ATP + rhodopsin (Reversibility: ?) [56] P ADP + phospho-rhodopsin S ATP + rhodopsin ( light-dependent deactivation of rhodopsin involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase [2]; enzyme is required for normal rhodopsin deactivation. Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase [8]; null mutations in the rhodopsin kinase gene are a cause of Oguchi disease and extend the known genetic heterogeneity in congenital stationary night blindness [5]) (Reversibility: ?) [2, 4, 5, 6, 8] P ADP + phosphorylated rhodopsin S ATP + rhodopsin ( Cterminus is required for phosphorylation of photo-activated rhodopsin and may be involved in interaction with it [25]; 4 mol phosphate/ mol rhodopsin [32]; specific for photoactivated rhodopsin, time-dependent phosphorylation, light-dependent translocation, i.e. association of the kinase with photoactivated rhodopsin [25]; the autophosphorylation region of RK is involved in binding of ATP to the catalytic site and may regulate selectivity of the site of phosphorylation [40]; regulation of GRK1 [43]; domain structure, catalytic domain of 270 amino acids in the center of the sequence [2]; phosphorylates multiple serine and threonine residues in the C-terminal region of opsin peptide in the sequence 334-343, incorporation of up to 7 phosphates, rate of incorporation of the first phosphates is slower than the rate of formation of more highly phosphorylated species [17]; substrates: octopus rhodopsin, rhodopsin-containing phospholipid vesicles [38]; phosphorylates rhodopsin in the disc-membrane [12,19]; highly specific for rhodopsin [2,12,13]; specificity of ATP-binding site [15,21]; rhodopsin of rhabdomeric membranes [28]; recombinant RK 375

Rhodopsin kinase

2.7.11.14

expressed in SF9 cells catalyzes high-gain phosphorylation in which photoactivation of one rhodopsin molecule causes incorporation of up to several hundred phosphates into the total rhodopsin pool [26,39]; the interaction of RK-ATP complex with photoactivated rhodopsin leads to the formation, presumably due to the reorganization of the protein structure, of a soluble active kinase species which reverts to the inactive resting state in a time-dependent fashion, the active kinase catalyzes a photoactivated rhodopsin-independent peptide phosphorylation and dark-phosphorylation of rhodopsin, two-step model for enzyme activation and catalysis [29]; fully bleached rhodopsin [26]; catalyzes multisite phosphorylation of purified rhodopsin in phospholipid vesicles [22]; incorporation of 1.8 mol phosphate/mol of RK [29]; only phosphorylates C-terminal sites of rhodopsin, role of the cytoplasmic loops and C-terminal region of rhodopsin in binding and activating enzyme, V-VI loop is crucial for kinase binding, truncated forms of rhodopsin as substrates [16]; substrates: metarhodopsin II and III [32]; light-dependent phosphorylation [2, 12, 13, 14, 22, 23, 27, 30, 32, 38, 39]; preferred substrate: ATP [12, 15, 21, 31]; phosphorylates rhodopsin from Musca domestica, Lucilia cuprina and Drosophila melanogaster [23]; phosphorylation sites of bleached rhodopsin, hierarchical order [30]; phosphorylates bovine rhodopsin [11, 12, 13, 15, 16, 17, 18, 19, 22, 24, 29, 31, 34]; phosphorylates rhodopsin solubilized in dodecyl maltoside [19]; highly specific for photobleached rhodopsin [2, 3, 11, 17, 19, 20, 21, 22, 31, 32, 34, 36, 40, 41, 43]; preferred substrate: photobleached rhodopsin [14, 43]; preferred substrate: light-activated form of rhodopsin, i.e. metarhodopsin II [23, 24, 29, 30, 33]; RK normally exists in an inactive resting state and is only activated following interaction with photoactivated rhodopsin [29, 30, 32]; blocking of SH- and aminogroups of rhodopsin by chemical modification does not affect phosphorylation, except for succinylated rhodopsin, the binding or recognition site of enzyme contains multiple regions of rhodopsin [19]; 5-6 mol phosphate/mol rhodopsin [21]; RK binds to the cytoplasmic loops of photolyzed rhodopsin, forming a stable complex, and then phosphorylating it at the C-terminus, phosphorylation at different sites, including Ser-334, Ser-338 and Ser342, may play different roles in phototransduction [31]; incorporation of 5-7 phosphate groups/mol rhodopsin [13]; substrate of pineal RK: pineal rod opsin [6]; phosphorylates rhodopsin from cattle, rabbit, pig, alligator, best substrate: bovine rhodopsin [19]; phosphorylation sites of photolyzed rhodopsin [40,43]; rhodopsin with multiphosphorylation sites [12,24]; effect of bovine rhodopsin mutants with disulfide cross-links between different cytoplasmic regions on the possibility to serve as substrate, only substrate is a rhodopsin mutant containing a disulfide cross-link between Cys-65 and Cys-316 [33]; substrate of pineal RK: pineal blue cone opsin [6]; binds tightly to its substrate metarhodopsin and partially dissociates from rhodopsin [23]; substrates: R135K, R135Q, R135A 376

2.7.11.14

Rhodopsin kinase

and R135L mutants of bovine rhodopsin, R135A is phosphorylated even in the absence of 11-cis-retinal [42]; catalyzes the transfer of the terminal g phosphate group of ATP to the opsin protein [13]; high-gain phosphorylation of rhodopsin [3,26,29,30,32,39,43]; phosphorylates serine and threonine residues in the carboxy-terminal region of opsin peptide [2,13,14,27]; domain structure [3,31,40,43]; phosphorylation of enzyme may represent one of the control mechanisms for rhodopsin phosphorylation [20]; specific and Ca2+ -dependent recoverin/RK interaction may play an important role in photoreceptor light adaptation [39]; RK partially terminates the biochemical events that follow photon absorption [40]; deactivation of photoexcited rhodopsin by its phosphorylation by RK, in vivo, since ATP is present, RK exists in an autophosphorylated state [30]; natural substrate is photoactivated rhodopsin [25]; important enzyme of phototransduction [31,34,36]; major regulatory mechanism for the control of photorhodopsin transduction pathway [22]; the deactivation of photoexcited rhodopsin requires multiple phosphorylations [30]; light-dependent initiating of deactivation of rhodopsin [2,34,36]; initiation of deactivation of photoexcited visual pigments in rod and cone photoreceptors, recoverin is a Ca2+ -dependent negative regulator of RK in vertebrate phototransduction [27]; involved in a mechanism for quenching or terminating the visual signal involving the interaction of metarhodopsin II with RK and arrestin, phosphorylation of light-activated rhodopsin by RK is the key step in the signal-termination reaction [24,31]; second messenger-independent protein kinase, involved in the deactivation of photolyzed rhodopsin [3]; involved in quenching of the excitational pathway of phototransduction [17]; dual role of RK in the inactivation of the squid visual system [28]; enzyme in vivo is probably inactive in the dark, but is almost fully activated in the light [12]; phosphorylation of rhodopsin may control passive permeability to certain ions in rod outer segments, so mediating the responsiveness to a light impulse [11]; involved in the inactivation of light-sensitive opsins in pineal, which contains a functional photoreceptive system [6]; localization of enzyme enables it to quench immediately the activated form of the photopigment [23]; activity is increased in diabetes due to upregulation of the enzyme and downregulation of inhibitory recoverin and transducin [47]; differential spatial and temporal phosphorylation of the visual receptor rhodopsin at the primary phosphorylation sites Ser334 and Ser338 in mice exposed to light, phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin [45]; GRK1 and GRK7 [44]; the enzyme phosphorylates activated rhodopsin, light causes phosphorylation of nonactivated rhodopsin in intact rod photoreceptor cells [49]; bovine retina rhodopsin, GRK1 and GRK7 [44]; light-activated or dark-adapted rhodopsin isolated from bovine retina, phosphorylation of rhodopsin in mice exposed to light at Ser334 and Ser338 [45]; rhodopsin in rod outer segment 377

Rhodopsin kinase

P

S P S P S P S

P

378

2.7.11.14

membranes [54]) (Reversibility: ir) [2, 3, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 54] ADP + phosphorhodopsin [2, 3, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43] ATP + rhodopsin 1 (Reversibility: ?) [52] ADP + phosphorhodopsin 1 ATPgS + rhodopsin ( ATPgS is a good substrate, 2-3 mol phosphate/mol rhodopsin [21]) (Reversibility: ?) [21] ? GTP + rhodopsin ( very poor substrate [15,31]; can replace ATP to a lesser extent [12,13,21]) (Reversibility: ?) [12, 13, 15, 21, 31] GDP + phosphorhodopsin Additional information ( not: succinylated rhodopsin [19]; not: glycogen synthetase [19]; not: protamine [12,13]; not: apoprotein opsin [32,40]; not: unbleached rhodopsin [11, 40]; not: casein, phosvitin, histones [11, 12, 13]; b-adrenergic receptor kinase, EC 2.7.1.126, is also capable of rhodopsin phosphorylation in a light-dependent manner [14]; almost inactive toward histone [23]; enzyme quenches light activation of cGMP phosphodiesterase in a reconstituted system [18]; kinase plays a role in human visual signaling [9]; RK phosphorylates other color opsins in vivo [2]; substrate specificity, the enzyme depends on basic residues for substrate recognition, the residues at the substrate phosphorylation site greatly influence the enzyme activity, autoregulation by a pseudosubstrate mechanism, overview [1]; analysis of the deactivation of the cone phototransduction cascade in mouse retina and the role of GRK1, GRK1-dependent phosphorylation of cone opsins and binding to cone arrestin leads to association of cone arrestins to the membrane in a light-dependent manner [50]; cone-specific kinase GRK7 is essential for cone vision [51]; dark-adapted retina rhodopsin is also phosphorylated in transgenic photoreceptor cells overexpressing the human S opsin mutant K296E [49]; for cone vision GRK1 is not essential [51]; absence of PrBP/d, a ubiquitous prenyl binding protein, in Pde6d knockout mice retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy, overview [59]; the enzyme performs autophosphorylation at Ser491 and Thr492 of the C-terminal domain [58]; the enzyme performs autophosphorylation in a light-dependent manner [54]) (Reversibility: ?) [1, 2, 9, 11, 12, 13, 14, 18, 19, 23, 32, 40, 49, 50, 51, 54, 58, 59] ?

2.7.11.14

Rhodopsin kinase

Inhibitors 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-b-d-furanuronamide ( good inhibitor [21]) [21] 2’,3’-dideoxyadenosine [15] 2-chloroadenosine [21] 3’-deoxyadenosine [15] 5’-(N-ethylcarbamoyl)adenosine [21] 5’-AMP ( 1 mM, 50% inhibition [12,13]; competitive inhibition with respect to ATP [15]) [12, 13, 15, 21] 5’-deoxyadenosine [15] 5’-[p-(fluorosulfonyl)benzoyl]adenosine ( pseudo-first-order kinetics, MgATP and ATP protect almost completely, rhodopsin only slightly, Mg2+ not at all [19]) [19] 5,6-dichloro-1-(b-ribofuranosyl)-benzimidazole [21] 8,2’-anhydro-8-mercapto-9-(b-d-arabinofuranosyl)adenine ( weak [21]) [21] 8,3’-anhydro-8-oxy-9-(b-d-xylofuranosyl)adenine ( good inhibitor [21]) [21] 8,5’-anhydro-8-oxy-9-(b-d-ribofuranosyl)adenine [21] 8-bromoadenosine ( good inhibitor [21]) [21] ADP ( competitive inhibition with respect to ATP [15]) [15, 21] ADPbS [21] AMPS [21] ATP-analogues ( weak or no inhibition, overview [15]) [15] ATPaS ( S-isomer [21]) [21] adenine [21] adenosine ( 1 mM, 50% inhibition [12,13]; competitive inhibition with respect to ATP [15]) [12, 13, 15, 21] adenosine 5’-monosulfate [15] Ca2+ ( at concentrations equal to Mg2+ , forms an unproductive CaATP complex, Mg2+ partially reverses [19]) [19] calmodulin ( very poor inhibitor [41]) [41] d-myo-Inositol 1-phosphate ( weak, above 0.1 mM, stimulates below [15]) [15] dextran sulfate ( strong [38]) [38] dibutyryl-cAMP ( weak, not in the dark [11]) [11] digitonin ( 0.1%, 50% inhibition [12]; 0.1%, about 60% inhibition [13]) [12, 13] diphosphate [21] EDTA [19] formycin monophosphate [21] G-protein ( competes for binding to rhodopsin [23]) [23] heparin ( potent inhibitor [23,38]; modest inhibition [40]) [23, 38, 40] K+ ( weak, only at high concentrations [19]) [19] mastoparan ( potent inhibitor [38]) [38]

379

Rhodopsin kinase

2.7.11.14

Mg2+ ( at high concentrations [15,31]; above 10 mM, presumably by formation of Mg-ATP-Mg [19]; requirement at lower concentrations [15, 19, 31]) [15, 19, 31] Na+ ( 0.1 M, 90% inhibition [12,13]; weak, only at high concentrations [19]) [12, 13, 19] papaverine ( weak, not in the dark [11]) [11] peptides from cytosolic surface of rhodopsin [19] phosphodiesterase inhibitor SQ 20009 ( light-dependent [11]) [11] polyglutamic acid ( weak [38]) [38] polylysine ( potent inhibitor [38]) [38] sangivamycin ( strong, in vivo and in vitro [21]) [21] spermidine ( potent inhibitor [38]) [38] spermine ( at higher concentrations, activates at low concentrations [38]) [38] synthetic peptide ( corresponding to sequences within opsin loops 3-4 and 5-6 and the C-terminus, bleached rhodopsin as substrate [22]) [22] theophylline ( light-dependent [11]) [11] toyocamycin ( strong [21]) [21] transducin ( inhibits the enzyme, recoverin expression is reduced in diabetic rat retina [47]) [47] triphosphate [21] tubercidin 5’-phosphate ( good inhibitor [21]) [21] urea [23] Zn2+ ( 1 mM, 90% inhibition [12,13]) [12, 13] arrestin ( competes for binding to rhodopsin [23]) [23] cAMP ( weak [15]) [15] chelator [31] emulphogene ( 0.1%, 50% inhibition [12]; BC720, 0.1%, about 60% inhibition [13]) [12, 13] inositol triphosphate ( weak [15]) [15] isoquinoline derivative [21] nucleoside analogue ( overview [21]) [21] polyanion ( e.g. heparin, dextran sulfate, polyglutamic acid [38]) [31, 38] polycation ( e.g. polyamines or polylysine, potent inhibitor [38]) [38] purine nucleotide ( overview [21]) [21] pyrrolopyrimidine derivative ( preferentially in anti-configuration, strong [21]) [21] recoverin ( Ca2+ -dependent inhibition [26, 27, 30, 39, 41, 43]; ATP inhibits and ADP enhances the RK-recoverin interaction, inhibition mechanism [30]; highly specific direct Ca2+ -dependent interaction with RK, N-terminal myristoyl residue of recoverin enhances RK inhibition and introduces cooperativity to the inhibitory effect, quenches high-gain phosphorylation of rhodopsin in the presence of Ca2+ [39]; Ca2+ is required for recoverin to bind RK, 0.1 mM ADP enhances, ATP causes RK autophosphorylation and strongly weakens inhibition, effect of N-myristoylation of 380

2.7.11.14

Rhodopsin kinase

recoverin on inhibition [41]; a neuronal calcium sensor, myristoylated, inhibits the enzyme in a Ca2+ -dependent manner, two Ca2+ binding sites in the EF- hand structure, high-affinity binding site mutant E121Q and low-affinity binding site mutant E85Q are unable to inhibit the enzyme, binding kinetics [46]; inhibits the enzyme Ca2+ -dependently in detergent-resistant membranes, that are insoluble in Triton X-100, cholesterol increases the inhibition by recoverin by facilitating the binding [48]; inhibits the enzyme, recoverin expression is reduced in diabetic rat retina [47]; binds exclusively to an amphipathic peptide at the N-terminus of rhodopsin kinase, inhibiting rhodopsin phosphorylation without affecting catalytic activity of the kinase, calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response [54]; calcium-induced inhibition, structural mechanism, recoverin serves as a calcium sensor that regulates rhodopsin kinase activity, binding structures, overview, NMR structure determination and analysis of the ternary complex RK25-Ca2+ -recoverin [55]) [26, 27, 30, 39, 41, 43, 46, 47, 48, 54, 55] Additional information ( not inhibited by cAMP [11, 12, 13, 15, 20]; not inhibited by Na+ [18]; not inhibited by cGMP [11, 12, 13, 15]; not inhibited by 2-deoxyadenosine, adenosine 2,3-monophosphate, adenosine 2-deoxy-3,5-monophosphate, inosine monophosphate, guanosine, GDP, xanthosine 5-monophosphate, hypoxanthine 9-arabinofuranoside, 1N6 -ethenoadenosine monophosphate, NAD+, NADH, NADP+, NADPH [15]; inhibition studies with adenosine analogues, not inhibited by ribose 5phosphate, ethenoadenosine, 8-bromo-AMP, 8,2-anhydro-8-oxy-9-(b-d-arabinofuranosyl)adenine [21]; not inhibited by a synthetic peptide corresponding to the major or minor autophosphorylation site of RK [3]; not inhibited by spermidine [13]; not inhibited by dibutyryl-GMP [11]; not inhibited by K+ [12,13]; not: protein inhibitor of cAMP-dependent protein kinase [23]; little inhibition by S-adenosyl-l-methionine, coenzyme A, methylene adenosine 5-triphosphate, imidoadenosine 5-triphosphate, adenosine 9-arabinofuranoside 5-monophosphate, GMP, adenosine N1 -oxide [15]; autoregulation by a pseudosubstrate mechanism, overview [1]; influence of complexing lipids on the activity of the solubilized enzyme, cholesterol increases the inhibition by recoverin by facilitating the binding [48]) [1, 3, 11, 12, 13, 15, 18, 20, 21, 23, 48] Cofactors/prosthetic groups ATP [1, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 57, 58] Activating compounds d-myo-inositol 1-phosphate ( about 20% activation, between 0.0003 mM and 0.1 mM, slightly inhibitory at higher concentrations [15]) [15] G-protein ( dependent on [52]) [52] mastoparan ( stimulates, mechanism [16]; enhances peptide phosphorylation [43]) [16, 43] spermidine ( slight stimulation [12]) [12] 381

Rhodopsin kinase

2.7.11.14

Spermine ( at low concentrations: up to 20% activation, inhibits at higher concentrations [38]) [38] bg-subunit of a photoreceptor G protein ( directly activates, 2.5fold [38]) [38] light ( stimulates rhodopsin activation by phosphorylation [49]) [45, 49, 50, 51] polycation ( activates approximately 2fold [31]) [31] rhodopsin ( activates [3]; activation, allosteric, the kinase binds to cytoplasmic loops of rhodopsin [16]; RK normally exists in an inactive resting state and is only activated following interaction with photoactivated rhodopsin [29,30,32]; metarhodopsin II and III, or their phosphorylated derivatives activate [32]; photolyzed rhodopsin stimulates [39,40,43]) [3, 16, 29, 30, 32, 39, 40, 43] Additional information ( not activated by cAMP [11,20]; not activated by cGMP, dibutyryl-cGMP [11]; molecular mechanisms of GRK1 activation [43]; activation of RK by metarhodopsin II and III, their phosphorylated derivatives, all the derivatives of opsin, which contain the Schiff base linkage with the all-trans-retinylidene moiety, but not opsin [32]; various enzymatically truncated forms of photolyzed rhodopsin stimulate, light-dependent [16]; not activated by cyclic nucleotides [20]; autoregulation by a pseudosubstrate mechanism, overview [1]; GRK1 and GRK7 are activated by autophosphorylation and by phosphorylation through cAMP-dependent protein kinase, EC 2.7.11.11 [44]; influence of complexing lipids on the activity of the solubilized enzyme [48]; enzyme activity requires light-activation, Ca2+ and membranes [56]; marked expression induction by transcription factors Crx and Otx2 [53]) [1, 11, 16, 20, 32, 43, 44, 48, 53, 56] Metals, ions Ca2+ ( Ca2+ -dependent phosphorylation of arrestin, Ca2+ concentrations above 0.0025 mM free Ca2+ stimulate, maximal at 0.05 mM Ca2+ , Ca2+ may facilitate arrestin-binding to RK, phosphorylation of rhodopsin is not Ca2+ -dependent [28]; controls the binding of recoverin to the enzyme [46]; dependent on, rhodopsin phosphorylation in rod outer segment membranes [48]; affects enzyme binding to rhodopsin, induces binding of recoverin to the enzyme inhibiting its activity, NMR structure determination and analysis of the ternary complex RK25-Ca2+ -recoverin [55]; enzyme activity requires light-activation, Ca2+ and membranes [56]; induces a conformational change, while in the calciumfree form, the myristoyl group is held within a hydrophobic cleft, addition of calcium leads to its release and to membrane binding, inhibits rhodopsin binding to the enzyme via recoverin, which binds to the amphipathic peptide at the N-terminus of the enzyme and blocks rhodopsin binding [54]) [28, 46, 48, 54, 55, 56] Mg2+ ( requirement [2, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 31]; inhibition at high concentrations [15]; 1 mM required [31];

382

2.7.11.14

Rhodopsin kinase

inhibitory in excess, presumably by formation of Mg-ATP-Mg [19]; actual substrate: Mg-ATP complex, plus activation by free Mg2+ -ions [19]; the optimum activation is obtained with a 10:1 ratio of Mg2+ /ATP [19]) [1, 2, 3, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 52, 54, 56, 57, 58] Additional information ( Ca2+ -independent phosphorylation of rhodopsin [39]) [39] Turnover number (min–1) 1 (rhodopsin, recombinant wild-type GRK7, pH 7.0, 30 C [44]) [44] 1.2 (rhodopsin, recombinant wild-type GRK1, pH 7.0, 30 C [44]) [44] Additional information ( ATP: 2.2 mol of phosphate bound/min, GTP: 0.12 mol of phosphate bound/min [12]) [12] Specific activity (U/mg) 0.01 ( pH 7.4 [22]) [22] 0.013 [14] 0.017 ( pH 7.4, 37 C [11]) [11] 0.026 ( pH 7.4, 37 C [13]) [12, 13] 0.028 ( pH 7.4, 37 C [13]) [13] 0.04 ( pH 7.4, 30 C, recombinant RK expressed in COS-1 cells, purified in absence or presence of ATP [35]) [35] 0.046 ( pH 7.4, 15 C [38]) [38] 0.062 ( pH 7.5, 23 C [18]) [18] 0.097 ( pH 7.5, 30 C [37]) [37] 0.1 [31] 0.18 ( 32 C [29]) [29] 0.5 ( pH 7.5, 30 C [21]) [21, 24] 0.555 ( pH 7.5 [3]) [3] 0.96 ( pH 7.5, 25 C [15]) [15] 1.2 ( recombinant RK expressed in SF9 cells [26]) [26] Additional information ( assay at dark [52]; quantitative determination of GRK mRNA and protein in the eye, initial reaction velocities at 25 C and 30 C, overview [57]) [30, 44, 52, 57] Km-Value (mM) 0.00062 (rhodopsin, pH 7.4, fully phosphorylated enzyme [22]) [22] 0.00064 (rhodopsin, pH 7.4, minimally phosphorylated enzyme [22]) [22] 0.0016-0.002 (ATP, pH 7.5, 25 C, at 1 mM Mg2+ [15]; pH 7.5, 30 C, cosubstrate rhodopsin, membrane-bound [21]) [15, 21] 0.0019 (rhodopsin, recombinant wild-type GRK7, pH 7.0, 30 C [44]) [44] 0.002-0.004 (rhodopsin, pH 7.5, 30 C, wild-type RK and mutants [40]) [40] 0.003 (ATP, pH 7.5, 25 C [19]) [19] 0.003 (rhodopsin, pH 7.5, 25 C [19]) [19]

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0.0035 (rhodopsin, recombinant wild-type GRK1, pH 7.0, 30 C [44]) [44] 0.004-0.0044 (rhodopsin, pH 7.5, 30 C [16,25]; pH 7.5, 25 C, freshly bleached [15]) [15, 16, 25] 0.005 (ATP, pH 7.5, 30 C, S488D mutant [40]) [40] 0.0052 (ATP, pH 7.5, 25 C, at 10 mM Mg2+ [15]) [15] 0.007 (ATP, pH 7.5, 30 C, wild-type RK [40]) [40] 0.008 (ATP, pH 7.4, 37 C [12,13]) [12, 13] 0.009 (ATP, pH 7.5, 20 C [23]) [23] 0.0106 (ATP, recombinant wild-type GRK1, pH 7.0, 30 C [44]) [44] 0.012-0.015 (ATP, pH 7.5, 30 C, K491A or T489A mutant [40]) [40] 0.0214 (ATP, recombinant wild-type GRK7, pH 7.0, 30 C [44]) [44] 0.025 (ATP, pH 7.5, 30 C, S488D/T489D double mutant [40]) [40] 0.027 (ATPgS, pH 7.5, 30 C [21]) [21] 0.14 (ATP, pH 7.5, 30 C, S488A/T489A double mutant [40]) [40] 0.166 (ATP, pH 7.5, 30 C, S488A mutant [40]) [40] 0.4 (GTP, pH 7.4, 37 C [12,13]) [12, 13] 1 (GTP, pH 7.5, 25 C, at 2 mM Mg2+ [15]) [15] 2 (RRREEEEESAAA, pH 7.5, 30 C [25]) [25] 6 (rhodopsin, farnesylated GRK1 [34]) [34] 7.1 (DDEASTTVSKTETSQVARRR, pH 7.5, 30 C, peptide C [16]) [16] 9 (rhodopsin, C588S mutant GRK1 [34]) [34] 30 (rhodopsin, geranylgeranylated GRK1 [34]) [34] Additional information ( kinetics [44]; Km values for truncated forms of rhodopsin [16]; kinetic parameters for various synthetic peptide substrates [17,19]; kinetic study of multiple phosphorylation of rhodopsin [17]; kinetic study of autophosphorylation, Km values for synthetic peptide substrates: 5 mM or greater, pH 7.4 [22]) [16, 17, 19, 22, 44] Ki-Value (mM) 0.00018 (sangivamycin, pH 7.5, 30 C [21]) [21] 0.0036 (tubercidin 5’-phosphate, pH 7.5, 30 C [21]) [21] 0.004 (5,6-dichloro-1-(b-ribofuranosyl)-benzimidazole, pH 7.5, 30 C [21]) [21] 0.004 (adenosine, pH 7.5, 25 C [15]; pH 7.5, 30 C [21]) [15, 21] 0.005 (5’-AMP, pH 7.5, 25 C [15]; pH 7.5, 30 C [21]) [15, 21] 0.0087 (adenine, pH 7.5, 30 C [21]) [21] 0.01 (5’-deoxyadenosine, pH 7.5, 25 C [15]) [15] 0.01 (8,3’-anhydro-8-oxy-9-(b-d-xylofuranosyl)adenine, pH 7.5, 30 C [21]) [21] 0.012 (ADP, pH 7.5, 25 C [15]; pH 7.5, 30 C [21]) [15, 21] 0.014 (triphosphate, pH 7.5, 30 C [21]) [21] 0.016 (1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-b-d-furanuronamide, pH 7.5, 30 C [21]) [21] 0.016 (3’-deoxyadenosine, pH 7.5, 25 C [15]) [15]

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0.02 (8-bromoadenosine, pH 7.5, 30 C [21]) [21] 0.022 (ADPbS, pH 7.5, 30 C [21]) [21] 0.022 (AMPS, pH 7.5, 30 C [21]) [21] 0.024 (diphosphate, pH 7.5, 30 C [21]) [21] 0.027 (8,5’-anhydro-8-oxy-9-(b-d-ribofuranosyl)adenine, pH 7.5, 30 C [21]) [21] 0.035 (ATPaS, pH 7.5, 30 C, S-isomer [21]) [21] 0.036 (formycin monophosphate, pH 7.5, 30 C [21]) [21] 0.065 (2’,3’-dideoxyadenosine, pH 7.5, 25 C [15]) [15] 0.08 (adenosine 5’-monosulfate, pH 7.5, 25 C [15]) [15] 0.3 (heparin, pH 7.5, 30 C [40]) [40] 0.52 (8,2’-anhydro-8-mercapto-9-(b-d-arabinofuranosyl)adenine, pH 7.5, 30 C [21]) [21] 0.9 (5’-[p-(fluorosulfonyl)benzoyl]adenosine, pH 7.5, 25 C [19]) [19] Additional information ( values for synthetic peptides [24]) [21, 24] pH-Optimum 6 ( His-tagged recombinant RK expressed in COS-1 cells [35]; 2 optima: pH 6 and pH 7.5 [13]) [13, 35] 6-8 [31] 6.5 ( recombinant RK expressed in Sf21 cells [37]) [37] 6.67 ( RK from rod outer segments [35]) [35] 7 ( assay at [20,44]; native RK [37]) [19, 20, 37, 44] 7.2 ( assay at [17]) [17] 7.4 ( assay at [11, 12, 13, 22, 24, 35, 54]) [11, 12, 13, 22, 24, 35, 38, 54] 7.5 ( assay at [2, 15, 16, 18, 19, 21, 23, 25, 33, 37, 40, 46, 48, 52, 57]; assay at, photolyzed rhodopsin substrate [31]; 2 optima, pH 6 and pH 7.5 [13]) [2, 13, 15, 16, 18, 19, 21, 23, 25, 31, 33, 37, 40, 46, 48, 52, 57] 8 ( assay at [56]) [56] 8.5 ( assay at, synthetic peptide substrates [31]) [31] Additional information ( activity/pH-profile with synthetic peptide 327-347 as substrate [19]) [19] pH-Range 6.3-8.7 ( pH 6.3: about 75% of maximal activity, pH 8.7: about halfmaximal activity [19]) [19] 6.67 ( RK from rod outer segments: 60% of activity is lost with one pH unit change, recombinant RK expressed in COS-1 cells shows broader activity profile [35]) [35] Temperature optimum ( C) 15 ( assay at [38]) [38] 20 ( assay at [23,32]) [23, 32] 23 ( assay at [18]) [18]

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25 ( assay at [2, 15, 19, 46, 48, 52, 54]) [2, 15, 19, 46, 48, 52, 54] 25-30 ( assay at [57]) [57] 26 ( assay at [56]) [56] 30 ( assay at [3, 16, 17, 20, 21, 24, 25, 31, 35, 37, 40, 44, 45]; peptide assay [3]) [3, 16, 17, 20, 21, 24, 25, 31, 35, 37, 40, 44, 45] 32 ( assay at [29]) [29] 36 ( about, native RK and recombinant RK expressed in Sf21 cells [37]) [37] 37 ( assay at [11,12,13,32]) [11, 12, 13, 32]

4 Enzyme Structure Molecular weight 53000 ( gel filtration [12]) [12] 67000 ( gel filtration [15]) [15] 68000 ( sucrose density gradient centrifugation [20]) [20] Additional information ( amino acid sequence [34]; amino acid sequence shows high degree of homology to b-adrenergic receptor kinase primary structure [2,30,38]) [2, 30, 34, 38] Subunits ? ( x * 62000, SDS-PAGE [37]; x * 65000, SDS-PAGE [29]; x * 64000, SDS-PAGE [39]; x * 66000 [41]; x * 80000, SDS-PAGE, predicted from the ork gene sequence [38]; x * 66000, autophosphorylated RK, SDS-PAGE [3,39]; x * about 61000, SDS-PAGE [34]; x * 63000, Western blot analysis [25]; x * 65000, dephosphorylated RK, SDS-PAGE [3]; x * 62900, calculated from the amino acid sequence [2]; x * 63500, recombinant FLAG-tagged GRK1, SDS-PAGE, x * 62200, recombinant FLAG-tagged GRK7, SDS-PAGE [44]) [2, 3, 25, 29, 34, 37, 38, 39, 41, 44] monomer ( 1 * 62000, SDS-PAGE [22]; 1 * 68000, SDS-PAGE [20]; 1 * 65000, SDS-PAGE [18]; 1 * 50000, SDS-PAGE [12]; 1 * 70000, SDS-PAGE [15]; 1 * 62000, unphosphorylated kinase, SDS-PAGE [22]; 1 * 64000, autophosphorylated kinase, SDS-PAGE [22]) [12, 15, 18, 20, 22, 54] Additional information ( GRK1 and GRK7 peptide mapping [44]; enzyme organization with an N-terminal region containing a myristoylation site and a rhodopsin binding site, with the central catalytic domain, and a C-terminal domain harboring an autophosphorylation site and a farnesylation site, overview [58]; NMR structure determination and analysis of the ternary complex RK25-Ca2+ -recoverin [55]) [44, 55, 58] Posttranslational modification lipoprotein ( the enzyme contains a myristoylation site at the Nterminus and a C-terminal farnesylation site, overview [58]; the enzyme

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is farnesylated [59]; the enzyme is myristoylated in a Ca2+ -dependent manner [54]) [54, 58, 59] phosphoprotein ( enzyme is autophosphorylated [2,29,35,37,39,41]; recombinant RK expressed in COS-1 cells is mainly diphosphorylated [35]; incubation of purified RK with ATP leads to its autophosphorylation, resulting in the modification of 3 serine residues in positions 21, 488 and 489, the presence of phosphoryl groups may play a regulatory role [30]; autophosphorylation of RK causes a lower affinity of enzyme for photolyzed rhodopsin allowing dissociation [31]; autophosphorylation of serine residues, uneffected by the presence of bleached rhodopsin, results in a transition of the molecular mass to 64 kDa, not a major regulatory mechanism for control of kinase activity [22]; autophosphorylation sites of RK, major sites are Ser-488 and Thr-489, minor site is Ser-21 [3,43]; autophosphorylation may lower the affinity of RK for photoactivated rhodopsin via repulsion between phosphorylated sites on photoactivated rhodopsin and RK [3]; native RK: mixture of monoand diphosphorylated forms, two main fractions of purified recombinant RK expressed in Sf21 cells differ in their phosphorylation state: one is monophosphorylated, the other is diphosphorylated [37]; major autophosphorylation sites: Ser-488, Thr-489, the autophosphorylation region of RK is involved in binding of ATP to the catalytic site, it may regulate selectivity of the site of phosphorylation and may influence the rate of RK dissociation from phosphorylated photolyzed rhodopsin, mechanism of RK regulation by autophosphorylation [40]; enzyme itself is phosphorylated by ATP [13]; enzyme is autophosphorylated in the absence of rhodopsin [18,20,22]; autophosphorylation, the site often depends more on structure than on primary sequence [1]; GRK1 and GRK7 perform autophosphorylations, GRK7 at Ser490 and GRK1 at Ser488/Thr489, and are phosphorylated in vivo in forskolin-stimulated cells by cAMP-dependent protein kinase, PKA, EC 2.7.11.11, in the dark, when cAMP levels are elevated, PKA phosphorylation of GRK1 at Ser21 and of GRK7 at Ser23 and Ser36, no phosphorylation of the respective alanine mutants [44]; the enzyme performs autophosphorylation at Ser491 and Thr492 of the C-terminal domain [58]; the enzyme performs autophosphorylation in a light-dependent manner [54]) [1, 2, 3, 13, 18, 20, 22, 29, 30, 31, 35, 37, 39, 40, 41, 43, 44, 54, 58] side-chain modification ( GRK1 is geranylgeranylated ex vivo [43]; native enzyme is farnesylated in vitro and in vivo [34,43]; prenylated and carboxyl-methylated at Cys-558 [43]; incorporation of a farnesyl moiety at the C-terminal cysteine residue of the mature protein [30]; enzyme is isoprenylated, recombinant RK expressed in COS-1 cells is mainly farnesylated [35]; native RK is isoprenylated, purified recombinant RK expressed in Sf21 cells: isoprenyl groups consist of mixtures of C5 , C10 , C15 and C20 isoprenyl moieties, no specific isoprenylation [37]; GRK1 contains a C-terminal consensus sequence for geranylgeranylation [34]; purified retinal RK is farnesylated [39]) [30, 34, 35, 37, 39, 43] Additional information ( putative posttranslational modification sites: C-terminal isoprenylation, N-terminal myristoylation [3]; 387

Rhodopsin kinase

2.7.11.14

the primary structure of enzyme suggests several posttranslational modifications, e.g. myristoylation, phosphorylation, isoprenylation sites [2]; RK is present predominantly in a nonphosphorylated form [29]; not isoprenylated [38]; enzyme may be posttranslationally modified [23]) [2, 3, 23, 29, 38]

5 Isolation/Preparation/Mutation/Application Source/tissue brain ( GRK7-2, almost exclusively in the brain [57]) [57] cone [57] eye ( adult [52]; retina [2, 3, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 27, 29, 30, 31, 34, 35, 36, 37, 38, 39, 42, 43]; eyespecific, not in brain or abdomen [23]; dark-adapted [11, 12, 13, 18, 20, 22]) [2, 3, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 39, 42, 43, 45, 52, 54, 56, 57, 58, 59] head [52] larva [51] photoreceptor ( most GPRK1 in the eye [52]; S and M cone photoreceptors, expression of GRK1 [50]) [47, 48, 49, 50, 51, 52] pineal gland ( contains RK mRNA [2]; young and adult [34]; GRK1 expression [43]; expression of retinal GRK1 [34, 36]; adult, expression of RK [6]; GRK1B and GRK7-2 are the dominant subtypes [57]) [2, 6, 31, 34, 36, 43, 57] retina ( rod cell outer segment [2, 3, 11, 12, 13, 14, 15, 16, 18, 19, 20, 22, 24, 26, 29, 30, 31, 35, 37, 39, 42]; from dark-adapted eyes [11, 12, 13, 18, 20, 22]; in rod and cone photoreceptors [27, 34, 36, 43]; photoreceptors also transcribe the splice variant of GRK1 named GRK1b, which is not conespecific [36]; photoreceptor microvillar membranes [38]; retina-specific expression [38, 43]; rod-dominant retina, use of cone-enriched fovea, GRK1 occurs mainly in cone outer segments, to a lesser degree in rod outer segments, in all classes of cone cells [36]; cone-dominant retina [34, 36]; expressed exclusively in rods [43]; GRK1 [44]; of healthy and diabetic Sprague-Dawley rats, expression of the enzyme is increased in the diabetic retina [47]; in rod outer segments [59]; rods and cones [57]) [2, 3, 6, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 27, 29, 30, 31, 34, 35, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 49, 50, 53, 54, 55, 57, 58, 59] retinal cone ( outer segment [51]; outer segment GRK1-homologue GRK7a [51]; outer segment GRK1-homologue GRK7b [51]) [9, 51] retinal rod ( outer segment [46, 47]) [46, 47, 55, 57] 388

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Rhodopsin kinase

Additional information ( not in brain, optic lobe, testis, liver, muscle, salivary gland, skin [38]; isozyme expression analysis, overview [57]) [38, 57] Localization cytosol ( recombinant RK, in both plasma membranes and cytosolic fractions [25]; cytosolic protein [24]) [24, 25] membrane ( membrane-bound [12, 13, 18, 23, 34, 40, 43]; binds to photobleached rod outer segment membranes [24]; rod disc membranes [18]; also found associated with the rhabdomeric membranes [28]; enzyme is released from rhodopsin-containing eye membranes [23]; photoreceptor microvillar membranes, in both the soluble and membrane fractions [38]; located on the external surface of the disc membrane, the affinity of membrane for the enzyme increases upon photobleaching of rhodopsin [13]; phosphorylation occurs preferentially in newly formed discs [12]; rod membranes [2, 3, 12, 13, 16, 22, 29]; Ca2+ -bound enzyme [54]) [2, 3, 12, 13, 16, 18, 22, 23, 24, 28, 29, 34, 38, 40, 43, 46, 47, 49, 51, 52, 54] photoreceptor outer segment [46, 51] photoreceptor outer segment membrane [44, 48] plasma membrane ( recombinant RK, in both plasma membranes and cytosolic fractions [25]) [25] soluble ( behaves as a soluble protein [15]; in both the soluble and membrane fractions [38]; soluble active kinase [29]; in the soluble fraction of eye homogenates [28]; Ca2+ -free enzyme [54]) [15, 28, 29, 38, 54] Additional information ( GRK1-dependent phosphorylation of cone opsins and binding to cone arrestin leads to association of cone arrestins to the membrane in a light-dependent manner [50]) [50] Purification (recombinant FLAG-tagged wild-type and mutant GRK1 and GRK7 from HEK-293 cells) [44] (recombinant His-tagged GRK1 and GRK1b, expressed in Escherichia coli M15) [36] (partial) [31] [3, 13, 16, 19, 22, 29, 31] (1055fold, to near homogeneity) [15] (87-110fold) [12] (native RK, recombinant RK and mutants expressed in COS-7 cells) [40] (native and recombinant RK purified from baculovirus-infected Sf21 cells: 63fold) [37] (native and recombinant RK, expressed in SF9 cells, recovering affinity chromatography) [26, 39] (partial) [11, 14, 18, 20] (partially by isolation of the rod outer segment membrane, partial solubilization by Triton X-100 in presence of Ca2+ ) [48]

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(recombinant GST-tagged enzyme peptide fragments by glutathione affinity chromatography and gel filtration, recombinant His6-tagged truncation mutant 32-562 from Escherichia coli by nickel affinity chromatography and gel filtration, recombinant enzyme N-terminal fragment from Spodoptera frugiperda Sf9 cells by recoverin affinity chromatography and gel filtration to homogeneity) [54] (recombinant His-tagged enzyme fragment RK25 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [55] (recombinant RK expressed in SF9 cells) [41] (recombinant RK purified from baculovirus-infected Sf21 cells) [33] (recombinant RK with N-terminal hexahistidine tag, expressed in Pichia pastoris GS115, COS-1 cells: 210-222fold, and HEK-293 cell line) [35] (partial, 55fold) [23] [28] (1000fold) [2] (from whole retina) [34] (17.2fold) [38] Cloning (overexpression of myc-tagged wild-type and K220R mutant enzyme fused to heat shock inducible promotors in photoreceptor cells of flies, cells overexpressing the wild-type enzyme show increased phosphorhodopsin content after heat shock activation) [52] (GRK1 and GRK1b are cloned from fovea, sequenced and expressed in Escherichia coli M15, GRK1b is a splice variant of GRK1 with low catalytic activity transcribed in photoreceptors, GRK1b is produced by retention of the last intron 6 and differs in its C-terminal region next to the catalytic domain) [36] (RK gene encodes a 564 amino acids polypeptide) [30] (RK gene is located on chromosome 13q34) [30, 31] (cDNAs encoding full-length RK and C-terminus-truncated mutant RK lacking the last 59 amino acids are cloned and expressed in HEK-293 cells) [25] (expression of FLAG-tagged wild-type and mutant GRK1 and GRK7 in HEK-293 cells) [44] (gene GRK1, DNA and amino acid sequence determination and analysis, spatial profile and transcriptional mechanism, interactions with cone-rod homeodomain transcription factor Crx, as well as transcription factors Otx2, Nr2e3, and Nrl at the homeodomain in the 5’ flanking sequence of the promoter region, overview, phylogenetic analysis) [53] (gene GRK1, DNA and amino acid sequence determination and anaylsis of wild-type enzyme mutant genes) [58] (RK gene expression in baculovirus-infected Sf21 cells) [33, 37] (RK gene is cloned and expressed in Pichia pastoris GS115, COS-1 cells and HEK-293 stable cell line, best in COS-1 cells with correct posttranslational modifications) [35] (RK is cloned and expressed in SF9 cells) [26, 31, 39]

390

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Rhodopsin kinase

(cDNA encoding RK is characterized and sequenced, 561 amino acids protein) [30] (expression of GST-tagged enzyme peptide fragments, expression of residues 1-562 of rhodopsin kinase in Spodoptera frugiperda Sf9 cells, expression of the truncation mutant comprising residues 32-562 in Escherichia coli as C-terminally His6-tagged protein) [54] (expression of RK and mutants in COS-7 cells) [40] (expression of functional residues 1-25 of the enzyme as His-tagged peptide RK25 in Escherichia coli strain BL21(DE3)) [55] (GRK1 is cloned) [43] [2] (cDNA encoding enzyme is cloned and expressed in COS-7 cells, sequence of the 561 amino acids protein) [2] [6] (GRK1 gene with 7 exons is located on chromosome 13q34 and encodes a 561 amino acids protein) [43] (cDNA encoding RK is cloned, amino acid sequence) [6] [6] (cDNA encoding RK is cloned from pineal gland and retina, amino acid sequence) [6] (GRK7) [9] [43] (GRK1 from retina and pineal gland is cloned and sequenced) [34] (cloning of one GRK1 from cone-dominant retina) [36] (GRK1 gene encodes a 689 amino acids protein) [43] (ork gene encoding RK is cloned and sequenced) [38] (GRK1 gene encodes a 689 amino acids protein) [43] (gene grk7a, DNA and amino acid sequence determination and analysis) [51] (gene grk7b, DNA and amino acid sequence determination and analysis) [51] (isozyme GRK1 1A, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression of GRK proteins in HEK-293S cells) [57] (isozyme GRK1B, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression of GRK proteins in HEK-293S cells) [57] (isozyme GRK7 7-1, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in HEK-293S cells) [57] (isozyme GRK7-2, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in HEK-293S cells) [57] Engineering A11R ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] C588S ( unprenylated mutant [34]) [34]

391

Rhodopsin kinase

2.7.11.14

D2A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] E7A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] F15A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] F3A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] G4A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] K219R ( kinase inactive mutant of GRK1 [44]) [44] K220R ( kinase inactive mutant of GRK7 [44]; site-directed mutagenesis, ATP binding site mutation, inactive mutant [52]) [44, 52] K491A ( mutant is unable to phosphorylate acidic peptides, residue participates in substrate binding [40]) [40] L6A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] N12A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] P391H ( a naturally occuring homozygous mutation in gene GRK1 leading to the Oguchi disease, a stationary blindness with autosomal recessive transmission, with markedly reduced cone response [58]) [58] S13A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] S21A ( site-directed mutagenesis of GRK1 PKA phosphorylation site, the mutant shows 87% of wild-type enzyme activity [44]) [44] S21E ( site-directed mutagenesis of GRK1 PKA phosphorylation site, the mutant shows 80% of wild-type enzyme activity [44]) [44] S23A ( site-directed mutagenesis of GRK7 PKA phosphorylation site, the mutant shows 67% of wild-type enzyme activity [44]) [44] S23A/S36A ( site-directed mutagenesis of GRK7 PKA phosphorylation sites, the mutant shows 81% of wild-type enzyme activity [44]) [44] S23E ( site-directed mutagenesis of GRK7 PKA phosphorylation site, the mutant shows 67% of wild-type enzyme activity [44]) [44] S23E/S36E ( site-directed mutagenesis of GRK7 PKA phosphorylation sites, the mutant shows 79% of wild-type enzyme activity [44]) [44] S333A ( site-directed mutagenesis of GRK7, mutant is similar to the wild-type enzyme [44]) [44]

392

2.7.11.14

Rhodopsin kinase

S36A ( site-directed mutagenesis of GRK7 PKA phosphorylation site, the mutant shows 76% of wild-type enzyme activity [44]) [44] S36E ( site-directed mutagenesis of GRK7 PKA phosphorylation site, the mutant shows 77% of wild-type enzyme activity [44]) [44] S441A ( site-directed mutagenesis of GRK7, mutant is similar to the wild-type enzyme [44]) [44] S488A ( autophosphorylation site mutant with increased activity for the phosphorylation of rhodopsin in the dark [30,40]; S488A/T489A double mutant with almost eliminated autophosphorylation and increased ability to phosphorylate rhodopsin in the dark [40]; autophosphorylation site mutant with 50% reduced autophosphorylation [40]) [30, 40] S488D ( autophosphorylation site mutant with 50% reduced autophosphorylation and increased ability to phosphorylate rhodopsin in the dark [40]; S488D/T489D double mutant with almost eliminated autophosphorylation [40]) [40] S489A ( autophosphorylation site mutant with increased activity for the phosphorylation of rhodopsin in the dark [30]) [30] S490A ( site-directed mutagenesis of GRK7, the mutant is unable to autophosphorylate [44]) [44] S490E ( site-directed mutagenesis of GRK7, the mutant is unable to autophosphorylate [44]) [44] S5A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] T353A ( site-directed mutagenesis of GRK7, mutant is similar to the wild-type enzyme [44]) [44] T489A ( S488A/T489A double mutant with almost eliminated autophosphorylation and increased ability to phosphorylate rhodopsin in the dark [40]; autophosphorylation site mutant with 50% reduced autophosphorylation [40]) [40] T489D ( autophosphorylation site mutant with 50% reduced autophosphorylation [40]; S488D/T489D double mutant with almost eliminated autophosphorylation [40]) [40] T8A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] V10A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] V9A ( site-directed mutagenesis, the mutation of the residues leads to alterd binding of recoverin to the N-terminal fragment compared to the wild-type enzyme, kinetics constant, overview [54]) [54] Additional information ( 50 kDa C-terminus-truncated mutant RK lacking the last 59 amino acids shows abolished light-dependent translocation and is unable to phosphorylate photoactivated rhodopsin, but phosphorylates the small peptide substrate RRREEEEESAAA like wild-type RK [25]; mutations at the autophosphorylation region affect the Km for 393

Rhodopsin kinase

2.7.11.14

ATP and change the initial site of phosphorylation on photolyzed rhodopsin, influence of mutations on the affinity for heparin-Sepharose [40]; RK knockout mice [27,43]; GRK1 mutations causing the Oguchi disease [43]; construction of grk7a-knockout fish mutants, mutant larvae are delayed in electroretinographic measurements and temporal contrast sensitivity is reduced compared to the wild-type, overview [51]; construction of rhodopsin-kinase-knockout mice [45]; construction of transgenic mouse photoreceptor cells overexpressing the human S opsin mutant K296E still show the trans-phosphorylation phenomenon and phosphorylation of nonactivated rhodopsin, overview [49]; a kinase mutant lacking the Nterminal recoverin binding site is unable to phosphorylate light-activated rhodopsin [54]) [25, 27, 40, 43, 45, 49, 51, 54] Application medicine ( mutations occuring in the variable region of RK C-terminus and catalytic domain cause Oguchi disease, an inherited form of stationary night blindness [25]; Oguchi disease patients suffering from congenital stationary night blindness have defective RK or arrestin genes [27]; mutation in RK is associated with the Oguchi disease [31,36,43]) [25, 27, 31, 36, 43]

6 Stability Organic solvent stability ethanol ( marked sensitivity to organic solvents, e.g. 5% ethanol reduces the activity by 45% within 2-3 min [15]) [15] ethylene glycol ( used as stabilizer [38]) [38] urea ( 5 M, almost complete denaturation of enzyme [12,13]) [12, 13] General stability information , dilution inactivates [15] , freezing at liquid N2 temperatures or -20 C, in water or inositol leads to 80% loss of activity, in sucrose to 20% loss of activity [15] , glycerol does not protect against inactivation during purification [35] , instability of enzyme, protease inhibitors stabilize during purification [18] , monovalent cations, e.g. K+ or NH+4 , and 15% glycerol stabilize to some extent [12, 13] , stable in 6 mM dodecyl maltoside for at least 15 min [19] , ethylene glycol is used as stabilizer [38] Storage stability , -20 C, 20% adonitol, several months, stable [15] , -20 C, kinase solution, solid sucrose, initially loses 30% of activity, stable to further storage [15] , -70 C, several months, stable [26]

394

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Rhodopsin kinase

, 0 C, 20 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane, 2 mM MgCl2 , 0.1 mM EDTA, 1 mM dithiothreitol, pH 7.4, 280 mM NaCl, 0.004% Tween 80, 1 mM benzamidine, 0.1 mM phenylmethanesulfonylfluoride, 5 days, 10% loss of activity [29] , 3 C, 0.1 M KCl, 1 week, 50% loss of activity [12, 13] , 4 C, crude extract, t1=2 : 30 days, highly purified enzyme, t1=2 : 3-5 h [15] , 4 C, highly unstable, partially purified preparation, within 4-5 days, 90% loss of activity, mixture of protease inhibitors stabilizes for several weeks [18] , on ice, RK mutants, 2 days, stable [40] , -70 C, at least 3 months, stable [23]

References [1] Kemp, B.E.; Parker, M.W.; Hu, S.; Tiganis, T.; House, C.: Substrate and pseudosubstrate interactions with protein kinases: determinants of specificity. Trends Biochem. Sci., 19, 440-444 (1994) [2] Lorenz, W.; Inglese, J.; Palczewski, K.; Onorato, J.J.; Caron, M.G.; Lefkowitz, R.J.: The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the b-adrenergic receptor kinase. Proc. Natl. Acad. Sci. USA, 88, 8715-8719 (1991) [3] Palczewski, K.; Buczylko, J.; Van Hooser, P.; Carr, S.A.; Huddleston, M.J.; Crabb, J.W.: Identification of the autophosphorylation sites in rhodopsin kinase. J. Biol. Chem., 267, 18991-18998 (1992) [4] Khani, S.C.; Abitbol, M.; Yamamoto, S.; Maravic-Magovcevic, I.; Dryja, T.P.: Characterization and chromosomal localization of the gene for human rhodopsin kinase. Genomics, 35, 571-576 (1996) [5] Yamamoto, S.; Sippel, K.C.; Berson, E.L.; Dryja, T.P.: Defects in the rhodopsin kinase gene in the Oguchi form of stationary night blindness. Nat. Genet., 15, 175-178 (1997) [6] Zhao, X.; Haeseleer, F.; Fariss, R.N.; Huang, J.; Baehr, W.; Milam, A.H.; Palczewski, K.: Molecular cloning and localization of rhodopsin kinase in the mammalian pineal. Vis. Neurosci., 14, 225-232 (1997) [7] Tokumitsu, H.; Enslen, H.; Soderling, T.R.: Characterization of a Ca2+ /calmodulin-dependent protein kinase cascade. Molecular cloning and expression of calcium/calmodulin-dependent protein kinase kinase. J. Biol. Chem., 270, 19320-19324 (1995) [8] Chen, C.K.; Burns, M.E.; Spencer, M.; Niemi, G.A.; Chen, J.; Hurley, J.B.; Baylor, D.A.; Simon, M.I.: Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase. Proc. Natl. Acad. Sci. USA, 96, 3718-3722 (1999) [9] Weiss, E.R.; Ducceschi, M.H.; Horner, T.J.; Li, A.; Craft, C.M.; Osawa, S.: Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction. J. Neurosci., 21, 9175-9184 (2001)

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[10] Cassill, J.A.; Whitney, M.; Joazeiro, C.A.; Becker, A.; Zuker, C.S.: Isolation of Drosophila genes encoding G protein-coupled receptor kinases. Proc. Natl. Acad. Sci. USA, 88, 11067-11070 (1991) [11] Weller, M.; Virmaux, N.; Mandel, P.: Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin. Proc. Natl. Acad. Sci. USA, 72, 381-385 (1975) [12] Shichi, H.; Somers, R.L.: Light-dependent phosphorylation of rhodopsin. Purification and properties of rhodopsin kinase. J. Biol. Chem., 253, 70407046 (1978) [13] Shichi, H.; Somers, R.L.; Yamamoto, K.: Rhodopsin kinase. Methods Enzymol., 99, 362-366 (1983) [14] Benovic, J.L.; Mayor, F.; Somers, R.L.; Caron, M.G.; Lefkowitz, R.J.: Lightdependent phosphorylation of rhodopsin by b-adrenergic receptor kinase. Nature, 321, 869-872 (1986) [15] Palczewski, K.; McDowell, J.H.; Hargrave, P.A.: Purification and characterization of rhodopsin kinase. J. Biol. Chem., 263, 14067-14073 (1988) [16] Palczewski, K.; Buzylko, J.; Kaplan, M.W.; Polans, A.S.; Crabb, J.W.: Mechanism of rhodopsin kinase activation. J. Biol. Chem., 266, 12949-12955 (1991) [17] Adamus, G.; Arendt, A.; Hargrave, P.A.; Heyduk, T.; Palczewski, K.: The kinetics of multiphosphorylation of rhodopsin. Arch. Biochem. Biophys., 304, 443-447 (1993) [18] Sitaramayya, A.: Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system. Biochemistry, 25, 5460-5468 (1986) [19] Palczewski, K.; McDowell, J.H.; Hargrave, P.A.: Rhodopsin kinase: substrate specificity and factors that influence activity. Biochemistry, 27, 2306-2313 (1988) [20] Lee, R.H.; Brown, B.M.; Lolley, R.N.: Autophosphorylation of rhodopsin kinase from retinal rod outer segments. Biochemistry, 21, 3303-3307 (1982) [21] Palczewski, K.; Kahn, N.; Hargrave, P.A.: Nucleoside inhibitors of rhodopsin kinase. Biochemistry, 29, 6276-6282 (1990) [22] Kelleher, D.J.; Johnson, G.L.: Characterization of rhodopsin kinase purified from bovine rod outer segments. J. Biol. Chem., 265, 2632-2639 (1990) [23] Doza, Y.N.; Minke, B.; Chorev, M.; Selinger, Z.: Characterization of fly rhodopsin kinase. Eur. J. Biochem., 209, 1035-1040 (1992) [24] Onorato, J.J.; Palczewski, K.; Regan, J.W.; Caron, M.G.; Lefkowitz, R.J.; Benovic, J.L.: Role of acidic amino acids in peptide substrates of the b-adrenergic receptor kinase and rhodopsin kinase. Biochemistry, 30, 5118-5125 (1991) [25] Yu, Q.M.; Cheng, Z.J.; Zhao, J.; Zhou, T.H.; Wu, Y.L.; Ma, L.; Pei, G.: Carboxyl terminal of rhodopsin kinase is required for the phosphorylation of photo-activated rhodopsin. Cell Res., 8, 303-310 (1998) [26] Chen, C.-K.; Hurley, J.B.: Purification of rhodopsin kinase by recoverin affinity chromatography. Methods Enzymol., 315, 404-410 (2000)

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[27] Chen, C.K.: Recoverin and rhodopsin kinase. Adv. Exp. Med. Biol., 514, 101-107 (2002) [28] Mayeenuddin, L.H.; Mitchell, J.: Squid visual arrestin: cDNA cloning and calcium-dependent phosphorylation by rhodopsin kinase (SQRK). J. Neurochem., 85, 592-600 (2003) [29] Dean, K.R.; Akhtar, M.: Novel mechanism for the activation of rhodopsin kinase: Implications for other G protein-coupled receptor kinases (GRK’s). Biochemistry, 35, 6164-6172 (1996) [30] Senin, I.I.; Koch, K.-W.; Akhtar, M.; Philippov, P.P.: Ca2+ -dependent control of rhodopsin phosphorylation: Recoverin and rhodopsin kinase. Adv. Exp. Med. Biol., 514, 69-99 (2002) [31] Sokal, I.; Pulvermller, A.; Buczylko, J.; Hofmann, K.-P.; Palczewski, K.: Rhodopsin and its kinase. Methods Enzymol., 343, 578-600 (2001) [32] McCarthy, N.E.M.; Akhtar, M.: Activation of rhodopsin kinase. Biochem. J., 363, 359-364 (2002) [33] Cai, K.; Klein-Seetharaman, J.; Hwa, J.; Hubbell, W.L.; Khorana, H.G.: Structure and function in rhodopsin: Effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase. Biochemistry, 38, 12893-12898 (1999) [34] Zhao, X.; Yokoyama, K.; Whitten, M.E.; Huang, J.; Gelb, M.H.; Palczewski, K.: A novel form of rhodopsin kinase from chicken retina and pineal gland. FEBS Lett., 454, 115-121 (1999) [35] Bruel, C.; Cha, K.; Reeves, P.J.; Getmanova, E.; Khorana, H.G.: Rhodopsin kinase: Expression in mammalian cells and a two-step purification. Proc. Natl. Acad. Sci. USA, 97, 3004-3009 (2000) [36] Zhao, X.; Huang, J.; Khani, S.C.; Palczewski, K.: Molecular forms of human rhodopsin kinase (GRK1). J. Biol. Chem., 273, 5124-5131 (1998) [37] Cha, K.; Bruel, C.; Inglese, J.; Khorana, H.G.: Rhodopsin kinase: Expression in baculovirus-infected insect cells, and characterization of post-translational modifications. Proc. Natl. Acad. Sci. USA, 94, 10577-10582 (1997) [38] Kikkawa, S.; Yoshida, N.; Nakagawa, M.; Iwasa, T.; Tsuda, M.: A novel rhodopsin kinase in Octopus photoreceptor possesses a pleckstrin homology domain and is activated by G protein bg-subunits. J. Biol. Chem., 273, 74417447 (1998) [39] Chen, C.-K.; Inglese, J.; Lefkowitz, R.J.; Hurley, J.B.: Ca2+ -dependent interaction of recoverin with rhodopsin kinase. J. Biol. Chem., 270, 18060-18066 (1995) [40] Palczewski, K.; Ohguro, H.; Premont, R.T.; Inglese, J.: Rhodopsin kinase autophosphorylation. Characterization of site-specific mutations. J. Biol. Chem., 270, 15294-15298 (1995) [41] Satpaev, D.K.; Chen, C.-K.; Scotti, A.; Simon, M.I.; Hurley, J.B.; Slepak, V.Z.: Autophosphorylation and ADP regulate the Ca2+ -dependent interaction of recoverin with rhodopsin kinase. Biochemistry, 37, 10256-10262 (1998) [42] Shi, W.; Sports, C.D.; Raman, D.; Shirakawa, S.; Osawa, S.; Weiss, E.R.: Rhodopsin arginine-135 mutants are phosphorylated by rhodopsin kinase and bind arrestin in the absence of 11-cis-retinal. Biochemistry, 37, 4869-4874 (1998) 397

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[43] Maeda, T.; Imanishi, Y.; Palczewski, K.: Rhodopsin phosphorylation: 30 years later. Prog. Retin. Eye Res., 22, 417-434 (2003) [44] Horner, T.J.; Osawa, S.; Schaller, M.D.; Weiss, E.R.: Phosphorylation of GRK1 and GRK7 by cAMP-dependent protein kinase attenuates their enzymatic activities. J. Biol. Chem., 280, 28241-28250 (2005) [45] Adams, R.A.; Liu, X.; Williams, D.S.; Newton, A.C.: Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light. Biochem. J., 374, 537-543 (2003) [46] Komolov, K.E.; Zinchenko, D.V.; Churumova, V.A.; Vaganova, S.A.; Weiergraeber, O.H.; Senin, I.I.; Philippov, P.P.; Koch, K.-W.: One of the Ca2+ binding sites of recoverin exclusively controls interaction with rhodopsin kinase. Biol. Chem., 386, 285-289 (2005) [47] Kim, Y.H.; Kim, Y.S.; Noh, H.S.; Kang, S.S.; Cheon, E.W.; Park, S.K.; Lee, B.J.; Choi, W.S.; Cho, G.J.: Changes in rhodopsin kinase and transducin in the rat retina in early-stage diabetes. Exp. Eye Res., 80, 753-760 (2005) [48] Senin, I.I.; Hoeppner-Heitmann, D.; Polkovnikova, O.O.; Churumova, V.A.; Tikhomirova, N.K.; Philippov, P.P.; Koch, K.-W.: Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments. J. Biol. Chem., 279, 48647-48653 (2004) [49] Shi, G.W.; Chen, J.; Concepcion, F.; Motamedchaboki, K.; Marjoram, P.; Langen, R.: Light causes phosphorylation of nonactivated visual pigments in intact mouse rod photoreceptor cells. J. Biol. Chem., 280, 41184-41191 (2005) [50] Zhu, X.; Brown, B.; Li, A.; Mears, A.J.; Swaroop, A.; Craft, C.M.: GRK1-dependent phosphorylation of S and M opsins and their binding to cone arrestin during cone phototransduction in the mouse retina. J. Neurosci., 23, 6152-6160 (2003) [51] Rinner, O.; Makhankov, Y.V.; Biehlmaier, O.; Neuhauss, S.C.F.: Knockdown of cone-specific kinase GRK7 in larval zebrafish leads to impaired cone response recovery and delayed dark adaptation. Neuron, 47, 231-242 (2005) [52] Lee, S.-J.; Xu, H.; Montell, C.: Rhodopsin kinase activity modulates the amplitude of the visual response in Drosophila. Proc. Natl. Acad. Sci. USA, 101, 11874-11879 (2004) [53] Young, J.E.; Kasperek, E.M.; Vogt, T.M.; Lis, A.; Khani, S.C.: Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene. Genomics, 90, 236-248 (2007) [54] Higgins, M.K.; Oprian, D.D.; Schertler, G.F.: Recoverin binds exclusively to an amphipathic peptide at the N terminus of rhodopsin kinase, inhibiting rhodopsin phosphorylation without affecting catalytic activity of the kinase. J. Biol. Chem., 281, 19426-19432 (2006) [55] Ames, J.B.; Levay, K.; Wingard, J.N.; Lusin, J.D.; Slepak, V.Z.: Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin. J. Biol. Chem., 281, 37237-37245 (2006) [56] Swardfager, W.; Mitchell, J.: Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase. J. Neurochem., 101, 223-231 (2007) 398

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[57] Wada, Y.; Sugiyama, J.; Okano, T.; Fukada, Y.: GRK1 and GRK7: unique cellular distribution and widely different activities of opsin phosphorylation in the zebrafish rods and cones. J. Neurochem., 98, 824-837 (2006) [58] Hayashi, T.; Gekka, T.; Takeuchi, T.; Goto-Omoto, S.; Kitahara, K.: A novel homozygous GRK1 mutation (P391H) in 2 siblings with Oguchi disease with markedly reduced cone responses. Ophthalmology, 114, 134-141 (2007) [59] Zhang, H.; Li, S.; Doan, T.; Rieke, F.; Detwiler, P.B.; Frederick, J.M.; Baehr, W.: Deletion of PrBP/d impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments. Proc. Natl. Acad. Sci. USA, 104, 8857-8862 (2007)

399

b-Adrenergic-receptor kinase

2.7.11.15

1 Nomenclature EC number 2.7.11.15 Systematic name ATP:[b-adrenergic receptor] phosphotransferas Recommended name b-adrenergic-receptor kinase Synonyms G protein-coupled receptor kinase [45, 56] G protein-coupled receptor kinase 2 [14, 15, 40, 42, 43, 44, 46, 50, 57, 58, 59, 60, 62, 65, 66, 67, 68, 69, 70, 71, 73, 76, 77, 82, 87] G protein-coupled receptor kinase 3 [55, 59, 68, 69, 73] G protein-coupled receptor kinase-2 [41, 61] G protein-coupled receptor regulatory kinase [85] G-protein coupled receptor kinase 2 [52, 80] G-protein receptor kinase 2 [63] G-protein-coupled receptor kinase 2 [51, 72, 74, 79, 86] G-protein-coupled receptor kinase-2 [53] GPCR kinase [65] GPCR kinase 2 [77, 79] GRK [56] GRK2 [40, 41, 42, 43, 44, 45, 46, 50, 51, 52, 53, 57, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 79, 80, 81, 82, 83, 84, 86, 87] GRK3 [45, 52, 55, 59, 68, 69, 73, 83, 87] Gprk2 [15, 85] ark [5] b-AR kinase b-ARK b-ARK 1 b-ARK 2 b-adrenergic receptor kinase [3, 24, 47, 86] b-adrenergic receptor kinase 1 [4, 5, 6, 7, 8, 9, 13] b-adrenergic receptor kinase 2 [8, 10, 11, 12] b-adrenergic receptor-specific kinase b-receptor kinase bARK [54, 64] bARK1 [24, 45, 47, 49]

400

2.7.11.15

b-Adrenergic-receptor kinase

bARK2 [24, 45] bb-adrenergic receptor kinase 1 [49] guanine nucleotide-binding protein-coupled receptor kinase [70] kinase (phosphorylating), b-adrenergic-receptor Additional information ( enzymes belong to the GRK family, GRK2 and GRK3, i.e. bARK1 and bARK2, form the bARK subfamily [45]; the enzyme belongs to the bARK subfamily of the GRK family [65]; the enzymes belong to the family of G protein-coupled receptor kinases [24]; the enzyme belongs to the b-adrenergic receptor kinase subfamily [73]; the enzyme belongs to the G-protein-coupled receptor kinase family [86]; the enzyme belongs to the GRK2 subfamily, consisting of GRK2, b-ARK1, GRK3, and b-ARK2 [87]; the nezyme belongs to the serine/threonine kinase family [80]) [24, 45, 65, 73, 80, 86, 87] CAS registry number 102925-39-3

2 Source Organism Drosophila melanogaster (no sequence specified) [42, 85] Mammalia (no sequence specified) [1] Mus musculus (no sequence specified) [18, 28, 38, 45, 49, 50, 66, 68, 72, 74, 75, 77, 79, 84] Homo sapiens (no sequence specified) [5, 23, 28, 30, 36, 45, 46, 47, 51, 52, 54, 56, 57, 65, 67, 69, 73, 74, 78, 80, 81, 86, 87] Rattus norvegicus (no sequence specified) [13, 29, 35, 45, 46, 50, 53, 55, 60, 63, 76, 83, 84] Sus scrofa (no sequence specified) [49, 64] Bos taurus (no sequence specified) [2, 3, 5, 12, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 37, 39, 40, 41, 43, 44, 48, 58, 59, 61, 62, 66, 70, 82] Mesocricetus auratus (no sequence specified) [13, 18, 29] Bos taurus (UNIPROT accession number: P21146) [3] Homo sapiens (UNIPROT accession number: P25098) [4, 5, 6, 7] Rattus norvegicus (UNIPROT accession number: P26817) [8, 9] Bos taurus (UNIPROT accession number: P26818) [10] Rattus norvegicus (UNIPROT accession number: P26819) [8] Homo sapiens (UNIPROT accession number: P35626) [11, 12] Mesocricetus auratus (UNIPROT accession number: Q64682) [13] Drosophila melanogaster (UNIPROT accession number: P32866) [14, 15] Didelphis marsupialis virginiana (UNIPROT accession number: O97627) [71]

401

b-Adrenergic-receptor kinase

2.7.11.15

3 Reaction and Specificity Catalyzed reaction ATP + [b-adrenergic receptor] = ADP + phospho-[b-adrenergic receptor] ( mechanism [17]; sequential mechanism [24]; regulation mechanism [45]; substrate and activator binding site structure, active site structure, structure-function relationship, overview [70]) Reaction type phospho group transfer Natural substrates and products S ATP + DREAM ( i.e. downstream regulatory element antagonist modulator protein, GRK2 mediates phosphorylation of DREAM/potassium channel interacting protein KChIP3, a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+ -binding proteins with specific roles in different cell compartments, regulating membrane trafficking of Kv4.2 potassium channel, phosphorylation of Ser95 affects cell surface localization, but not Kv4 channel tetramerization, overview [78]) (Reversibility: ?) [78] P ADP + phosphorylated DREAM S ATP + G protein-coupled receptor ( desensitization by GRK2 of the ligand-activated receptor [66]; desensitization of agonist-activated receptor [58]; GRK2 performs desensitization of the ligand-activated receptor by phosphorylation [43]; phosphorylation has a regulatory role, regulation of the signal transduction involving GRK2 and b-arrestin, overview [68]; regulation mechanism of GRK2, overview, regulation by phosphorylation at specific sites via distinct specific kinases, overview [45]) (Reversibility: ?) [43, 45, 55, 58, 66, 67, 68] P ADP + phosphorylated G protein-coupled receptor S ATP + M1 muscarinic acetylcholine receptor ( phosphorylationdependent and -independent mechanisms in the regulation of M1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 in hippocampal neurons, GRK2 can inhibit the receptor-dependent signaling via phospholipase C, overview [60]) (Reversibility: ?) [60] P ADP + phosphorylated M1 muscarinic acetylcholine receptor S ATP + Nedd4 ( GRK2 interacts with and phosphorylates the ubiquitin protein ligase Nedd4 preventing the binding to proline-rich motifs present in the C-termini of epithelial Na+ channel subunits and inhibition of the channels, overview [72]) (Reversibility: ?) [72] P ADP + phosphorylated Nedd4 S ATP + Nedd4-2 ( GRK2 interacts with and phosphorylates the ubiquitin protein ligase Nedd4-2 preventing the binding to proline-rich motifs present in the C-termini of epithelial Na+ channel subunits and inhibition of the channels, overview [72]) (Reversibility: ?) [72] P ADP + phosphorylated Nedd4-2

402

2.7.11.15

b-Adrenergic-receptor kinase

S ATP + PDEg ( phosphorylation of PDEg possibly stimulates EGFR-mediated ERK activation [86]) (Reversibility: ?) [86] P ADP + phosphorylated PDEg S ATP + PDGFRb (Reversibility: ?) [86] P ADP + phosphorylated PDGFRb S ATP + Smo ( GPRK2 participates in Hedgehog signaling in Drosophila melanogaster, and plays a key role in the Smo signal transduction pathway, when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced, overview [85]) (Reversibility: ?) [85] P ADP + phosphorylated Smo S ATP + Smoothened protein ( GRK2 promotes smoothened signal transduction involved in regulation of cellular proliferation and differentiation through activation of the transcription factor Gli, overview [82]) (Reversibility: ?) [82] P ADP + phosphorylated Smoothened S ATP + [TSH receptor] ( GRK2 and GRK3, receptor activation [52]) (Reversibility: ?) [52] P ADP + [TSH receptor]phosphate S ATP + a protein (Reversibility: ?) [1] P ADP + a phosphoprotein S ATP + a-synuclein ( colocalization of GRK2, GRK5, a-synuclein, and tau in neurodegenerative disorders characterized by fibrillary tau inclusions and/or a-synuclein-enriched Lewy bodies, overview [80]) (Reversibility: ?) [80] P ADP + phosphorylated a-synuclein S ATP + a1 b-adrenergic receptor ( substrate specificities of GRK2 and GRK3 in cardiac myocytes, overview [83]) (Reversibility: ?) [83] P ADP + phosphorylated a1 b-adrenergic receptor S ATP + a2 A-adrenergic receptor (Reversibility: ?) [58] P ADP + phosphorylated a2 A-adrenergic receptor S ATP + angiotensin receptor ( phosphorylation by GRK2 preceeds the binding of arrestins, which inhibits the seven-transmembrane receptor, but initiates internalization, overview [86]) (Reversibility: ?) [86] P ADP + phosphorylated angiotensin receptor S ATP + b-adrenergic receptor ( general role in the desensitization of synaptic receptors [8]; specifically phosphorylates and inactivates b-AR after stimulation by receptor agonists, facilitating the binding of the inhibitor protein b-arrestin to the receptor, during myocardial ischemia the membrane activity of b-ARK is increased [35]; b-ARK 1 might be involved in uncoupling and down-regulation of b-AR, presumably both b1 - and b2 -AR, in failing hearts via receptor phosphorylation [13]; presumably modulates some receptormediated immune functions [5]; role of b-ARK 1 in heart failure, myocardial development and function [28]; natural substrate: b2 adrenergic receptor [23, 25]; b-ARK 1 and 2 may have a similar substrate specificity in vivo [24]; regulation of the b-AR function in vivo 403

b-Adrenergic-receptor kinase

P S

P S

P S

404

2.7.11.15

[21]; desensitization of b-adrenergic receptor [19,37]; plays, together with cAMP-dependent protein kinase, an important role in agonist-promoted receptor desensitization, coordinated regulatory mechanism involving sequential depalmitoylation and phosphorylation of the b2 -AR by the two kinases [31]; agonist-occupied form of the receptor [2, 3, 5, 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 37, 39]; involved in homologous desensitization of b-adrenergic receptor [2, 3, 5, 8, 17, 18, 20, 21, 22, 23, 25, 28, 29, 39]; functional role of the b-ARK/b-arrestin mechanism of receptor desensitization in immune cells [12]) (Reversibility: ?) [2, 3, 5, 8, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 31, 35, 37, 39] ADP + phospho-b-adrenergic receptor [2, 3, 5, 8, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 31, 35, 37, 39] ATP + b-adrenergic receptor ( agonist-activated receptor substrate [24]; bARK is involved in myocardial b-adrenergic receptor signaling, enzyme dysfunction can cause heart failure, regulation mechanism and physiology, overview [64]; bARK1 is responsible for desensitization and down regulation of b-adrenergic receptors [49]; desensitization of the receptor by GRK2 and GRK3 [68]; G proteincoupled receptor kinase phosphorylation mediates b-1 adrenergic receptor endocytosis via clathrin-coated pits [56]; G-protein-coupled receptor kinase-2 and b-arrestin-2 are involved in exercise-induced b-adrenergic receptor trafficking from cytosol to membranes in adipocytes, role in b-adrenergic receptor-ubiquitination in the ubiquitin-proteasome pathway [53]; GRK3 and GRK2 are involved in down-regulation of the a2 B-adrenoceptor, regulation of the pathway, overview [81]; in human heart failure, impaired b-adrenergic receptor signaling compromises cardiac sensitivity to inotropic stimulation, overview [87]; phosphorylation and internalization of the receptor requires clathrin, GRK2 specifically phosphorylates the activated form of the receptor that promotes the translocation of b-arrestins to the plasma membrane, overview [76]; phosphorylation by GRK2 preceeds the binding of arrestins, which inhibits the seven-transmembrane receptor, but initiates internalization, overview [86]; the enzyme is involved in regulation of the badrenergic receptor signaling by inhibiting arrestin recruitment to the receptor and subsequent desensitization and internalization, regulation of GRK2 by S-nitrosylation, molecular mechanism, overview [74]) (Reversibility: ?) [24, 49, 53, 56, 64, 68, 74, 76, 81, 84, 86, 87] ADP + phosphorylated b-adrenergic receptor ATP + b2 -adrenergic receptor ( desensitization of the receptor with subsequent decline in the stimulatory effects of b2 -adrenergic agonists over time, the receptor is involved in alveolar Na+ and water clearance [63]) (Reversibility: ?) [63] ADP + phosphorylated b2 -adrenergic receptor ATP + corticotropin-releasing factor receptor type 1 ( i.e. CRFR1, phosphorylation leads to desensitization and downregulation of the receptor [50]) (Reversibility: ?) [50]

2.7.11.15

b-Adrenergic-receptor kinase

P ADP + phosphorylated corticotropin-releasing factor receptor type 1 S ATP + dopamine D1 receptor ( phosphorylation by GRK2 has a regulatory role as part of the PI3K-PKC-GRK2 cascade [71]) (Reversibility: ?) [71] P ADP + phosphorylated dopamine D1 receptor S ATP + dopamine D3 receptor ( the receptor is activated by GRK2 and GRK3 phosphorylation involving b-arrestins, GRK-mediated regulation of receptor-filamin complex stability and receptor-G protein signaling potential, GRK2 reduces the dopamine D3 receptor signaling, overview [59]) (Reversibility: ?) [59] P ADP + phosphorylated dopamine D3 receptor S ATP + epithelial Na+ channel ( channel inactivation [86]) (Reversibility: ?) [86] P ADP + phosphorylated epithelial Na+ channel S ATP + ezrin ( phosphorylation of ezrin affects the 7TM receptor mediated cytoskeletal reorganization [86]) (Reversibility: ?) [86] P ADP + phosphorylated ezrin S ATP + insulin receptor substrate 1 ( role of GRK2 in insulin receptor IR signaling [79]) (Reversibility: ?) [79] P ADP + phosphorylated insulin receptor substrate 1 S ATP + p38 MAP kinase ( GRK2 inactivates and regulates MAP kinase p38 modulating p38-dependent physiological processes, p38 and GRK2 are localized in a multimolecular complex [75]) (Reversibility: ?) [75] P ADP + phosphorylated p38 MAP kinase S ATP + phosducin ( phosducin is activated to inhibit Gbg protein [86]) (Reversibility: ?) [86] P ADP + phosphorylated phosducin S ATP + platelet-derived growth factor receptor-b ( feedback inhibition mechanism, overview [61]) (Reversibility: ?) [61] P ADP + phosphorylated platelet-derived growth factor receptor-b S ATP + protein ( specifically phosphorylates the agonistoccupied forms of the b 2-adrenergic receptor and related G proteincoupled receptors [7]; the enzyme mediates agonist-dependent phosphorylation of the b 2-adrenergic and related G protein-coupled receptors [4]; specifically phosphorylates the agonist-occupied form of the b-adrenergic and related G protein-coupled receptors [10]) (Reversibility: ?) [4, 7, 10] P ATP + phosphoprotein S ATP + protein M33 ( GRK2 is a potent regulator of the mouse cytomegalovirus GPCR protein M33-induced Gq/11 signaling through its ability to phosphorylate M33 and sequester Gaq/11 proteins dependent on an intact RH domain, the protein M33 is able to induce inositol phosphate accumulation, activate NF-kB, and promote smooth muscle cell migration, viral GPCRs like M33 play a role in viral dissemination in vivo, M33 is required for efficient murine cytwlovirus replication in the mouse,

405

b-Adrenergic-receptor kinase

P S P S P S P S

406

2.7.11.15

and induces several signlaing pathways, overview [77]) (Reversibility: ?) [77] ADP + phosphorylated protein M33 ATP + rhodopsin ( light-activated rhodopsin [24,76]) (Reversibility: ?) [24, 74, 76, 78, 83] ADP + phosphorylated rhodopsin ATP + ribosomal protein P2 ( activation of P2 [86]) (Reversibility: ?) [86] ADP + phosphorylated ribosomal protein P2 ATP + synuclein (Reversibility: ?) [86] ADP + phosphorylated synuclein Additional information ( enzyme is important in mediating rapid agonist-specific desensitization [3]; role for b ARK in modulating some receptor-mediated immune functions [5]; general role in the desensitization of synaptic receptors [8]; Gprk2 is required for egg morphogenesis [15]; b-ARK is probably a general adenylate cyclase-coupled receptor kinase [2]; b-ARK 1 is a key regulatory enzyme involved in the regulation of G proteincoupled receptors which associate with microsomal and plasma membranes [32]; plays a pivotal role in phosphorylating and desensitizing G protein-coupled receptors by vitue of pleckstrin homology domainmediated membrane translocation [27]; b-ARK activity is regulated by endogenous G proteins in different intracellular locations [37]; phosphorylates and regulates receptors coupled to either stimulation or inhibition of adenylate cyclase [2,19]; general function in desensitizing of many G protein-coupled receptor systems [8]; involved in the regulation of G protein-coupled receptor function, b-ARK 1 appears to be the predominant GRK in early embryogenesis and plays a fundamental role in cardiac development, enzyme participates in intracellular signal transduction mechanisms, which regulate cardiogenesis [38]; adenovirus-mediated overexpression of phosphoinositide 3-kinase restore contractile function of cardiac myocytes isolated from failing hearts, the recombinant PIK replaces the endogenous PIK in the transgenic pigs, the endogenous PIK shows abnormally increased activity in complex with bARK1, overview [49]; bARK enhances the contractility in heart myocardium via inhibition of Gbg subunits [54]; bARK1 binds phosphoinositide 3-kinase, which is by this way targeted to agonist-stimulated b-adrenergic receptors, where it regulates endocytosis, disruption of the bARK1-PIK complex leads to restoration of b-adrenergic receptor signaling and contractile function in heart failure, overview [49]; bARK1 inhibition improves b-adrenergic signaling and contractile function in failing human myocytes [47]; brain death induction, as well as sham-operation of pigs, lead to uncoupling of the b-adrenergic receptor with acutely increased myocardial b-adrenergic receptor kinase activity leading to left ventricular dysfunction [64]; dopamine D3 receptor binds to G proteins, the coupling is regulated by the filamin expression level [59]; G protein-coupled receptors are involved in the

2.7.11.15

b-Adrenergic-receptor kinase

regulation of diverse physiological processes, mechanisms of G proteincoupled receptor desensitization, e.g. by phosphorylation or feedback inhibition, overview [68]; GRK2 and GRK3 are involved in methacholine-stimulated inositol 3-phosphate production [69]; GRK2 expression plays a role in the fly development, GRK2 expression is required in the germline for proper formation of the anterior egg structures, egg hatching, and for early and late embryogenesis [42]; GRK2 is involved in A2 adenosine receptor response to agonists [44]; GRK2 mediates endothelin-1-induced insulin resistance via the inhibition of both Gaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes, GRK2 does not affect insulin receptor tyrosine phosphorylation [67]; GRK2 regulation mechanism of ERK activation involving interaction with mitogen-activated protein kinase, GRK2 diminishes the level of activating phosphorylation of ERK by CCL2 binding to chemokine receptor CCR2 in endothelial cells [66]; GRK3 is essential for induction of germinal vesicle breakdown, GRK3 forms a complex with b-arrestin-2 causing G protein-coupled receptor desensitization [55]; GRKs are involved in diverse physiological processes and pathologies, overview [45]; phosphorylation of heptahelical receptors by GRK2 is a universal regulatory mehanism leading to desensitization of G protein signaling and to the activation of alternative signaling pathways [70]; the enzyme is involved in G protein-coupled receptor signal transduction pathways and desensitization, GRK2 is a multiple domain kinase regulating by multiple mechanisms, overview [65]; adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure, adrenal gland-specific GRK2 inhibition reverses a2 b-adrenergic receptor dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac b-adrenergic signaling and function, and increased sympatholytic efficacy of a a2 b-adrenergic agonist [84]; GPCR-dependent kinases play a major role in agonist-induced phosphorylation and desensitization of G-protein coupled receptors, GRK2 is a component of neuronal and glial fibrillary t deposits with no preference in t isoform binding, GRK2 may play a role in hyperphosphorylation of t in tauopathies [80]; GRK activity is regulated by phosphorylation through several kinases and by interactions with several cellular proteins, e.g. calmodulin, caveolin or RKIP, GRK also interacts with PI3K, Akt, GIT or MEK, the interactions occur at the RH and PH domains, overview, the GRK interactome: role of GRKs in GPCR regulation and signaling, detailed overview [73]; GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization, b-arrestinmediated receptor internalization, activity of GRKs and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calcium-sensitive proteins, selective binding of activated Gaq and Ga-11 to RH domains of GRK2 and GRK3 selectively inhibits Gq signaling [87]; GRK2 activity leads to translocation of parts of b-adrenergic receptors to endocytic vesicles [76]; GRK2 interacts with multiple signaling proteins and is involved 407

b-Adrenergic-receptor kinase

2.7.11.15

in several cellular processes, e.g. expression and regulation of key cardiac seven-transmembrane receptors, 7TM receptors, such as the b-adrenergic and angiotensin receptors, GRK2 interacts with NCS-1, mechanism, overview, GRK2 inhibition can ameliorate heart failure, molecular mechanism of GRK2 activity regulation, GRK2 is probably involved in regulation of hypertension, overview [86]; GRK2 is essential, and GRK2-deficient mice are embryonically lethal [75]; GRK2 negatively regulates glycogen synthesis in mouse liver FL83B cells, regulates basal and insulin-stimulated glycogen synthesis via a post-IR signaling mechanism, and GRK2 may contribute to reduced IR expression and function during chronic insulin exposure [79]; GRK2 plays a role in sodium transport regulation and is involved in the development of essential hypertension, overview [72]; GRK2 regulates 7TM G-protein-coupled receptor activity, GRK2 promotes the association between active Smoothened and b-arrestin 2, overview [82]; insulin-mediated dopamine D1 receptor desensitization and underlying molecular mechanism in opossum kidney cells, overview [71]) (Reversibility: ?) [2, 3, 5, 8, 15, 19, 27, 32, 37, 38, 42, 44, 45, 47, 49, 54, 55, 59, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 79, 80, 82, 84, 86, 87] P ? Substrates and products S ATP + DREAM ( i.e. downstream regulatory element antagonist modulator protein, GRK2 mediates phosphorylation of DREAM/potassium channel interacting protein KChIP3, a multifunctional protein of the neuronal calcium sensor subfamilyof Ca2+ -binding proteins with specific roles in different cell compartments, regulating membrane trafficking of Kv4.2 potassium channel, phosphorylation of Ser95 affects cell surface localization, but not Kv4 channel tetramerization, overview [78]; i.e. downstream regulatory element antagonist modulator protein, GRK2 phosphorylates Ser95 [78]) (Reversibility: ?) [78] P ADP + phosphorylated DREAM S ATP + G protein-coupled receptor ( desensitization by GRK2 of the ligand-activated receptor [66]; desensitization of agonist-activated receptor [58]; GRK2 performs desensitization of the ligand-activated receptor by phosphorylation [43]; phosphorylation has a regulatory role, regulation of the signal transduction involving GRK2 and b-arrestin, overview [68]; regulation mechanism of GRK2, overview, regulation by phosphorylation at specific sites via distinct specific kinases, overview [45]; the GPCRs possess multiple phosphorylation sites for serine/threonine kinases [68]) (Reversibility: ?) [43, 45, 55, 58, 66, 67, 68] P ADP + phosphorylated G protein-coupled receptor S ATP + LEESSSSDHAERPPG (Reversibility: ?) [17] P ? S ATP + M1 muscarinic acetylcholine receptor ( phosphorylationdependent and -independent mechanisms in the regulation of M1 mus-

408

2.7.11.15

P S

P S

P S P S P S P S P S P S P S P S P S P S

P

b-Adrenergic-receptor kinase

carinic acetylcholine receptors by G protein-coupled receptor kinase 2 in hippocampal neurons, GRK2 can inhibit the receptor-dependent signaling via phospholipase C, overview [60]) (Reversibility: ?) [60] ADP + phosphorylated M1 muscarinic acetylcholine receptor ATP + Nedd4 ( GRK2 interacts with and phosphorylates the ubiquitin protein ligase Nedd4 preventing the binding to proline-rich motifs present in the C-termini of epithelial Na+ channel subunits and inhibition of the channels, overview [72]; recombinant GST-tagged wild-type Nedd-4, no activity with Nedd-4 mutant T466A, GRK2 interacts with and phosphorylates the ubiquitin protein ligase Nedd4 at multiple sites, mapping of phosphorylation sites, overview [72]) (Reversibility: ?) [72] ADP + phosphorylated Nedd4 ATP + Nedd4-2 ( GRK2 interacts with and phosphorylates the ubiquitin protein ligase Nedd4-2 preventing the binding to proline-rich motifs present in the C-termini of epithelial Na+ channel subunits and inhibition of the channels, overview [72]; GRK2 interacts with and phosphorylates the ubiquitin protein ligase Nedd4-2 at multiple sites, mapping of phosphorylation sites, overview [72]) (Reversibility: ?) [72] ADP + phosphorylated Nedd4-2 ATP + PDEg ( phosphorylation of PDEg possibly stimulates EGFR-mediated ERK activation [86]) (Reversibility: ?) [86] ADP + phosphorylated PDEg ATP + PDGFRb (Reversibility: ?) [86] ADP + phosphorylated PDGFRb ATP + R-smad ( GRK2 [73]) (Reversibility: ?) [73] ADP + phosphorylated R-smad ATP + RRRAEAAASAAA (Reversibility: ?) [25] ? ATP + RRRAEASAA ( poor peptide substrate [25]) (Reversibility: ?) [25] ? ATP + RRRASAAASAA ( poor peptide substrate [25]) (Reversibility: ?) [25] ? ATP + RRRASASAA ( poor peptide substrate [25]) (Reversibility: ?) [25] ? ATP + RRRASpAAASAA ( poor peptide substrate, higher catalytic efficiency than RRRASAAASAA [25]) (Reversibility: ?) [25] ? ATP + RRRASpASAA ( poor peptide substrate [25]) (Reversibility: ?) [25] ? ATP + RRREEEEESAAA ( nonreceptor peptide substrate [39]; good peptide substrate, but the activated receptor is a much better substrate [25]) (Reversibility: ?) [17, 25, 36, 37, 39] ? 409

b-Adrenergic-receptor kinase

2.7.11.15

S ATP + Smo ( GPRK2 participates in Hedgehog signaling in Drosophila melanogaster, and plays a key role in the Smo signal transduction pathway, when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced, overview [85]) (Reversibility: ?) [85] P ADP + phosphorylated Smo S ATP + Smoothened protein ( GRK2 promotes smoothened signal transduction involved in regulation of cellular proliferation and differentiation through activation of the transcription factor Gli, overview [82]) (Reversibility: ?) [82] P ADP + phosphorylated Smoothened S ATP + Smoothened protein ( a seven-transmembrane signaling protein [82]) (Reversibility: ?) [82] P ADP + phopshorylated Smoothened S ATP + [TSH receptor] ( GRK2 and GRK3, receptor activation [52]; GRK2 and GRK3 [52]) (Reversibility: ?) [52] P ADP + [TSH receptor]phosphate S ATP + a-synuclein ( colocalization of GRK2, GRK5, a-synuclein, and t in neurodegenerative disorders characterized by fibrillary tau inclusions and/or a-synuclein-enriched Lewy bodies, overview [80]; phosphorylation at Ser129 [80]) (Reversibility: ?) [80] P ADP + phosphorylated a-synuclein S ATP + a1 b-adrenergic receptor ( substrate specificities of GRK2 and GRK3 in cardiac myocytes, overview [83]) (Reversibility: ?) [83] P ADP + phosphorylated a1 b-adrenergic receptor S ATP + a2 -adrenergic receptor ( dependent on agonist occupancy by (-)-epinephrine, equally effective as b-adrenergic receptor, incorporation of 7-8 mol phosphate/mol receptor [19]; agonist-induced phosphorylation [27]; from human platelets [2,17,19]) (Reversibility: ?) [2, 17, 19, 21, 22, 27] P ADP + phospho-a2 -adrenergic receptor S ATP + a2 A-adrenergic receptor ( interaction with GRK2 via the second and third intracellular loop of the receptor, determination of regions required for specific interaction and phosphorylation activity utilizing recombinant GST-tagged wild-type and several mutant a2 A AR substrates, residues R225, R226, R218, K320, R322, and K358 are important, overview [58]) (Reversibility: ?) [58] P ADP + phosphorylated a2 A-adrenergic receptor S ATP + angiotensin receptor ( phosphorylation by GRK2 preceeds the binding of arrestins, which inhibits the seven-transmembrane receptor, but initiates internalization, overview [86]) (Reversibility: ?) [86] P ADP + phosphorylated angiotensin receptor

410

2.7.11.15

b-Adrenergic-receptor kinase

S ATP + b-adrenergic receptor ( agonist-activated receptor substrate [24]; bARK is involved in myocardial b-adrenergic receptor signaling, enzyme dysfunction can cause heart failure, regulation mechanism and physiology, overview [64]; bARK1 is responsible for desensitization and down regulation of b-adrenergic receptors [49]; desensitization of the receptor by GRK2 and GRK3 [68]; G proteincoupled receptor kinase phosphorylation mediates b-1 adrenergic receptor endocytosis via clathrin-coated pits [56]; G-protein-coupled receptor kinase-2 and b-arrestin-2 are involved in exercise-induced b-adrenergic receptor trafficking from cytosol to membranes in adipocytes, role in b-adrenergic receptor-ubiquitination in the ubiquitin-proteasome pathway [53]; mouse wild-type receptor, cytosolic phosphorylation domain of the substrate, overview [56]; receptor desensitization by phosphorylation [64]; GRK3 and GRK2 are involved in down-regulation of the a2 B-adrenoceptor, regulation of the pathway, overview [81]; in human heart failure, impaired b-adrenergic receptor signaling compromises cardiac sensitivity to inotropic stimulation, overview [87]; phosphorlytaion and internalization of the receptor requires clathrin, GRK2 specifically phosphorylates the activated form of the receptor that promotes the translocation of b-arrestins to the plasma membrane, overview [76]; phosphorylation by GRK2 preceeds the binding of arrestins, which inhibits the seven-transmembrane receptor, but initiates internalization, overview [86]; the enzyme is involved in regulation of the b-adrenergic receptor signaling by inhibiting arrestin recruitment to the receptor and subsequent desensitization and internalization, regulation of GRK2 by S-nitrosylation, molecular mechanism, overview [74]; mapping of S-nitrosylation sites and regulatory locus in GRK2 [74]; recombinantly expressed b-adrenergic receptor in U2-OS cells, mapping of S-nitrosylation sites and regulatory locus in GRK2 [74]; ureatreated rod outer segments as substrate [76]) (Reversibility: ?) [24, 49, 53, 56, 64, 68, 74, 76, 81, 84, 86, 87] P ADP + phosphorylated b-adrenergic receptor S ATP + b-adrenergic receptor ( phosphorylation sites [17]; phosphorylation sites are located mainly at the Cterminal tail of the receptor [2]; incorporation of 7-8 mol phosphate/mol receptor [19]; incorporation of about 9 mol phosphate/ mol receptor [20]; b2 -adrenergic receptor [2, 3, 8, 17, 21, 22, 24, 33, 39]; specifically phosphorylates the agonist-occupied form of the receptor [2, 3, 5, 8, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 34, 35, 37, 39]; similar rates of b-ARK 1 and 2 for b2 -adrenergic receptor phosphorylation [24]; human b2 adrenergic receptor [23,25,30,31]; at a similar rate as muscarinic cholinergic receptor [22]; at a similar rate as a2 -adrenergic receptor [19]; substrates: wild-type and mutants of b2 -AR, synergistic action of bARK and cAMP-dependent protein kinase depends on the palmitoylation state of the receptor, putative phosphorylation sites of b2 -AR [31]; incorporation of up to 5 mol phosphate/mol receptor [23,39]; b-ARK 411

b-Adrenergic-receptor kinase

P

S P S

P

412

2.7.11.15

1, substrate specificity, the overall topological structure of the activated receptor plays a key role in regulating signal-dependent receptor phosphorylation [25]; b-ARK 1 is more active than b-ARK 2 [8]; phosphate is incorporated solely into Ser-residues [18]; b-ARK 2 has a 25% lower specific activity than b-ARK 1 towards rhodopsin and b2 -AR [33]; incorporation of 6-8 mol phosphate/mol receptor [17]; b-AR from Sf9 cells [23]; b-ARK 1: substrate recognition mechanism, consensus sequence required for substrates, 3-dimensional model structure of the catalytic domain, potential phosphorylation sites of human b2 -adrenergic receptor [30]; b-AR from hamster lung [2, 3, 8, 16, 18, 19, 20, 39]; b-AR is a much better substrate than rhodopsin [3, 16, 20]; general role in the desensitization of synaptic receptors [8]; specifically phosphorylates and inactivates bAR after stimulation by receptor agonists, facilitating the binding of the inhibitor protein b-arrestin to the receptor, during myocardial ischemia the membrane activity of b-ARK is increased [35]; b-ARK 1 might be involved in uncoupling and down-regulation of b-AR, presumably both b1 - and b2 -AR, in failing hearts via receptor phosphorylation [13]; presumably modulates some receptor-mediated immune functions [5]; role of b-ARK 1 in heart failure, myocardial development and function [28]; natural substrate: b2 -adrenergic receptor [23, 25]; bARK 1 and 2 may have a similar substrate specificity in vivo [24]; regulation of the b-AR function in vivo [21]; desensitization of badrenergic receptor [19,37]; plays, together with cAMP-dependent protein kinase, an important role in agonist-promoted receptor desensitization, coordinated regulatory mechanism involving sequential depalmitoylation and phosphorylation of the b2 -AR by the two kinases [31]; agonist-occupied form of the receptor [2, 3, 5, 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 37, 39]; involved in homologous desensitization of b-adrenergic receptor [2, 3, 5, 8, 17, 18, 20, 21, 22, 23, 25, 28, 29, 39]; functional role of the b-ARK/b-arrestin mechanism of receptor desensitization in immune cells [12]) (Reversibility: ?) [2, 3, 5, 8, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39] ADP + phospho-b-adrenergic receptor [2, 3, 5, 8, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39] ATP + b-casein ( recombinant HA-tagged wild-type and mutant GRK3 [55]) (Reversibility: ?) [55] ADP + phosphorylated b-casein ATP + b-tubulin ( purified substrate from porcine brain or human GST-tagged protein recombinantly expressed in Escherichia coli, phosphorylation of bI- and bIII-tubulin at Thr409, Ser420, and, in bIII-tubulin, also at Ser420 of the C-terminal outer surface of the substrate protein [51]) (Reversibility: ?) [51] ADP + phosphorylated b-tubulin

2.7.11.15

b-Adrenergic-receptor kinase

S ATP + b2 -adrenergic receptor ( desensitization of the receptor with subsequent decline in the stimulatory effects of b2 -adrenergic agonists over time, the receptor is involved in alveolar Na+ and water clearance [63]) (Reversibility: ?) [63] P ADP + phosphorylated b2 -adrenergic receptor S ATP + corticotropin-releasing factor receptor type 1 ( i.e. CRFR1, phosphorylation leads to desensitization and downregulation of the receptor [50]) (Reversibility: ?) [50] P ADP + phosphorylated corticotropin-releasing factor receptor type 1 S ATP + dopamine D1 receptor ( phosphorylation by GRK2 has a regulatory role as part of the PI3K-PKC-GRK2 cascade [71]; hyperserine phosphorylation by GRK2 [71]) (Reversibility: ?) [71] P ADP + phosphorylated dopamine D1 receptor S ATP + dopamine D3 receptor ( the receptor is activated by GRK2 and GRK3 phosphorylation involving b-arrestins, GRK-mediated regulation of receptor-filamin complex stability and receptor-G protein signaling potential, GRK2 reduces the dopamine D3 receptor signaling, overview [59]) (Reversibility: ?) [59] P ADP + phosphorylated dopamine D3 receptor S ATP + epidermal growth factor receptor (Reversibility: ?) [86] P ADP + phosphorylated epidermal growth factor receptor S ATP + epithelial Na+ channel ( channel inactivation [86]) (Reversibility: ?) [86] P ADP + phosphorylated epithelial Na+ channel S ATP + ezrin ( phosphorylation of ezrin affects the 7TM receptor mediated cytoskeletal reorganization [86]; GRK2 [73]) (Reversibility: ?) [73, 86] P ADP + phosphorylated ezrin S ATP + insulin receptor substrate 1 ( role of GRK2 in insulin receptor IR signaling [79]; phosphorylation at Ser307 by GRK2 [79]) (Reversibility: ?) [79] P ADP + phosphorylated insulin receptor substrate 1 S ATP + micro-opioid receptor ( mutant C221V GRK2 shows slightly higher effect on morphine-induced internalization of the microopioid receptor compared to the wild-type GRK2, while the effects of mutants L271G, L273Y, and L336F are reduced, overview [62]) (Reversibility: ?) [62] P ADP + phosphorylated micro-opioid receptor S ATP + muscarinic acetylcholine receptor ( agonist-dependent phosphorylation, subtype 2 [27]) (Reversibility: ?) [27] P ADP + phospho-muscarinic acetylcholine receptor S ATP + muscarinic cholinergic receptor ( in vitro as good as badrenergic receptor, phosphorylation depends on the presence of a muscarinic agonist ligand, not merely receptor occupancy, the agonist induces a conformational change, which allows phosphorylation, phosphorylation sites: 70% Ser- and 30% Thr-residues, incorporation of 3-4 mol phos-

413

b-Adrenergic-receptor kinase

P S P S

P S

P S P S P S

P S

P S

414

2.7.11.15

phate/mol receptor [22]; from chick heart [2, 17, 22]) (Reversibility: ?) [2, 17, 22] ADP + phospho-muscarinic cholinergic receptor ATP + myelin basic protein ( recombinant HA-tagged wild-type and mutant GRK3 [55]) (Reversibility: ?) [55] ADP + phosphorylated myelin basic protein ATP + p38 MAP kinase ( GRK2 inactivates and regulates MAP kinase p38 modulating p38-dependent physiological processes, p38 and GRK2 are localized in a multimolecular complex [75]; phosphorylation at Thr123 located in the docking groove [75]) (Reversibility: ?) [75] ADP + phosphorylated p38 MAP kinase ATP + peptide ( b-ARK 1 and 2 prefer peptide substrates with acidic amino acids N-terminal to a Ser-residue [24]; b-ARK 1 prefers peptides containing acidic residues on the N-terminal side of a serine or threonine, presence of activated receptor enhances peptide phosphorylation [25]; e.g. Leu-Glu-Glu-Ser-Ser-Ser-Ser-Asp-His-Ala-Glu-ArgPro-Pro-Gly or Arg-Arg-Arg-Glu-Glu-Glu-Glu-Glu-Ser-Ala-Ala-Ala, role of acidic amino acids in peptide substrates, preference for negatively charged amino acids localized to the N-terminal side of a Ser- or Thrresidue, Ser-containing peptides are 4fold better than Thr-containing [17]; synthetic [17, 24, 25]) (Reversibility: ?) [17, 24, 25] ADP + phosphopeptide ATP + phosducin ( phosducin is activated to inhibit Gbg protein [86]; GRK2 [73]) (Reversibility: ?) [51, 73, 86] ADP + phosphorylated phosducin ATP + platelet-activating factor receptor ( PAF receptor acts as substrate [5]) (Reversibility: ?) [5] ADP + phospho-platelet-activating factor receptor ATP + platelet-derived growth factor receptor-b ( feedback inhibition mechanism, overview [61]; active with the wild-type and mutant Y740F/Y751F PDGFRb, GRK2 phosphorylation desensitizes the PDGF receptor-b, feedback inhibition mechanism, overview [61]) (Reversibility: ?) [61] ADP + phosphorylated platelet-derived growth factor receptor-b ATP + protein ( specifically phosphorylates the agonistoccupied forms of the b2-adrenergic receptor and related G proteincoupled receptors [7]; the enzyme mediates agonist-dependent phosphorylation of the b2-adrenergic and related G protein-coupled receptors [4]; specifically phosphorylates the agonist-occupied form of the b-adrenergic and related G protein-coupled receptors [10]) (Reversibility: ?) [4, 7, 10] ADP + phosphoprotein ATP + protein ( specifically phosphorylates the agonistoccupied forms of the b2-adrenergic receptor and related G proteincoupled receptors [7]; the enzyme mediates agonist-dependent phosphorylation of the b2-adrenergic and related G protein-coupled receptors [4]; specifically phosphorylates the agonist-occupied form

2.7.11.15

P S

P S

P S

P S P S P S P S

P S

b-Adrenergic-receptor kinase

of the b-adrenergic and related G protein-coupled receptors [10]) (Reversibility: ?) [4, 7, 10] ATP + phosphoprotein ATP + protein M33 ( GRK2 is a potent regulator of the mouse cytomegalovirus GPCR protein M33-induced Gq/11 signaling through its ability to phosphorylate M33 and sequester Gaq/11 proteins dependent on an intact RH domain, the protein M33 is able to induce inositol phosphate accumulation, activate NF-kB, and promote smooth muscle cell migration, viral GPCRs like M33 play a role in viral dissemination in vivo, M33 is required for efficient murine cytwlovirus replication in the mouse, and induces several signlaing pathways, overview [77]; recombinant FLAG-tagged M33 expressed in HEK-293T cells [77]) (Reversibility: ?) [77] ADP + phosphorylated protein M33 ATP + rhodopsin ( light-activated rhodopsin [24,76]; dark-adapted and light-activated rhodopsin in urea-washed rod outer segment membranes [43]; light-activated rhodopsin, both isozymes ARK1 and ARK2 prefer acidic amino acids N-terminal to a serine for phosphorylation [24]; substrate in dark-adapted, urea-stripped rod outer segment membranes [83]) (Reversibility: ?) [24, 43, 57, 62, 64, 74, 76, 78, 83] ADP + phosphorylated rhodopsin ATP + rhodopsin ( b-ARK 2 has a 25% lower specific activity than b-ARK 1 towards rhodopsin and b2 -AR [33]; b-AR is a much better substrate than rhodopsin [3, 16, 19, 20]; lightdependent, actual substrate: light-bleached rhodopsin [2, 3, 5, 16, 19, 20, 21, 24, 25, 35, 37, 38]; incorporation of 0.15 mol phosphate/mol rhodopsin [19, 20]; in form of bovine rod outer segments [2, 3, 5, 12, 16, 19, 20, 21, 23, 25, 27, 33, 35, 36, 37, 38]; metarhodopsin II, poor substrate [17]; recombinant b-ARK 2: 40% of efficiency of bARK 1 in phosphorylating rhodopsin [12]) (Reversibility: ?) [2, 3, 5, 12, 16, 17, 19, 20, 21, 23, 24, 25, 27, 33, 35, 36, 37, 38] ADP + phosphorhodopsin [2, 3, 5, 12, 16, 17, 19, 20, 21, 23, 24, 25, 27, 33, 35, 36, 37, 38] ATP + ribosomal protein P2 ( activation of P2 [86]; GRK2 [73]) (Reversibility: ?) [73, 86] ADP + phosphorylated ribosomal protein P2 ATP + synuclein ( GRK2 [73]) (Reversibility: ?) [51, 73, 86] ADP + phosphorylated synuclein ATP + tubulin ( GRK2 [73]) (Reversibility: ?) [62, 73, 86] ADP + phosphorylated tubulin GTP + b-adrenergic receptor ( agonist-occupied form of b-AR from hamster lung, GTP can substitute ATP, 2% as effective as ATP [20]) (Reversibility: ?) [20] GDP + phospho-b-adrenergic receptor [20] Additional information ( domain structure [2, 3, 28, 30]; b-ARK interacts rapidly with a high 415

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affinity binding site present in salt-stripped rat liver microsomal membranes, modulation of binding of enzyme [37]; mechanism and significance of the PH-domain function [27]; phosphorylates G protein coupled receptors in an agonist-dependent manner [17,37]; role of the PH domain, ligand binding characteristics of the PH domain, distinct role for each ligand, i.e. bg subunits of G proteins and phosphatidylinositol 4,5-bisphosphate, in enzyme-mediated receptor phosphorylation [34]; b-ARK 1 phosphorylates b2 -AR and other G protein-coupled receptors, substrate recognition mechanism, consensus sequence required for substrates, 3-dimensional model structure of the catalytic domain, residues 188-436 [30]; not: a1 -adrenergic receptor [19]; not: casein, histones [18]; rhodopsin kinase, EC 2.7.11.14, is also capable of badrenergic receptor phosphorylation [16]; very poor substrates: casein, phosvitin [21]; enzyme is important in mediating rapid agonistspecific desensitization [3]; role for b ARK in modulating some receptor-mediated immune functions [5]; general role in the desensitization of synaptic receptors [8]; Gprk2 is required for egg morphogenesis [15]; b-ARK is probably a general adenylate cyclasecoupled receptor kinase [2]; b-ARK 1 is a key regulatory enzyme involved in the regulation of G protein-coupled receptors which associate with microsomal and plasma membranes [32]; plays a pivotal role in phosphorylating and desensitizing G protein-coupled receptors by vitue of pleckstrin homology domain-mediated membrane translocation [27]; b-ARK activity is regulated by endogenous G proteins in different intracellular locations [37]; phosphorylates and regulates receptors coupled to either stimulation or inhibition of adenylate cyclase [2,19]; general function in desensitizing of many G protein-coupled receptor systems [8]; involved in the regulation of G protein-coupled receptor function, b-ARK 1 appears to be the predominant GRK in early embryogenesis and plays a fundamental role in cardiac development, enzyme participates in intracellular signal transduction mechanisms, which regulate cardiogenesis [38]; adenovirus-mediated overexpression of phosphoinositide 3-kinase restore contractile function of cardiac myocytes isolated from failing hearts, the recombinant PIK replaces the endogenous PIK in the transgenic pigs, the endogenous PIK shows abnormally increased activity in complex with bARK1, overview [49]; bARK enhances the contractility in heart myocardium via inhibition of Gbg subunits [54]; bARK1 binds phosphoinositide 3-kinase, which is by this way targeted to agonist-stimulated b-adrenergic receptors, where it regulates endocytosis, disruption of the bARK1-PIK complex leads to restoration of b-adrenergic receptor signaling and contractile function in heart failure, overview [49]; bARK1 inhibition improves b-adrenergic signaling and contractile function in failing human myocytes [47]; brain death induction, as well as sham-operation of pigs, lead to uncoupling of the b-adrenergic receptor with acutely increased myocardial badrenergic receptor kinase activity leading to left ventricular dysfunction [64]; dopamine D3 receptor binds to G proteins, the coupling is regu416

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lated by the filamin expression level [59]; G protein-coupled receptors are involved in the regulation of diverse physiological processes, mechanisms of G protein-coupled receptor desensitization, e.g. by phosphorylation or feedback inhibition, overview [68]; GRK2 and GRK3 are involved in methacholine-stimulated inositol 3-phosphate production [69]; GRK2 expression plays a role in the fly development, GRK2 expression is required in the germline for proper formation of the anterior egg structures, egg hatching, and for early and late embryogenesis [42]; GRK2 is involved in A2 adenosine receptor response to agonists [44]; GRK2 mediates endothelin-1-induced insulin resistance via the inhibition of both Gaq/11 and insulin receptor substrate-1 pathways in 3T3L1 adipocytes, GRK2 does not affect insulin receptor tyrosine phosphorylation [67]; GRK2 regulation mechanism of ERK activation involving interaction with mitogen-activated protein kinase, GRK2 diminishes the level of activating phosphorylation of ERK by CCL2 binding to chemokine receptor CCR2 in endothelial cells [66]; GRK3 is essential for induction of germinal vesicle breakdown, GRK3 forms a complex with barrestin-2 causing G protein-coupled receptor desensitization [55]; GRKs are involved in diverse physiological processes and pathologies, overview [45]; phosphorylation of heptahelical receptors by GRK2 is a universal regulatory mehanism leading to desensitization of G protein signaling and to the activation of alternative signaling pathways [70]; the enzyme is involved in G protein-coupled receptor signal transduction pathways and desensitization, GRK2 is a multiple domain kinase regulating by multiple mechanisms, overview [65]; bARK1 interacts with recombinant phosphoinositide 3-kinase expressed in transgenic mice [49]; GRK2 functionally ineracts with clathrin, phosphoinositol 3-phosphate kinase-g, and G protein-coupled receptor kinase interacting protein, GIT [45]; GRK2 functionally ineracts with clathrin, phosphoinositol 3-phosphate kinase-g, and GIT [45]; GRK2 interacts by direct binding with Gaq/11, phosphorylation of agonist-activated seven-transmembrane receptors by GRK2 [67]; GRK2 interacts with calmodulin, substrate specificity, GRK2 phosphorylates serine and threonine residues with preceeding acidic amino acid residues [65]; GRK2 prefers acidic protein sequences for phosphorylation [51]; peptide phosphorylation study [24]; substrate specificity, the enzyme depends on basic residues for substrate recognition, the residues at the substrate phosphorylation site greatly influence the enzyme activity, autoregulation by a pseudosubstrate mechanism, overview [1]; the enzyme contains regulatory sites for Ca2+ /calmodulin, protein kinase C, and clathrin [70]; the GRKs are specific for GPCRs and arrestins, overview [68]; the M3 muscarinic acetylcholine receptor is no substrate for GRK2 and GRK3 [69]; adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure, adrenal gland-specific GRK2 inhibition reverses a2 b-adrenergic receptor dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac badrenergic signaling and function, and increased sympatholytic efficacy 417

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of a a2 b-adrenergic agonist [84]; GPCR-dependent kinases play a major role in agonist-induced phosphorylation and desensitization of Gprotein coupled receptors, GRK2 is a component of neuronal and glial fibrillary tau deposits with no preference in t isoform binding, GRK2 may play a role in hyperphosphorylation of t in tauopathies [80]; GRK activity is regulated by phosphorylation through several kinases and by interactions with several cellular proteins, e.g. calmodulin, caveolin or RKIP, GRK also interacts with PI3K, Akt, GIT or MEK, the interactions occur at the RH and PH domains, overview, the GRK interactome: role of GRKs in GPCR regulation and signaling, detailed overview [73]; GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization, b-arrestin-mediated receptor internalization, activity of GRKs and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calcium-sensitive proteins, selective binding of activated Gaq and Ga-11 to RH domains of GRK2 and GRK3 selectively inhibits Gq signaling [87]; GRK2 activity leads to translocation of parts of b-adrenergic receptors to endocytic vesicles [76]; GRK2 interacts with multiple signaling proteins and is involved in several cellular processes, e.g. expression and regulation of key cardiac seventransmembrane receptors, 7TM receptors, such as the b-adrenergic and angiotensin receptors, GRK2 interacts with NCS-1, mechanism, overview, GRK2 inhibition can ameliorate heart failure, molecular mechanism of GRK2 activity regulation, GRK2 is probably involved in regulation of hypertension, overview [86]; GRK2 is essential, and GRK2-deficient mice are embryonically lethal [75]; GRK2 negatively regulates glycogen synthesis in mouse liver FL83B cells, regulates basal and insulin-stimulated glycogen synthesis via a post-IR signaling mechanism, and GRK2 may contribute to reduced IR expression and function during chronic insulin exposure [79]; GRK2 plays a role in sodium transport regulation and is involved in the development of essential hypertension, overview [72]; GRK2 regulates 7TM G-protein-coupled receptor activity, GRK2 promotes the association between active Smoothened and b-arrestin 2, overview [82]; insulin-mediated dopamine D1 receptor desensitization and underlying molecular mechanism in opossum kidney cells, overview [71]; GRK2 and tau do not crossreact [80]; GRK2 binds to and inhibits Gaq protein via its N-terminal RGS domain, GRK2 interacts with NCS-1 [86]; GRK2 interacts with a component of the MAPK pathway, as well as with the PI- 3K substrate AKT, GRK2 and GRK3 bind the Gbg subunit complex, a process that induces activation of the GRKs [87]) (Reversibility: ?) [1, 2, 3, 5, 8, 15, 16, 17, 18, 19, 21, 24, 27, 28, 30, 32, 34, 37, 38, 42, 44, 45, 47, 49, 51, 54, 55, 59, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 79, 80, 82, 84, 86, 87] P ?

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Inhibitors 2,3-diphosphoglycerate ( weak [2,21]; inhibits in the millimolar range, IC50: 1.1 mM with rhodopsin as substrate [21]) [2, 21] 4-amino-1-tert-butyl-3-(1’-naphthyl)pyrrazolo[3,4-d]pyrimidine ( i.e. 1-Na-PP1, inhibits mutant C221V mediated morphine-induced internalization of the micro-opioid receptor, but not wild-type GRK2 and mutant L271G activities [62]) [62] actin [73] alprenolol ( b-adrenergic antagonist, blocks phosphorylation [18]) [18] atropine ( receptor antagonist [22]) [22] C-terminus of b-ARK ( bARKmini, inhibits [29]) [29] Ca2+ ( inhibits GRK2 [45]) [45] Ca2+ /calmodulin ( inhibit GRK2 with an IC50 of about 0.002 mM, inhibition mechanism via direct binding [68]) [68] calmodulin ( inhibits GRK2 [45]) [45, 73] chloropromazine ( IC50: 0.043 mM [2]) [2] concanavalin A ( inhibitor of clathrin-mediated receptor endocytosis via clathrin binding domain of b-arrestin-2, inhibitor reduces the GRK3/ b-arrestin-2 induction activity of germinal vesicle breakdown [55]) [55] d-sphingosine ( IC50: 0.027 mM [2]) [2] d-glucosamine 2,6-disulfate ( inhibits in the millimolar range, weak, IC50: 7.3 mM with rhodopsin as substrate [21]) [21] dextran sulfate ( strong, IC50: 0.00015 mM with rhodopsin as substrate [2,21]) [2, 21] digitonin ( IC50: 0.05 mM [2]; 0.05-0.1%, 93-95% inhibition [20]) [2, 20] EDTA [40] heparan sulfate ( less inhibitory than heparin [21]) [21] heparin ( most potent inhibitor to date [2,21,24]; strong, kinetics, IC50: 0.00003 mM with b-AR as substrate, 0.00015 mM with rhodopsin as substrate [21]; specific inhibitor, 1 mM/l: almost complete inhibition of rhodopsin phosphorylation [35]; b-ARK 1: IC50 is 0.0014 mM, b-ARK 2: IC50 is 0.0011 mM [24]; potent inhibitor of bARK 1 [39]; 0.001 mM, complete inhibition of muscarinic cholinergic receptor phosphorylation [22]; polylysine, spermine or spermidine at lower concentrations partially reverses [21]; de-N-sulfated heparin is 8fold less effective [21]; rhodopsin phosphorylation, in a dose-dependent manner [5]; inhibits both isozymes ARK1 and ARK2 potently with IC50 of 0.0014 mM and 0.0011 mM, respectively [24]) [2, 5, 12, 17, 21, 22, 24, 35, 37, 39] inositol hexaphosphate ( weak, IC50: 3.6 mM with rhodopsin as substrate [2,21]) [2, 21] inositol hexasulfate ( good inhibitor [2]; 270fold more potent than inositol hexaphosphate [21]; IC50: 0.0135 mM with rhodopsin as substrate [2,21]) [2, 21] McN-A343 ( weak, partial antagonist [22]) [22] 419

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NaCl ( 0.1 M, 90% inhibition [2]; 20 mM, 48% inhibition, as efficient as F-, I- , NO2- or acetate, less efficient than citrate, phosphate or sulfate [20]) [2, 20] NaF ( as efficient as Cl- , I- , NO2- or acetate, less efficient than citrate, phosphate or sulfate [20]) [20] NaI ( as efficient as Cl- , F-, NO2- or acetate, less efficient than citrate, phosphate or sulfate [20]) [20] NaNO2 ( as efficient as Cl- , I- , F- or acetate, less efficient than citrate, phosphate or sulfate [20]) [20] peptide ( synthetic peptides from a variety of intracellular regions of b2 -AR specifically inhibit phosphorylation of the intact receptor, but not of peptide substrates [17]; synthetic peptides derived from the receptor intracellular loop inhibit [34]) [17, 34] phosphatidylinositol 4,5-bisphosphate ( inhibits receptor phosphorylation, causes membrane association, 30% inhibition of phosphorylation of RRREEEEESAAA [39]) [39] polyaspartic acid ( good inhibitor [2]; IC50: 0.0013 mM with rhodopsin as substrate [2,21]) [2, 21] polyglutamic acid ( good inhibitor [2]; IC50: 0.002 mM with rhodopsin as substrate [21]) [2, 21] polylysine ( weak, more potent than spermine and spermidine [21]; IC50: 0.069 mM with rhodopsin as substrate [2,21]) [2, 21] protein kinase C inhibitor H7 ( weak [2,5]; IC50: 0.25 mM [2]) [2, 5] pyridoxal 5’-phosphate ( inhibits in the millimolar range [21]; weak, IC50: 0.9 mM with rhodopsin as substrate [2,21]) [2, 21] RKIP [73] S-nitrosothiols ( inhibit GRK2 by S-nitrosylation at Cys340, regulatory function of S-nitrosylation [74]) [74] sangivamycin ( IC50: 0.067 mM [2]) [2] sodium acetate ( as efficient as Cl- , I- , NO-2 or F-, less efficient than citrate, phosphate or sulfate [20]) [20] sodium citrate ( 20 mM, 97% inhibition, more efficient than phosphate, sulfate, Cl- , F-, I- , NO2- or acetate [20]) [20] sodium phosphate ( 20 mM, 76% inhibition, less efficient than citrate, as good as sulfate, more efficient than Cl- , F-, I- , NO2- or acetate [20]) [20] sodium sulfate ( less efficient than citrate, as good as phosphate, more efficient than Cl- , F-, I- , NO2- or acetate [20]) [20] spermidine ( weak, less potent than polylysine [21]) [21] spermine ( IC50: 1.6 mM [2]; weak, less potent than polylysine [2,21]) [2, 21] tamoxifen ( IC50: 0.04 mM [2]) [2] trifluoperazine ( IC50: 0.035 mM [2]) [2] Triton X-100 ( IC50: 0.054 mM [2]) [2] Tween 20 ( IC50: 0.027 mM [2]) [2]

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Yohimbine ( a2 -adrenergic antagonist, co-incubation completely blocks phosphorylation of a2 -adrenergic receptor [19]) [19] Zn2+ ( 0.1-0.2 mM ZnCl2 , 94-98% inhibition [20]) [20] actinin ( inhibits GRK2 [45]) [45] a-actinin [73, 86] a2 -adrenergic antagonist ( co-incubation completely blocks phosphorylation of a2 -adrenergic receptor [19]) [19] bARKct ( the isolated C-terminal sequence of the enzyme acts as a peptide inhibitor [47]) [47] calveolin [86] caveolin ( microsomal anchor protein, inhibits GRK2 [45]) [45, 73] chondroitin sulfate B ( weak [21]) [21] chondroitin sulfate C ( less inhibitory than heparin [21]) [21] corticotropin-releasing factor ( reduces GRK2 expression in pituitary cells, suppressed by astressin, opposite effect compared to corticotroph tumor cells AtT20 [50]) [50] microsomal membranes ( marked inhibition of rhodopsin or synthetic peptide phosphorylation in the presence of increasing amounts of microsomal membranes, bound enzyme is less able to interact with its substrate [37]) [37] monodansyl cadaverin ( inhibitor of clathrin-mediated receptor endocytosis via clathrin binding domain of b-arrestin-2, inhibitor reduces the GRK3/b-arrestin-2 induction activity of germinal vesicle breakdown [55]) [55] nitric oxide ( inhibits GRK2 by S-nitrosylation at Cys340, regulatory function of S-nitrosylation [74]) [74] propranolol ( b-adrenergic antagonist, in presence no phosphorylation [33]) [33] quinpirole ( inhibits GRK2 and GRK3 [59]) [59] rhodopsin ( intact light-activated rhodopsin slightly inhibits phosphorylation of RRREEEEESAAA [25]) [25] Additional information ( not inhibited by cAMP, cGMP, cAMP-dependent protein kinase inhibitor, Ca2+ /calmodulin, Ca2+ /phospholipid, phorbol esters [18]; not affected by Ca2+ or Co2+ [20]; not inhibited by protein kinase A inhibitor PKI [5]; not inhibited by staurosporine [5,35]; alprenolol is no inhibitor with a2 -adrenergic receptor as substrate [19]; autoregulation by a pseudosubstrate mechanism, overview [1]; clathrin inhibitors inhibit the internalization of the b-adrenergic receptor by GRK [56]; deletion mutants of the enzyme, comprising parts of the C-terminus or the N-terminus, are able to inhibit the full-length wild-type enzyme, overview [57]; isolated pleckstrin homology domains of GRK2 and GRK3 can act as enzyme inhibitors preventing interaction of the enzymes with the G protein subunits [68]; phosphorylation by ERK1/ 2 inhibits GRK2 [45]; platelet-derived growth factor receptor-b activates the enzyme by tyrosine phosphorylation, the wild-type PDGFRb is 60fold more active with GRK2 than PDGFRb mutant Y857F, GRK2 activation also 421

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increases GRK2 degradation and downregulation [61]; ERK phosphorylates and inhibits GRK2, protein kinase C phosphorylated GRK2 at Ser685 and induces RKIP binding, RKIP binds to GRK2 and inhibits it after it has been phosphorylated by protein kinase C [86]; quantitative determination of S-nitrosylation of GRK2 by the biotin-switch assay [74]) [1, 5, 18, 19, 20, 35, 45, 56, 57, 61, 68, 74, 86] Cofactors/prosthetic groups ATP ( requirement [20]; cofactor ATP [23]; as MgATP2- [24]) [1, 2, 3, 5, 8, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 45, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87] Activating compounds (-)-epinephrine ( requirement, agonist, a2 -adrenergic receptor as substrate [19]) [19] AlF-4 ( enhances b-ARK activity upon stimulation of heterotrimeric G proteins [37]) [37] carbachol ( requirement, agonist, muscarinic cholinergic receptor as substrate [22]) [22] cardiolipin ( activates, phosphorylation of b2 -AR [39]) [39] G protein ( binding and modulation of GRK2 and GRK3, Gbg subunits bind GRK2 required for lipid association and G protein-coupled receptor phosphorylation and are released afterwards [45]; binding of Gb/g dimers, activation is required for translocation from cytosol to plasma membrane [68]; GRK2 binds G protein b1 g2 subunits, binding structure, no correct complex formation with recombinant Gbg mutant C68S lacking the isoprenylation site, overview [40]; GRK2 binds G protein b1 g2 subunits, binding structure, overview [70]; interaction via GRK2 N-terminal RGS domain, binding of free Gbg subunits initiates GRK2 translocation from cytosol to the plasma membrane and its activation for receptor phosphorylation [65]; required for GRK2 activation [66]) [40, 45, 65, 66, 68, 70] G protein Gbg subunits ( activate both ARK1 and ARK2 about 2.5fold [24]; required for activity, 20fold stimulation with substrate rhodopsin, 2 regulatory Gbg binding sites: one N-terminal domain of GRK2, and one C-terminal within the pleckstrin homology domain, both sites are functionally different, overview [57]) [24, 57] G protein bg-subunit ( activates, binding domain is localized to the C-terminal region of b-ARK [26,27,37]; required for maximum activity, targets b-ARK 1 to the membrane, which presumably facilitates the precise orchestration of phosphorylation of only activated receptors [38]; from brain, binds to the C-terminal half of the PH domain [27]; from bovine brain, b-ARK 1 and 2: requirement, selectivity for bg subunits, both isoforms differentiate between defined bg subunits [33]; from brain, stimulates the phosphorylation of rhodopsin, but not of the peptide RRREEEEESAAA, an intact N-terminus of the g subunit is required for stimulation, but not for kinase binding, endoprotease Lys-C blocks stimulation, 422

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Gbg binds to the C-terminal region of b-ARK containing the pleckstrin homology domain, role of the G protein g-subunit [36]; 10fold activation, b-ARK 1 and 2, increases incorporation of phosphate from 4 to 10 mol phosphate/mol receptor [24]; requirement, binding plays an important role in specifically targeting the enzyme complex to its receptor substrate [34]) [24, 26, 27, 33, 34, 36, 37, 38, 39] Gbg subunits ( required for activity on G protein-coupled receptors, binding via enzyme C-terminal pleckstrin homology domain, binding does not induce large domain rearrangements, but small rotations of the enzyme domains, the domain interfaces remain intact upon GRK2 activation, structure analysis of GRK2 in complex with G protein b1 g2 subunits, overview [43]) [43] insulin ( initiates GRK2 membrane translocation [79]; leads to increase dopamine D1 receptor serine phosphorylation by GRK2 [71]) [71, 79] isoproterenol ( stimulates [39]; requirement, bagonist, b-adrenergic receptor as substrate [2, 3, 5, 16, 17, 18, 19, 20, 21, 22]) [2, 3, 5, 16, 17, 18, 19, 20, 21, 22, 39] oxytremorine ( requirement, agonist, muscarinic cholinergic receptor as substrate [22]) [22] PIP2 ( activates GRK2 through binding to its PH domain [86]) [86] phosphatidic acid ( activates, phosphorylation of b2 -AR [39]) [39] phosphatidylglycerol ( activates, phosphorylation of b2 -AR [39]) [39] phosphatidylinositol ( activates, phosphorylation of b2 -AR, 6fold activation of phosphorylation of RRREEEEESAAA [39]) [39] phosphatidylinositol 4,5-bisphosphate ( binding of GRK2 and GRK3 via their pleckstrin homology domains, activate the phosphorylation activity [45]) [45] phosphatidylserine ( activates, phosphorylation of b2 -AR [39]; binding of GRK2 and GRK3 via their pleckstrin homology domains, activate the phosphorylation activity [45]) [39, 45] phospholipid ( required for phosphorylation of b2 -AR, activation is associated with a conformational change in b-ARK 1, acidic phospholipid specificity, not activated by phosphatidylinositol 4,5-diphosphate, direct regulation of b-ARK 1 activity by phospholipids [39]) [39] platelet activating factor ( requirement, agonist, b-adrenergic receptor as substrate [5]) [5] a2 A-adrenergic receptor ( epinephrine-activated a2 A-adrenergic receptor activates GRK2, interaction with GRK2 via the second and third intracellular loop of the receptor, determination of regions required for specific interaction utilizing recombinant GST-tagged wild-type and several mutant a2 A ARs, residues R225, R226, R218, K320, R322, and K358 are important, overview [58]) [58] arrestins ( arrestins modulate the enzyme activity having a regulatory role, regulation of arrestins, overview [68]) [68]

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b-arrestin ( required for phosphorylation activity and receptor substrate endocytosis [56]) [56] b-arrestin-2 ( forms a complex with GRK3 causing G proteincoupled receptor desensitization [55]; required, membrane protein [53]) [53, 55] b-arrestin2 ( mediates GRK activity, forms a signaling complex with filamin and dopamine D3 receptor, interactions, overview [59]) [59] b2 -adrenergic receptor ( interaction of enzyme with human agonistoccupied receptor specifically and significantly enhances peptide phosphorylation, at lower concentrations, enhances both the affinity and catalytic efficiency for peptide phosphorylation [25]; interaction of b-ARK 1 with the agonist-occupied receptor activates [39]) [25, 39] cAMP-dependent protein kinase ( PKA-mediated phosphorylation favors subsequent phosphorylation of b2 -AR by b-ARK, PKA increases the phosphorylation rate of b-ARK [31]) [31] corticotropin-releasing factor ( induces GRK2 expression in pituitary cells, suppressed by astressin, opposite effect compared to pituitary cells [50]) [50] mastoparan/guanosine 5’-(3-O-thio)triphosphate ( enhances b-ARK activity upon stimulation of heterotrimeric G proteins [37]) [37] platelet-derived growth factor receptor-b ( activates the enzyme by tyrosine phosphorylation at Y13, Y86, and Y92, required, the wild-type PDGFRb is 60fold more active with GRK2 than PDGFRb mutant Y857F, GRK2 activation also increases GRK2 degradation and downregulation, independent of Gbg subunits and phosphoinositide 3-kinase [61]) [61] rhodopsin ( interaction of enzyme with light-activated rhodopsin or truncated rhodopsin lacking its C-terminal phosphorylation sites activates peptide phosphorylation, at lower concentrations, enhances both the affinity and catalytic efficiency for peptide phosphorylation, but intact light-activated rhodopsin slightly inhibits the phosphorylation of RRREEEEESAAA [25]) [25] Additional information ( partial agonists promote reduced receptor phosphorylation [2]; not effected by Ca2+ or Co2+ [20]; not activated by cAMP, cGMP, Ca2+ /calmodulin, Ca2+ /phospholipid, phorbol esters [18]; myocardial ischemia: b-ARK is activated, rapid induction of bARK 1 activity in membranes, presumably caused by the release of the receptor agonist noradrenaline [35]; not activated by polycations [2]; a2 AR phosphorylation is not stimulated by isoproterenol [19]; agonists induce specific conformational changes allowing phosphorylation [22]; autoregulation by a pseudosubstrate mechanism, overview [1]; phosphorylation activates the enzyme [73]; phosphorylation by c-Src activates GRK2 [45]; GRK2 is activated by phosphorylation through c-Src in a positive feedback reaction, Hsp90 secures proper folding and maturation and protects GRK2 from ubiquitination and proteasomal degradation, it induces GRK2 expression, phosphoinositol kinase 3 binds GRK2 and enhances receptor internalization [86]; GRK3 bind the Gbg subunit complex, a process that induces activation of the GRKs by phosphorylation [87]) [1, 2, 18, 19, 20, 22, 35, 45, 73, 86, 87] 424

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Metals, ions Ca2+ ( inhibits GRK2 [45]) [45] Mg2+ ( requirement [2, 3, 5, 12, 16, 17, 18, 19, 20, 21, 22, 23]; increases binding of b-ARK to microsomal membranes [37]; optimal concentration: 2-6 mM [20]; as MgATP2[24]) [1, 2, 3, 5, 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 37, 40, 43, 51, 53, 55, 57, 58, 62, 63, 64, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 85, 86, 87] Mn2+ ( requirement, can replace Mg2+ with 50% efficiency, optimal concentration: 1-3 mM [20]; Mn2+ can partially substitute Mg2+ in increasing the binding of b-ARK to microsomal membranes [37]) [20, 37, 55] Additional information ( no activation by Ca2+ [78]; not activated by Ca2+ , Co2+ or Zn2+ [20]) [20, 78] Turnover number (min–1) 0.0001 (ATP, pH 7.8, 23 C, recombinant GRK2 L271G mutant, with substrate tubulin [62]) [62] 0.0029 (ATP, pH 7.8, 23 C, recombinant wild-type GRK2, with substrate tubulin [62]) [62] 0.0041 (ATP, pH 7.8, 23 C, recombinant GRK2 C221V mutant, with substrate tubulin [62]) [62] Specific activity (U/mg) 0.00873 ( pH 7.5, 30 C [20]) [20] 0.05 ( pH 7.5, 30 C [22]) [22] 1 ( about, recombinant b-ARK 1, expressed in SF9 cells [25]) [25] Additional information ( b-ARK 2 has a 25% lower specific activity than b-ARK 1 towards rhodopsin and b2 -AR [33]; quantitative enzyme level determination [83]) [33, 37, 56, 60, 83] Km-Value (mM) 0.00025 (b-adrenergic receptor, pH 7.5, 30 C [20]) [20] 0.0038 (rhodopsin, pH 7.5, 30 C, recombinant b-ARK 1 [23]) [23] 0.0053 (rhodopsin, pH 7.4, 30 C, in absence of heparin [21]) [21] 0.006 (rhodopsin, pH 7.5, 30 C [20]) [20] 0.014 (rhodopsin, b-ARK 1 and 2 [24]; isozymes ARK1 and ARK2 [24]) [24] 0.017 (ATP, pH 7.8, 23 C, recombinant wild-type GRK2, with substrate tubulin [62]) [62] 0.022 (ATP, pH 7.5, 30 C, recombinant b-ARK 1, rhodopsin as substrate [23]) [23] 0.033 (ATP, pH 7.5, 30 C, b-adrenergic receptor as substrate [20]) [20] 0.037 (ATP, pH 7.5, 30 C, rhodopsin as substrate [20]) [20] 0.06-0.09 (ATP, MgATP2-, b-ARK 1 and 2 [24]) [24] 0.06-0.09 (MgATP2-, isozymes ARK1 and ARK2 [24]) [24] 0.062 (ATP, pH 7.8, 23 C, recombinant GRK2 L271G mutant, with substrate tubulin [62]) [62]

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0.149 (ATP, pH 7.8, 23 C, recombinant GRK2 C22 1V mutant, with substrate tubulin [62]) [62] 0.72 (RRREEEEESAAA, pH 7.4, 30 C, recombinant b-ARK 1 [25]) [25] 0.9-1.3 (Peptide, acid-rich synthetic peptides [17]) [17] 1.34 (RRREEEEESAAA, pH 7.4, 30 C, recombinant b-ARK 1 [39]) [39] 1.5-4.8 (Peptide, synthetic peptides containing a single Glu-residue [17]) [17] 3.3 (RRRASAAASAA, pH 7.4, 30 C, recombinant b-ARK 1 [25]) [25] 3.4 (RRRASpASAA, pH 7.4, 30 C, recombinant b-ARK 1 [25]) [25] 4.6 (RRRASpAAASAA, pH 7.4, 30 C, recombinant b-ARK 1 [25]) [25] 5.1 (RRRAEASAA, pH 7.4, 30 C, recombinant b-ARK 1 [25]) [25] 5.4 (RRRASASAA, pH 7.4, 30 C, recombinant b-ARK 1 [25]) [25] Additional information ( kinetics of peptide phosphorylation, influence of activated rhodopsin and b2 -AR on the Km value for different peptide substrates [25]; kinetics of isozymes ARK1 and ARK2 [24]) [24, 25] Ki-Value (mM) 0.00015 (heparin) [22] Additional information [59] pH-Optimum 6-7.5 [20] 7.3 ( assay at [55]) [55] 7.4 ( assay at [5, 17, 18, 21, 25, 31, 39, 56, 57, 69, 81]) [5, 17, 18, 21, 25, 31, 39, 56, 57, 69, 81] 7.5 ( assay at [2, 3, 8, 16, 19, 20, 22, 37, 43, 58, 76, 78]) [2, 3, 8, 16, 19, 20, 22, 37, 43, 58, 76, 78] 7.8 ( assay at [62]) [62] 8 ( assay at [83]) [83] Temperature optimum ( C) 22 ( assay at room temperature [55,57]) [55, 57] 23 ( assay at [62]) [62] 25 ( assay at [81]) [81] 30 ( assay at [2, 3, 5, 8, 16, 17, 18, 19, 20, 21, 22, 23, 25, 31, 33, 34, 36, 37, 39, 43, 58, 76, 78]; assay at, rhodopsin phosphorylation by recombinant mutant b-ARK [27]) [2, 3, 5, 8, 16, 17, 18, 19, 20, 21, 22, 23, 25, 27, 31, 33, 34, 36, 37, 39, 43, 58, 76, 78] 37 ( assay at [56, 69]; assay at, intact cell phosphorylation of muscarinic receptor subtype 2 and a2 -adrenergic receptor by recombinant mutant b-ARK [27]) [27, 56, 69]

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4 Enzyme Structure Molecular weight 77000 ( b-ARK 2, Western blot analysis [8]) [8] 80000 ( gel filtration [20]; Western blot analysis [29, 32, 38, 39]; expected size of native b-ARK [29]) [20, 29, 32, 38, 39] 82000 ( b-ARK 1, Western blot analysis [8]) [8] Additional information ( b-ARK 1: amino acid sequence, 689 amino acids protein [30]; amino acid sequences of human and bovine b-ARK 1 and 2 [12]; equilibrium sedimentation measurements on GRK2-Gbg complexes, overview [43]) [12, 30, 43] Subunits ? ( x * 80000, SDS-PAGE [3]; x * 79700, calculated from the amino acid sequence [3]; x * 79800, predicted from the amino acid sequence [8]; x * 79900, predicted from the amino acid sequence [8]; x * 80000, recombinant GRK2, gel filtration and SDS-PAGE [40]; x * 79000, GRK3, SDS-PAGE, x * 81000, GRK2, SDS-PAGE [83]; x * 80000, GRK2, SDS-PAGE [73]) [3, 8, 40, 73, 83] monomer ( 1 * 80000, SDS-PAGE [20]) [20] Additional information ( bARK subfamily multidomain structure, overview [45]; GRK family domain structure, the N-terminal RGS domain of GRK2 has regulatory function on G protein binding and receptor phosphorylation and activity, overview [65]; GRK2 consists of 3 modular domains: a predominantly N-terminal RGS homology domain, a central protein kinase domain, and a C-terminal pleckstrin homology domain, domain functions, overview [40,70]; GRK2 consists of 3 modular domains: a predominantly N-terminal RGS homology domain, a central protein kinase domain, and a C-terminal pleckstrin homology domain, the interfaces of the domains are important for stability, and enzyme expression and activity, and remain intact upon GRK2 activation, structure analysis of GRK2 in complex with G protein b1 g2 subunits, overview [43]; GRK3 wild-type and mutant domain structures, overview [55]; domain structure of GRK2, overview [73]) [40, 43, 45, 55, 65, 70, 73] Posttranslational modification phosphoprotein ( enzyme appears to autophosphorylate [20]; autophosphorylation, the site often depends more on structure than on primary sequence [1]; epinephrine-activated a2 A-adrenergic receptor activates GRK2, interaction with GRK2 via the second and third intracellular loop of the receptor, determination of regions required for specific interaction and phosphorylation activity utilizing recombinant GST-tagged wild-type and several mutant a2 A ARs, residues R225, R226, R218, K320, R322, and K358 are important, overview [58]; GRK2 is phosphorylated by c-Src kinase, ERK1 and ERK2, protein kinase C, and PKA, overview [45]; GRK2 is phosphorylated by MAP kinase at S670 [43]; regulation of GRKs by other kinases, such as PKA, PKC, ERK1 and ERK2, e.g. GRK2

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is phosphorylated at Ser670 by ERK1 or ERK2, phosphorylation of the badrenergic receptor-activated GRK2 by c-SRC at tyrosine residues mediated by binding of b-arrestin, which rapidly activates the GPCR phosphorylation activity of GRK2 and its degradation via the ubiquitine/proteosome pathway, overview [68]; GRK2 is activated by phosphorylation through c-Src, while phosphorylation by ERK inhibits GRK2 [86]; GRKs are regulated by phopshorylation [87]; phosphorylation, e.g. by PKA, PKC, ERK1/2 or c-SRC, activates the enzyme [73]) [1, 20, 43, 45, 58, 61, 68, 73, 86, 87] Additional information ( no autophosphorylation of b-ARK 1 [39]; GRK2 is ubiquinated after being tyrosine phosphorylated leading to its proteolytic degradation via the ubiquitine/proteasome pathway, regulation overview [68]; mechanisms of regulation of GRK protein stability and degradation, e.g. via ubiquination or protease cleavage, overview [45]; mechanisms of regulation of GRK protein stability and degradation, e.g. via ubiquitination or protease cleavage, overview [45]) [39, 45, 68]

5 Isolation/Preparation/Mutation/Application Source/tissue AtT-20 cell ( corticotroph tumor cell line, GRK2, no expression of GRK3 [50]) [50] BN17 cell [81] FL83B cell ( a hepatocyte cell line [79]) [79] HEK-293 cell [56, 74, 86] HL-60 cell ( high expression of b-ARK 1 [5]) [5, 86] IMR-32 cell ( b-ARK 2 [12]; b-ARK 1 [5]) [5, 12] JURKAT cell ( high expression of b-ARK 1 [5]) [5, 86] MOLT-4 cell ( high expression of b-ARK 1 [5]) [5] Ng-108-15 cell [81] OK cell ( a renal proximal tubular cell line [71]) [71] S49 cell ( kin- mutant cell line of S49 lymphoma cells [18]) [18] SH-SY5Y cell ( neuroblastoma cell line [69]) [69] T-lymphocyte [45] U-937 cell ( high expression of b-ARK 1 [5]) [5] U2-OS cell ( an osteosarcoma cell line [74]) [74] adipocyte [53] adrenal gland [84] alveolar cell type I ( i.e. AT1 cells, from transdifferentiation of AT2 cells [63]) [63] alveolar cell type II ( i.e. AT2 cells, primary cells, transdifferentiation to AT1 cells [63]) [63] anterior pituitary gland ( GRK2, no expression of GRK3 [50]; no expression of GRK3 [50]) [50] brain ( cerebral cortex [2, 3, 16, 17, 19, 20, 21, 22]; synapses [8]; b-ARK mRNA is expressed intensely in the cerebellar granule cell layer and moderately in the hippocampal pyramidal cells 428

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and dentate granule cells. The neocortex and piriform cortex express it moderately to weakly, whereas the thalamus and hypothalamus express it weakly to faintly. No significant expression of the mRNA is detected in the caudateputamen. Weak expression of b-ARK mRNA in several nuclei of the brainstem and in the spinal gray matter [9]; highest b-ARK mRNA concentrations in brain and spleen, brain: highest levels in cerebral cortex and cerebellum, with significant lower levels in basal ganglia, brain stem, pituitary and hypothalamus [3]; highest b-ARK activity in cerebral cortex [20]; regional and cellular distribution of b-ARK 1 and 2 in brain [8]; 40-50% co-localization of GRK2 with neurofibrillary tangles in Alzheimers disease [80]) [2, 3, 8, 9, 12, 16, 17, 18, 19, 20, 21, 22, 23, 80] cardiac myocyte [83, 86] cardiomyoblast ( H9c2 cardiomyolbasts, ATCC CRL 1446 [29]) [29] cell culture [71] central nervous system [58] cerebellum ( brain: highest b-ARK mRNA concentrations in cerebral cortex and cerebellum [3]) [3, 12] cerebral cortex ( highest b-ARK activity [20]; brain: highest bARK mRNA concentrations in cerebral cortex and cerebellum [3]) [2, 3, 16, 17, 19, 20, 21, 22] chromaffin cell [84] corticotropic cell ( GRK2 [50]) [50] embryo ( whole embryo extract [38]) [38] epithelium ( analysis of GRK2 localization in alveolar epithelium [63]) [63, 72] gut [72] heart ( enhanced expression of b-adrenergic receptor kinase 1 in the hearts of cardiomyopathic Syrian hamsters, BIO53.58 [13]; moderate expression of b-ARK 2 [12]; moderate expression of b-ARK 1 [5]; significantly increased expression of b-ARK 1 in the hearts of BIO53.58 hamsters compared to control hamsters F1b [13]; during myocardial ischemia the membrane activity of b-ARK is increased [35]; b-ARK 1 is the predominant myocardial GRK [28]; adult heart [38]; about 40% of b-ARK mRNA concentration in brain [3]; GRK3 expression is not increased in heart failure [87]) [3, 5, 12, 13, 18, 28, 35, 38, 45, 46, 47, 49, 54, 64, 83, 84, 86, 87] heart ventricle [46] hepatocyte [79] hippocampus [60] kidney ( about 20% of b-ARK mRNA concentration in brain [3]) [3, 71, 72] leukemia cell ( myeloid and lymphoid leukemia cell lines, high expression of b-ARK 1 [5]) [5] leukocyte ( peripheral blood leukocytes [5 ,12]; mononuclear, peripheral blood leukocytes, high expression [5, 12]; b-ARK 2 [12]; b-ARK 1 [5]) [5, 12] liver [79] 429

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lung ( moderate expression of b-ARK 2 [12]; about 40% of b-ARK mRNA concentration in brain [3]; analysis of GRK2 localization in alveolar epithelium, determination of expression level [63]) [3, 12, 18, 63, 72, 74] lymphocyte ( peripheral [46]) [46, 86] macrophage [75] myelomonocytic leukemia cell ( GRK2 [45]) [45] myocardium [47, 49, 64] myocyte ( cardiac myocytes isolated from failing hearts [49]; primary isolated from hearts of patients in the end-stage of heart failure undergoing heart transplantation [47]) [47, 49] neuron ( b-ARK 1 and 2 are expressed primarily in neurons distributed throughout the CNS [8]; hippocampal [60]; colocalization of GRK2 and tau in intracellular neurofibrillary tangles and isolated paired helical filaments [80]) [8, 60, 80] ovary ( about 20% of b-ARK mRNA concentration in brain [3]) [3] retina [4] salivary gland [72] smooth muscle ( DDT-MF2 smooth muscle cells derived from ductus deferens leiomyosarcoma [29]) [18, 29] spleen ( highest b-ARK mRNA concentrations in brain and spleen [3]) [3] splenocyte ( primary [66]) [66] thyroid gland ( increased expression of GRK3 in hyperfunctioning thyroid nodule [87]) [87] thyroid nodule ( prepared without lymphocytic infiltrations in the tumor, GRKs expression patterns, low expression of GRK2, and high expression level of GRK3 [52]) [52] wing [85] Additional information ( tissue distribution [3,20]; ubiquitous enzyme [2,20]; tissue distribution of b-ARK 1 [5]; not in liver, muscle and adrenal gland [3]; tissue distribution of b-ARK 2 [12]; analysis of GRK2 expression rate and mRNA level in heart and lymphocytes by RT-PCR [46]; GRK2 and GRK3 are expressed in a wide variety of tissues, expression patterns, overview [45]; GRK2 expression analysis in different developmental stages, the expression is required in the germline [42]; Lewy bodies were negative for both GRK2 and GRK5 in Lewy body disease [80]; quantitative enzyme level determination [83]; the enzyme is ubiquitously expressed, developmental regulation of GRK2 expression, overview [86]) [2, 3, 5, 12, 20, 42, 45, 46, 80, 83, 86] Localization cytoplasm ( predominantly cytoplasmic, enzyme activity depends upon its translocation from the cytoplasm to the membrane, the bg subunits of G proteins bind to enzyme and recruit it to the membrane, bARK 1 binds to Gb2 [26]; GRK2 and GRK3 [45]) [26, 45]

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cytosol ( cytosolic enzyme, isoproterenol and platelet-activating factor induce translocation of b-ARK from cytosol to membrane [5]; cardiac, total cytosolic activity amount to values almost 3times those of membrane activity, during myocardial ischemia the membrane activity of b-ARK is increased, but the cytosolic activity is not altered [35]; in unstimulated cells enzyme is mainly located in the cytosol [29]; GRK2 translocation from cytosol to the plasma membrane [79]) [5, 8, 16, 20, 21, 23, 24, 29, 32, 35, 52, 56, 65, 68, 79] membrane ( cardiac, total cytosolic activity amount to values almost 3times those of membrane activity, during myocardial ischemia the membrane activity of b-ARK is increased, but the cytosolic activity is not altered [35]; G protein bg-subunit targets b-ARK 1 to the membrane [38]; isoproterenol and platelet-activating factor induce translocation of b-ARK from cytosol to membrane [5]; enzyme is localized to the specific membrane compartment by bg subunits of G proteins and phosphatidylinositol phosphates that specifically and coordinately bind to the C- and N-terminal half, respectively, of the PH domain [27]; enzyme activity depends upon its translocation from the cytoplasm to the membrane, the bg subunits of G proteins bind to enzyme and recruit it to the membrane, bARK 1 binds to Gb2 [26]; after b-AR agonist stimulation b-ARK is partially translocated to the membranes [29]; the PH domain ligands bg subunits of G proteins and phosphatidylinositol 4,5-bisphosphate affect membrane localization of enzyme, simultaneous presence of both ligands is required for effective membrane localization, cooperative binding of the ligands, membrane translocation [34]; microsomal and plasma membrane, anchoring of b-ARK to cellular membranes under basal conditions is independent of the availability of heterotrimeric G protein subunits, additional anchoring mechanisms, Gbg subunits may play a role in agonistmediated targeting of b-ARK to the membrane in intact cells [32]; associated with, membrane association is required for activity on G proteincoupled receptors, and mediated by the C-terminal Gbg binding domain [55]; associated, GRK2 in complex with G protein b1 g2 subunits [40, 43, 70]; GRK2 [53]; GRK2 membrane translocation is induced by insulin [79]; insulin causes GRK2 translocation to the membranes, which is blocked by genistein, wortmannin, chelerythrine chloride, and GRK2-specific siRNA [71]) [5, 26, 27, 29, 32, 34, 35, 38, 40, 43, 52, 53, 55, 58, 65, 70, 71, 74, 79] microsome ( microsomal membranes [32]; associates with intracellular microsomal membranes, mechanism, modulation of binding of enzyme, main determinants of binding appear to be localized to an 60 amino acid residue stretch, residues 88 to 145, G protein bg subunits are not the main anchor in the membranes [37]) [32, 37] microtubule [51] plasma membrane ( soluble enzyme, that transiently translocates to the plasma membrane [37]; b-AR may serve as membrane anchor for enzyme, translocation of b-ARK from the cytosol to the plasma membrane [20]; GRK2 and GRK3, transient translocation upon G 431

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protein-coupled receptor activation [45]; transient recruitment of GRK2 and GRK3 after GPCR activation, the enzymes bind via pleckstrin homology domains to phosphoinositol-4,5-bisphosphate in the plasma membrane [68]; recruitment of GRk2 to the plasma membrane is assisted by phospholipids and Gbg proteins binding to the PH domain and another N-terminal site [86]; the wild-type GRK2 is translocated to the plasma membrane [76]) [20, 32, 37, 45, 49, 68, 76, 84, 86] sarcolemma [64] soluble ( predominantly [18]; soluble enzyme, that transiently translocates to the plasma membrane [37]) [18, 37] Additional information ( subcellular distribution of b-ARK 1 [32]; complex modulation of the subcellular distribution of b-ARK [37]; subcellular distribution of b-ARK 1 and 2 [8]; subcellular distribution analysis of GRK2 and GRK3 [83]) [8, 32, 37, 83] Purification (partial, 50-100fold, from kin- mutants of S49 lymphoma cells) [18] (recombinant GRK2 from insect Sf9 cells by ammonium sulfate fractionation, hydrophobic interaction and heparin affinity chromatography) [51] (recombinant GST-tagged wild-type and truncation mutant GRK2s from Escherichia coli by glutathione affinity chromatography, recombinant wildtype GRK2 from Sf9 insect cells by heparin affinity chromatography and gel filtration) [57] (recombinant b-ARK 1, expressed in Sf9 cells) [23, 36] [17, 19, 21, 22] (20300fold, to near homogeneity) [20] (about 20000fold, from brain) [2, 3] (partial) [16] (recombinant ARK1 and ARK2 from Sf9 insect cells by sequential chromatography, including heparin affinity chromatography) [24] (recombinant GRK2 from Spodoptera frugiperda Sf9 cells to near homogeneity by ultracentrifugation, 3 steps of cation exchange chromatography, heparin affinity chromatography, another ion exchange chromatography step, and gel filtration, the recombinant His6-tagged G protein b1 g2 subunits by solubilization from membranes, Ni2+ affinity and ion exchange chromatography, and gel filtration) [40] (recombinant His-tagged wild-type and mutant GRK2s from Spodoptera frugiperda Sf9 cells by nickel affinity chromatography) [62] (recombinant b-ARK 1 and 2 overexpressed in Sf9 cells) [24, 33] (recombinant b-ARK 1 expressed in Sf9 cells) [23, 25, 27, 37, 39] (recombinant b-ARK expressed in Sf9 cells) [34] (expression in COS 7 cells) [10] Renaturation (reconstitution of the GRK2-G protein b1 g2 subunits complex by mixing in a 2:3 ratio) [40]

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Crystallization (purified recombinant GRK2 S670A mutant, hanging drop vapour diffusion method, 4 C, 0.002 ml protein solution containing 15 mg/ml GRK2, 20 mM HEPES, pH 8.0, 0.2 M NaCl, and 2 mM DTT, mixed with 0.002 ml well solution containing 0.1 M HEPES, pH 7.5-8.0, 1 M NaCl, 1 M urea, 10-40 mM phosphoserine, pH 7.0, 5% v/v glycerol, and 9.5-11.5% PEG 8000, 2-3 weeks, X-ray diffraction structure determination and analysis at 4.5 A resolution) [43] (purified recombinant GRK2 in complex with purified recombinant G protein b1 g2 subunits, X-ray diffraction structure determination and analysis at 2.5 A resolution, modeling) [70] (purified recombinant GRK2 in complex with purified recombinant bovine G protein b1 g2 subunits, two-dimensional hanging drop vapor diffusion method screen, 0.001 ml protein solution containing 5-20% PEG3350 versus pH 5.0-7.5, 1 M NaCl, 12 mg/ml protein, mixed with 0.001 ml of well solution containing 0.1 M MES, pH 5.25, 0.2 M NaCl, 1 mM inositol-3,4,5-trisphosphate, 5 mM MgCl2 , and 6.9-7.8% PEG3350, versus 1 ml of well solution, inositol-3,4,5-trisphosphate can be substituted by EDTA and phosphatidylserine, both crystal types diffract differently but show identical unit-cell parameters, X-ray diffraction structure determination and analysis at 3.2 A resolution, Cu Ka radiation) [40] Cloning (expression of GRK2-fusion constructs in transgenic flies, expression analysis, overview) [85] (cloning and disruption of the b-ARK 1 gene by homologous recombination, effects of gene disruption on the embryos) [38] (expression of Myc-tagged wild-type and mutant GRK2s in HEK-293T cells, co-expression with M33 in wild-type and Gaq/11-/- mouse embryonic fibroblasts, M33 couples directly to the Gq/11 signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner) [77] (expression of a dominant negative GRK2 mutant in AtT20 cells showing reduced CRFR1 desensitization) [50] (studies with b-ARK 1 knockout mice) [28, 38] (studies with transgenic mice overexpressing b-ARK 1) [28] (transient expression of GRK2 in HEK-293T cells, co-expression with GST-tagged epithelial Na+ channel, and GST-tagged Nedd4 and Nedd4-2) [72] (DNAs encoding the C-terminal domains Gly556-Ser670 and Pro466Leu689 are cloned and expressed in Escherichia coli) [36] (adenovirus-mediated gene transfer and expression of inhibitor bARKct in primary myocytes isolated from hearts of patients in the end-stage of heart failure undergoing heart transplantation, bARK1 activity is inhibited but badrenergic signaling and contractile function are improved) [47] (b-ARK 1 is cloned and expressed in SF9 cells using the baculovirus expression system) [23]

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(cDNA encoding b-ARK 1 is cloned and sequenced, very similar sequences of bovine and human kinases) [5] (co-expression of rat thyrotropin receptor with GRK2 and GRK3 in HEK-293 cells, receptor phosphorylation occurs with both enzymes) [87] (expression of GRK2 in Spodoptera frugiperda Sf9 cells) [51] (expression of GST-tagged wild-type and truncation mutant GRK2s in Escherichia coli, expression of wild-type GRK2 in Spodoptera frugiperda Sf9 cells using the baculovirus infection system, expression of wild-type and truncation mutant GRK2s in HEK-293 cells) [57] (expression of kinase-dead GRK2 mutant and of the N-terminal region of GRK2, residues Ala2-Thr187, in HEK-293 cells) [65] (overexpression of the C-terminus of bARK in ventricular cardiomyocytes and in the myocardium of rabbits suffering heart failure by adenoviral gene transfer, co-expression of N-terminal deletion mutant of phosducin, leading to increase in contractility of the cells due to inhibition of Gbg subunits rather than to b-adrenergic receptor resensitization, bARK additionally stimulates cAMP production) [54] (overexpression of wild-type and kinase-dead mutant GRK2 in murine 3T3-L1 adipocytes via adenovirus infection system leading to inhibition of Gaq/11 signaling, including tyrosine phosphorylation of Gaq11 and cdc42associated phosphatidylinositol 3-kinase activity, overexpression of the inactive muant, but not of wild-type enzyme, inhibits endothelin-1-induced Ser612 phosphorylation of insulin receptor substrate-1, and restores activation of the pathway) [67] (regulation of GRK2 expression, analysis, overview) [86] (stable overexpression of GFP-fusion K220R mutant GRK2 and GFP-fusion K220R mutant GRK3 in SH-SY5Y neuroblastoma cells via adenovirus infection system) [69] (co-expression of HA-tagged M1 muscarinic acetylcholine receptor and of GRK2 wild-type and mutants in CHO-K1 cells, suppression of 80% of endogenous GRK2 in HEK293 cells and in hippocampal neurons by antisense construct expression of GRK2, co-expression of wild-type and mutant GRK2s with eGFP-tagged inositol 1,4,5-trisphosphate biosensor eGFP-PHPLCd in HEK293 cells) [60] (cytosolic expression of wild-type and mutant GRK2 in HEK-293 cells, co-expression with the b-adrenergic receptor) [76] (expression of GRK2 and GRK3 in cardiac myocytes via adenoviral transfection, overview) [83] (functional expression of HA-tagged wild-type and mutant GRK3, and coexpression with b-arrestin-2, in Xenopus laevis oocytes via injection of mRNA, leads to induction of maturation of the oocytes) [55] (nucleotide sequence of b-ARK 1, 95% homology to Syrian hamster bARK 1) [13] (adenovirus-mediated expression of b-adrenergic receptor kinase C-terminus in pigs in endothelial cells and smooth muscle cells, overview) [48] (b-ARK 1 and 2 are cloned and expressed in SF9 cells using the baculovirus expression system) [24, 33] 434

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(b-ARK 1 is cloned and expressed in SF9 cells using the baculovirus expression system) [23, 25, 37, 39] (b-ARK 1 is cloned and expressed in yeast strain L40) [26] (b-ARK 1 is stably overexpressed in HEK-293 cells) [32] (b-ARK expression in SF9 cells) [34] (cDNA encoding b-ARK is cloned from brain, sequenced and expressed in COS-7 cells, cDNA encodes a 689 amino acids protein) [3] (cloning of GST-b-ARK fusion proteins and expression in Escherichia coli AG1) [37] (co-overexpression of GRK2 and G protein b1 g2 subunits in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [70] (co-overexpression of GRK2 and His6-tagged G protein b1 g2 subunits in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [40] (co-overexpression of GRK2 mutants and G protein b1 g2 subunits in Spodoptera frugiperda Sf9 cells using the baculovirus infection system, and in COS-1 cells) [43] (expression of GRK2 in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [58] (expression of HA-tagged GRK2 wild-type and mutants and of FLAGtagged platelet-derived growth factor receptor-b wild-type and mutants in HEK-293 cells) [61] (expression of wild-type GRK2 and mutants in HEK-293 cells, co-expression of GRK2 and CCR2B produces a 65-70% reduction for the wild-type GRK2 and 52% for the mutant GRK2 K220R in ERK phosphorylation by inhibition of CCL2) [66] (expression of wild-type and mutant GRK2 in C3H10T1/2 cells, transient expression in HEK-293 cells, expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription) [82] (functional GRK2 overexpression in transgenic mice, tissue-specific transgenic mRNA expression analysis, tissue-specific overexpression of GRK2 in mouse osteoblasts by usage of the osteocalcin gene-2 promoter) [41] (overexpression of ARK1 and ARK2 in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [24] (overexpression of GRK2 in HEK-293 cells and in COS-7 cells, co-expression with FLAG-tagged dopamine D3 receptor, and filamin A and/or b-arrestin, recombinant b-arrestin is accumulated in the plasma membrane in presence of GRK2 and absence of agonist) [59] (stable co-expression of C-terminally HA-tagged wild-type GRK2 and murine micro-opioid receptor in HEK-293 cells, transient expression of Cterminally HA-tagged wild-type and mutant GRK2s in HEK-293 cells, expression of His-tagged wild-type and mutant GRK2s in Spodoptera frugiperda Sf9 cells using the baculovirus infection system) [62] (transient overexpression of wild-type GRK2 in NG108-15 mouse neuroblastoma x rat glioma hybrid cells) [44] (very similar cDNA sequences of bovine and human kinases) [5, 12]

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(wild-type b-ARK 1 is expressed in SF9 and COS-7 cells, mutant b-ARK 1 is cloned and expressed in COS-7 cells) [27] (cDNA encoding b-ARK 1 is cloned from heart and sequenced, 689 amino acids protein, 95% homology to rat and 90% homology to human b-ARK 1) [13] (expression in COS-7 cells) [3] (b ARK locus segregated with the long arm of chromosome 11, centromeric to 11q13) [4] (cloning and sequencing) [5] (cDNAs encoding b-ARK 1, 689 amino acids, and b-ARK 2, 688 amino acids, are cloned, expressed in COS-7 cells and sequenced) [8] (cDNAs encoding b-ARK 1, 689 amino acids, and b-ARK 2, 688 amino acids, are cloned, expressed in COS-7 cells and sequenced) [8] (cDNA encoding b-ARK 2 is cloned, expressed in COS-7 cells and sequenced) [12] (expression in COS7 cells) [12] (very similar sequences of bovine and human kinases) [12] [13] Engineering C221V ( site-directed mutagenesis, mutant GRK2 activity is slightly increased compared to the wild-type enzyme, the requirement for initial ligand-induced internalization of a G protein-coupled receptor with subsequent rounds of internalization is different for the mutant GRK2 compared to the wild-type enzyme [62]) [62] D110A ( site-directed mutagenesis, GRK2 mutant deficient in Gaq/11 binding [60]; the mutant shows impaired binding to Gaq proteins, mutant GRK2 inhibits ERK phosphorylation similar to the wild-type enzyme when co-expressed with CCR2B in HEK-293 cells [66]; the point mutation within the RH domain abrogates GRK2 sequestration of activated GTP-boundGaq/11 [77]) [60, 66, 77] D110A/K220R ( site-directed mutagenesis, inactive GRK2 mutant deficient in Gaq/11 binding [60]; the mutant, which exhibits no RH or kinase activity, is completely defective in its ability to attenuate M33 signaling [77]) [60, 77] D635K ( triple mutant D635K/S636K/D637K, mutation in the Gbgbinding region of the PH domain [27]) [27] D637K ( triple mutant D635K/S636K/D637K, mutation in the Gbgbinding region of the PH domain [27]) [27] E520A ( site-directed mutagenesis, very low recombinant expression level, mutant GRK2 shows a dramatic loss of activity with rhodopsin as substrate [43]) [43] E646K ( mutation in the Gbg-binding region of the PH domain [27]) [27] E96A ( site-directed mutagenesis, slightly increased recombinant expression level compared to the wild-type GRK2, mutant GRK2 shows activity with rhodopsin as substrate similar to the wild-type enzyme [43]) [43]

436

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K220R ( mutant GRK2 inhibits ERK phosphorylation in a slightly reduced rate compared to the wild-type enzyme when co-expressed with CCR2B in HEK-293 cells [66]; site-directed mutagenesis, inactive mutant GRK2 [60]; site-directed mutagenesis, inactive, dominant negative mutants of GRK3 and GRK2, overexpression in SH-SY5Y cells does not influence endogenous M3 muscarinic acetylcholine receptor phosphorylation and desensitization by the endogenous wild-type GRK6, but inhibits methacholine-stimulated inositol 3-phosphate production by 75% [69]; sitedirected mutagenesis, kinase-dead GRK2 mutant, overexpression in HEK293 cells leads to partially desensitization of G protein-coupled receptor [65]; site-directed mutagenesis, kinase-dead mutant of GRK3, the mutant is unable to induce germinal vesicle breakdown [55]; inactive GRK2 mutant [75,82]; the point mutation within the kinase domain inhibits GRK2 catalytic activity [77]) [55, 60, 65, 66, 69, 75, 77, 82] K567E ( mutation in the PIP2-binding region of the PH domain [27]) [27] K645E ( mutation in the Gbg-binding region of the PH domain [27]) [27] L271G ( site-directed mutagenesis, mutant GRK2 shows highly reduced activity compared to the wild-type enzyme [62]) [62] L273Y ( site-directed mutagenesis, mutant GRK2 shows highly reduced activity compared to the wild-type enzyme [62]) [62] L336F ( site-directed mutagenesis, mutant GRK2 shows highly reduced activity compared to the wild-type enzyme [62]) [62] L647G ( residue of the PH domain, mutation completely abolishes bARK activity and activation by the G protein bg-subunit [27]) [27] P638D ( site-directed mutagenesis, RH-PH domain interface residue mutant, 40% reduced recombinant expression level compared to the wildtype GRK2, mutant GRK2 shows 15% of wild-type activity with rhodopsin as substrate [43]) [43] Q642G ( mutation in the Gbg-binding region of the PH domain [27]) [27] R516A ( site-directed mutagenesis, 60% reduced recombinant expression level compared to the wild-type GRK2, mutant GRK2 shows slightly reduced activity with rhodopsin as substrate [43]) [43] R578N ( double mutant R578N/R579N, mutation in the PIP2-binding region of the PH domain [27]) [27] R579N ( double mutant R578N/R579N, mutation in the PIP2-binding region of the PH domain [27]) [27] S636K ( triple mutant D635K/S636K/D637K, mutation in the Gbgbinding region of the PH domain [27]) [27] S670A ( site-directed mutagenesis, the MAP kinase phosphorylation site S670 is eliminated in the GRK2 mutant enzyme [43]; the GRK2 mutant shows reduced phosphorylation by ERK and is less degraded [86]) [43, 86] V42E ( site-directed mutagenesis, RH-PH domain interface residue mutant, 60% reduced recombinant expression level compared to the wild437

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type GRK2, mutant GRK2 shows 25% of wild-type activity with rhodopsin as substrate [43]) [43] W576A ( mutation in the PIP2-binding region of the PH domain [27]) [27] W643A ( residue of the PH domain, mutation completely abolishes b-ARK activity and activation by the G protein bg-subunit [27]) [27] Y13F ( site-directed mutagenesis, required activating tyrosine phosphorylation by platelet-derived growth factor receptor-b kinase activity is reduced with the mutant GRK2, reduced activity compared to the wild-type [61]) [61] Y13F/Y86F/Y92F ( site-directed mutagenesis, required activating tyrosine phosphorylation by platelet-derived growth factor receptor-b kinase activity is eliminated with the mutant GRK2, reduced activity compared to the wild-type [61]) [61] Y46A ( site-directed mutagenesis, RH-PH domain interface residue mutant, 80% reduced recombinant expression level compared to the wildtype GRK2, mutant GRK2 shows 50% of wild-type activity with rhodopsin as substrate [43]) [43] Y86F ( site-directed mutagenesis, required activating tyrosine phosphorylation by platelet-derived growth factor receptor-b kinase activity is reduced with the mutant GRK2, reduced activity compared to the wild-type [61]) [61] Y86F/Y92F ( site-directed mutagenesis, required activating tyrosine phosphorylation by platelet-derived growth factor receptor-b kinase activity is highly reduced with the mutant GRK2, reduced activity compared to the wild-type [61]) [61] Y92F ( site-directed mutagenesis, required activating tyrosine phosphorylation by platelet-derived growth factor receptor-b kinase activity is reduced with the mutant GRK2, reduced activity compared to the wild-type [61]) [61] Additional information ( mutant, gprk2(6936) disrupts expression of a putative member of the GRK family, the G proteincoupled receptor kinase 2 gene Gprk2. This mutation affects Gprk2 gene expression in the ovaries and renders mutant females sterile. The mutant eggs contain defects in several anterior eggshell structures that are produced by specific subsets of migratory follicle cells. In addition, rare eggs that become fertilized display severe defects in embryogenesis [15]; effects of mutations in the pleckstrin homology domain of b-ARK on activity, Ala-insertion following Trp-643 completely abolishes b-ARK activity and activation by the G protein bg-subunit [27]; adenovirus-mediated expression of b-adrenergic receptor kinase C-terminus reduces intimal hyperplasia and luminal stenosis of arteriovenous polytetrafluoroethylene grafts in pigs [48]; construction and expression of a mouse mutant b-adrenergic receptor lacking the GRK phosphorylation sites in HEK-293 cells, and coexpression of murine GFPtagged b-arrestin [56]; construction of a deletion GRK2 mutant comprising residues 438-689, which does not inhibit ERK phosphorylation when coexpressed with CCR2B in HEK-293 cells [66]; construction of a phosphor438

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ylation-defective CCR2 mutant mice, in the mutant mice GRK2 inhibits ERK activation similar to the wild-type mice, the inhibitory effect is eliminated by co-expression of Gaq-binding RGS-like RH domain of GRK2 or its Gbg-binding domain [66]; construction of bARKct mutant comprising the C-terminus of the enzyme [54]; construction of deletion mutants comprising residues 1-485, 1-185, 561-689, 1-53, 1-200, or 54-185, analysis of activity with rhodopsin and binding of G protein bg subunits, overview [57]; construction of GRK2-depleted hippocampal neurons and expression of a GRK2 mutant deficient in Gaq/11 binding, overexpression of eGFP-tagged inositol 1,4,5trisphosphate biosensor eGFP-PHPLCd leads to suppression of M1 muscarinic acetylcholine receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism via binding of Gaq/11 to the RGS homology domain of the enzyme [60]; construction of transgenic tissue specific or knockout mice [45]; isolated mutant grk26936 shows developmental defects throughout the life cycle of the fly, phenotype overview, construction of several transgenic flies expressing grk2 under control of the hsp70 or germline-specific promoter, analysis of germline mosaic [42]; phenotypes of several GRK and arrestin knockout mice mutants and of transgenic mice overexpressing GRK2 or GRK3, overview [68]; targeted tissue-specific overexpression of GRK2 in mouse osteoblasts attenuates parathyroid hormone receptor PTH signaling, and promotes bone loss with decreased osteoclastic activity and decreased osteoblasts and trabecular bone, equal effects in female and male mice, overview [41]; transient overexpression of wild-type GRK2 in NG108-15 mouse neuroblastoma x rat glioma hybrid cells leads to selective inhibition of A2 adenosine receptor responsiveness, but does not affect secretin-stimulated cyclic AMP accumulation [44]; truncated GRK3DC lacking the C-terminal Gbg binding domain is unable to induce germinal vesicle breakdown, and is defective in membrane association [55]; construction of GRK2-deficient cells by expression of siRNA leads to increased insulin-induced glycogen synthesis and basal and insulin-stimulated phosphorylation of Ser21 in glycogen synthase kinase-3a, phosphorylation of insulin receptor substrate 1 ia increased in case of GRK2 depletion [79]; expression of GRK2-specific siRNA leads to downregulation of the enzyme [71]; homozygous GRK2-deficient mice are embryonically lethal, heterozygous deficient mice show reduced activity an an altered phenotype, overview [75]; knockdown of endogenous GRK2 by short hairpin RNA significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2 [82]; lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function, phenotype, overview [85]; mutation of the clathrin-binding motif of GRK2 results in a dominant negative form of GRK2 without affecting kinase activity itself and the ability to interact with b2 -adrenergic receptor, no translocation of b-adrenergic receptors to endocytic vesicles as well as GRK2 translocation to the plasma membrane with the dominant-negative mutant and mutant GRK2-5A, overview [76]) [15, 27, 41, 42, 44, 45, 48, 54, 55, 56, 57, 60, 66, 68, 71, 75, 76, 79, 82, 85] 439

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Application biotechnology ( engineering of a GRK2 mutant sensitive to a specific inhibitor [62]) [62] diagnostics ( GRK2 levels in heart and peripheral lymphocytes correlate well, therefore the lymphocytic enzyme level might be a very suitable marker for determination for the sympathetic drive to heart failure during clinical course and treatment of human congestive heart failure patients [46]) [46] medicine ( heart failure: therapeutic strategy by manipulating bAR signaling, specifically through the inhibition of b-ARK 1, elevated levels of b-ARK 1 are an early ubiquitous consequence of myocardial injury [28]; effectiveness of in vivo applications of b-ARK 1-targeted gene therapy at ameliorating heart failure, b-ARK 1 upregulation often precedes the development of measurable heart failure and may represent an indicator for cardiac injury and potential therapeutic intervention prior to clinical dysfunction [28]) [28]

6 Stability Organic solvent stability Triton X-100 ( stabilizes [2,17]; 0.02% [17]) [2, 17] General stability information , glycerol stabilizes [51] , Triton X-100 stabilizes [2, 17] Storage stability , -80 C, purified recombinant His-tagged wild-type and mutant GRK2s, 50 mM NaH2 PO4, pH 8.0, 300 mM NaCl, 250 mM imidazole, 30% glycerol, stable for 2 months [62] , 4 C, crude enzyme: several months, stable, purified enzyme: t1=2 of 5-10 days [20] , 4 C, in presence of Triton X-100, 1 year, stable [2] , 4 C, several months, stable [21]

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[57] Eichmann, T.; Lorenz, K.; Hoffmann, M.; Brockmann, J.; Krasel, C.; Lohse, M.J.; Quitterer, U.: The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gb g binding site. J. Biol. Chem., 278, 8052-8057 (2003) [58] Pao, C.S.; Benovic, J.L.: Structure/function analysis of a2 A-adrenergic receptor interaction with G protein-coupled receptor kinase 2. J. Biol. Chem., 280, 11052-11058 (2005) [59] Kim, K.M.; Gainetdinov, R.R.; Laporte, S.A.; Caron, M.G.; Barak, L.S.: G protein-coupled receptor kinase regulates dopamine D3 receptor signaling by modulating the stability of a receptor-filamin-b-arrestin complex. A case of autoreceptor regulation. J. Biol. Chem., 280, 12774-12780 (2005) [60] Willets, J.M.; Nahorski, S.R.; Challiss, R.A.: Roles of phosphorylation-dependent and -independent mechanisms in the regulation of M1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 in hippocampal neurons. J. Biol. Chem., 280, 18950-18958 (2005) [61] Wu, J.H.; Goswami, R.; Kim, L.K.; Miller, W.E.; Peppel, K.; Freedman, N.J.: The platelet-derived growth factor receptor-b phosphorylates and activates G protein-coupled receptor kinase-2. A mechanism for feedback inhibition. J. Biol. Chem., 280, 31027-31035 (2005) [62] Kenski, D.M.; Zhang, C.; von Zastrow, M.; Shokat, K.M.: Chemical genetic engineering of G protein-coupled receptor kinase 2. J. Biol. Chem., 280, 35051-35061 (2005) [63] Liebler, J.M.; Borok, Z.; Li, X.; Zhou, B.; Sandoval, A.J.; Kim, K.J.; Crandall, E.D.: Alveolar epithelial type I cells express b2 -adrenergic receptors and Gprotein receptor kinase 2. J. Histochem. Cytochem., 52, 759-767 (2004) [64] Pandalai, P.K.; Lyons, J.M.; Duffy, J.Y.; McLean, K.M.; Wagner, C.J.; Merrill, W.H.; Pearl, J.M.; Akhter, S.A.: Role of the b-adrenergic receptor kinase in myocardial dysfunction after brain death. J. Thorac. Cardiovasc. Surg., 130, 1183-1189 (2005) [65] Picascia, A.; Capobianco, L.; Iacovelli, L.; De Blasi, A.: Analysis of differential modulatory activities of GRK2 and GRK4 on Gaq-coupled receptor signaling. Methods Enzymol., 390, 337-353 (2004) [66] Jimenez-Sainz, M.C.; Murga, C.; Kavelaars, A.; Jurado-Pueyo, M.; Krakstad, B.F.; Heijnen, C.J.; Mayor, F., Jr.; Aragay, A.M.: G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits. Mol. Biol. Cell, 17, 25-31 (2006) [67] Usui, I.; Imamura, T.; Babendure, J.L.; Satoh, H.; Lu, J.C.; Hupfeld, C.J.; Olefsky, J.M.: G protein-coupled receptor kinase 2 mediates endothelin-1induced insulin resistance via the inhibition of both Gaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes. Mol. Endocrinol., 19, 2760-2768 (2005) [68] Kohout, T.A.; Lefkowitz, R.J.: Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol. Pharmacol., 63, 918 (2003) [69] Willets, J.M.; Mistry, R.; Nahorski, S.R.; Challiss, R.A.: Specificity of G protein-coupled receptor kinase 6-mediated phosphorylation and regulation of

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[70] [71]

[72] [73]

[74]

[75]

[76]

[77] [78]

[79] [80]

[81]

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single-cell M3 muscarinic acetylcholine receptor signaling. Mol. Pharmacol., 64, 1059-1068 (2003) Lodowski, D.T.; Pitcher, J.A.; Capel, W.D.; Lefkowitz, R.J.; Tesmer, J.J.: Keeping G proteins at bay: a complex between G protein-coupled receptor kinase 2 and Gbg. Science, 300, 1256-1262 (2003) Banday, A.A.; Fazili, F.R.; Lokhandwala, M.F.: Insulin causes renal dopamine D1 receptor desensitization via GRK2-mediated receptor phosphorylation involving phosphatidylinositol 3-kinase and protein kinase C. Am. J. Physiol. Renal Physiol., 293, F877-F884 (2007) Sanchez-Perez, A.; Kumar, S.; Cook, D.I.: GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2. Biochem. Biophys. Res. Commun., 359, 611-615 (2007) Ribas, C.; Penela, P.; Murga, C.; Salcedo, A.; Garcia-Hoz, C.; Jurado-Pueyo, M.; Aymerich, I.; Mayor, F.: The G protein-coupled receptor kinase (GRK) interactome: Role of GRKs in GPCR regulation and signaling. Biochim. Biophys. Acta, 1768, 913-922 (2007) Whalen, E.J.; Foster, M.W.; Matsumoto, A.; Ozawa, K.; Violin, J.D.; Que, L.G.; Nelson, C.D.; Benhar, M.; Keys, J.R.; Rockman, H.A.; Koch, W.J.; Daaka, Y.; Lefkowitz, R.J.; Stamler, J.S.: Regulation of b-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2. Cell, 129, 511-522 (2007) Peregrin, S.; Jurado-Pueyo, M.; Campos, P.M.; Sanz-Moreno, V.; Ruiz-Gomez, A.; Crespo, P.; Mayor, F.; Murga, C.: Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK. Curr. Biol., 16, 2042-2047 (2006) Mangmool, S.; Haga, T.; Kobayashi, H.; Kim, K.M.; Nakata, H.; Nishida, M.; Kurose, H.: Clathrin required for phosphorylation and internalization of b2 -adrenergic receptor by G protein-coupled receptor kinase 2 (GRK2). J. Biol. Chem., 281, 31940-31949 (2006) Sherrill, J.D.; Miller, W.E.: G protein-coupled receptor (GPCR) kinase 2 regulates agonist-independent Gq/11 signaling from the mouse cytwlovirus GPCR M33. J. Biol. Chem., 281, 39796-39805 (2006) Ruiz-Gomez, A.; Mellstroem, B.; Tornero, D.; Morato, E.; Savignac, M.; Holguin, H.; Aurrekoetxea, K.; Gonzalez, P.; Gonzalez-Garcia, C.; Cena, V.; Mayor, F.; Naranjo, J.R.: G protein-coupled receptor kinase 2-mediated phosphorylation of downstream regulatory element antagonist modulator regulates membrane trafficking of Kv4.2 potassium channel. J. Biol. Chem., 282, 1205-1215 (2007) Shahid, G.; Hussain, T.: GRK2 negatively regulates glycogen synthesis in mouse liver FL83B cells. J. Biol. Chem., 282, 20612-20620 (2007) Takahashi, M.; Uchikado, H.; Caprotti, D.; Weidenheim, K.M.; Dickson, D.W.; Ksiezak-Reding, H.; Pasinetti, G.M.: Identification of G-protein coupled receptor kinase 2 in paired helical filaments and neurofibrillary tangles. J. Neuropathol. Exp. Neurol., 65, 1157-1169 (2006) Desai, A.N.; Salim, S.; Standifer, K.M.; Eikenburg, D.C.: Involvement of G protein-coupled receptor kinase (GRK) 3 and GRK2 in down-regulation of the a2 B-adrenoceptor. J. Pharmacol. Exp. Ther., 317, 1027-1035 (2006)

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[82] Meloni, A.R.; Fralish, G.B.; Kelly, P.; Salahpour, A.; Chen, J.K.; WechslerReya, R.J.; Lefkowitz, R.J.; Caron, M.G.: Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol. Cell. Biol., 26, 75507560 (2006) [83] Vinge, L.E.; Andressen, K.W.; Attramadal, T.; Andersen, G.?.; Ahmed, M.S.; Peppel, K.; Koch, W.J.; Freedman, N.J.; Levy, F.O.; Skomedal, T.; Osnes, J.B.; Attramadal, H.: Substrate specificities of G protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function. Mol. Pharmacol., 72, 582-591 (2007) [84] Lymperopoulos, A.; Rengo, G.; Funakoshi, H.; Eckhart, A.D.; Koch, W.J.: Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure. Nat. Med., 13, 315-323 (2007) [85] Molnar, C.; Holguin, H.; Mayor, F.; Ruiz-Gomez, A.; de Celis, J.F.: The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila. Proc. Natl. Acad. Sci. USA, 104, 7963-7968 (2007) [86] Hansen, J.L.; Theilade, J.; Aplin, M.; Sheikh, S.P.: Role of G-protein-coupled receptor kinase 2 in the heart–do regulatory mechanisms open novel therapeutic perspectives?. Trends Cardiovasc. Med., 16, 169-177 (2006) [87] Yang, W.; Xia, S.: Mechanisms of regulation and function of G-proteincoupled receptor kinases. World J. Gastroenterol., 12, 7753-7757 (2006)

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1 Nomenclature EC number 2.7.11.16 Systematic name ATP:[G-protein-coupled receptor] phosphotransferase Recommended name G-protein-coupled receptor kinase Synonyms G protein-coupled receptor kinase [13, 25] G protein-coupled receptor kinase 4 [11, 19, 20, 22, 23, 24] G protein-coupled receptor kinase 5 [10, 12, 14, 15, 16, 20, 26, 29] G protein-coupled receptor kinase 6 [11, 12, 16, 21] G protein-coupled receptor kinase GRK4 [1, 2, 3] G protein-coupled receptor kinase GRK5 [4, 5, 9] G protein-coupled receptor kinase GRK6 [6, 8] G protein-coupled receptor kinase-6 [28] G-protein coupled receptor kinase [17] G-protein-coupled receptor kinase 5 [30] G-protein-coupled receptor kinase 6 [18] GPCR kinase [10, 19] GRK [25] GRK-6 [28] GRK4 [11, 13, 17, 19, 20, 22, 23, 24, 26, 31] GRK5 [4, 5, 10, 12, 13, 14, 15, 16, 17, 20, 26, 29, 30, 31] GRK6 [6, 7, 11, 12, 13, 16, 17, 18, 21, 26, 27, 29, 31] by G protein-coupled receptor kinase 6 [27] Additional information ( enzymes belong to the GRK family, GRK4, GRK5, and GRK6 form the GRK4 subfamily [13]; GRK4 belongs to the GRK4 subfamily of the GRK family [22]; GRK5 is a member of the GRK family [10]; GRK6 belongs to the GRK4 subfamily of the GRK family [18]; the enzyme belongs to the GRK4 subfamily of the GRK family [19]; the enzyme belongs to the GRK4-6 family [16]; GRK6 is a member of the GRK4 subfamily of GRKs [28]; the enzyme belongs to the GRK4 subfamily [26]; the enzyme belongs to the GRK4 subfamily, consisting of GRK4, GRK5 and GRK6 [31]) [10, 13, 16, 18, 19, 22, 26, 28, 31] CAS registry number 127407-08-3

448

2.7.11.16

G-Protein-coupled receptor kinase

2 Source Organism Mus musculus (no sequence specified) [10, 13, 18, 20, 25, 29] Homo sapiens (no sequence specified) [12, 13, 14, 17, 19, 21, 22, 23, 24, 25, 26, 28, 30, 31] Rattus norvegicus (no sequence specified) [11, 14, 16, 27] Bos taurus (no sequence specified) [15] Homo sapiens (UNIPROT accession number: P32298) [1,2,3] Homo sapiens (UNIPROT accession number: P34947) [4] Bos taurus (UNIPROT accession number: P43249) [5] Homo sapiens (UNIPROT accession number: P43250) [6,7] Rattus norvegicus (UNIPROT accession number: P97711) [8] Rattus norvegicus (UNIPROT accession number: Q62833) [9]

3 Reaction and Specificity Catalyzed reaction ATP + [G protein-coupled receptor] = ADP + [G protein-coupled receptor]phosphate ( regulation mechanism [13]) Natural substrates and products S ATP + BLT1 receptor ( GRK6, ablation of GRK6 leads to augmented signaling by leukotriene B4 acting through the BLT1 receptor [25]) (Reversibility: ?) [25] P ADP + phosphorylated BLT1 receptor S ATP + CXCR4 receptor ( GRK6, the pathway is important in facilitating neutrophil retention in the bone marrow [25]) (Reversibility: ?) [25] P ADP + phosphorylated CXCR4 receptor S ATP + G protein-coupled receptor ( phosphorylation has a regulatory role [20]; regulation mechanism of GRK5, overview, regulation by phosphorylation at specific sites via distinct specific kinases, overview [13]) (Reversibility: ?) [13, 2] P ADP + phosphorylated G protein-coupled receptor S ATP + G-protein-coupled receptor ( leading to receptor endocytosis [31]) (Reversibility: ?) [31] P ADP + phosphorylated G-protein-coupled receptor S ATP + [G protein-coupled receptor] ( GRK4-6 [22]) (Reversibility: ?) [22] P ADP + [G protein-coupled receptor]phosphate S ATP + [G-protein-coupled receptor] (Reversibility: ?) [18] P ADP + [G-protein-coupled receptor]phosphate

449

G-Protein-coupled receptor kinase

2.7.11.16

S ATP + [M3 muscarinic acetylcholine receptor] ( GRK6 plays a major role in specific M3 muscarinic acetylcholine receptor regulation [21]) (Reversibility: ?) [21] P ADP + phospho-[M3 muscarinic acetylcholine receptor] S ATP + [TSH receptor] ( GRK4-6, receptor activation [17]) (Reversibility: ?) [17] P ADP + [TSH receptor]phosphate S ATP + [dopamine D1 receptor] ( GRK4 constitutively phosphorylates active and inactive receptor, in the latter case diminishing stimulation of the receptor by dopamine, phosphorylation reduces receptor desensitization and internalization, followed by reduced cAMP accumulation [22]) (Reversibility: ?) [22] P ADP + [dopamine D1 receptor]phosphate S ATP + a G protein-coupled receptor ( the GRKs are important in the cardiovascular system, the major G protein-coupled receptor regulatory pathway involves phosphorylation of activated receptors by GRKs, followed by binding of arrestin proteins, which prevent receptors from activating downstream heterotrimeric G protein pathways while allowing activation of arrestin-dependent signaling pathways, general mechanisms of GRK-arrestin regulation, overview, physiological functions and potential pathophysiological roles of GRKs and arrestins in human disorders, overview [25]) (Reversibility: ?) [25] P ADP + a phosphorylated G protein coupled receptor S ATP + a-synuclein ( GRK5 phosphorylates Ser-129 of a-synuclein at the plasma membrane and induces translocation of phosphorylated a-synuclein to the perikaryal area, GRK5 promotes a-linolenic acid-induced oligomerization of a-synuclein, a-synuclein phosphorylation by GRK5 plays a crucial role in the pathogenesis of sporadic Parkinsons disease, sPD [30]) (Reversibility: ?) [30] P ADP + phosphorylated a-synuclein S ATP + b-adrenergic receptor ( desensitization of the receptor by GRK4, GRK5, and GRK6 [20]) (Reversibility: ?) [20] P ADP + phosphorylated b-adrenergic receptor S ATP + central M2 muscarinic receptor ( desensitization of the receptor by GRK5, GRK5 regulates pulmmonary responses by activation of the airway receptor, but does not regulate the peripheral cardiac muscarinic receptors [10]) (Reversibility: ?) [10] P ADP + phosphorylated central M2 muscarinic receptor S ATP + dopamine D1 receptor ( rapid desensitization of the receptor by GRK4 and GRK6, Na+ /H+ exchanger activity of the receptor, overview [11]; dopamine D1 receptors in IEC-6 rapidly desensitize to D1-like agonist stimulation and GRK 6 isozymes A and B, but not GRK 4, appear to be involved in agonist-mediated responsiveness and desensitization [27]; GRK4 [25]) (Reversibility: ?) [11, 25, 27] P ADP + phosphorylated dopamine D1 receptor S ATP + protein p105 (Reversibility: ?) [29] P ADP + phosphorylated protein p105 450

2.7.11.16

G-Protein-coupled receptor kinase

S ATP + rhodopsin (Reversibility: ?) [28] P ADP + phosphorylated rhodopsin S Additional information ( enzyme is involved in fertilization [1]; enzyme mainly involved in homologous desensitization of the TSH receptor [9]; G protein-coupled receptors are involved in the regulation of diverse physiological processes, mechanisms of G protein-coupled receptor desensitization, e.g. by phosphorylation or feedback inhibition, overview [20]; GRK5 and especially GRK6 have selective regulatory roles in cAMP accumulation response of the secretin receptor to agonists like b-arrestin-1 or forskolin, overview [12]; GRK5 defects can cause obstructive airway diseases such as asthma [10]; GRK5 elevates blood pressure in vascular smooth muscle via Gi signaling involing the b1 -adrenergic receptor, mechanism overview [15]; GRKs are involved in diverse physiological processes and pathologies, overview [13]; the enzyme is involved in GPCR signal transduction pathways and desensitization [19]; the GRK4 isozymes are differentially regulated, overview [22]; the GRK4-6 family member enzymes mediate b-adrenergic receptor desensitization, the b-adrenergic receptor is stimulated by agonists such as isoproterenol, cholera toxin, or forskolin, and induces cAMP production [16]; the variable C-terminal extension of GRK6 constitutes an accessorial autoregulatory domain [18]; arrestin-2 and GRK5, not GRK6, interact with NFkB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages, overview [29]; GRK activity is regulated by phosphorylation through several kinases and by interactions with several cellular proteins, e.g. calmodulin, caveolin or RKIP, GRK also interacts with PI3K, Akt, GIT or MEK, the interactions occur at the RH and PH domains, overview, the GRK interactome: role of GRKs in GPCR regulation and signaling, detailed overview [26]; GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization, b-arrestin-mediated receptor internalization, activity of GRKs and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calcium-sensitive proteins [31]; GRK4 is involved in activity of dopamine receptors in renal proximal tubules and mediates sodium reabsorption and blood pressure regulation [23,24]; GRK6 is a key regulator of dopaminergic signaling and lymphocyte chemotaxis [28]) (Reversibility: ?) [1, 9, 10, 12, 13, 15, 16, 18, 19, 20, 22, 23, 24, 26, 28, 29, 31] P ? Substrates and products S ATP + BLT1 receptor ( GRK6, ablation of GRK6 leads to augmented signaling by leukotriene B4 acting through the BLT1 receptor [25]; GRK6 [25]) (Reversibility: ?) [25] P ADP + phosphorylated BLT1 receptor

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G-Protein-coupled receptor kinase

2.7.11.16

S ATP + CXCR4 receptor ( GRK6, the pathway is important in facilitating neutrophil retention in the bone marrow [25]; GRK6 [25]) (Reversibility: ?) [25] P ADP + phosphorylated CXCR4 receptor S ATP + G protein-coupled receptor ( phosphorylation has a regulatory role [20]; regulation mechanism of GRK5, overview, regulation by phosphorylation at specific sites via distinct specific kinases, overview [13]; the GPCRs possess multiple phosphorylation sites for serine/threonine kinases [20]) (Reversibility: ?) [13, 2] P ADP + phosphorylated G protein-coupled receptor S ATP + G-protein-coupled receptor ( leading to receptor endocytosis [31]) (Reversibility: ?) [31] P ADP + phosphorylated G-protein-coupled receptor S ATP + [G protein-coupled receptor] ( GRK4-6 [22]) (Reversibility: ?) [22] P ADP + [G protein-coupled receptor]phosphate S ATP + [G-protein-coupled receptor] (Reversibility: ?) [18] P ADP + [G-protein-coupled receptor]phosphate S ATP + [M2 muscarinic receptor] ( GRK4-6 [22]) (Reversibility: ?) [22] P ADP + [M2 muscarinic receptor]phosphate S ATP + [M3 muscarinic acetylcholine receptor] ( GRK6 plays a major role in specific M3 muscarinic acetylcholine receptor regulation [21]) (Reversibility: ?) [21] P ADP + phospho-[M3 muscarinic acetylcholine receptor] S ATP + [TSH receptor] ( GRK4-6 [17]; GRK4-6, receptor activation [17]) (Reversibility: ?) [17] P ADP + [TSH receptor]phosphate S ATP + [b2 -adrenergic receptor] ( GRK4-6 [22]) (Reversibility: ?) [22] P ADP + [b2 -adrenergic receptor]phosphate S ATP + [dopamine D1 receptor] ( GRK4 constitutively phosphorylates active and inactive receptor, in the latter case diminishing stimulation of the receptor by dopamine, phosphorylation reduces receptor desensitization and internalization, followed by reduced cAMP accumulation [22]; GRK4 phosphorylates active and inactive receptor [22]) (Reversibility: ?) [22] P ADP + [dopamine D1 receptor]phosphate S ATP + [follicle-stimulating hormone receptor] ( GRK4-6 [22]) (Reversibility: ?) [22] P ADP + [follicle-stimulating hormone receptor]phosphate S ATP + [luteinizing hormone/chorionic gonadotropin receptor] ( GRK4-6 [22]) (Reversibility: ?) [22] P ADP + [luteinizing hormone/chorionic gonadotropin receptor]phosphate S ATP + [rhodopsin] ( GRK4-6 [22]) (Reversibility: ?) [22] P ADP + [rhodopsin]phosphate

452

2.7.11.16

G-Protein-coupled receptor kinase

S ATP + a G protein-coupled receptor ( the GRKs are important in the cardiovascular system, the major G protein-coupled receptor regulatory pathway involves phosphorylation of activated receptors by GRKs, followed by binding of arrestin proteins, which prevent receptors from activating downstream heterotrimeric G protein pathways while allowing activation of arrestin-dependent signaling pathways, general mechanisms of GRK-arrestin regulation, overview, physiological functions and potential pathophysiological roles of GRKs and arrestins in human disorders, overview [25]; cell surface localized receptors [25]) (Reversibility: ?) [25] P ADP + a phosphorylated G protein-coupled receptor S ATP + activated form of G protein-coupled receptors (Reversibility: ?) [3] P ADP + phosphorylated G protein-coupled receptors S ATP + a-synuclein ( GRK5 phosphorylates Ser-129 of a-synuclein at the plasma membrane and induces translocation of phosphorylated a-synuclein to the perikaryal area, GRK5 promotes a-linolenic acid-induced oligomerization of a-synuclein, a-synuclein phosphorylation by GRK5 plays a crucial role in the pathogenesis of sporadic Parkinsons disease, sPD [30]; phosphorylation at Ser129 [30]) (Reversibility: ?) [30] P ADP + phosphorylated a-synuclein S ATP + b2-adrenergic receptor ( phosphorylation in an agonist-dependent manner, phosphorylates the C-terminal tail regions of both receptor proteins [5]) (Reversibility: ?) [5, 7] P ADP + phosphorylated b2-adrenergic receptor S ATP + b-adrenergic receptor ( desensitization of the receptor by GRK4, GRK5, and GRK6 [20]) (Reversibility: ?) [20] P ADP + phosphorylated b-adrenergic receptor S ATP + central M2 muscarinic receptor ( desensitization of the receptor by GRK5, GRK5 regulates pulmmonary responses by activation of the airway receptor, but does not regulate the peripheral cardiac muscarinic receptors [10]; GRK5 [10]) (Reversibility: ?) [10] P ADP + phosphorylated central M2 muscarinic receptor S ATP + dopamine D1 receptor ( rapid desensitization of the receptor by GRK4 and GRK6, Na+ /H+ exchanger activity of the receptor, overview [11]; GRK4 and GRK6 [11]; dopamine D1 receptors in IEC-6 rapidly desensitize to D1-like agonist stimulation and GRK 6 isozymes A and B, but not GRK 4, appear to be involved in agonist-mediated responsiveness and desensitization [27]; GRK4 [25]) (Reversibility: ?) [11, 25, 27] P ADP + phosphorylated dopamine D1 receptor S ATP + protein ( major autophosphorylation sites are Ser484 and Thr485 [5]) (Reversibility: ?) [5] P ADP + phosphoprotein

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G-Protein-coupled receptor kinase

2.7.11.16

S ATP + protein p105 ( recombinant GST-tagged p105, residues 497-968, substrate, GRK5 binds and phosphorylates p105, to which arrestin-2 is already C-terminally bound [29]) (Reversibility: ?) [29] P ADP + phosphorylated protein p105 S ATP + rhodopsin ( GRK5 phosphorylates rhodopsin in a light-dependent manner, phosphorylates the C-terminal tail region [5]; reaction only with GRK4a isoform, no reaction with GRK4b, GRK4g, GRK4d [1]; light-activated rhodopsin, recombinant isoforms mGRK6-A, mGRK6-B, and mGRK-C, the latter showing highest activity [18]; urea treated outer segments of rhodopsin [16]) (Reversibility: ?) [1, 5, 7, 16, 18, 28] P ADP + phosphorylated rhodopsin S Additional information ( substrate specificity [19]; GRK6, but not other GRKs tested, incorporated tritium after incubation with [3 H]palmitate in Sf9 and in COS-7 cells overexpressing the kinase [6]; enzyme is involved in fertilization [1]; enzyme mainly involved in homologous desensitization of the TSH receptor [9]; G protein-coupled receptors are involved in the regulation of diverse physiological processes, mechanisms of G protein-coupled receptor desensitization, e.g. by phosphorylation or feedback inhibition, overview [20]; GRK5 and especially GRK6 have selective regulatory roles in cAMP accumulation response of the secretin receptor to agonists like barrestin-1 or forskolin, overview [12]; GRK5 defects can cause obstructive airway diseases such as asthma [10]; GRK5 elevates blood pressure in vascular smooth muscle via Gi signaling involing the b1 adrenergic receptor, mechanism overview [15]; GRKs are involved in diverse physiological processes and pathologies, overview [13]; the enzyme is involved in GPCR signal transduction pathways and desensitization [19]; the GRK4 isozymes are differentially regulated, overview [22]; the GRK4-6 family member enzymes mediate badrenergic receptor desensitization, the b-adrenergic receptor is stimulated by agonists such as isoproterenol, cholera toxin, or forskolin, and induces cAMP production [16]; the variable C-terminal extension of GRK6 constitutes an accessorial autoregulatory domain [18]; GRK5 performs autophosphorylation [13]; structure-activity relationships of the enzyme C-terminus, overview [18]; the GRKs are specific for GPCRs and arrestins, overview [20]; arrestin-2 and GRK5, not GRK6, interact with NFkB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages, overview [29]; GRK activity is regulated by phosphorylation through several kinases and by interactions with several cellular proteins, e.g. calmodulin, caveolin or RKIP, GRK also interacts with PI3K, Akt, GIT or MEK, the interactions occur at the RH and PH domains, overview, the GRK interactome: role of GRKs in GPCR regulation and signaling, detailed overview [26]; GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization, b-arrestin-mediated receptor internalization, activity of GRKs 454

2.7.11.16

G-Protein-coupled receptor kinase

and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calciumsensitive proteins [31]; GRK4 is involved in activity of dopamine receptors in renal proximal tubules and mediates sodium reabsorption and blood pressure regulation [23,24]; GRK6 is a key regulator of dopaminergic signaling and lymphocyte chemotaxis [28]; GRK6 structure-function relationship determination and analysis [28]; no binding to RH domain of G protein Gaq and Ga-11 by GRK4 [31]) (Reversibility: ?) [1, 6, 9, 10, 12, 13, 15, 16, 18, 19, 20, 22, 23, 24, 26, 28, 29, 31] P ? Inhibitors actin [26] Ca2+ ( inhibits GRK5 [13]) [13] Ca2+ /calmodulin ( inhibit GRK5 with an IC50 of 40-50 nM, inhibition mechanism via inducing inhibitory autophosphorylation and blocking of membrane association [20]) [20] calmodulin ( reaction of isoenzyme GRK4a with rhodopsin, IC50: 80 nM [1]; inhibits GRK5 [13]) [1, 13, 26] heparin ( GRK inhibitor [16]; inhibits GRK6 [27]) [11, 16, 27] RKIP [26] actinin ( inhibits GRK5 [13]) [13] a-actinin [26] caveolin ( inhibits GRK5 [13]) [13, 26] Additional information ( GRK6 activity on the M3 muscarinic acetylcholine receptor is not affected by phorbol-12,13-dibutyrate and Ro 31-8220 [21]; phosphorylation by PKC inhibits GRK5 [13]; the variable C-terminal extension of GRK6 constitutes a domain with autoinhibitory function involving residues D560, S566, and L576 of 3 inhibitory elements and an intervening stimulatory element [18]) [13, 18, 21] Cofactors/prosthetic groups ATP [10, 11, 13, 16, 17, 18, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31] Activating compounds phosphatidylinositol 4,5-bisphosphate ( activates the receptor phosphorylation activity of GRK5, but not its autophosphorylation or peptide phosphorylation activity [13]) [13] phospholipids ( activate GRK5 [13]) [13] arrestins ( arrestins modulate the enzyme activity having a regulatory role, regulation of arrestins, overview [20]) [20] methacholine ( stimulation of M3 muscarinic acetylcholine receptor phosphorylation is reversible by atropine [21]) [21] Additional information ( autophosphorylation of GRK5 is stimulated by phospholipids, such as phosphatidylinositol-4,5-bisphosphate [20]; GRK6 activity on the M3 muscarinic acetylcholine receptor is not affected by phorbol-12,13-dibutyrate and Ro 31-8220 [21]; no interaction

455

G-Protein-coupled receptor kinase

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with G protein Gbg subunits by GRK4, which contains no N-terminal RGS domain [19]; phosphorylation activates the enzyme [26]) [19, 20, 21, 26] Metals, ions Ca2+ ( inhibits GRK5 [13]) [13] Mg2+ [16, 18, 20, 21, 22, 26, 27, 28, 29, 31] Specific activity (U/mg) Additional information ( physiologic activity of lungs in wild-type and GRK5-deficient mice, overview [10]) [10, 21] pH-Optimum 7.4 ( assay at [21]) [21] 7.5 ( assay at [16,18,29]) [16, 18, 29] Temperature optimum ( C) 30 ( assay at [16,18,29]) [16, 18, 29] 37 ( assay at [21]) [21]

4 Enzyme Structure Subunits ? ( x * 69000, GRK5, SDS-PAGE [26]) [26] dimer ( 2 * 66050, recombinant acetylated, full-length, palmitoylation-deficient GRK6, mass spectrometry [28]) [28] Additional information ( GRK family domain structure [19]; GRK4 subfamily multidomain structure, overview [13]; the variable C-terminal extension of GRK6 constitutes an accessorial autoregulatory domain consisting of 3 autoregulatory elements involving residues D560, S566, and L576 and an intervening stimulatory element [18]; domain structure of GRK4-6, overview [26]; GRK4, GRK5, and GRK6 lack the G protein bg-subunit binding domain but use direct PIP2 binding and/or covalent lipid modification with palmitate to reside primarily at the plasma membrane [25]; GRK6 structure-function relationship determination and analysis, the structure involves the RH-kinase domain core, and small and large kinase lobes, comparison to the structure of GRK2, EC 2.7.11.15, overview [28]) [13, 18, 19, 25, 26, 28] Posttranslational modification Lipoprotein ( palmitoylation of GRK6 appears essential for membrane association [6]; GRK4 is palmitoylated at the C-terminus [22]; GRK4-6 are permanently modulated by fatty acids and/or phospholipids [20]; the C-terminus of GRK6 is palmitoylated, not required for phosphorylation activity [18]; GRK6 is palmitoylated at Cys561, Cys562, and Cys565 [28]) [6, 18, 20, 22, 28] phosphoprotein ( major autophosphorylation sites are Ser484 and Thr485 [5]; GRK5 performs autophosphorylation and is phos-

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phorylated by protein kinase C [13]; regulation of GRK5 by phosphorylation through PKC, GRK5 is activated by autophosphorylation, autophosphorylation is stimulated by phospholipids, such as phosphatidylinositol4,5-bisphosphate, overview [20]; GRKs are regulated by phopshorylation [31]; phosphorylation, e.g. by PKA, PKC, ERK1/2 or c-SRC, activates the enzyme [26]) [5, 13, 20, 26, 31] Additional information ( mechanisms of regulation of GRK protein stability and degradation, e.g. via ubiquitination or protease cleavage, overview [13]) [13]

5 Isolation/Preparation/Mutation/Application Source/tissue IEC-6 cell ( intestinal epithelial cell line [11,27]) [11, 27] Purkinje cell ( GRK4 [25]) [25] SH-SY5Y cell ( neuroblastoma cell line, contains no GRK5 [21]) [21] T-lymphocyte [13] aorta [8] brain ( very weak activity [5]; expression of GRK4 in the brain is limited to cerebellar Purkinje cells [25]; GRK4 predominantly [31]; GRK5 accumulates in Lewy bodies and colocalizes with asynuclein in the pathological structures of the brains of sporadic Parkinsons disease patients [30]) [2, 5, 7, 8, 25, 30, 31] cell culture ( spermatogonia cell line GC-1 spg [1]) [1] cerebellum [25] colon [9] crypt [27] epithelium [11] germ [1] heart ( high activity [5]; GRK5 expression is increased in heart failure [31]) [5, 7, 8, 13, 14, 25, 31] kidney ( very weak activity [5]; GRK4 predominantly [31]) [5, 8, 9, 31] liver ( very weak activity [5]) [5, 7, 8] lung ( high activity [5]; GRK5 [10]) [5, 7, 8, 9, 10, 25] lymphocyte ( peripheral [14]) [14, 25] macrophage [29] myelomonocytic leukemia cell ( GRK6 [13]) [13] myometrium ( primary cells [16]) [16] neuron ( GRK4, GRK5, and GRK6 [25]) [25, 30] neutrophil [25] pancreas [7] placenta [7] retina ( high activity [5]) [5] skeletal muscle ( predominantly expressed in [8]) [7, 8] 457

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small intestine [8, 11] smooth muscle ( airway, GRK5 [10]) [10] sperm ( GRK4g is the only detectable isoform in human sperm [1]) [1] spermatozoon [1] stomach [8] substantia nigra [30] testis ( very weak activity [5]; GRK4, mainly [13]; GRK4 predominantly [31]) [1, 5, 13, 31] thymus [8] thyroid gland ( increased expression of GRK4 in hyperfunctioning thyroid nodule [31]) [9, 31] thyroid nodule ( prepared without lymphocytic infiltrations in the tumor, GRKs expression patterns, moderate expression level of GRK4 [17]) [17] tongue epithelium [5] trachea ( GRK5 [10]) [10] uterus [8] ventriculus [14] villus [27] Additional information ( most abundantly in lung, heart, retina, and lingual epithelium, but expressed very little in brain, liver, kidney, or testis [5]; analysis of GRK5 expression rate and mRNA level in heart and lymphocytes by RT-PCR [14]; GRK5 and GRK6 are expressed in a wide variety of tissues, expression patterns, overview [13]; wide tissue distribution of GRK5 and GRK6 [31]) [5, 13, 14, 31] Localization cell membrane ( association of GRK6 with the cell membrane is mediated in part by the palmitoylation of cysteine residues that lie in a Cterminal region [28]) [28] cytoplasm [25] cytosol [17] membrane ( GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do b-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively [5]; anchoring of GRK4 by palmitoyl residue at the C-terminus [22]; anchoring via palmitoylation [19]) [5, 17, 19, 22] plasma membrane ( constitutive association, location of GRK4-6 near the activated receptors, covalent attachment by fatty acids to the C-terminus, e.g. palmitoylated GRK4 and GRK6, or by electrostatic interactions with phospholipids, e.g. for GRK5 [20]; bound to GPC receptors [25]) [20, 25, 30] Additional information ( GRK4, GRK5, and GRK6 lack the G protein bg-subunit binding domain but use direct PIP2 binding and/or covalent lipid modification with palmitate to reside primarily at the plasma membrane [25]) [25]

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Purification (GRK2 by gel filtration from Sf9 cells) [20] (recombinant isozymes mGRK6-A, mGRK6-B, and mGRK-C from Sf9 insect cells by sequential gel filtration and heparin affinity chromatography) [18] [5] Cloning (expression of isoforms mGRK6-A, mGRK6-B, mGRK-C, and mGRK-D in Spodoptera frugiperda Sf9 cells via baculovirus infection system, transient expression of isoforms mGRK6-A, mGRK6-B, mGRK-C, and mGRK-D in COS-7 cells, recombinant isoforms mGRK6-A, mGRK6-B, and mGRK-C are membrane-associated, recombinant isoform mGRK6-D is inactive and located in the nucleus) [18] (GRK4, DNA and amino acid sequence determination of polymorphisms, genotyping of American twins) [23] (GRK4, DNA and amino acid sequence determination of polymorphisms, genotyping of Han Chinese individuals) [24] (co-expression of isozyme GRK4g and dopamine D1 receptor in HEK293 cells leads to phosphorylation of the receptor by GRK4, this effect is not seen with isozyme GRK4b and GRK4d) [22] (co-expression of rat thyrotropin receptor with GRK4, GRK5, and GRK6 in HEK-293 cells, receptor phosphorylation occurs with GRK5 and GRK6) [31] (expression of GRK6 in both Spodoptera frugiperda Sf9 and Trichoplusia ni High5 insect cells as a soluble, palmitoylation-deficient mutant in which three potential palmitoylation sites, located at Cys561, Cys562, and Cys565, are converted to Ser) [28] (expression of kinase-dead GRK4 mutant in HEK-293 cells) [19] (gene GRK5, co-expression with human a-synuclein in HEK-293 cells and co-localization in the plasma membrane, functional co-expression of GRK5 and a-synuclein in primary cortical neurons from the cerebral cortex of fetal mice) [30] (stable overexpression of wild-type and K215R mutant GRK6 in SHSY5Y neuroblastoma cells via adenovirus infection system) [21] (transient overexpression of wild-types and dominant negative mutants of GRK5 and GRK6 in NG108-15 mouse neuroblastoma x rat glioma hybrid cells) [12] (transient overexpression of wild-types and dominant negative mutants of GRK5 and GRK6 in COS-7 cells) [16] (gene encoding GRK5 with SM22a promoter for construction of transgenic mice expressing the enzyme in vascular smooth muscle) [15] Engineering A142V ( expression of the GRK4g mutant in transgenic mice leads to development of hypertension and lack of D1 agonist-induced diuresis and natriuresis in the mice [22]; naturally occuring functional polymorph-

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ism, the mutation leads to increased GRK4 activity and phosphorylation of dopamine receptors, phenotype [23,24]) [22, 23, 24] A486V ( naturally occuring functional polymorphism, the mutation leads to increased GRK4 activity and phosphorylation of dopamine receptors, phenotype [23,24]) [23, 24] C561S/C562S/C565S ( site-directed mutagenesis, mutation of palmitoylation sites, the mutant protein retains its ability to phosphorylate rhodopsin, albeit with a 5fold higher Km and 2fold lower Vmax compared with those of wild-type GRK6 [28]) [28] F527D ( site-directed mutagenesis, mutant structure in comparison to the wild-type enzyme [28]) [28] I165E ( site-directed mutagenesis, mutant structure in comparison to the wild-type enzyme [28]) [28] I165E/F527D ( site-directed mutagenesis, mutant structure in comparison to the wild-type enzyme [28]) [28] I39E ( site-directed mutagenesis, mutant structure in comparison to the wild-type enzyme [28]) [28] I39E/I165E ( site-directed mutagenesis, mutant structure in comparison to the wild-type enzyme [28]) [28] K215R ( inactive dominant negative mutants of GRK5 or GRK6, overexpression of the GRK5 mutant in myometrial cells does not influence badrenergic receptor sensitivity, while overexpression of GRK6 mutant leads to a 70% increase in isoproterenol-stimulated b-adrenergic signaling via the badrenergic receptor [16]; site-directed mutagenesis, dominant negative mutants of GRK5 or GRK6, overexpression of mutant GRK6 or mutant GRK5 in NG108-15 mouse neuroblastoma x rat glioma hybrid cells results in selective increase in secretin-stimulated cyclic AMP response, the dominant negative mutant of GRK5 has no effect on cAMP response [12]; site-directed mutagenesis, inactive, dominant negative mutant of GRK6, 30fold overexpression in SH-SY5Y cells produces a 50% suppression of endogenous M3 muscarinic acetylcholine receptor phosphorylation and desensitization by the wild-type enzyme, GRK5 K215R mutant overexpression has no effect [21]; the GRK5 mutant binds to a-synuclein but does not phosphorylate it [30]) [12, 16, 21, 30] K216M/K217M ( site-directed mutagenesis, kinase-dead GRK4 mutant, overexpression in HEK-293 cells leads to partially desensitization of GPCR [19]) [19] R65L ( naturally occuring functional polymorphism, the mutation leads to increased GRK4 activity and phosphorylation of dopamine receptors, phenotype [24]; naturally occuring functional polymorphism, the mutation leads to increased GRK4 activity and phosphorylation of dopamine receptors, the mutation plays a role in blood pressure regulation in adolescents and young adults, phenotype [23]) [23, 24] R65L/A142V/A486V ( co-expression in CHO cells with the dopamine D1 receptor causes enhanced desensitization and agonist-independent phosphorylation of the receptor [22]) [22]

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Additional information ( construction of GRK5 deletion mutant mice [10]; construction of isozymes mGRK6-A, mGRK6-B, mGRK-C, and mGRK-D by alternative splicing, recombinant isoforms mGRK6-A, mGRK6-B, and mGRK-C are membrane-associated, recombinant isoform mGRK6-D is inactive and located in the nucleus, structure-activity relationships of the enzyme C-terminus, overview [18]; construction of transgenic mice overexpressing GRK5 2fold in vascular smooth muscle leading to hypertension in the mutant mice with a 25-35% increase in blood pressure, which segregates with sex, male show higher blood pressure than female mice, and is dependent on Gi-mediated signaling, inhibition of the latter by pertussis toxin or inhibition of b1 -adrenergic receptor activity restore blood pressure to normal level [15]; construction of transgenic tissue specific or knockout mice [13]; overexpression of wild-type GRK6 increase the endogenous M3 muscarinic acetylcholine receptor phosphorylation and desensitization level [21]; phenotypes of several GRK and arrestin knockout mice mutants and of transgenic mice overexpressing GRK4 or GRK5, overview [20]; transient overexpression of wild-type GRK6 in NG10815 mouse neuroblastoma x rat glioma hybrid cells results in highly selective inhibition of secretin-stimulated cyclic AMP accumulation without afffecting the A2 adenosine receptor responsiveness to cAMP, while overexpression of wild-type GRK5 leads to a partly selective inhibition of secretin-stimulated cyclic AMP response and to inhibition of A2 adenosine receptor responsiveness [12]; GRK short hairpin RNA knockdown, GRK5 knockout results in altered central and lung M2 muscarinic receptor regulation, with normal heart M2 receptor regulation, while GRK4 knockout does not result in an altered phenotype, GRK6 knockout leads to altered central dopamine receptor regulation deficient lymphocyte chemotaxis, increased acute inflammation and neutrophil chemotaxis, positive correlation between certain GRK4 polymorphisms, or haplotypes, and hypertensive disease [25]; GRK short hairpin RNA knockdown, GRK5 knockout results in altered central and lung M2 muscarinic receptor regulation, with normal heart M2 receptor regulation, while GRK4 knockout does not result in an altered phenotype, GRK6 knockout leads to altered central dopamine receptor regulation deficient lymphocyte chemotaxis, increased acute inflammation and neutrophil chemotaxis, positive correlation between certain GRK4 polymorphisms, or haplotypes, and hypertensive disease, knockout mice phenotypes, detailed overview [25]; GRK5 knockdown results in enhanced IkB kinasemediated p105 phosphorylation and degradation, and correlates well with an enhanced LPS-stimulated ERK1/2 phosphorylation [29]; haplotypic association of the GRK5 gene with susceptibility to sporadic Parkinsons disease, the haplotype contains two functional single-nucleotide polymorphisms, m22.1 and m24, in introns of the GRK5 gene, which bind to Yin Yang1, YY1, and cAMP response element-binding protein 1, CREB-1, respectively, and increases transcriptional activity of the reporter gene, overview [30]) [10, 12, 13, 15, 18, 20, 21, 25, 29, 30]

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Application diagnostics ( GRK5 levels in heart and peripheral lymphocytes correlate well, therefore the lymphocytic enzyme level might be a very suitable marker for determining the sympathetic drive to heart failure during clinical course and treatment of human congestive heart failure patients [14]) [14]

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[26] Ribas, C.; Penela, P.; Murga, C.; Salcedo, A.; Garcia-Hoz, C.; Jurado-Pueyo, M.; Aymerich, I.; Mayor, F.: The G protein-coupled receptor kinase (GRK) interactome: Role of GRKs in GPCR regulation and signaling. Biochim. Biophys. Acta, 1768, 913-922 (2007) [27] Fraga, S.; Luo, Y.; Jose, P.; Zandi-Nejad, K.; Mount, D.B.; Soares-da-Silva, P.: Dopamine D1-like receptor-mediated inhibition of Cl/HCO3 -exchanger activity in rat intestinal epithelial IEC-6 cells is regulated by G proteincoupled receptor kinase 6 (GRK 6). Cell Physiol. Biochem., 18, 347-360 (2006) [28] Lodowski, D.T.; Tesmer, V.M.; Benovic, J.L.; Tesmer, J.J.: The structure of G protein-coupled receptor kinase (GRK)-6 defines a second lineage of GRKs. J. Biol. Chem., 281, 16785-16793 (2006) [29] Parameswaran, N.; Pao, C.S.; Leonhard, K.S.; Kang, D.S.; Kratz, M.; Ley, S.C.; Benovic, J.L.: Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages. J. Biol. Chem., 281, 34159-34170 (2006) [30] rawaka, S.; Wada, M.; Goto, S.; Karube, H.; Sakamoto, M.; Ren, C.H.; Koyama, S.; Nagasawa, H.; Kimura, H.; Kawanami, T.; Kurita, K.; Tajima, K.; Daimon, M.; Baba, M.; Kido, T.; Saino, S.; Goto, K.; Asao, H.; Kitanaka, C.; Takashita, E.; Hongo, S.; Nakamura, T.; Kayama, T.; Suzuki, Y.; Kobayashi, K.; Kat, K.a.t.a.: The role of G-protein-coupled receptor kinase 5 in pathogenesis of sporadic Parkinsons disease. J. Neurosci., 26, 9227-9238 (2006) [31] Yang, W.; Xia, S.: Mechanisms of regulation and function of G-proteincoupled receptor kinases. World J. Gastroenterol., 12, 7753-7757 (2006)3

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