Calcium Regulation of Cellular Function, Volume 30 (Advances in Second Messenger & Phosphoprotein Research) [1 ed.] 0120361302, 9780120361304, 9780080552736

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ADVANCES IN SECOND MESSENGER AND PHOSPHOPROTEIN RESEARCH Volume 30

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Calcium Regulation of Cellular Function

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Advances in Second Messenger and Phosphoprotein Research

Series Editors

Paul Greengard, New York, New York Angus C. Nairn, New York, New York Shirish Shenolikar, Durham, North Carolina

International Advisory Board Michael J. Berridge, Cambridge, England (United Kingdom) Ernesto Carafoli, Zurich, Switzerland E. Costa, Washington, D.C. Pedro Cuatrecases, Ann Arbor, Michigan Raymond L. Erikson, Cambridge, Massachusetts Alfred G. Gilman, Dallas, Texas Joel G. Hardman, Nashville, Tennessee Tony Hunter, San Diego, California Claude B. Klee, Bethesda, Maryland Edwin G. Krebs, Seattle, Washington Yasutomi Nishizuka, Kobe, Japan Ira H. Pastan, Bethesda, Maryland G. Alan Robinson, Houston, Texas Martin Rodbell, Research Triangle Park, North Carolina Michael J. Welsh, Iowa City, Iowa Keith R. Yamamoto, San Francisco, California

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ADVANCES IN SECOND MESSENGER AND PHOSPHOPROTEIN RESEARCH Volume 30

Calcium Regulation of Cellular Function

Editor

Anthony R. Means, PH.D. Department of Pharmacology Duke University Medical Center Durham, North Carolina

Raven Press

~

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New York

Raven Press, Ltd., 1185 Avenue of the Americas, New York, New York 10036 9 1995 No part form or without

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by Raven Press, Ltd. All rights reserved. This book is protected by copyright. of it may be reproduced, stored in a retrieval system, or transmitted, in any by any means, electronical, mechanical, photocopying, recording, or otherwise, the prior written permission of the publisher.

Made in the United States of America

International Standard Book Number 0-7817-0233-X The material contained in this volume was submitted as previously unpublished material, except in the instances in which credit has been given to the source from which some of the illustrative material was derived. Great care has been taken to maintain the accuracy of the information contained in the volume. However, neither Raven Press nor the editors can be held responsible for errors or for any consequences arising from the use of the information contained herein. 987654321

Contents

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Contributing Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.

Intracellular Calcium Waves

1

...........................

David E. Clapham and James Sneyd 2.

ix

Regulation of Calcium Channels in the Heart . . . . . . . . . . . . . .

25

Donald L. Campbell and Harold C. Strauss 3.

Determinants that Govern High Affinity Calcium Binding

...

89

Calcium Regulation of Smooth Muscle Contractile Proteins . . . .

153

Sara Linse and Sture Forsen 4.

J. David Johnson and Christopher H. Snyder 5.

Calcium-Dependent Protein Kinases in Learning and Memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

175

Thomas R. Soderling 6.

Calcium-Dependent Regulation of Protein Synthesis . . . . . . . .

191

H. Clive Palfrey and Angus C. Nairn 7.

Calcium Regulation of Gene Expression . . . . . . . . . . . . . . . . . .

225

Laura B. Rosen, David D. Ginty, and Michael E. Greenberg 8.

Calcium Regulation of Apoptosis

.......................

255

Diane R. Dowd 9.

Role of Calcium in T-Lymphocyte Activation . . . . . . . . . . . . .

281

Maria E. Cardenas and Joseph Heitman 10.

Regulation of the Cell Division Cycle by Inositol Triphosphate and the Calcium Signalling Pathway . . . . . . . . . .

299

Michael Whitaker 11.

The Regulation of Calcium in Paramecium . . . . . . . . . . . . . . . Robert D. Hinrichsen, Dean Fraga, and Chris B. Russell

311

12.

Calcium in Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . .

339

Trisha N. Davis 13.

Calcium Regulation of Drosophila Development . . . . . . . . . . .

359

Kathy Beckingham Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

395

This Page Intentionally Left Blank

zyxwvu zyxwvu Contributing Authors

Kathy Beckingham, PH.D. Department of Biochemistry and Cell Biology, Wiess School of Natural Sciences, Rice University, P.O. Box 1892, Houston, Texas 77251 Donald L. Campbell, PH.D. Department of Pharmacology, Duke University Medical Center, Box 3845, Durham, North Carolina 27710

Maria E. Cardenas, PH.D. Department of Genetics, Duke University Medical Center, Box 3054, Durham, North Carolina 27710 David E. Clapham, M.D., PH.D. Department of Pharmacology, Mayo Clinic and Foundation, 200 First Street, S.W., Rochester, Minnesota 55905 Trisha N. Davis, PH.D. Department of Biochemistry, University of Washington, SJ-70, Seattle, Washington 98195-0001 Diane R. Dowd, PH.D. Departments of Biochemistry and Molecular Biology, St. Louis University Medical Center, 1402 South Grand Street, St. Louis, Missouri 63104 Sture Forsen, PH.D. Physical Chemistry 2, Lund University Chemical Centre, P.O. Box 124, S-221 O0 Lund, Sweden Dean Fraga, PH.D. Division of Basic Science, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104 David D. Ginty, PH.D. Department of Neurology, Harvard Medical School and Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115

Michael E. Greenberg, PH.D. Department of Neurology, Harvard Medical School and Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115 Joseph Heitman, M.D., PH.D. Departments of Genetics and Pharmacology, Howard Hughes Medical Institute, Box 3546, Duke University Medical Center, Durham, North Carolina 27710 Robert Hinrichsen, PH.D. Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104 J. David Johnson, PH.D. Department of Medical Biochemistry, Ohio State University Medical Center, 1645 Neil Avenue, Columbus, Ohio 43210

Sara Linse, PH.D. Physical Chemistry 2, Lund University Chemical Centre, P.O. Box 124, S-221 O0 Lund, Sweden vii

viii

zyxw zyxwvut CONTRIBUTING A UTHORS

Angus C. Nairn, PH.D. Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021 H. Clive Palfrey, PH.D.

Department of Pharmacological and Physiological Sciences, The University of Chicago, 947 East 58th Street (MC 0926), Chicago, Illinois 60637

Laura B. Rosen Department of Neurology, Harvard Medical School and Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115 Chris B. Russell, PH.D. Division of Basic Science, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104 James Sneyd, PH.D. Department of Biomathematics, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, California Christopher H. Snyder Department of Medical Biochemistry, Ohio State University Medical Center, 1645 Neil Avenue, Columbus, Ohio 43210 Thomas R. Soderling, PH.D. Vollum Institute, L474, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97201-3098 Harold C. Strauss, M.D. Department of Medicine, Box 3845, Duke University Medical Center, Durham, North Carolina 27710 Michael Whitaker, M.D., PH.D. Department of Physiology, University College London, Gower Street, London WC1E 6BT, England

Preface

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The prominent role played by calcium as an intraceUular second messenger has long been puzzling to scientists working in widely disparate fields. Why would the earth's fifth most abundant element have been chosen and elevated to this lofty position? In truth, it is not only intracellular calcium homeostasis but also the control of the extracellular concentration of calcium that is important in biology. Three hormones--vitamin D3, parathyroid hormone, and calcitonin--are charged with the responsibility for maintaining appropriate calcium concentrations in the extracellular fluids of mammals. Specific receptors for calcium have been identified on the surfaces of parathyroid gland cells, within all eukaryotic cells (calmodulin), and within specialized differentiated cells, such as striated muscle (troponin c). More than 100 calcium-binding proteins have been described, many of which demonstrate remarkable specificity for this ion. Selective ion channels that can be stimulated by a variety of agonists to transiently increase the intracellular calcium concentration may be found in the plasma membranes of both excitable and nonexcitable cells. Conversely, calcium pumping ATPases in eukaryotic cells serve to release Ca 2§ from intracellular stores or to eliminate calcium from the cell. At least for the plasma membrane pumps, another calcium receptor, calmodulin, is required to activate the pump, which must also be charged with bound calcium. It is the transient and spatial nature of calcium fluxes that impart exquisite control to many compartmentalized cellular processes. High concentrations of intracellular calcium can result in programmed cell death or apoptosis. In addition to the channels, pumps, and calcium-binding proteins, intracellular organelles, such as mitochondria and vacuoles, store remarkably high concentrations of this ion. The conundrum is why calcium, which could be viewed as a general cellular poison, controls an intricate network of cellular events. The idea behind the present volume was to present a catholic picture of calc i u m - f r o m how it enters and moves through the cell and interacts with its many binding proteins, to the exploitation of genetically tractable organisms to unravel specific physiological roles of its ubiquitous intracellular receptor, calmodulin. The book begins with two chapters that discuss how calcium transients can be amplified in a living cell (Clapham and Sneyd) and the properties of the membrane proteins that gate its entry into the cytoplasm (Campbell and Strauss). Calcium then encounters a formidable array of binding proteins. Linse and Forsen present a thorough discourse of the structural determinants that provide the framework for its high affinity binding. Johnson and Snyder then dissect the events by which calcium, bound to calmodulin, regulates the contractile apparatus present in smooth muscle cells. Just as the array of calcium-binding proteins present in a cell can orchestrate responses of the cell to changes in calcium concentration, so can the calcium/cal/x

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PREFACE

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modulin complex signal via a large number of target proteins. More than 30 calmodulin-binding proteins have been described, but perhaps the largest number regulate protein phosphorylation/dephosphorylation. The next segment of the book deals with how calcium- and calmodulin-dependent protein kinases or phosphatases are involved in vital cellular processes. These processes are synaptic signal transduction (Soderling), protein synthesis (Palfrey and Naim), gene expression (Rosen, Ginty, and Greenberg), programmed cell death (Dowd), activation of quiescent T-lymphocytes (Cardenas and Heitman), and the general control of cell cycles (Whitaker). The major targets required to transduce the calcium signal in these six very different systems include calmodulin-dependent protein kinases II, III, and IV, as well as the calmodulin-dependent protein phosphatase 2B known as calcineurin. The final three chapters discuss genetically manipulable organisms that have been effectively utilized to identify mutations that disrupt calcium-dependent events. Paramecium was utilized to identify mutations in calmodulin that result in defective Na + and K + transport in ciliary membranes. These defects alter the ability of the organism to respond to environmental stimuli and are discussed by Hinrichsen. Davis evaluates experiments carried out primarily in budding yeasts that reveal the importance of calcium in the growth of this organism. Even though calcium is clearly important in yeast, the calmodulin gene is essential in at least some events that do not require calcium binding! Calmodulin targets have been identified that are involved in nuclear division and should provide clues to address the same process in mammalian cells. In the final chapter, Beckingham describes what is known about calcium-dependent processes in the fruit fly. She discusses an amazing variety of calcium-dependent pathways involving early development, vision, learning, and memory, as well as muscle function. What is very satisfying about the studies carried out in all three experimental organisms described in this book is the similarity with less mechanistic experiments performed in mammals or mammalian cells. There is little doubt that the paradigms and processes discovered in these lower organisms will prove to be extremely relevant in the general quest to understand the importance of calcium signalling in more advanced mammals, from mice to humans. Anthony R. Means

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ADVANCES IN SECOND MESSENGER AND PHOSPHOPROTEIN RESEARCH Volume 30

Calcium Regulation of Cellular Function

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Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York9 1995.

Intracellular Calcium Waves David E. Clapham* and James Sneydt *Departments of Pharmacology, Physiology, and Biophysics, Mayo Foundation, Rochester, Minnesota 55905; tDepartment of Biomathematics, UCLA School of Medicine, Los Angeles, California 90024

This review will summarize calcium control mechanisms in nonexcitable cells, explain regenerative calcium release and calcium waves, and provide examples of the biological significance of calcium waves. The Xenopus oocyte will be used to illustrate how relatively simple calcium control mechanisms can give rise to complex regenerative waves and calcium oscillations. At least four fundamental calcium control mechanisms are present in cells. First, calcium is released from intracellular stores such as the endoplasmic reticulum (ER) into the cytosol. Calcium in the ER is at least 10 IxM and is largely bound to specialized calcium-binding proteins. Second, calcium stores are repleted by calcium entry from 1 to 2 mM extracellular calcium, crossing the membrane through several types of calcium-permeant channels. Third, calcium in the cytoplasm is taken up by two energy-requiring transport systems, the smooth endoplasmic reticulum Ca 2 + ATPases (SERCA pumps) and the plasma membrane Ca 2 + ATPases (PMCA pumps). Finally, calcium is buffered by a number of binding sites that do not actively transport calcium but localize and dampen the time course of calcium change. Calcium buffers may be fixed or mobile and are the most complex and least understood of the calcium control mechanisms. In many nonexcitable cells, calcium release is initiated by surface receptors (1) (Fig. 1). There are two major types of receptors that control calcium release through second messengers. The first is the G protein-linked receptor such as the et-adrenergic, serotonin, purinergic, histaminergic, and bradykinin receptors. The second class is the growth factor or tyrosine kinase-linked receptor class (2). The G protein-linked receptors activate the heterotrimeric plasma membrane-associated G proteins to stimulate phosphatidyl 4,5-bisphosphate (PIP2) phosphodiesterase (PDE), otherwise known as PIP2PDE or more commonly, phospholipase C. The four classes of PLC include 13, "y, ~, and ~. Of these, G proteins activate PLCI3 to split PIP2 into diacylglycerol (DAG) and inositol 1, 4,5 trisphosphate (InsP3, or IP3). DAG activates protein kinase C (PKC). IP3 binds and gates a distinct class of intracellular, endoplasmic reticulum-bound IP3 receptor (IP3R) chan1

t~

FIG. 1. Schematic diagram of the model. Agonist binds to a receptor (R) and activates the G protein (G) by catalyzing the exchange of GTP for GDP. Phosphotidylinositol-(4,5)-bisphosphate PIP2 is hydrolyzed by phospholipase C (PLC) to diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors (IP3R) on the endoplasmic reticulum, opening a calcium-permeable channel and allowing calcium to flow out of the endoplasmic reticulum. Calcium coming out of the channel modulates the IP3 receptor in two ways. It activates it quickly and inactivates it slowly. It can then diffuse to neighboring IP3 receptors to initiate further calcium release there, forming a propagating wave. The calcium pumps resequester calcium into the endoplasmic reticulum, and there is a continual leak of calcium into the cytoplasm, both from outside the cell, as well as from the endoplasmic reticulum, possibly via the calcium pump protein itself. (From J. Amundson and Clapham, ref. 1, with permission.)

INTRA CELLULAR CALCIUM WAVES

nel. Once the IP3R channel is open, calcium flows down the large calcium concentration gradient from the ER into the cytoplasm. The calcium concentration in the cytoplasm rises rapidly, within seconds, to 1-2 I~M levels. Calcium pouring out of the channel inactivates the IP3R (negative feedback loop). Calcium diffuses to adjacent IP3R (and ryanodine receptor in many cells) channels, initiating the release of more calcium. The exact details of the propagative release of calcium depend on the cell type. This area of the calcium release mechanism will be discussed in more detail. Each of the steps briefly outlined above will be described in more detail in the next section. CELLULAR COMPONENTS OF REGENERATIVE CALCIUM RELEASE

Receptors

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Key examples of these receptors linked to phosphatidyl inositol turnover are listed in Table 1. G-protein receptors have seven putative transmembrane domains which span the membrane in serpentine fashion (3). These receptors are two-sided, having an extraceUular site for selecting the agonist and an intracellular site for specifying the G protein. Agonist binding has been adapted to be activated by light, an array of small molecules (hormones, olfactory compounds), enzymes (thrombin), and peptides. No common theme has emerged for agonist binding, nor is it known how agonist binding alters the conformation of the molecule to interact with the heterotrimeric G protein. Once bound by agonist, the G protein-linked receptor undergoes a conformational change to enable it to act as a catalyst for the generation of effective messen-

TABLE 1. RepresentativePLC-linked

G-protein receptors Muscarinic ml, m3 Purinergic P2x, P2y Serotonin 5HT1C Histamine H-1 GnRH TRH Glucagon Cholecystokinin Vasopressin V-1 a, V-1 b Oxytocin Angiotensin II Thrombin Bombesin IgE Bradykinin Tachykinin Thromboxanes Platelet-activating factor

4

INTRACELLULAR CALCIUM WAVES

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ger molecules (4,5). Each receptor is most highly conserved in the putative transmembrane-spanning domains while the intracellular second, third, and carboxyl tail regions are variable. Variations in amino acid sequence give rise to poorly understood conformational differences that specify the G protein interacting with the receptor (3). For example, the muscarinic type 2 receptor (m2) inhibits adenylyl cyclase and activates a K +-selective channel, but weakly stimulates PI turnover. The muscarinic type 3 receptor (m3) strongly stimulates PI turnover. The difference in G-protein specificity appears to be due to variations in the first 21 amino acids of the amino terminus of the third cytoplasmic loop (3,6-9). Chimeras made by swapping 21 amino acids in this region from m2 to m3 show that the m3 receptor can be made to behave as if it were an m2 receptor. Alternatively, by exchanging only nine amino acids, the m2 receptor can be "converted" into an m3 receptor which strongly stimulates PI turnover. Full understanding of the three-dimensional interactions between intracellular receptor interface and G protein will require extensive use of crystallography and two-dimensional NMR imaging approaches.

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G Proteins

The G-protein complex is composed of a dimer of a (35-52 kD) and 13"y(itself a dimer of 35-36 kD 13 and 8-kD ~/) subunits. The 13~/subunit is presumed to exist only in its dimeric form in vivo. Figure 2 shows the well-known G-protein cycle. At rest, the heterotrimer is probably anchored to the intracellular surface of the memReceptor , ~ J ~t,

a

|

GTPase

Pi ~,,I

' (~..~=_ GDP

/-

Effector

N Q Agonist

b ;~DP GTP

Mg 2+

FIG. 2. G-protein cycle. The receptor, when bound by agonist (b), interacts with the heterotrimeric G protein (o~13~/)to exchange guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on cxand dissociate the heterotrimer into o~and ~/subunits (c). Both subunits activate effectors. The oLsubunit hydrolyzes GTP to GDP, causing reassociation of the heterotrimer (a). (From Luckhoff and Clapham, ref. 31, with permission.)

INTRA CELLULAR CALCIUM WAVES

5

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brane by fatty acid modifications of both the et and ~/subunits. The agonist-bound receptor interacts with the et[3~/heterotrimer to catalyze the exchange of guanosine diphosphate (GDP) on the ot subunit for guanosine triphosphate (GTP). [3~/-et interaction is required for et[3~/association with the receptor. The presumed role of [3~/ during these initial steps is to prevent spontaneous GTP replacement of GDP, since GTP is normally at higher concentration (100 txM) than GDP (10 IxM) intracellularly. When GTP replaces GDP, et and [3~/have lower affinity for each other and become functionally active. There is now ample evidence that both ot-GTP and [3-y activate effectors, first reported in the [3~/activation of a cardiac muscarinicgated K + channel (10). In the case of phospholipase C(PLC)[31-3, both ct and [3~/ activate the enzyme, binding at separate sites in the molecule (11-16). Each et-GTP has a unique rate of GTP hydrolysis. Once ot-GTP binds PLCI3, PLC[3 enhances the hydrolytic rate of ot to terminate PLC[3 activation (17). Once hydrolyzed, et-GDP and 13~/presumably reassociate. There are --~20 subtypes of G-protein et subunits, 4 [3 subunits, and 7 or more ~/ subunits. Thus there are combinatorially 28 [3~/ dimers (although [31~/2 does not appear to be formed), yielding relatively equal numbers of ot and [3~/subtypes. It seems probable that [3~/subtype specificity is determined by selective association of [3~/, et, and receptor rather than by dynamic release of ot and [3~/subtypes which then find a particular effector. PLC[3 reconstitutions with et and [3~/are rapidly changing our views of G-protein effector interactions. To date, G-protein et and [3~/subunits both activate PLC[3 1-3, with G,~ binding at the carboxy half of PLC[3 and [3~/ binding the amino half of the molecule. Gq family (G 16, Gq- and perhaps Gi/o) G proteins activate PLC[3 1-4. Ga~ activates PLC[3 3>PLC[3 2>PLC[3 1. It should also be pointed out that PLCI3 is not only G protein-dependent, but PLC rates are also altered by calcium concentration (18). Pertussis toxin, which catalyzes the transfer of ADP-ribose from NAD to the carboxy terminus of the et subunit, blocks interaction of the receptor with the et subunit. The eti/o and ott subunits are sensitive to PTX but it is clear that both PTXsensitive and insensitive stimulation of PLC occurs. There are two main possibilities. First, Ctq may stimulate PLCI3 in a PTX-insensitive manner, and G-protein eti/o would then account for PTX-sensitive stimulation. Alternatively, activated Otvo protein may release [3~/, which stimulates PLC[3 1-3. Which of these alternatives takes place in the cell is a subject of investigation.

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Growth Factors---PLC~/1 Growth factors such as platelet-derived growth factor (PDGF) also initiate calcium release from stores, albeit more slowly than the pathways mentioned above. Tyrosine kinase-linked receptors, often single membrane-spanning domain receptors, dimerize upon ligand binding, inducing autophosphorylation and phosphorylation of other accessory proteins on tyrosine groups. PLC'y generates IP 3 to release calcium from internal stores.

6

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INTRACELLULAR CALCIUM WAVES PLC

Subtypes

Phospholipase C 13 and -y are the major receptor-linked pathways for releasing calcium from IP3R stores. PLC subtypes have two domains in common, called X and Y, which are responsible for PIP2 hydrolysis. PLC~/1 and ~/2 have 2 SH2 (src homology) domains and one SH3 domain lying between the X and Y regions. The SH2 binding domain binds PLC~/to the receptor while the SH3 domain function may associate PLC~/with the cytoskeleton. PLC~ and 9 are not well understood by comparison. PLC8 activation mechanism is distinguished from that of other PLCs in that it possesses an EF-hand calcium binding motif (19).

IP3 Receptor A family of IP3R (IP3R1-3: Table 2) has been cloned (21). The IP3R is a homotetramer of 260-kD proteins comprising a relatively high-conductance (~/= 40 pS in 50 mM Ca2+), cationic, voltage-independent channel pore. Apparently one IP3 binds to the pore to activate it (22). Attempts to correlate IP3 binding to channel function have been complicated by an apparent unusual property of the reconstituted IP3R known as quantal release. Hill coefficients of the IP3R in a lipid bilayer are estimated to be low (---2). Three separate genes produce IP3R; the hierarchy of IP3R sensitivity to IP3 is IP3R 2>IP3R 1>IP3R 3. Alternative splicing produces more subtypes which express in a tissue-dependent fashion. A key feature of the IP3R is that it is sensitive to both IP3 and calcium. An ---100 amino acid segment of the receptor (22) near the calcium pore lining region binds calcium. The IP3R can be phosphorylated by protein kinase A and calmodulin kinase II and has two ATP binding sites. The IP3R is competitively blocked by heparin. Caffeine, which activates the ryanodine receptor, can block or activate the IP3R in a calcium-dependent fashion, confusing studies in which both IP3R and ryanodine receptor coexist. TABLE 2. Intracellular calcium release channels

Activator

Inhibitor

Permeability

~/(pCa = 6.7)

MW

45 pS

260 kD x 4

200 pS

500 kD x 4

IP3 Receptor IPa (IP3 2> 1>3)

Heparin

Ba>Sr>Ca>Mg

Ca2+ cADPR (?) Heparin (and other polyanions) at Ca>50 nM

Ryanodine Ruthenium red

Ba>Sr>Ca>Mg

RyR Receptor

Mg2 +

IP3, inositol trisphosphate; RyR, ryanodine receptor; cADPR, cyclic ADP ribose. Data from refs. 20-22.

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INTRA CELLULAR CALCIUM WAVES

Ryanodine Receptors

7

The ryanodine receptor (RyR: Table 2) is a homotetramer of 450-kD proteins (2). There are three isoforms (RyR1-3) of the RyR. The RyR is activated by calcium, blocked by ryanodine, and also has ATP binding sites. RyRs mediate the fast calcium release seen in muscle cells, but both IP3R and RyR coexist in many tissues. One hypothesis is that the RyR is adapted for fast intracellular calcium release by virtue of its close association with the dihydropyridine binding site in muscle and its sensitivity to calcium. Moreover, H. Lee and colleagues (reviewed in 23) have developed substantial evidence that cyclic ADP ribose (cADPR) is a natural agonist of nonskeletal muscle RyR (24). Figure 3 shows the broad dependence on calcium of the RyR, unlike the IP3R, suggesting that RyRs do not inactivate quickly due to accumulation of local calcium levels. In one sense, the IP3R is a modified RyR whose activation is conditional upon IP3 binding.

Calcium Pumps SERCA 1-4 and PMCA 1-4 CaATPase proteins pump calcium into the ER or across the plasma membrane (25). The SERCA CaATPase molecules have 12 putative transmembrane-spanning domains, hydrolyze ATP, and pump calcium against the electrochemical gradient. The SERCA pumps are blocked by thapsigargin, ditert butyl hydroxyquinone, cyclopiazonic acid, and by the plant diterpine. Once blocked, a steady leak of calcium from stores depletes the stores and somehow activates a transmembrane calcium entry pathway.

Calcium Entry Mechanisms Unlike excitable cells in which voltage-activated calcium channels rapidly increase cytoplasmic calcium levels upon depolarization, nonexcitable cells bring in

pCa = 6.7

pCa = 5.0

0L_

FIG. 3, The IP3R and ryanodine receptor (RyR) both release calcium from the endoplasmic reticulum. The IP3R is gated by IP3

g

while the RyR is gated by calcium and some types by cADP ribose. Both receptors are modulated by calcium levels, but the IP3R has a much sharper apparent dependence on calcium level. (From Clapham, ref. 71, pCa

with permission.)

8

INTRACELL ULAR CALCIUM WAVES

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calcium via slower, low-conductance channels which are relatively voltage-independent. Hyperpolarization increases the calcium gradient and accelerates calcium entry. Thus, K § channels play an important role in the extent of hyperpolarization and regulate the net calcium influx. Alternatively, depolarization slows influx since many nonexcitable cells have no voltage-activated Ca 2 + channels. There are multiple calcium entry pathways, the best defined being calcium release activated current (IcRAC) (26,27). This small (--~20 pA at hyperpolarized potentials), inward calcium flux is somehow gated by depletion of stores. Its singlechannel conductance is estimated to be ---20 fA, if indeed it is an ion channel. Depletion of ER calcium by ionomycin, thapsigargin, B APTA dialysis, or receptors activates the current. ICRAC, once activated, lasts for many minutes and can readily restore pool calcium levels. Several hypotheses for the message released from the stores have been proposed. They include an unknown, low-molecular-weight ( (.3

10 mM B~,PTA 500 pA 100 ms

(1)

0.5

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O3 I >.. "O ~3 (l.)

/

U3 ~ 1 0 mM BAPTA

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,

,

-40

,

,

0 mV

(c)

,

,

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,

// /

,

,

....,

80

"T's~l0

100 "/-fast

O

'~ .~_

,

40

300 E "-" t-

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20 40 mV FIG. 3. Whole-cell properties of Ca 2 +-dependent inactivation of Ica L in ferret right ventricular myocytes (1.8 mM [Ca 2+]o in all recordings except lower pane/, A). A: Effect of 10 mM intracellular BAPTA and 1.0 mM extracellular Ba 2 + on ICa,L recorded at 0 mV. Both maneuvers increase peak ICa,L and greatly slow inactivation. B: Mean peak IC-,L steady-state inactivation relationship in control and in the presence of 10 mM intracellular BAPTA. Note that the relationship =bends up" depolarized to 0 mV, i.e., inactivation is incomplete in the range of membrane potentials corresponding to the cardiac action potential plateau (see Fig. 5). C: Voltage _. ,_ _ , . _ z__, __-J ..~ .... :--..,;....,;..,, ,; . . . . . ~t~nt~ kilt-that th,r,-~lc=tinnehin~ ~r~ I I-_eh~n~.d And Rnnrnximatelv parallel

38

CALCIUM CHANNELS IN THE HEART

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dent phosphorylation-dephosphorylation reactions. The major evidence can be summarized as follows: 1. Although nonmonotonic U-shaped inactivation curves are consistently observed in various cardiac myocytes, these curves never approach a value of 1.00 at depolarized potentials but rather reach saturating values of ---0.30-0.60 (see Fig. 3B). This is true even for potentials positive to the reversal potential for ICa,L (e.g., 15,30,31,72-74). 2. While Sr 2 +, Ba 2 +, and monovalent cation flow through L channels reduce both the rate and extent of inactivation, these maneuvers do not completely prevent inactivation (e.g., 15,73,77). 3. Influx of Ca 2 + ions through the cardiac L channel appears not to be an obligatory prerequisite for development of inactivation. The following lines of experimental evidence support this conclusion: (a) outward currents through L channels carded by Na + and K + at potentials depolarized to the ICa,L reversal potential exhibit time- and voltage-dependent inactivation (e.g.,72,73,75,76,84-88); (b) these outward monovalent currents can be inhibited by depolarizing prepulses that produce no net inward Ca 2 + flux. This phenomena can also be demonstrated in Ba 2 + and Ca 2 +-free solutions (e.g., 72); (c) inactivation can still be observed when monovalent cations cma3, current (72,73,76); and (d) single-channel studies have demonstrated that small depolarizing prepulses that failed to give any detectable channel openings produced significant inactivation of single-channel events recorded during subsequent test pulses (e.g., 15,20,89,90) and have failed to demonstrate any consistent correlation between the amount of charge flowing through individual Ca channels and the duration of their subsequent closed periods (91; however, see below). 4. Studies of Ba + flow through single L channels isolated from calf ventricle and expressed in lipid bilayers clearly indicate that cardiac L channels can inactivate in a voltage-dependent manner (92). These bilayer results are interesting since they were obtained in a completely cell-free system, i.e., inactivation still occurred in the absence of ATP and without possible contamination from intracellular Ca 2 + stores, Ca 2 + accumulation, and Ca 2 +-dependent enzymes (see below). It is possible that the Ca 2 +-dependent component of L channel inactivation could reflect Ca 2 +-dependent modulation of the inherent voltage-dependent component (e.g., 75). However, this seems unlikely since two recent gating current studies in guinea pig ventricular myocytes have demonstrated that Ca 2 + does not affect the voltage-dependent component of inactivation (93,94). For example, voltage-dependent inactivation reduces the putative gating charge movements associated with L channels, while Ca 2 +-dependent inactivation does not (94). These gating current results again strongly argue for separate voltage- and current-dependent components. In summary, a consensus seems to have been reached (a rare condition indeed in cardiac cellular electrophysiology!) that inactivation of the cardiac L channel depends upon both voltage and Ca 2 + flux through the channel. This has an important implication for both the continuous and modal gating models: L channels

CALCIUM CHANNELS IN THE HEART

39

have states that are not only governed by purely voltage-dependent transitions but also display states that are dependent upon current flow through the channel (e.g., 23). When present, the Ca 2 +-dependent component displays much faster kinetics than the voltage-dependent component. While the relative contributions of the two components of inactivation remain to be experimentally quantified, a recent model of cardiac L-channel activation has attempted to theoretically address this issue, as will be described below (95).

Possible Models and Mechanisms of Ca2 +-Dependent Inactivation

The underlying intracellular and biochemical mechanisms of Ca 2+-dependent inactivation of cardiac L channels are presently far from clear. However, based upon work from a number of different tissue types, four basic types of functional models have been proposed. The spatial domains encompassed by these models range from the entire cell myoplasm to the level of the single L-channel pore (Fig. 4). Following the nomenclature of Imredy and Yue (96), these different models can be categorized as follows: (a) "Global" models, wherein the net bulk myoplasmic [Ca2 +]i exerts a negative feedback on inactivation, presumably through secondary Ca 2 +-dependent metabolic pathways; (b) "Interdomain" models, wherein Ca 2 § induces negative feedback among local Ca channels ("channel crosstalk") due to limited diffusion in a space immediately adjacent to the sarcolemma ("fuzzy space"[97]) without significantly perturbing myoplasmic [Ca2+]i, in the strictest case of this model a single channel cannot inactivate itself, but rather inactivation only occurs through channel crosstalk (e.g., 98); (c) "Intradomain" models, wherein Ca channels can inactivate themselves by Ca 2 § diffusion in a local domain not in the immediate vicinity of the channel pore; and (d) high-affinity "pore" models, wherein a Ca channel inactivates itself by Ca 2 § binding directly within or very near the permeation pathway. As summarized above, it is quite clear that both increases and decreases in global myoplasmic [Ca2+ ]i can alter whole-cell cardiac L-channel gating. However, such macroscopic current measurements do not allow unambiguous determination of the factors governing Ca 2+-dependent inactivation at the single-channel level. While there is still controversy concerning the "reality" of some of the underlying assumptions of the remaining three single-channel model types (interdomain, intradomain, pore), recent results have begun to shed some light on their possible applicability to cardiac L channels. Due to the rapid steplike nature of Ca 2 + influx through a single L channel, Ca 2+ influx could possibly quickly saturate and overwhelm physiologically important Ca 2 +-binding proteins and molecules (e.g., 85) located in the immediate vicinity of the pore. Theoretical modeling of Ca 2 § diffusion suggests that the [Ca2+] in the immediate vicinity of the intracellular mouth of the pore (---50 nM) could (a) equilibrate within I~secs of channel opening or closing and (b) reach very high concentrations (---0.5 mM) within 1-2 nm of the pore (99) and --~1 ~M at 100-200 nm from the pore (100). These theoretical results raise the

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regions, with the relative concentration of Ca2+ being highest adjacent to the channel pore and progressively decreasing with distance. A." "Global" models (inactivation due to increases in bulk myoplasmic [Ca2+]~). B-"lnterdomain" models (inactivation due to overlapping channel [Ca24] microdomains, i.e., "channel crosstalk"). C" "lntradomain" models (individual channels capable of autoinactivation). D: =Pore" models (individual channels capable of autoinactivation due to binding to a site within the channel pore). Modified from Imredy and Yue (96).

CALCIUM CHANNELS IN THE HEART

41

possibility that interactions between adjacent L channels could influence their inactivation kinetics. Elegant evidence that such interactions may indeed take place at the level of single cardiac L channels has recently been supplied by two independent laboratories, although the conclusions reached are somewhat different. These resuits can be summarized as follows: 1. Yue and colleagues (96,101) applied conditional probability analysis to study Ca 2 +-dependent inactivation of single L channels (160 mM Ca 2 +) in guinea pig ventricular myocytes. This work demonstrated that in patches containing only a single L channel: (a) Ca 2 + flow through a single channel could produce inactivation of that channel; and (b) at depolarized potentials (e.g., + 20 mV), where channel openings are more frequent and closely spaced, previous channel openings favored acceleration of subsequent channel closure. The latter phenomenon was termed gating plasticity. However, at less depolarized potentials (e.g., + 15 mV), where channel openings are less frequent and more widely spaced, individual channels did not display gating plasticity. Since the peak [Ca 2+ ] near the channel mouth should be higher at less depolarized potentials (due to the larger driving force for Ca 2 + flow through the channel), these results suggest that Ca 2 + is not exerting its effect directly on the Ca 2 + pore but is rather acting through a slightly more distant site. To address this point, measurements were repeated in patches containing two channels. These measurements revealed that (a) ensemble averages of two-channel patches inactivated more rapidly than did those of one-channel patches, (b) at less depolarized potentials channel gating plasticity was restored, and (c) BAPTA abolished gating plasticity in two-channel patches but only partially inhibited it in one-channel patches (Fig. 5A). Therefore, under the conditions employed (160 mM Ca 2 +), Ca 2 + influx through one channel can influence the inactivation of a neighboring channel. These results (96,101) would appear to strongly argue for an intradomain type of model wherein Ca 2 + mainly exerts its effects at a region some distance away from the channel pore, and suggest that adjacent channels can modulate the inactivation of their neighbors through channel crosstalk via overlapping [Ca 2 +]i microdomains. However, while this work suggests that a strict pore model cannot be the exclusive mechanism of Ca 2 +-mediated inactivation, Imredy and Yue (96) point out that a pore mechanism could still be a secondary contributory mechanism (see below). 2. DeFelice and colleagues (23,59) have also demonstrated the phenomenon of channel crosstalk for L channels in chick embryonic heart cells. When five or more Ca channels were present in a patch single channel Ba 2 + currents (20 mM) inactivated more rapidly than currents recorded with only two channels in the patch (see Fig. 5B). These results indicate that channel density can influence the inactivation kinetics of L channels. The effect was also voltage-dependent; at HP = - 80 mV the channels inactivated rapidly, while at HP = - 4 0 mV (which will produce slow inactivation of L channels [26-29]), fewer channels opened and inactivation was slowed. While these results qualitatively agree with those of Yue et al. (96,101), DeFelice and colleagues propose that Ca 2 + influx through a single L channel would not establish the necessary localized concentration to produce autoinactivation ex-

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FIG. 5. Demonstration of Ca 2+-dependent inactivation and =channel crosstalk" for single cardiac L channels. A: Guinea pig ventricular myocytes, cell-attached recording, 160 mM [Ca 2+]o. The left panel illustrates the mean ensemble average behavior of a single-channel patch and a two-channel patch. Note that the two-channel patch inactivates faster. The right panel illustrates the effect of BAPTA on ensemble average behavior of two-channel patches. Note that BAPTA reduces inactivation, i.e., reduces =channel crosstalk." B- Embryonic chick ventricular myocytes, cell-attached recording, 20 mM Ba2 +, patch areas approximately 7-10 i~m2. The patch on the left contained at least five L channels, while that on the right contained only two. Each trace is a compilation of five sweeps to the indicated potentials from a HP = -80mV. Note that inactivation was more pronounced in the five-channel patch. From Mazzanti et al. (59) and Imredy and Yue (96).

CALCIUM CHANNELS IN THE HEART

43

cept in very high [Ca2+]o (e.g., 160 mM, as used in the studies of Yue et al. [96, 101]). Instead, DeFelice et al. have proposed (23,59,95) that under more physiologically relevant conditions L channels would show very little Ca 2 +-dependent inactivation unless the channels exist in clusters (i.e., "channel crosstalk" is obligatory). This could possibly account for the previous observations of Lux and Brown (91), who concluded from studies of patches containing 40 mM [Ca2+ ]o and only one or two channels that individual channels do not inactivate themselves. Risso and DeFelice (95) have proposed a mathematical model of cardiac L-channel gating that involves both voltage- and current-dependent components. In this stochastic model the L channel is proposed to possess both (a) voltage-dependent states that have intrinsic voltage-dependent rate constants that are the same for both Ca 2+ and Ba 2+, and (b) current-dependent states whose rate constants vary depending upon whether Ca 2+ or Ba 2 + carry current (for quantitative details see [95]). This model also incorporates possible changes in the Ca 2 + equilibrium potential, Eca, due to changes in Ca 2+ concentration near the inner mouth of the pore. This model is interesting in that it can predict, depending upon the assumed number of channels in a patch, the different behaviors of L channels observed in both low- and highdensity channel patches (23,59). For example, this model predicts that in a lowdensity, two-channel patch the voltage-dependent component of inactivation would predominate, and that Ca 2 +-dependent inactivation would be minor. However, in a high-density, 40-channel patch both voltage- and Ca2+-dependent components would exert approximately equal influences. The relative contribution of the two components would therefore depend upon the number of channels that interact in a sarcolemmal cluster. While these studies have elegantly demonstrated the existence of Ca 2+-dependent inactivation at the single L-channel level, the exact details under more physiological conditions clearly need further elucidation. However, these studies strongly suggest that the gating characteristics of cardiac L channels could be profoundly affected by their local densities in the sarcolemma. As DeFelice and colleagues have hypothesized (23,59,95), the population of available L channels in a cardiac myocyte may not gate homogeneously, but rather there may be subpopulations of channels within the sarcolemma that display different inactivation characteristics based upon their density. Channels in high-density clusters could display rapid, short-lived bursts of activity (mode-l-like? active-early-like?), while channels less densely clustered could display slower, longer-lasting openings (mode-2like? active-late-like?). As a result, mode 2 could represent a relaxation of the current-dependent component (23). It may be that the gating characteristics of cardiac L channels are not only dependent upon the internal gating characteristics of the channel subunits but also upon local spatial characteristics such as their distribution in the sarcolemma and the Ca 2 +-buffeting capacity of the immediate myoplasmic environment. However, while these speculations are interesting, we are presently unaware of any data indicating that L channels can occur in clusters in the sarcolemma of cardiac myocytes. Furthermore, while the model of Risso and De-

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Felice (95) suggests approximately equal roles for voltage- and current-dependent inactivation, most macroscopic voltage clamp results indicate that the Ca 2+-dependent component of inactivation is quite rapid and prominent at depolarized potentials around the peak of the IC,,L I-V relationship (e.g., 15,20,30,31,72). These issues clearly deserve more experimental attention. What could be the physical substrates corresponding to these intradomain Ca 2 + effects? This is presently one of the major unanswered questions in the field of cardiac L-channel regulation. However, a recent hypothesis based upon work in molluscan neurons (102) and GH3 pituitary cells (103,104) has suggested one possibility. In these preparations it appears that (a) L channels must be phosphorylated before they can open in response to depolarization, and (b) Ca2+-dependent inactivation can be attributed to desphosphorylation of the L channel by the Ca 2 +-dependent phosphatase calcineurin (phosphatase 2B [discussed below]; however, see [ 105]). Applying this hypothesis to cardiac myocytes, the more distant intradomain substrate of the L channel (96) could therefore potentially be a Ca 2 +-dependent phosphatase that inactivates L channels by dephosphorylating them in response to Ca 2+ influx. However, such a hypothesis may not be directly applicable to cardiac L channels. As will be described later, there is now a large body of evidence strongly suggesting that the basal cardiac L channel may reside largely in the dephosphorylated state and that it does not have to be phosphorylated to activate and enter the open state. Furthermore, Frace and Hartzell (106) have recently demonstrated that micromolar concentrations of the substrate inhibitor peptide for calcineurin (CNIP) produced no effect on basal ICa,L in amphibian myocytes. Only very high concentrations (1 mM) of CNIP were capable of producing small and slow (e.g., 20 min) increases in basal ICa,L with only a slight acceleration in inactivation kinetics. Therefore, the Ca2+-dependent calcineurin hypothesis would therefore appear not to be applicable to cardiac L channels. The differences between cardiac and neuronal L-channel inactivation may be due to differences in channel subunits and their degree of phosphorylation (see below). With final regard to the pore model, the results of Rosenberg et al. (92) on L channels incorporated into bilayers would seem to create a major difficulty for the literal applicability of this model to cardiac L channels: raising the [Ca2+]i to as high as 10 mM did not produce any appreciable effect on either the steady-state inactivation relationship or the rate of inactivation. However, these bilayer results clearly do not rule out either (a) metabolic modulation of inactivation in intact cardiac myocytes (e.g., by phosphorylation altering the affinity of a Ca 2+-binding site [94]) and/or (b) involvement of another membrane component associated with the L channel that possess a high-affinity Ca 2 +-binding site and which promotes Ca 2+-dependent inactivation (92). In summary, the physical substrates corresponding to the intradomain effectors that produce Ca 2§ inactivation of ICa,L still remain undetermined in cardiac myocytes. From a functional viewpoint, this qualifies as possibly the most important unanswered question in the entire field of regulation of cardiac L channels.

CALCIUM CHANNELS IN THE HEART Functional Whole-Cell Implications of Ca 2 +-Dependent Inactivation

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Regardless of the underlying mechanisms governing Ca 2 +-dependent inactivation, the fact that both the kinetics and degree of ICa,L inactivation depend upon previous Ca 2+ influx has important theoretical and functional implications for the influence of ICa,L during the cardiac action potential plateau, early phases of repolarization, and the EC coupling process. In particular, during the action potential plateau the membrane potential is both in (a) the region of"bend up" of the inactivation relation (see Fig. 3B) and (b) a range where ICa,L inactivates most slowly (see Fig. 3C). Figure 6A illustrates the behavior of ICa,L during the action potential predicted by a recent mathematical model of pacemaker activity and Ca 2+-homeostasis in single bullfrog sinus venosus cells (4,107,108). This model predicts that ICa,L would be activated during the approximate latter one-third of the diastolic depolarization. During the upstroke ICa,L displays rapid but incomplete inactivation during the early plateau phase. However, instead of progressively declining (as would be predicted by cardiac action potential models where ICa,L is modeled using more conventional Hodgkin-Huxley-like kinetics; e.g., [109]), ICa,L is predicted to display a secondary inward "hump" during the later phases of the plateau and early repolarization. This complex behavior can be understood as follows: (a) during the plateau, ICa,L slowly and incompletely inactivates since it is in the range where the inactivation relationship bends up; (b) during early repolarization, the driving force for Ca 2+ flow through noninactivated channels increases, thereby producing the secondary inward hump; and (c) as repolarization progresses further, ICa,L rapidly deactivates and/or inactivates. The slow and incomplete inactivation kinetics of ICa,L are predicted to produce the maintained inward current component that both sustains the action potential plateau and modulates the EC coupling process. These theoretical model predictions (4,107,108) have been recently verified: in both rabbit sinoatrial node cells and guinea pig ventricular myocytes (whole-cell "action potential clamp" [ 110,111]) and in embryonic chick heart myocytes (cell attached singlechannel recordings [33]), a secondary inward hump of noninactivating ICa,L during the repolarizing phase of the action potential has been recorded before the channels subsequently inactivate and/or deactivate (see Fig. 6B,C). These macroscopic and single-channel results provide direct experimental verification of the predicted functionally important macroscopic behavior of ICa,L due to the Ca 2+-dependent component of inactivation.

zyxwvu Selectivity of L- Channels

A large amount of work on isolated myocytes has now clearly indicated that cardiac L channels are highly selective for Ca 2+ ions over monovalent ions. For example, within the framework of constant field formalism (which formally is not applicable to descriptions of Ca 2 § channel permeation properties, but is nonetheless very useful for comparative and macroscopic modeling purposes [15]), in physi-

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CALCIUM CHANNELS IN THE HEART

47

ological solutions (mM [Ca 2 +]o) cardiac L channels have permeability ratios of PNa/Pca and PK/Pca of approximately 0.001 (e.g., 15,84,86,87). In other words, under physiological conditions the cardiac Ica.L is essentially a pure Ca 2 + current. The underlying biophysical mechanisms governing this high selectivity of L channels for Ca 2 + have been extensively studied over the last decade. The now classic experimental observations on L-channel selectivity and permeation characteristics including the "anomalous mole fraction effect" (e.g., 32, 112-114), saturation of peak ICa,L with high external divalent concentrations (e.g., 115), block of current flow through the L channel by low (l~M) [Ca2+ ]o (e.g., 112,116) and "nonspecific" monovalent cation fluxes (e.g., 72,112,117,118), as well as the theoretical obstacles that an L channel has to overcome to simultaneously be highly selective for Ca 2 + while allowing a high ionic flux through the channel, have all been extensively reviewed in numerous previous articles to which the reader is referred to for details (e.g., 8,15,20). These experimental results have led to three basic models of L-channel selectivity which have been frequently discussed in the literature. These models can be briefly summarized as follows (Fig. 7): 1. Allosteric external regulatory site model. This model was proposed by Kostyuk et al. (119,120), and hypothesizes that the high-affinity divalent cation binding site that regulates Ca 2 + selectivity is not located in the Ca channel pore, but is rather externally located. Through allosteric mechanisms, this site is proposed to control conformations of the channel pore which alter its selectivity. When Ca 2 + is bound to the site the pore is highly Ca 2 +-selective, while in the absence of Ca 2 + it becomes nonselective. 2. Symmetrical intrapore two-site model. This now classic model was proposed simultaneously in 1984 by Almers and McCleskey (117) for the skeletal muscle L

FIG. 7. Schematic representations of previous models of L-channel permeation. Putative locations of high-affinity binding sites are indicated by the black boxes. Left: =AIIosteric" external binding site model. Middle: Classic intrapore two-site model with binding sites symmetrically located within the pore at effective electrical distances (~) of approximately 0.33 and 0.67. Right: Intrapore one-site model with a single binding site located near the external mouth of the pore. In the allosteric model, selectivity is determined by Ca 2 § ions binding to the external allosteric site, while in the two-intrapore-site models, selectivity arises from multi-ionic interactions at the intrapore binding site(s). Adapted from Hess and Tsien (112), Almers and McCleskey (117), Kostyuk and Mironov (119), and Kostyuk et al. (120).

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48

CALCIUM CHANNELS IN THE HEART

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channel and Hess and Tsien (112) for the cardiac L channel. The details of this very prevalent model have been extensively reviewed (e.g., 8,15,114). For the purposes of this chapter, it is important to note that this model assumes two discrete highaffinity Ca 2+-binding sites within the L-channel pore that are symmetrically located at effective electrical distances of approximately ~ = 0.33 and 0.67 (112). 3. Intrapore one-site model. This simpler intrapore model has recently been proposed by Armstrong and Neyton (121). Based upon purely electrostatic arguments (for quantitative details see [ 121]), this model proposes that L-channel selectivity is due to a single cation binding site located near the external mouth of the pore. The essence of this model is that the single site can either bind (a) a single Ca / + ion with high affinity, or (b) two Ca 2+ ions with lower affinity. For conduction to occur, both the one- and two-site intrapore models require (a) multiple-ion occupancy within the pore, (b) single-file cationic flow within the pore, and (c) electrostatic repulsion between cations bound to the intrapore high-affinity site(s). As will be described, convincing evidence against the validity of the allosteric model has accumulated. However, since both the one- and two-site models can formally reproduce all of the classic permeation phenomena displayed by L channels (112,117,121), they would appear to be plausible alternative models. However, recent electrophysiological results to be described below strongly suggest that the features of both intrapore models may need to be significantly revised.

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Tests Among Models: Recent Electrophysiological Evidence Recent electrophysiological results have begun to shed some light on the general applicability of the these three models of L-channel permeation. The most relevant electrophysiological results to date would appear to be the very elegant and detailed studies of Kuo and Hess (122-125) on the possible location and number of highaffinity ion binding sites in L channels of rat pheochromocytoma (PC12) cells. This analysis has important implications not only for L-channel selectivity, but also for possible channel mechanisms of Ca 2 +-dependent inactivation and possible regulatory effects of intracellular Mg 2+ on ICa,L (e.g., 126-129). The extensive results of this very detailed work can be summarized as follows: 1. Both the size and surface potential associated with the external and internal pore mouths were estimated by studying current saturation and the effects of varying solution viscosity (glycerol, dextran, sucrose) and ionic concentration (122). These measurements convincingly lead to the following surprising conclusions regarding L-channel architecture: (a) the pore mouth entrances are asymmetrical, with the derived functional estimates of pore mouth radii being 5.29 .A, for the external and 2.70/~ for the internal mouth; and (b) the estimated negative surface charge density at both mouths are similar, but the resulting surface charge potentials would be insignificant in ionic strengths of 110 mM and greater. These results therefore indicate that surface charge effects in the pore mouth regions would not signifi-

CALCIUM CHANNELS IN THE H E A R T

49

cantly contribute to Ca 2 + selectivity by causing localized increases in divalent ion concentration (e.g., 130). Rather, the large external pore mouth appears to be the major mechanism for allowing nondiffusion limited inward Ca 2 § flow into the pore. 2. In a series of detailed experiments designed to characterize block of both inward and outward Li + currents by both external and internal Ca 2 + (123) it was found that the unblocking off-rates for Ca 2 + measured at a fixed potential were the same regardless of which side Ca 2 + entered the channel provided that Li + current flow was in the same direction. These elegant results unequivocally demonstrate that the high-affinity Ca 2+-binding site is located in the channel pore, and that regardless of which side it enters the pore, Ca 2 + produces block by binding to the same high-affinity sites. The fact that the exit of blocking Ca 2 + ions from the pore was always in the same direction as Li + flow indicated the existence of a "long pore effect" (131,132), suggesting the existence of ion-ion interactions within the pore. Based upon kinetic analysis of these Ca 2 +-Li + interactions and the long pore effect, it was convincingly demonstrated that the L-channel pore contains two types of binding sites: (a) one "set" of high-affinity Ca 2 +-binding sites composed of at least two sites separated by insignificant energy barriers; and (b) at least two low-affinity (nonspecific) sites located internally to the set of high-affinity sites. 3. In a series of experiments designed to characterize the high-affinity binding sites (124), the multi-ionic nature of the L channel and the existence of ion-ion interactions within it were unequivocally demonstrated. "Lock in" is an electrophysiological term describing the process wherein an ion at a given site in a pore cannot move if the neighboring site is occupied by another ion, while "enhancement" is the process wherein an ion that occupies a given site can promote the exit of an ion in a neighboring site (e.g., 133-135). Kinetic analysis demonstrated that the exit rates of Ca 2+ unblock produced by Li + flow through the channel could be reduced by increasing the [Li + ] on the opposite side of the channel, thereby demonstrating that Ca 2 + could be "locked in" the high-affinity binding sites by Li + . "Enhancement" was verified by demonstrating that the off-rates of Ca 2 + unblock increased linearly with increasing external Li + concentration (75-850 mM). Extrapolation of the relationship to zero external Li + indicated that Ca 2 + exit from the pore was negligible in the absence of Li + , i.e., enhancement was required for Ca 2 + to exit the pore. The linear relationship over the concentration range studied indicates that the affinity of Li + to the site producing enhancement of Ca 2 + exit was very low when a Ca 2 + ion was present in the neighboring site. To determine the approximate location of the enhancement site its effective electrical distance, ~, was measured by determining the voltage dependence of the Kd of Cd 2 § block of Ba 2 + flux through the channel. It was demonstrated that the Kd was the same for different membrane potentials (Kd ---45 mM), arguing very strongly for a small value of 8 (8 ---0). This indicates that there is a very short electrical distance between the high-affinity sites and the large external mouth of the pore. This conclusion was further strengthened by the fact that the high-affinity sites could be easily titrated from the extracellular surface by changing the pH in the patch electrode:

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50

CALCIUM CHANNELS IN THE HEART

zyx

inward Ba 2 + currents (110 mM) could be reduced with a pKd of approximately 5, suggesting that carboxylate groups may be involved in the high-affinity sites. 4. Millimolar external Mg 2+ is known to produce block of both whole-cell ICR,L and unitary currents carded by Ba 2+ (e.g., 126-129). To more carefully quantify these effects at the single-channel level, Kuo and Hess (125) measured the block produced by both external and internal Mg 2+ on inward and outward Li + and Na + flux. Their analysis indicated that both external and internal Mg 2+ ions can block the pore and that internal Mg 2+ ions may be producing two types of block. Resolvable discrete block of inward Li + currents could be produced by 3-8 IxM external Mg 2+ and was demonstrated to result from binding to the same external highaffinity sites that Ca 2 + binds to. However, even though Mg 2+ and Ca 2 + have a charge of + 2, Mg 2+ exerted a much weaker repulsive effect (i.e., enhancement) on Li + than did Ca 2 +. (For example, when Mg 2+ was present the apparent Kd for loading Li + into the adjacent enhancement site was approximately 300 mM, while when Ca 2 + was present the similar apparent Kd was 2-3 M [ 124]). In addition, in contrast to Ca 2 +, Mg 2 + could (a) exit the high-affinity blocking sites in the absence of any enhancement effect, and (b) exit much more easily to the outside of the pore than into it. This latter effect indicates a much higher energy barrier for Mg 2+ on the inner side of the high affinity sites compared to Ca 2 +. In support of this notion, it was demonstrated that internal Mg 2+ was much more effective in blocking outward currents than inward currents. For example, 5-10 mM internal Mg 2+ was required to block inward Na + currents. In addition, 5-10 mM internal Mg 2+ also reduced the size of the unitary inward Na + currents, presumably due to a very rapid (i.e., unresolvable) binding and unbinding reaction. It was therefore argued that internal Mg 2+ produced the very rapid block that reduced unitary current amplitude by binding to the low-affinity sites located internally in the pore, and the slower discrete block by binding to the high-affinity external pore sites. Due to the high energy barrier for Mg 2+ between the two sets of sites, Mg 2+ binding to the lowaffinity internal sites could be considered as being in equilibrium with the bulk internal Mg 2+ . The voltage dependence of the apparent Kd ( " 10 mM) for Mg 2+ binding to these internal sites gave an effective electrical distance of ~ ---0.3 from the internal pore mouth. In summary, the results of Kuo and Hess (122-125) convincingly rule out the external allosteric model (119,120), while they support the essential assumptions of the intrapore one-site (121) and two-site (112,117) models in demonstrating that L channels are multi-ion pores capable of having at least three ions in them at a time (see also [32]) and that significant ion-ion interactions (enhancement) do occur within the pore. However, they also strongly suggest that both the one-site and classic two-site models have to be significantly revised in two major respects: (a) the channel pore is not functionally symmetrical, i.e., there are not two discrete sets of high-affinity sites symmetrically located at effective electrical distances of approximately 0.33 and 0.67. This disagrees with previous results obtained from isolated L channels in bilayers (136); and (b) The very different effects of Ca 2+ and

CALCIUM CHANNELS IN THE HEART

51

Mg 2+ in producing enhancement of Li + flux indicates that an electrical repulsion hypothesis based purely upon electrostatic considerations is probably too simplistic. Rather than being just dependent upon the charge of the interacting ions, the magnitude of the enhancement effect also seems to be very dependent upon both the affinity of the interacting ions for the sites and possible local deformations of the pore in the binding site region, similar to the known divalent cation binding properties of Ca 2+-chelating compounds such as EDTA (e.g., 137) and BAPTA (e.g., 138). Thus, Kuo and Hess (124,125) suggest that certain anionic groups such as carboxylate side chains may form an interacting array of closely spaced ligand groups in the L-channel pore rather than one single discrete binding site which selects electrostatically. In other words, there is no "fixed" site per se but rather a closely spaced "set" of high-affinity sites. Both the position and number of ions in the high-affinity set would be determined by both the ionic size and the stability of the ion-ligand complex. In analogy to B APTA, ions which form a more stable complex with the ligand groups would have a greater influence in changing the local pore conformation, which in turn would influence the binding process in other regions of the set. The analysis of Kuo and Hess (122-125) has led to a revised model of the L-channel pore (Fig. 8A). This new model proposes that the channel has a large external pore mouth which does not have significant surface potential for concentrating divalent cations near it under physiological conditions. Rather, the large size of the mouth assures a high frequency of Ca 2+ interactions with the pore. Highaffinity Ca 2+-binding occurs inside the external mouth immediately before the major narrowing part of the channel pore. These external pore sites are never completely empty in physiological [Ca2 +]o but always have some tightly bound Ca 2 + ions which exclude monovalent cations from the pore (see Fig. 8B). As described above, high-affinity binding results from an array of coordinating ligand groups made up of negatively charged carboxylate side chain groups. This arrangement produces negligible energy barriers between the "sites," therefore allowing free transfer of Ca 2+ to occur among the ligands and the enhancement effect to take place in the presence of high-affinity Ca 2 + binding. The narrow region beyond the external high-affinity sites is responsible for single-file flow and most of the potential drop across the pore. However, this region must be wide enough to allow for the various cations which can permeate the channel in the absence of Ca 2 + (e.g., tetramethylammonium, which has a diameter of ---6,~ [139]). This narrower region of the pore presents a much higher energy barrier to Mg 2 + than to Ca 2 + (see Fig. 8A). This latter fact, in combination with the small enhancement effect of Mg 2+ at the high-affinity sites, makes Mg 2+ functionally act as a "permeant blocker." A second set of much lower affinity binding sites (Kd in the range of approximately 10 mM) is located in the narrow region at an electrical distance of -~0.3 from the internal mouth. This set of sites could potentially modulate both inward and outward currents through fast intracellular Mg 2+ block, and could conceivably produce some degree of inward rectification at depolarized potentials. However, this mechanism awaits verification in cardiac myocytes under physiological conditions. As will be

zy

External

Energy profile of Mg 2§ Internal

AG

(RT) -10 -20

I

I

I

I

'

J

0

0-2

0-4

0.6

0-8

1

zyxwvutsrq 1 Energy profile of Ca2§

AG (RT)

-10

Q

Cation

-20

,,,,O Low affinity binding site /O

l

I

I

0

0.2

0.4

I

0.6

i

J

0.8

1

Electrical d i s t a n c e (5)

High affinity binding site

(B) Extracellular

(i)

(ii)

(iii)

(iv)

(v)

Intracellular

r.~

CALCIUM CHANNELS IN THE HEART

53

described later, recent molecular biological evidence is in remarkable agreement with some of the key features of this revised model of the L-channel pore. Selected Aspects of Modulation of Cardiac L Channels Classic neuromodulatory agents such as 13-adrenergic and muscarinic agents have long been recognized to be importantly involved in modulation of the voltage-dependent cardiac ICa,L. However, possibly in no other area of Ca channel regulation does as much controversy and confusion exist regarding possible basic mechanisms and their manifestations at the single L-channel level, as will be made obvious in the following sections. While the application of patch clamp technology has revolutionized our understanding of mechanisms involved in modulation of cardiac L channels, its inherent limitations are quite possibly one major reason for the wide variability in reported results in the cardiac L-channel neuromodulatory literature. For example, in the whole-cell configuration, the normal intracellular contents of the myoplasm can be dramatically altered during the course of a typical recording. Whole-cell ICa,L frequently displays a "run down" phenomenon, wherein the peak ICa,L progressively declines with time. Run down is a particularly frustrating problem in the "torn off" patch single-channel recording configuration, where single cardiac L-channel activity ceases within seconds to minutes after patch excision. While the mechanisms governing run down are not yet well understood, its existence is important since it demonstrates that L channels are regulated by intracellular factors which can be "washed out" of the myoplasm into the much larger volume of the patch pipette. The results of any given study must therefore always be put

FIG. 8. Present modified working model of the L-channel pore and permeation. A: Left panel: The L channel is believed to have a wide external vestibule for Ucapturing" Ca 2 + ions (surface potential effects appear to be negligible), a set of external high-affinity Ca z +-binding sites (carboxylate residues on glutamic acids) located at an electrical distance of &--O from the external surface, and a set of internal low-affinity binding sites located in a narrow "single-file" region of the pore at an electrical distance of &-~ 0.3 from the internal surface. Block by Mg 2 + can occur at both sets. Right panel: Energy profiles for binding of Ca 2 + and Mg 2+ within the pore. Mg2+: energy profile when there is a Li + simultaneously bound at the enhancement site (solid line); energy profile when Mg 2+ is alone in the high affinity sites (dotted line). Ca 2 +: average free energy level of Ca 2+ in the high affinity sites (dashed line); estimated energy level when Ca 2 + is alone in the high-affinity sites (dotted line); energy level of a C a 2 + when there is a Li + simultaneously bound at the enhancement site (solid line). B: Highly schematic representation of cation selectivity and permeation in the multi-ionic L-channel pore. The external high-affinity Usites~ are represented by the two Y-shaped binding domains. The L-channel pore can either contain (i) one divalent cation with high affinity or (ii) two divalent cations with lower affinity. In physiological [Ca2+]o the site is nearly always occupied by at least one Ca 2 + ion. Divalent permeation occurs when a second divalent cation approaches the "site" and simultaneously binds to it. This reduces the affinity of the ~site," thereby allowing the previously bound Ca 2 + to unbind (iii; =enhancement"). Ca 2+ is much more effective at producing enhancement than Mg 24. Bound Ca 2 + ions (iv) prevent monovalent cation flux. Although a monovalent cation can share the "site" with a bound Ca 2 + (v) its affinity is much less. As a result, it unbinds before Ca 2 + and returns to the extracellular side. Modified from Kuo and Hess (125) and Tang et al. (252).

zy

54

CALCIUM CHANNELS IN THE HEART

zyx

within the framework of the composition of the "intracellular" pipette solution used (15). While the problem of run down of whole-cell ICa,L can be alleviated by applying "perforated patch" techniques, wherein the patch of membrane under the recording pipette is not physically ruptured but is rather made permeable by application of iontophoretic antibiotic agents (e.g., nystatin [140]), this approach is limited in that it does not allow manipulation of the intracellular contents except by extracellular application of a presently limited number of membrane permeable compounds. Ideally, more future studies should combine both the conventional whole-cell configuration and the permeabilized patch technique for comparative purposes. An additional potential problem exists in standard configurations for recording single L channels (both cell attached and torn off). Upon formation of a gigaseal the patch of membrane under the pipette is typically deformed into a characteristic "omega figure" which can extend for quite some distance into the shaft of the pipette (141, 142). This can potentially create a significant diffusion barrier for intracellutar second messengers, as well as possibly complicate and/or distort channel gating kinetics due to shear or stretch forces. Finally, different receptor types, enzyme isoforms, and L-channel subunits and their degree of phosphorylation (see below) may exist among different cardiac tissue types and species. All of these factors must be kept in mind when evaluating the results of any given study on cardiac L-channel modulation. In the following section we briefly review the major basic mechanisms now recognized to be of importance in modulation of ICa,L by classic neuromodulators and some of the present controversies in the field. We emphasize 13-adrenergic modulation and the probable role of phosphorylation in modulating cardiac ICa,L gating, since this subject has received the most experimental attention. Due to space limitations, many details have been admittedly generalized or overlooked. Readers interested in diving into the frequently muddied waters of detail in this important but often confusing branch of the field are referred to the excellent reviews which will be cited below. Readers who are interested in other important aspects of modulation of cardiac ICa,L not discussed below (e.g., ct-adrenergic effects, protein kinase C [PKC], cyclic GMP [cGMP]-dependent effects and phosphodiesterases) are referred to the following recent reviews (143-148).

zyxwv

Phosphorylation: Possible Kinases and Phosphatases Involved in Regulation of Cardiac L Channels

In biochemical studies several different protein kinases have been demonstrated to be capable of phosphorylating both cardiac and skeletal muscle L channels. These kinases include cyclic AMP-dependent protein kinase A (cAMP-PKA; see below), PKC, cGMP-dependent kinase, and calmodulin-dependent kinase II (149, 150). While we will restrict our discussion to cAMP-PKA, both PKC and cGMPdependent kinases have reported effects on mammalian cardiac ICa,L (e.g., 24,81, 151,152). With regard to dephosphorylation, at least four types of serine-threoninespecific phosphoprotein phosphatases (PP) have been identified, termed PP-1,

zyxwvut CALCIUM CHANNELS IN THE HEART

55

PP-2A, PP-2B (calcineurin), and PP-2C. All have been found in cardiac nm~ele (153). PP-2B is dependent upon both Ca 2+ and calmodulin; PP-2C is Mg2+-delmw dent, while neither PP-1 nor PP-2A appear to be markedly divalent cation-dependent. PP-1 can also be ~ ' b i t e d by cytosolic inhibitmy subunits t e r n ~ inhibiwr-I and inhibiwr-2. Of pmlicular interest is the fact that inhibitor-1 is active only when phosphorylated, and that ~adrenergic stinmlafion enhances inhibitor-1 ptmsphorylation in cardiac muscle, presumably through cAMP-PKA activation (154,155). In turn, inhibitor-1 can also itself be dephosphorylated by PP-2A and PP-2B (153). The compounds okadaie acid (OA) and microcystin (MC) have been found to be relatively selective blockers of PP-1 and PP-2A, with a higher potency for PP-2A than PP-I (e.g., 156,157). The exact biophysical/biochemical mechanisms by which phosphorylation/dephosphorylation alters L-channel gating characteristics are presently unclear (e.g., electrostatic effects may be involved, since phosphorylation adds fixed negative charges). However, any covalent modification of a region of a channel protein that governs voltage-dependent conformational changes, or other channel regions that are physically located close to such voltage-sensing regions, will undoubtedly contribute to the stability of one conformational state in relation to another (e.g., 158). Summary of Classic Neuromodulatory Compound Effects and Present Controversies Figure 9 summarizes the present mechanisms believed to be involved in regulation of cardiac ICa,Lby 13-adrenergic and muscarinic agents. With regard to 13-adrenergic modulation, under physiological conditions three basic mechanisms have been proposed to be of prime importance: (a) cAMP-dependent phosphorylation of the channel ("indirect pathway"; e.g., [159]); (b) direct channel activation of the L channel by the activated as subunits of the trimeric GTP-binding protein Gs ("direct pathway"; e.g., [160]); and (c) a combination of both phosphorylation by cAMPdependent kinase and activated as subunits ("combined mechanism"; e.g., [161]). Both 13l- and 132-adrenergic receptor subtypes exist in cardiac tissue. 131 subtypes apparently predominate in mammals and 132 predominate in amphibians (21,143). Upon application and binding of 13 agonists such as isoproterenol, whole-cell ICa,L can increase, depending upon species, from two- to threefold to more than sevenfold (the larger increase being observed in amphibian cardiac myocytes [ 143]). An overwhelming amount of evidence for the involvement of cAMP-PKA in modulating cardiac ICa,L has accumulated. The enzymatic cascade in this pathway has been well worked out and can be summarized as follows: (a) the 13receptor is coupled to the GTP-binding protein Gs. Activation of the receptor produces GTP-GDP exchange on the as subunit, causing it to dissociate from the 13~/complex; (b) activated as then stimulates the sarcolemmal bound enzyme adenylyl cyclase (AC), which leads to synthesis of cAMP; (c) elevated levels of cAMP in turn activate cAMPPKA. The inactive cAMP-PKA holoenzyme is a tetrameric complex consisting of two regulatory subunits which bind cAMP and two catalytic subunits which cata-

FIG. 9. Schematic summary of the mechanisms governing 13-adrenergic and muscarinic modulation of cardiac ICa, L. Stimulation of 13-adrenergic receptors coupled to the GTP-binding protein Gs leads to dissociation of activated ors subunits from 13",/subunits. Activated ~ subunits can then activate adenylate cyclase, which in turn triggers the cAMP-PKA enzymatic cascade that ultimately results in phosphorylation of either the L channel or a closely related subunit (e.g., 21 ). This pathway is counteracted by the activities of various phosphodiesterases and phosphatases. Alternatively, activated ors subunits may directly activate the L channel through a phosphorylation-independent direct membrane delimited pathway. Stimulation of M2-type muscarinic receptors coupled to the GTP-binding protein G~ leads to activated ot~subunits, which then can deactivate adenlylate cyclase through several possible mechanisms. Modified from Brown (170) and Hartzell and Duchatelle-Gourdon (21).

CALCIUM CHANNELS IN THE HEART

57

lyze phosphorylation. Binding of cAMP to the regulatory subunits (each binds two cAMP molecules) leads to dissociation of the catalytic subunits, which in turn catalyze phosphorylation of a substrate which is believed to be either the L channel or a tightly associated subunit (see above); and (d) phosphorylation of the channel then leads to changes in channel gating characteristics (summarized below) that produce a net increase in whole-cell Ica,L. The phosphorylated substrate is dephosphorylated by phosphatases, possibly PP-1 and PP-2A (see below), cAMP is degraded by numerous phosphodiesterases (PDEs), one of which may include a cGMP-dependent PDE (e.g., 21). The relative rates and capacities of these various processes would determine both the extent and percentage of phosphorylated channels in the population, as would any experimental perturbation of these systems by internal perfusion of different patch pipette solutions. Acetylcholine (ACh) is generally believed to only inhibit ICa,L after it has been stimulated by 13 agonists (21,143). In cardiac muscle ACh exerts its effects by binding primarily to M2-type muscarinic receptors which are coupled to the inhibitory GTP-binding protein Gi. Activation of Gi in turn leads to inhibition of AC, although the exact mechanism appears to be unclear. Possibilities include: (a) direct inhibition of AC by activated oti; (b) free [~i subunits associate with and therefore neutralize activated as subunits previously activated by [3 stimulation; and (c) ~/i directly interacts with AC or alters its association with as subunits (21). In addition to its effect on AC, numerous reports have suggested that ACh-mediated inhibition of ICa,L may also involve cGMP, possibly through involvement of either a cGMPdependent phosphodiesterase or kinase (reviewed in [21]). However, these latter effects of cGMP are still contested and poorly understood (21,24). Finally, a recent abstract (162) has suggested that nitric oxide may be an obligatory intermediate in the ACh-mediated inhibition of B-stimulated ICa.L. While the details of muscarinic inhibition of ICa,L are still unclear, neither the existence nor the individual steps involved in the indirect cAMP-dependent pathway in mediation of [3-adrenergic effects are seriously in question. However, much evidence exists suggesting that G proteins can exert direct actions on L channels. The major four lines of evidence can be briefly summarized as follows: (a) G proteins can have direct effects on L channels incorporated into bilayers (e.g., 163-165); (b) G proteins can bind to purified Ca channels (e.g., 166); (c) G proteins can affect DHP binding to Ca channels (e.g., 167,168); and (d) run down of L channels in excised patches from guinea pig ventricular myocytes has been reported to be slowed by the addition of GTP, GTP~/S, activated Gs, and activated as subunit (e.g., 163). As a result of these and other findings the "indirect hypothesis" proposes that after stimulation of 13receptors, diffusion of activated as subunits would not only lead to activation of AC but would also produce rapid activation of L channels by as binding directly to the channel or a tightly associated subunit. The present controversy in the field exists over the relative contribution and importance under physiological conditions of the indirect cAMP-PKA-mediated pathway versus direct activation of the L channel by as subunits. Summarizing both the data described above and additional experimental results (e.g., demonstration of fast

zy

58

CALCIUM CHANNELS IN THE HEART

zyx

['r= 150 msec] and slow ['r= 36 sec] phases in the increase of ICa,L in guinea pig ventricular myocytes in response to rapid perfusion of isoproterenol [160]), Brown (169,170) has strongly argued that the direct G protein-mediated pathway (i.e., the fast component) is the major physiologically relevant pathway, and that the indirect cAMP-PKA pathway would be too slow to account for the rapid effects observed for [3-adrenergic compo~ds. However, in an independent series of experiments utilizing a different perfusion apparatus that produced rapid changes in isoproterenol concentration w ~ Lys mutants is mainly caused by a reduced on-rate, but also in part by an increased off-rate (161). In the case of unidentate side-chain ligands a charged versus uncharged residue can work in either direction toward the total calcium affinity in an EF-hand pair. A recent study of CaM illustrates this fact: seemingly identical substitution in sites II and III lead to opposite shifts in both calcium affinity and cooperativity within the respective globular domains (148). In sites II and III the aspartic acid in loop position 3 was replaced by asparagine, and in terms of the separated globular domains (fragments TR1C and TR2C) the first macroscopic calcium-binding constant (K1) was reduced by a similar amount (a factor of 2.0). The second macroscopic constant (K2) of TR2C was also reduced (by a factor of 7.8), leading to an average fourfold reduction in affinity (per site). However, K2 of TR~C was increased more than K~ was reduced, and the overall effect of the substitution was a slight increase in affinity, although the net domain charge as well as net ligand charge was reduced. This slight increase in total affinity was hence caused by a large increase in the cooperativity and clearly shows that affinity and cooperativity cannot be analyzed separately. It also shows how important the pairing of EF-hands is for maintenance of high-affinity calcium binding. It is remarkable that the affinity of the single calcium site in the D-galactose binding protein is decreased by four orders of magnitude when the ligating Gln142 (corresponding to loop position 9 in EF-hand sites) is replaced by a Glu residue (129). This site already bears a high negative ligand charge ( - 4.1) which in the mutant would be - 4 . 8 if all ligands are retained in the calcium coordination sphere. This value is more negative than for any of the naturally occurring sites as listed in Table 2. In addition, this site has lost most of its ability to discriminate against magnesium (129), which suggests that one of the charged side-chain ligands is retracted from the coordination sphere in the mutant. Thus, there appears to be an optimum value for the ligand charge, above which high affinity is no longer favored due to an increasingly unfavorable contribution to the binding enthalpy from ligand repulsion. Electrostatic interactions are extremely important for tuning the Ca 2+ affinity of a site. Charged side chains that are present on the surface of the protein can have a strong influence even if they are not directly involved as ligands. Studies of calbindin D9k have shown that removal of three negative surface charges at positions 17, 19, and 26 in the vicinity of the calcium sites (cf. Fig. 15) leads to a 45-fold decrease in average affinity (per site) at low ionic strength and a fivefold reduction

zy

t~ oo

TABLE 5. Effects on calcium affinity from substitutions of loop residues in EF-hands Position: Coordinate: Consensus residues:

2

3 Y D,N

4

Protein, ref. a

5 Z D,N, S

6 G

7 Y

8 I,L, V

9 X S,T, G,N, D,E

10

11

12 Z E

&lgK1

&lgK2

AIgK1K2

|

TnC, 145

N/D

n.d.


Cys + Leu31->Cys mutant) which was concluded to stabilize the EF-hand monomer since it induced a fair amount of et-helicity in the absence of calcium. The Ca 2 + affinity was, however, reduced threefold, probably due to impaired packing of the hydrophobic core in the dimer. No Ca 2 + binding could be detected when both cysteines were methylated, most likely due to even worse hydrophobic packing. The influence of charge-charge repulsion between ligands has been addressed by mutation of loop residues in a homodimer of EF-hand III of CaM (193). In this case the Ca 2 + affinity per site increased 12-fold if the first three ligating side chains (in positions 1, 3, and 5) were changed from Asp, Asp.

zy

140

DETERMINANTS OF CALCIUM BINDING

zyx

Asn to Asp, Asn, Asp. This shows that a proper distribution of the charged side chains is important. When in addition two extra surface charges per dimer were provided by substituting Asp for Ser in position 7, the affinity was 39-fold higher than in the wild-type homodimer. The stoichiometry of high-affinity Ca 2 + binding to the six EF-hand protein calbindin D28k still remains to be solved. Four of its EF-hands have loops that are reconcilable with the EF-hand consensus sequence (see above; 19). However, the sixth and especially the second EF-hand have loop sequences that deviate so much that they are not likely to bind calcium unless they adopt variant folds. Some attempts to determine the stoichiometry of calcium binding to calbindin D28k have yielded numbers as low as 4 (86) or even 3-4 (194). A study based on eight individual Ca 2+ titrations of the protein led to the conclusion that it binds 5 or 6 calcium ions, and that all sites have an affinity close to 108 M -~ at low ionic strength (85). A fragment comprising the two N-terminal EF-hands (sites I and II) has recently been characterized in terms of calcium binding at 0.15 M NaC1 (195). The stoichiometry of high-affinity calcium binding was not measured directly, but was inferred from lanthanide binding experiments as 1.0. Since this fragment contains one of the variant EF-hands in calbindin D28k it would be of great value if the stoichiometry of calcium binding could be measured in a more direct fashion.

zy

Mutagenesis and Fragment Studies of Other Proteins The calcium-binding sites present in many EGF-like modules of blood-clotting factors have gained much attention due their importance in the coagulation cascade and its regulation. One of the calcium ligands in these type of modules is a 13-hydroxylated Asp or Asn residue (Hya or Hyn) but the extra hydroxyl group does not appear to coordinate calcium (cf. Fig. 10) (82). It has recently been shown that the 13-hydroxylation has only a modest influence on calcium affinity in both factors IX and X (29). In addition, an isolated EGF-like module binds calcium a factor of 10100 weaker as compared to when it is present in the intact protein. Studies of fragments of varying length have helped to identify which of the neighboring modules influence the Ca 2+ affinity. The N-terminal module, which is often directly preceding the EGF module, is rich in ~/-carboxy glutamic acid (Gla) residues and has a strong influence on the Ca 2+-binding strength of the EGF-like module (196198). Mutational studies of the EGF-like module from factor IX have identified three residues, two Asps and the Hya, which are important for maintaining the calcium affinity (199,200). Two of them are calcium ligands in the highly homologous module from factor X (nr 49 and 63) and one of the Asps is interspersed between two Ca 2+-coordinating residues and provides a surface charge (nr 48) (82). In protein S there are four EGF-like domains arranged in tandem. Two to four of these have several orders of magnitude higher affinity than observed for factors IX and X (30). A question that remains to be solved is whether the calcium affinity of each of these EGF-like domains is high per se or if packing of several EGF modules

DETERMINANTS OF CALCIUM BINDING

,141

leads to favorable interactions in the presence of calcium that enhance the affinities of the individual sites. Lysozyme is highly homologous to ot-lactalbumin; however, lysozymes from most organisms lack the high-affinity calcium binding site as found in ot-lactalbumin with lg K ~ 9 at low ionic strength (45,46). Human lysozyme naturally lacks such a site, but one can be created by just a few residue changes in the region homologous to the ot-lactalbumin Ca 2 § site. The sequences of the Ca 2 +-coordinating loop in bovine et-lactalbumin (with O indicating backbone carbonyl ligands and * side-chain ligands) and the corresponding segment in human lysozyme follows: 0

*

0

*

*

Asp-Lys-Phe-Leu-Asp-Asp-Asp-Leu-Thr-Asp-Asp-Ile1 2 3 4 5 6 7 8 9 10 Ser-Ala-Leu-Leu-Gln-Asp-Asn-Ile-Ala-Asp-Ala-Val-

ot-lactalbumin lysozyme

A mutant human lysozyme with aspartic acid residues substituted for Gln and Ala in loop positions 4 and 10 binds calcium with an affinity of 5.106 M -1 (201). Crystal structures of this mutant have been obtained both in the absence and presence of calcium (120) and show that the Ca 2§ ion is bound to the ten-residue loop as predicted. The calcium ion as coordinated is found to be highly similar to that in ot-lactalbumin. Further mutational studies have shown that it is possible to increase the affinity slightly (to 1.107 M - l ) if four loop positions are mutated: Alal->Lys, Gln4->Asp, Asn6->Asp, and Alal0->Asp (202). The resulting mutant has almost the same loop sequence as et-lactalbumin, yet the affinity is a factor of 100 lower, implying that interactions outside the coordination sphere are important determinants for the affinity. The single mutant with Alal0 changed to Asp binds calcium with an affinity as low as 8-103 M-~ (in comparison with 2-102 M-~ in the wildtype lysozyme [202]).

Interaction with Other Macromolecules: Effects on Calcium Affinities

Some proteins, e.g., CaM, TnC, and the blood-clotting factors, undergo Ca 2+induced conformational changes that enhance their interactions with other biological macromolecules or entities ("target molecules"). Thermodynamics then tells us that the Ca 2 + affinity must be higher when the Ca 2 +-binding protein is present in a complex with the target molecule. As illustrated in Fig. 20, the free energy coupling (AAG) between binding of Ca 2+ ions and macromolecule can be calculated either from measurements of the affinity for Ca 2+ in the absence and presence of the target (AAG = AGIv - AGI), or from measurements of the affinity for the target molecule in the absence and presence of Ca 2 + (AAG = AGIII - AGn). The value of the free energy coupling is often large. In the TnC-Ca 2 +-TnI ternary complex it is found to be 23 kJ-mol- 1 when the binding of four Ca 2 + ions is considered (203). Thus the binding of TnI to Caa-TnC is 1.2.10a-fold stronger than binding to apo

zy

zy

142

DETERMINANTS OF CALCIUM BINDING

P + CaM + 4Ca 2§

,xGf, ....

P

9CaM + 4Ca 2§

AGt

,SGiv

I

P + CaM(Ca2+)4 AGIII I

P . CaM(Ca2+)4

FIG. 20. Free energy diagram of the equilibria involving CaM, target enzyme (P) and Ca 2 +.

zy

TnC, and the average Ca 2 + affinity (per site) is a factor of 10.6 higher in the TnCTnI complex than in free TnC. The binding of calcium in the TnC-TnI complex is semi-sequential as in free TnC, i.e., the two sites in the C-terminal domain bind calcium about 100-fold stronger than the other two sites. The situation in CaM is slightly different. In free CaM the two domains differ by as little as a factor of 5 - 6 in calcium affinity per site (31) but in the presence of target (mastoparan or a caldesmon fragment) all four sites have similar affinities (204). The average increase in affinity per site was 16-fold in the presence of mastoparan, but only threefold in the presence of the caldesmon fragment. Other studies confirm that the free energy coupling varies with target (205,206). The variations in Ca 2 + affinity of CaM in the presence of different CaM-regulated enzymes leads to different Ca 2 + sensitivities of the enzymes. In molecular terms, the higher Ca 2 + affinities of CaM and TnC when complexed with target enzymes are most likely, at least in part, derived from mutual interactions of hydrophobic residues. The exposure of a hydrophobic protein surface to solvent water is accompanied by a free energy penalty. In CaM and TnC this is paid for by a reduced Ca 2 + affinity. The penalty becomes smaller if the exposed hydrophobic side chains are either replaced by polar ones (171,172) or retracted from water due to formation of a complex with another molecule.

CONCLUSIONS High-affinity calcium binding is governed by several different factors. The collective picture emerging from a wealth of research on calcium-binding proteins and their fragments is that high-affinity calcium binding is favored if:

DETERMINANTS OF CALCIUM BINDING

143

zy

9 The protein is able to provide all calcium-coordinating oxygens without invoking strain in the polypeptide chain. 9 The protein surface within about 5-15 ~ of the binding site has a net negative chargemthe more negative the better as long as it does not cause disruptions of the Ca 2 + coordination sphere. 9 Minor changes in the average conformation, as well as in the number of available conformations, occur on calcium binding. Binding to a "preformed" site (i.e., a highly populated species) is most favorable. 9 A minimum of factors counteract the calcium-induced structural changes, if any, or there are factors that could promote binding. For example, a smaller hydrophobic surface area is exposed in the presence than in the absence of calcium. 9 The hydrophobic protein core is more favorably packed in the presence than in the absence of calcium. If all of these factors were taken to their extremes the resulting site would have an affinity well above 101~ M - ~ at physiological ionic strength. Therefore, only a subset of the determinants that govern high-affinity calcium binding need to be explored in order to create a site with reasonably high affinity ( l g K = 6 - 8 at I 0.15). In other words, breaking one or a few of these rules does not necessarily lead to a low-affinity calcium site. One could also suggest that one strategy explored by nature is to sacrifice some of the available free energy of Ca 2 § binding to a group of ligands in order to drive a desired conformational change, like in the regulatory proteins calmodulin and troponin C.

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145. Babu A, Su H, Ryu Y, Gulati J. Determination of residue specificity in the EF-hand of troponin C for calcium coordination, by genetic engineering. J Biol Chem 1992;267:15469-15474. 146. Negele JC, Dotson DG, Liu W, Sweeney HL, Putkey JA. Mutation of the high affinity calcium binding sites in cardiac troponin C. J Biol Chem 1992;267:825-831. 147. Putkey JA, Sweeney HL, Campbell ST. Site-directed mutation of the trigger calcium binding sites in cardiac troponin C. J Biol Chem 1989;264:12370-12378. 148. Waltersson Y, Linse S, Brodin P, Grundstr6m T. Mutational effects on the cooperativity of Ca2+ binding in calmodulin. Biochemistry 1993;32:7866-7871. 149. Wembedey B, Thulin E, Linse S, Chazin WJ. Ca2+-binding constants and NMR characterization of (Ca)~ N56A calbindin D9k: an improved model from the N-terminal half-saturated state. Manuscript. 150. Dotson DG, Putkey JA. Differential recovery of Ca2+ binding activity in mutated EF-hands of cardiac troponin C. J Biol Chem 1993;268:24067-24073. 151. Linse S, Bylsma N, Thulin E, Fors6n S, Svensson A, Zaitsev V, Zaitseva I, Marek J. A charge conservation Glu60->Asp mutation leading to increased cooperativity of Ca 2+ binding to ealbindin D9k: Ca 2+ binding, stability, IH NMR and X-ray structural studies. Biochemistry [in press]. 152. Golden LF, Corson DC, Sykes BD, Banville D, MacManus JP. Site-specific mutants of oncomodulin: proton NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59->Glu59 substitution in the calcium-specific site. J Biol Chem 1989;264:2031420319. 153. Haiech J, Kilhoffer MC, Lukas TJ, Craig TA, Roberts, DM, Watterson, DM. Restoration of the calcium binding activity of mutant calmodulins toward normal by the presence of a calmodulin binding structure J Biol Chem 1991;266:3427-3431. 154. Maune JF, Klee CB, Beckingham K. Calcium binding and conformational change in two series of point mutations to the individual calcium-binding sites of calmodulin. J Biol Chem 1992a;267: 5286-5295. 155. Maune JF, Beckingham K, Martin SR, Bayley PM. Circular dichroism studies on calcium binding to two series of calcium binding site mutants of Drosophila melanogaster calmodulin. Biochemistry 1992b ;31:7779- 7786. 156. Starovasnik MA, Su DR, Beckingham K, Klevit RE. A series of poifit mutations reveal interactions between the calcium-binding sites of calmodulin. Protein Sci 1992;1:245-253. 157. Carlstr6m G, Chazin WJ. Two-dimensional ~H NMR studies of half saturated (Ca2+)l state of calbindin D9k: further implications for the molecular basis of cooperative Ca 2+ binding. J Mol Biol 1993;231:415-430. 158. Johansson C, Brodin P, Grundstr6m T, Thulin E, Fors6n S, Drakenberg T. Biophysical studies of engineered mutant proteins based on calbindin D9k modified in the pseudo EF-hand. Eur J Biochem 1990a; 187:455-460. 159. Johansson C, Brodin P, Grundstr6m T, Fors6n S, Drakenberg T. Mutation of the pseudo-EF-hand of calbindin D9k into a normal EF-hand: biophysical studies. Eur J Biochem 1991 ;202:1283-1290. 160. Linse S, Johansson C, Brodin P, Grundstrfm T, Thulin E, Fors6n S. The role of protein surface charges in ion binding. Nature 1988;335:651-652. 161. Martin SR, Maune JF, Beckingham K, Bayley PM. Stopped-flow studies of calcium dissociation from calcium-binding-site mutants of Drosophila melanogaster calmodulin. Eur J Biochem 1992; 205:1107-1114. 162. Johansson C, Drakenberg T, Comparison of IH NMR assignments and secondary structure of five mutant proteins of calbindin D9k. Biochemistry 1993;32:8429-8438. 163. Brodin P, Johansson C, Fors6n S, Drakenberg T, Grundstr6m T. Functional properties of calbindin D9k mutants with exchanged Ca 2+ binding sites. J Biol Chem 1990;265:11125-11130. 164. Matsuura I, Ishihara K, Nakai Y, Yazawa M, Toa H, Yagi K. A site-directed mutagenesis study of yeast calmodulin. J Biochem (Tokyo) 1991 ;109:190-197. 165. George SE, Su Z, Fan D, Means AR. Calmodulin-cardiac troponin C chimeras. J Biol Chem 1993; 268:25213-25220. 166. Brzeska H, Venyaminov SV, Grabarek Z, Drabikowski W. Comparative studies on thermostability of calmodulin, skeletal muscle troponin C and their tryptic fragments. FEBS Lett 1983;153:169173. 167. Matsuura I, Kimura E, Tai K, Yazawa M. Mutagenesis of the fourth calcium-binding domain of yeast calmodulin. J Biol Chem 1993;268:13267-13273. 168. Ikura M, Clore M, Gronenborn AM, Zhu G, Klee CB, Bax A. Solution structure of a calmodulintarget peptide complex by multidimensional NMR. Science 1992;256:632-638.

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169. Meador WE, Means AR, Florante AQ. Target enzyme recognition by calmodulin: 2.4/~ structure of a calmodulin-peptide complex. Science 1992;257:1251-1255. 170. Herzberg O, Moult J, James MNG. A model for the Ca2+-induced conformational transition of troponin C: a trigger for muscle contraction. J Biol Chem 1986;261:2638-2644. 171. Pearlstone JR, Borgford T, Chandra M, Oikawa K, Kay CM, Herzberg O, Moult J, Herklotz A, Reinach FC, Smillie LB. Construction and characterization of a spectral probe mutant of troponin C: application to analyses of mutants with increased calcium affinity. Biochemistry 1992a;31: 6545-6553. 172. da Silva ACR, de Araujo AHB, Herzberg O, Moult J, Sorenson M, Reinach FC. Troponin-C mutants with increased calcium affinity. Eur J Biochem 1993;213:599-604. 173. Johnson JD, Nakkula RJ, Smillie LB. Modulation of calcium exchange with the calcium specific sites of troponin C. Biophys J 1994;66:A124. 174. Wendt B, Martin SR, Bayley PM, Brodin P, Grundstr6m T, Thulin E, Linse S, Fors6n S. Effect of amino acid substitutions and deletions on the thermal stability, the pH-stability and unfolding by urea of bovine calbindin D9k. Eur J Biochem 1988;175:439-445. 175. Golosinska K, Pearlstone JR, Borgford T, Oikawa K, Kay CM, Carpenter MR, Smillie LB. Determination of and corrections to sequences of turkey and chicken troponins-C: effects of Thr-130 to lie mutation on calcium affinity. J Biol Chem 1991;266:15797-15809. 176. Trigo-Gonzales G, Awang G, Racher K, Neden K, Borgford T. Helix variants of troponin C with tailored calcium affinities. Biochemistry 1993;32:9826-9831. 177. Grabarek Z, Tan RY, Wang J, Tao T, Gergely J. Inhibition of mutant troponin C activity by an intra-domain disulfide bond. Nature 1990;345:132-135. 178. Grabarek Z, Tan RY, Head J. Blocking of the Ca2+-induced opening of interhelical interfaces in either of the two domains of calmodulin renders the protein inactive. Biophys J 1991;59:23a. 179. Linse S, Thulin E, Sellers P. Disulfidc bonds in homo- and heterodimers of EF-hand subdomains of calbindin D9k: stability, calcium-binding and NMR studies. Protein Sci 1993;2:985-1000. 180. Trevino CL, Boschi JM, Henzl MT. Interactions between residues in the oncomodulin CD domain influence calcium ion-binding affinity. J Biol Chem 1991;266:11301-11308. 181. Fujimori K, Sorenson M, Herzberg O, Moult J, Reinach FC. Probing the calcium-induced conformational transition of troponin C with site-directed mutants. Nature 1990;345:182-184. 182. Thulin E,, Andersson A. Drakenberg T, ForsEn S, Vogel HJ. Metal ion and drug binding to proteolytic fragments of calmodulin: proteolytic, cadmium-113, and proton nuclear magnetic resonance studies. Biochemistry 1984;23:1862-1870. 183. Grabarek Z, Grabarek J, Leavis PC, Gergely J. Cooperative binding to the calcium-specific sites of troponin C in regulated actin and actinomyosin. J Biol Chem 1983;258:14098-14102. 184. Li MX, Chandra M, Pearlstone JR, Racher KI, Trigo-Gonzales G, Borgford T, Kay CM, Smillie LB. Properties of isolated recombinant N and C domains of chicken troponin C. Biochemistry 1994; 33:917-925. 185. Durussel I, Luan-Rilliet Y, Petrova T, Takagi T, Cox JA. Cation binding and conformation of tryptic fragments Nereis sarcoplasmic calcium binding protein: calcium-induced homo- and heterodimerization. Biochemistry 1993;32:2394-2400. 186. Permyakov EA, Medvekin VN, Mitin YV, Kretsinger RH. Noncovalent complex between domain AB and domains CD*EF of parvalbumin. Biochem Biophys Acta 1991;1976:67-70. 187. Reid RE. A synthetic 33-residue analogue of bovine brain calmodulin calcium binding site III: synthesis, purification and calcium-binding. Biochemistry 1987;26:6070-6073. 188. Shaw GS, Hodges RS, Sykes BD. Calcium-induced peptide association to form an intact protein domain: tH NMR structural evidence. Science 1990;249:280-283. 189. Finn BE, K6rdel J, Thulin E, Sellers P, Fors6n, S. Dissection of calbindin D9k into two subdomains by a combination of mutagenesis and chemical cleavage. FEBS Lett. 1992;298:211-214. 190. Tsuji T, Kaiser ET. Design and synthesis of the pseudo-EF hand in calbindin D9k: effect of amino acid substitutions in the a-helical regions. Proteins Struct Func Genet 1991;9:12-22. 191. Kay LE, Forman-Kay JD, McCubbin WD, Kay CM. Solution structure of a polypeptide dimer comprising the fourth Ca2+ -binding site of troponin C by a nuclear magnetic resonance spectroscopy. Biochemistry 1991;30:4323-4333. 192. Shaw GS, Hodges RS, Sykes BD. Determination of the solution structure of a synthetic two-site calcium binding homodimeric protein domain by NMR spectroscopy. Biochemistry 1992;31:957280. 193. Reid RE. Synthetic fragments of calmodulin calcium-binding site III: a test of the acid pair hypothesis. J Biol Chem 1990;265:5971-5976.

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194. Gross MD, Nelsestuen GL, Kumar R. Observation of the binding of lanthanides and C a 2 + to vitamin D-dependent chick intestinal calbindin D28k. J Biol Chem 1987;262:6539-6545. 195. Gross MD, Gosnell M, Tsarbopoulos A, Hunzikcr W. A functional and degenerate pair of EF hands containing the very high affinity calcium-binding site of calbindin D28k. J Biol Chem 1993 ;268:20917-20922. 196. Astermark J, Bj6rk I, Ohlin A-K, Stenflo J. Structural requirements for Ca2+ binding to the T-carboxy glutamic acid and epidermal growth factor like regions of factor IX: studies using intact domains isolated from controlled proteolytic digests of bovine factor IX. J Biol Chem 1991;266: 2430-2437. 197. Persson E, Bj6rk I, Stenflo J. Protein structural requirements for Ca2+ binding to the light chain of factor X. J Biol Chem 1991;266:2444-2452. 198. Valcarce C, Selander-Sunnerhagen M, T~nlitz AM, Drakenberg T, Bj6rk I, Stenflo J. Calcium affinity of the aminoterminal epidermal growth factor-like module of factor X. Effect of the gamma-carboxyglutamic acid-containing module. J Biol Chem 1993;268:26673. 199. Handford PA, Mayhew M, Baron M, Winship PR, Campbell ID, Brownlee GG. Key residues involved in calcium-binding motifs in EGF-like domains. Nature 1991;351:164-167. 200. Mayhew M, Handford P, Baron M, Tse AGD, Campbell ID, Brownlee GG. Ligand requirements for Ca2§ binding to EGF-like domains. Protein Eng 1992;5:489-494. 201. Kuroki R, Taniyama Y, Seko C, Nakamura H, Kikuchi M, lkehara M. Design and creation of a Ca2§ binding site in human lysozyme to enhance structural stability. Proc Natl Acad Sci USA

1989;86:6803-6807. 202. Haezebrouck P, De Baetselier A, Joniau M, Van Dael H, Rosenberg S, Hanssens I. Stability effects associated with the introduction of a partial and a complete Ca2 + -binding site into human lysozyme. Protein Eng 1993;7:643-649. 203. Wang C-K, Cheung HC. Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle. Biophys J 1985;48:727-739. 204. Yazawa M, Ikura M, Hikichi K, Ying L, Yagi K. Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. Calcium binding and proton NMR. J Biol Chem 1987;262:10951-10954. 205. Olwin BB, Storm DR. Calcium binding to complexes of calmodulin and calmodulin binding proteins. Biochemistry 1985;24:8081-8086. 206. Keller CH, Olwin BB, LaPorte DC, Storm DR. Determination of the free-energy coupling for binding of calcium ions and troponin I to calmodulin. Biochemistry 1982;21:156-162.

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Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1995.

4 Calcium Regulation of Smooth Muscle Contractile Proteins J. David Johnson and Christopher H. Snyder

Department of Medical Biochemistry, The Ohio State University College of Medicine, Columbus, Ohio 43210-1218

An increase in intracellular calcium (Ca) ion concentration is fundamental to the contraction of smooth muscle. An extensive series of hormone receptors, G proteins, ion channels, antiporters, and ATPases modulate ion flux across the cell and sarcoplasmic reticulum membranes, allowing a precise regulation of Ca homeostasis within the muscle cell (see 1-4 for review). The contraction-relaxation cycle of skeletal muscle is tightly coupled to a rise and fall, respectively, of cytosolic [Ca]. In smooth muscle, however, certain agonists produce tension with only subtle changes in [Ca], suggesting a dissociation of the contraction-relaxation cycle from the Ca transient (3-9). The Ca signal transduction mechanisms in smooth muscle further diverge at the level of the Ca receptors or Ca binding proteins which "witness" the Ca transient and transduce the "Ca signal" into a contraction-relaxation cycle. Calmodulin (CAM) controls the contractility of smooth muscle while troponin C controls the contractility of both skeletal and cardiac muscle. After this point, the contraction-relaxation mechanisms of smooth muscle become vastly different from skeletal muscle, involving both thick and thin filament regulatory mechanisms. In this chapter we will present an overview of the Ca-regulated contractile proteins of smooth muscle including: CaM, myosin light-chain kinase (MLCK), caldesmon (CAD), calponin (Calp), and caltropin. We will review the structural, functional, and biochemical aspects of these proteins. We will examine the kinetics of CaM's interaction with its smooth muscle target proteins; MLCK, CaD, and Calp. We will discuss the role of these contractile proteins in the thin and thick filament regulation of the contraction-relaxation cycle of smooth muscle. Finally, we will review several mechanisms by which hormone-receptor signal transduction pathways (and specific protein kinases) can modulate the Ca sensitivity of the contractile apparatus and alter the contraction-relaxation cycle of smooth muscle. 153

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OVERVIEW OF SMOOTH MUSCLE CONTRACTILE PROTEINS

The contraction-relaxation cycle of smooth muscle is controlled by Ca and phosphorylation-dephosphorylation-dependent processes on both the thick (myosin) filament and the thin (actin) filament.

Thick Filament Control of Actomyosin ATPase and Crossbridge Cycling It is now accepted that Ca-CaM binding and activation of MLCK and its subsequent phosphorylation of the 20-kDa myosin light chain (MLC) is a primary determinant of smooth muscle contraction (6-9 for review) (Fig. 1). Phosphorylation of MLC is closely correlated with an increase in actin-activated myosin ATPase activity and the initiation of smooth muscle contraction (see 10-12). As cytosolic Ca falls, CaM dissociates from MLCK, resulting in its inactivation and inhibition of MLC phosphorylation. MLC phosphatases, which are thought to be Ca-insensitive, dephosphorylate MLC, and the interaction of actin with myosin is again inhibited. Details of CaM's Ca-dependent interaction with MLCK will be discussed below.

Thin Filament Control of Actomyosin ATPase and Crossbridge Formation Both CaD and Calp bind to the thin filament and alter phosphorylated myosin's interaction with actin. This results in inhibition of both actomyosin ATPase activity

FIG. 1. A schematic representation of the calcium-dependent regulation of smooth muscle contraction-relaxation. The Ca-dependent exposure of hydrophobic sites on the N- and C-terminal lobes of calmodulin (CAM) allow it to bind myosin light-chain kinase (MLCK) and remove a pseudosubstrate inhibitory peptide from its catalytic site. This activates MLCK and it binds and phosphorylates (P) myosin light chain (MLC). Phosphorylation of MLC allows myosin to bind actin; actomyosin ATPase activity is increased and contraction occurs. MLC phosphatase (MLCPhtase) dephosphorylates MLC, resulting in an inhibition of myosin binding to actin and smooth muscle relaxation. Caldesmon and calponin bind to actin or the actin-tropomyosin complex to alter myosin binding to actin and inhibit actomyosin ATPase activity. In the presence of Ca, CaM (or caltropin, not shown) can bind to CaD and relieve its inhibition of actomyosin ATPase. Phosphorylation of both caldesmon and calponin by cellular kinases can also relieve their inhibition of actomyosin ATPase.

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155

and muscle contraction (7,8,13-15 for review and Fig. 1). In the presence of Ca, CaM can bind to both CaD and Calp and reverse their inhibition of actomyosin ATPase activity. While CaM's reversal of CaD inhibition appears to be physiologically relevant, its reversal of Calp's inhibition may not be. In addition, a number of cellular kinases phosphorylate both CaD and Calp to reverse their inhibition of actomyosin ATPase activity (13-15) and facilitate smooth muscle contraction. Thus, Ca-CaM apparently works both at the level of the thick filament via activating MLCK to phosphorylate MLC and initiate crossbridge cycling and at the level of the thin filament to relieve CaD inhibition of crossbridge cycling. Further, caltropin, a recently discovered Ca-binding protein, has been shown to induce a Cadependent reversal of CaD's inhibition of actomyosin ATPase (16). Details of CaM's interaction with these thin filament proteins and their role in modulating the contraction-relaxation cycle of smooth muscle is discussed below.

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Myosin Light Chain Kinase Smooth muscle MLCK is a --~110-kDa protein consisting of---972 amino acids (Fig. 2). It contains a high-affinity CaM binding sequence near its catalytic domain, and MLCK is responsible for the highly specific phosphorylation of MLC at Ser 19. MLCK activity is totally dependent on the Ca-dependent binding of 1 mole of CaM per mole of kinase with an affinity of --~1 nM (7,8,17). We have shown that halfmaximal CaM binding and activation of MLCK occurs near pCa 6.5 (17,18), and a number of investigators have found a similar Ca dependence for MLC phosphorylation and the development of force in intact and chemically skinned smooth muscle (5,10-12). Taylor et al. (11) have used the fluorescent Ca indicator fura-2 to demonstrate a strict correlation between cytosolic [Ca] and the extent of MLC phosphorylation in smooth muscle contracted with a variety of agonists. Rembold and Murphy (10) have used the photoprotein aequorin to measure [Ca] in smooth muscle and relate it to MLC phosphorylation, crossbridge cycling, and active stress. These studies demonstrate that cytosolic [Ca] is the primary determinant for MLC phosphorylation, crossbridge cycling, and active stress in smooth muscle. These and numerous other studies (6-8 for review) clearly show that the primary mechanism for smooth muscle contraction involves a rise in cytosolic [Ca], CaM activation of MLCK, phosphorylation of MLC, and the subsequent cycling of crossbridges and force generation (Fig. 1).

Mechanism of CaM Activation of MLCK Since CaM binding is essential for MLCK activity this complex has been studied extensively as a model for CaM activation of its various target enzymes. Kemp et al. (19) and Kennelly et al. (20) made the seminal observation that the CaM-binding domain of both smooth and skeletal muscle MLCK overlaps a region of the kinase which is homologous to the phosphorylation site domain (residues surrounding Ser

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19) of MLC (Fig. 2). This region of MLCK was postulated to occupy the catalytic site of MLCK and competitively inhibit MLC binding and phosphorylation. Thus, this region has been called the pseudosubstrate inhibitory (PSI) region of MLCK. Ca-CaM was proposed to bind and remove this PSI sequence from the catalytic site, resulting in MLCK activation (Fig. 3). Consistent with this mechanism, synthetic peptides containing this PSI sequence (residues 783-804 of SM-MLCK and a homologous sequence in skeletal muscle MLCK) were potent competitive (with respect to MLC) inhibitors of MLCK phosphorylation of MLC (19,20). This theory of a CaM-dependent removal of a PSI region has been supported by extensive experimental evidence: (a) synthetic peptides of the PSI region are potent competitive inhibitors of both the CaM-stimulated and constitutively active kinase (19,20, and see 21-23 for review); (b) removal of the PSI region (by limited proteolysis or truncation) results in a constitutively active enzyme that does not bind CaM (see 21-23) while removal of portions of the CaM-binding domain (which leave the PSI region intact) produce an irreversibly inhibited enzyme (21-23); (c) when part of the PSI sequence in MLCK was replaced with 5 amino acids of the phosphorylatable MLC substrate sequence, it was autophosphorylated in the absence of Ca-CaM (24); (d) Knighton et al. (21) have shown that the interaction of the PSI region of MLCK with its catalytic site can be easily modeled with the crystallographic coordinates of the cAMP-dependent kinase and its inhibitory peptide, suggesting that the PSI peptide model is structurally feasible.

REGULATION OF SMOOTH MUSCLE PROTEINS

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FIG. 3. A schematic representation of how calmodulin (CAM) binding to myosin light-chain kinase (MLCK) results in the activation of MLCK and might result in the simultaneous inactivation of myosin light chain (MLC) phosphatase. In the absence of Ca, the pseudosubstrate (PSI) domain of MLCK lies buried in its catalytic site and the kinase is inhibited (MLCK Inactive). As Ca rises, CaM is activated and binds near the PSI region of MLCK and reverses its inhibition of MLCK activity (Active K/nase). When the PSI region is removed from the active site, substrate protein (MLC) can bind and become phosphorylated by the kinase. It is proposed that this PSI region is also recognized by MLC phosphatase and acts as a competitive inhibitor of phosphatase (Phosphatase Inactivation). This would allow a simultaneous activation of MLCK and inhibition of MLC phosphatase. As Ca falls, CaM, would dissociate from CaM, and the PSI region would again occupy MLCK's catalytic site and competitively inhibit kinase. This could result in the rapid release and disinhibition of MLC phosphatase (PhosphataseActive), which would then dephosphorylate MLC, resulting in muscle relaxation.

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While this evidence supports the general concept of a CaM-dependent PSI sequence within MLCK, the precise boundaries of this inhibitory region and its interactions with the catalytic site are still being defined. The modeling studies of Knighton et al. (21) predicted that the 14 amino acids N-terminal to the PSI region might have extensive electrostatic interactions with the catalytic core of MLCK that could stabilize the PSI peptide in the active site. Consistent with this, they demonstrated that a peptide containing residues 774-807 (a 14 amino acid connecting peptide and the PSI region, see Fig. 2) competitively inhibited constitutively active MLCK ---30-fold more effectively (Ki =0.33 nM) than a peptide containing only residues 787-807 (K i = 11.7 nM). The extended nature of the connecting peptide and the PSI region and their more extensive electrostatic contacts with a large surface area of MLCK may explain why some site-directed mutagenesis experiments gave results that were apparently inconsistent with the pseudosubstrate hypothesis (21,23,25,26). Extensive studies by many investigators have proven the feasibility of this intrasteric or PSI mechanism and have shown that CaM removal of a PSI region from the catalytic site of many kinases and enzymes (including MLCK, CaM kinase II, the

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REGULATION OF SMOOTH MUSCLE PROTEINS

CaM-activated Ca-ATPase, and calcineurin) may be responsible for their activation (22,23). It is apparent that this type of intrasteric regulation represents a common on/off switch for the regulation of many enzymes.

CaM's Removal of the PSI Region from MLCK's Catalytic Site Activates MLCK and May Inhibit MLC Phosphatase The PSI region of MLCK is homologous to the region of MLC that MLCK phosphorylates and MLC phosphatase dephosphorylates. Since MLC phosphatase is able to bind and dephosphorylate this sequence of MLC, it follows that the homologous PSI region of MLCK should also be recognized by MLC phosphatase. Thus, nature has designed both MLCK and MLC phosphatase to have a high specificity and affinity for the sequence motif surrounding Ser 19 of MLC, and it has placed a similar sequence motif in the PSI domain of MLCK (Fig. 3). It has already been demonstrated that the PSI region of MLCK acts as a high-affinity competitive inhibitor of MLCK (19-22). We feel that it is likely that this PSI region of MLCK is also a competitive inhibitor of MLC phosphatase. Consistent with this idea, Strauss et al. (27) have shown that a peptide (MKI, residues KKRAARATS) based on the sequence motif N-terminal to ser-19 of MLC (and therefore homologous to regions in the PSI region of MLCK) was a potent myosin kinase inhibitor (MKI) and was equally effective as a competitive inhibitor of MLC phosphatase. This clearly demonstrates that a peptide designed around the MLC phosphorylation site can competitively inhibit both MLCK and MLC phosphatase, and MLCK possesses such a peptide in its PSI domain. In the presence of Ca, CaM binds and removes the PSI region from MLCK's active site allowing MLCK to phosphorylate MLC. If our hypothesis is correct, this PSI region will be exposed by CaM binding and competitively inhibit MLC phosphatase (Fig. 3). In the absence of Ca, CaM would dissociate from the PSI region of MLCK and it would return to its inhibitory position in the catalytic site of MLCK. This burying of the PSI region in MLCK's active site could result in the release of active phosphatase and the rapid dephosphorylation of MLC in the absence of Ca (Fig. 3). While this is currently hypothesis, the simplicity of this model is appealing: as Ca rises, CaM binding to the PSI region of MLCK might simultaneously activate MLCK and inhibit MLC phosphatase, dramatically favoring MLC phosphorylation. As Ca falls, CaM dissociation from the PSI region of MLCK would inhibit MLCK and could disinhibit MLC phosphatase, dramatically favoring the dephosphorylation of MLC. While MLCK phosphorylates MLC only in the presence of Ca-CaM, MLC phosphatase activity is thought to be Ca-independent (6,8). A constitutively active Caindependent phosphatase working against and competing with an active kinase (in the presence of Ca) would result in a wasteful and inefficient expenditure of ATP. A turning off of phosphatase when kinase is turned on and a turning on of phosphatase

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only when kinase is turned off would provide an energy-efficient mechanism consistent with the high efficiency of smooth muscle contraction. The Latch State in Smooth Muscle

The initial rise in smooth muscle tension is clearly associated with a rise in cytosolic [Ca], activation of CaM-MLCK, increased MLC phosphorylation, rapid crossbridge cycling, and increased energy consumption (28). This phasic rise in tension is often followed by the phenomena of the latch state. The latch state is characterized by the maintenance of contractile force in spite of decreased cytosolic [Ca], MLC phosphorylation, crossbridge cycling rate, and energy consumption (6,9). Attempts to explain the latch state have often evoked mechanisms where the thin and thick filaments are crosslinked by protein connections other than crossbridges. Candidates for this crosslink have included leiotonin, CaD, and Calp (4,6-8,29, 30). Growing evidence suggests that the latch state need not involve other crosslinks and that it is maintained by latch bridges" dephosphorylated, attached crossbridges (see 9 for review). Latch bridges are thought to be formed by rapid dephosphorylation of attached (phosphorylated) crossbridges or by cooperative interactions between crossbridges which allow the attachment of dephosphorylated crossbridges (9). Can Parallel Decreases in MLCK and MLC-Phosphatase Activity Induce a Latch State?

Peptide analogues of the MLC phosphorylation site and analogues of MLCK pseudosubstrate domain both induce a "latchlike" state in smooth muscle. Moreland et al. (31) have demonstrated that peptides based on the PSI domain of MLCK and on MLC's phosphorylation domain were capable of producing a latchlike state where superprecipitation (the biochemical equivalent of force) was maintained, although actomyosin ATPase activity (and presumably crossbridge cycling) was reduced. Strauss et al. (27) have conducted extensive studies with a similar peptide (based on MLC's phosphorylation domain) which was designed to be an MKI. They reported that this peptide induced a latchlike state in that it did not reduce tension (crossbridge attachment) in a Ca-contracted skinned muscle preparation, yet it inhibited unloaded shortening velocity (crossbridge cycling rate) and actomyosin ATPase activity. This peptide also decreased relaxation rate and exhibited suprabasal force maintenance as observed in the latch state. They discovered that this peptide was equally effective at inhibiting MLCK and MLC phosphatase. This peptide (like most other phosphatase inhibitors) induced tension and MLC phosphorylation at low [Ca] and dramatically slowed the rate and extent of relaxation in Ca-contracted fibers. Thus, this peptide inhibitor of MLCK and MLC phosphatase induced many of the characteristics of the latch state except that it did not significantly reduce the

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REGULATION OF SMOOTH MUSCLE PROTEINS

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levels of phosphorylated MLC (27). Many investigators have not, in fact, found a good correlation between MLC phosphorylation levels and induction of the latch state (see 6,27). As Strauss et al. (27) point out, their peptide might not be expected to decrease MLC phosphorylation because it is equally effective at inhibiting both MLCK and MLC phosphatase at peak [Ca]. There would, therefore, be no net change in the level of MLC phosphorylation but turnover of phosphorylated MLC would be dramatically reduced. This raises the very intriguing possibility that the latch state could possibly be induced even at different levels of MLC phosphorylation if net turnover of phosphorylated MLC is inhibited or reduced. If our hypothesis of a parallel Ca-CaM activation of MLCK and inhibition of MLC phosphatase via the PSI domain of MLCK is correct, then as [Ca] falls in smooth muscle, kinase activity would be turned down as phosphatase activity is turned up. Depending on the level of free [Ca] achieved, this could produce a state where MLCK activity and MLC phosphatase activity were essentially balanced. When this was achieved there would be little or no net turnover of MLC phosphorylation, and a latchlike state similar to that produced by the MKI peptide could result. Whether this parallel decrease in MLCK activity and increase in MLC phosphatase activity is mediated by CaM's interaction with MLCK's pseudosubstrate domain and whether this mechanism can explain the phenomena of latch state at submaximal [Ca], remains to be tested.

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CaM can bind and activate more than 20 different proteins in eukaryotic cells in a Ca-dependent manner. In the presence of Ca, CaM is a dumbbell-shaped molecule containing an N- and C-terminal lobe connected by an 8 turn central helix. Each lobe contains two Ca-binding sites (EF-hands) and exposes hydrophobic pockets with Ca-binding. CaM uses this Ca dependent exposure of its N- and C-terminal hydrophobic pockets to recognize and bind peptide regions which form an amphipathic helical motif on its target proteins (see 23, 32 for review). In SM-MLCK, Ca-CaM finds this motif in residues 796-815 which it binds with high ( - 1 nM) affinity, resulting in enzyme activation. Recently the X-ray crystallographic structure of CaM complexed with its SMMLCK-binding peptide (RS-20, residues 796-815) and the solution NMR structure of CaM bound to its 26-residue (M-13) skeletal muscle MLCK binding peptide have been determined (33,34). The structure determined by both techniques was quite similar and the X-ray crystallographic studies of Meador et al. (33) are summarized below. These investigators found that upon peptide binding there is an unfolding of the central helix, a -100-degree bend and a -120-degree twist between the N- and C-terminal lobes of CaM. This allows the hydrophobic pockets of the N- and C-terminal lobes of CaM to form a "hydrophobic tunnel" which engulfs and holds the Cand N-terminal of the peptide, respectively. There were -185 contacts between peptide and CaM, ---80 percent were van der Waal contacts and - 1 5 percent were

REGULATION OF SMOOTH MUSCLE PROTEINS

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hydrogen bonds. All nine Met residues of CaM were involved in hydrophobic interactions with peptide and all seven basic residues of the peptide formed salt bridges with CaM. CaM's C-terminal hydrophobic pocket was occupied by W5 and T8 of the peptide which (corresponding to W800 and T803 of MLCK) and its N-terminal hydrophobic pocket was occupied by A14,I15, and L18 (corresponding to A809, 1810, and L813 in MLCK). Further, R17 (corresponding to R812 of MLCK) of the peptide interacted extensively with the bend and the helices surrounding the bend in CaM's central helix. These crystallographic and NMR studies more fully explained site-directed mutagenesis studies of MLCK which showed that mutation of W800, R812, and L813 of MLCK inhibited its CaM-dependent activation (35) and that mutation of only three CaM residues (El4, T34, and $38) could convert it into a high-affinity competitive antagonist of MLCK (36). These crystallographic and NMR studies give the first three-dimensional picture of CaM bound to the peptide region which it encounters in MLCK and elucidate the specific contacts that occur within this complex. They suggest a mechanism by which the two lobes of CaM can fold together to remove the PSI region from the catalytic site of MLCK, resulting in its activation.

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Effect of MLCK Peptides on Ca dissociation from CaM's N- and C-Terminal Ca Binding Sites Target protein or peptide binding to CaM produce dramatic increases in its affinity for Ca (17). The X-ray crystallographic and NMR studies cited above detail the extensive contacts of the RS-20 and M-13 peptides with both the N- and C-terminal hydrophobic pockets of CaM. We wished to determine the effect of these peptides on the rates of Ca dissociation from CaM. When Quin 2 is rapidly mixed with a Ca-Ca binding protein complex, its fluorescence increases at the rate of Ca dissociation from that Ca-binding protein. Figure 4A shows Ca dissociation from the N- and C-terminal sites of CaM as monitored by Quin 2 fluorescence. Approximately 1.3 mol of Ca dissociate from the low-affinity N-terminal sites of CaM at a rate of --~400/sec (see 4A inset) and ---2 mols of Ca dissociate from CaM's higher affinity C-terminal Ca-binding sites at a rate of ---2.4/ sec. RS-20 and M-13 (two-fold molar excess) reduced the rate of Ca dissociation from CaM's N-terminal Ca-binding sites from 400/s to --~1.8/sec. RS-20 and M-13 reduced the rate of Ca dissociation from CaM's C-terminal Ca-binding sites from 2.4/sec to --~0.l/sec (Fig. 4B). Thus, both of these high-affinity CaM-binding peptides produce a --~220-fold decrease in the rate of Ca dissociation from CaM's N-terminal Ca-binding sites and a ---24-fold decrease in the rate of Ca dissociation from CaM's higher affinity C-terminal Ca-binding sites. Thus, Ca induces the exposure of hydrophobic binding sites on both the N- and C-terminal lobes of CaM, and when these hydrophobic sites are occupied by a high-affinity peptide the resulting peptide-CaM interactions (as defined in the X-ray and NMR structure of these com-

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CALCIUM REGULATION OF PROTEIN SYNTHESIS

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data from another report (107). For example, these authors claimed that purified hsp90 did not have EF-2 kinase activity until it was dephosphorylated and then rephosphorylated by PKA. However, hsp90 does not have a kinase domain, though it does bind ATP (and even CAM), thus it seems intrinsically unlikely that the protein would exhibit significant phosphotransferase activity. Ironically, a close association between hsp90 and HCR, in terms of molecular size and possible physical association, led to a similar confusion of these species in early studies (see 44 for review). Authentic CaMK III can autophosphorylate (Fig. 7) and is also a substrate for PKA (93,108). While both of these phosphorylation events tend to make the enzyme activity at least partially independent of Ca 2 +/CAM in vitro, in a manner reminiscent of CaMK II (cf. 109), it is not known if this is physiologically relevant in the case of CaMK III. Treatments that elevate cAMP levels in cells do not seem to alter EF-2 phosphorylation state (99,100; M. J. Brady and H. C. Palfrey, unpublished data, and see below), though they presumably lead to CaMK III phosphorylation. More detailed studies of the enzyme will be possible once its molecular structure is known and antibodies are prepared. Further regulation of the CaMK III-EF-2 system may involve modulation of enzyme levels. While it is apparent that the primary mechanism of regulation is presumably via Ca 2 +/CaM binding to the enzyme, in principle it would be possible to limit the amount of EF-2 phosphorylated during periods of elevated [Ca 2 +]cyt by reducing the total amount of CaMK III available. Such a situation arises in PC12 cells where CaMK 1II levels are down-regulated over a period of several hours following NGF treatment. This phenomenon was originally described by End et al. (110); however, they did not identify the kinase as a Ca 2 +/CaM-regulated enzyme. As shown by Nairn et al. (111) and Mitsui et al. (93), this was because the assays of End et al. were carded out at pH 6.2, under which conditions CaMK HI activity is virtually independent of Ca 2+/CAM. The mechanism of down-regulation is still unclear, but seems to involve a destruction of the enzyme as new protein synthesis is needed for recovery of enzyme levels after NGF treatment (111). The signaling pathway from the NGF receptor to CaMK HI is still under investigation. However, the cAMP system seems to be involved as agents that elevate cAMP also lead to CaMK HI inactivation (111), and PC12 cells that are deficient in PKA fail to respond to NGF or forskolin (112). Although PKA does phosphorylate CaMK III (see above) it leads to an activation rather than an inhibition of enzyme activity in vitro. Down-regulation in intact cells is a slow process, whereas cAMP activation of PKA is rapid, though loss of CaMK III activity may still be a consequence of PKA-mediated phosphorylation of the enzyme. ~ NGF-induced down-regulation of CaMK III involves the tyrosine kinase activity of the highaffinity NGF receptor Trk and may require ras activation (113). PC12 cells that do ~The stimulation of protein synthesis in reticulocyte lysates by high concentrations of cAMP (115) is often quoted as indicative of cAMP-dependent inhibition of CaM kinase III. However, this effect is entirely due to cAMP competition at the ATP substrate binding site of CaM kinase III (107; Palfrey and Nairn; unpublisheddata), not to an interaction between cAMP-dependentprotein kinase and CaM kinase III.

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not express Trk (PC12 nnr) fail to down-regulate CaMK III in response to NGF; down-regulation in normal cells can be blocked by the nonspecific kinase inhibitor K252a that is selective for the Trk family of receptor tyrosine kinases at low concentrations. Importantly, this inhibitor does not affect down-regulation induced either by forskolin or EGF. In addition, PC12 cells expressing a dominant negative form of ras (H-raSAsn 17) down-regulate CaMK III poorly in response to NGF but still respond effectively to cAMP elevation (113). By contrast, down-regulation appears independent of the well-studied MAP kinase pathway and PKC, both of which are also activated by the NGF in PC12 cells. Phorbol esters have little effect on CaMK III activity (though they do elevate MAP kinases), and PC12 cells that are substantially depleted of PKC by long-term TPA treatment still respond to NGF (111-113). The physiological significance of CaMK III down-regulation is currently unknown but may occur in other cells of nervous system origin (114; H. C. Palfrey, unpublished data) and during development (135). The obvious implication is that the protein synthetic apparatus would be less sensitive to Ca 2 +-mediated elongation block after down-regulation. Such an event could be important in developing neurons where Ca 2 + influx via voltage-sensitive Ca 2 + channels controls many processes from growth-cone migration to neurotransmitter release. The possibility that this is the case is presently under investigation.

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Currently, it is only possible to speculate as to the function of EF-2 phosphorylation. As the event appears to be of short duration with most stimuli (see above), it is unlikely to be a strategy the cell uses to control protein synthesis in the long term, in contrast to blockade of initiation. One rather prosaic explanation is that a transient elongation block (extending into a lengthier initiation block?) is a maneuver the cell uses to conserve energy when other Ca 2 +-dependent processes such as secretion or contraction are stimulated by hormones or neurotransmitters. Protein synthesis is energy-intensive; thus, it may be worthwhile for cells to inhibit this function temporarily while other energy-demanding processes take precedence. It is worth bearing in mind that the economy under consideration here may revolve as much around guanine nucleotide availability as that of adenine nucleotides. Functions such as signal transduction and intracellular trafficking events, including secretion, are heavily dependent on GTP. It is not immediately obvious how one would test the energy conservation hypothesis, but it might be possible to measure energy (i.e., GTP and ATP) consumption during imposed Ca 2 + loads in cells engineered to express nonphosphorylatable EF-2 mutants. A more intriguing possibility is that EF-2 phosphorylation is the cellular equivalent of treatment with the elongation-blocking drug cycloheximide. Cycloheximide has several effects on gene expression, the most prominent of which is the phenomenon of "superinduction." An example of this effect occurs in the expression of immediate-early gene mRNAs such as c-fos, c-jun and c-myc that normally have very short half-lives. Treatment of cells with cycloheximide leads to an apparent

CALCIUM REGULATION OF PROTEIN SYNTHESIS

215

stabilization of these mRNA species (see 116 for review). The mechanisms responsible for regulating mRNA stability are still being debated and include, in some cases, the existence of characteristic AU-rich sequences in the 3' UTRs of susceptible species (117). Translation appears to be important in controlling the process of mRNA degradation, possibly by producing a labile nuclease that attacks such mRNA populations. Inhibition of translation then slows synthesis of the nuclease, protecting the mRNA from degradation. On the other hand, ribosome stalling during elongation block appears to destabilize certain messages by allowing "gaps" to appear in polysomes that could be targeted by nonspecific RNases (e.g., 118). Could EF-2 phosphorylation serve a similar, though presumably not as spectacular, regulatory role as cycloheximide in the expression of these genes? A difficulty with this proposal is that an elongation block engendered by EF-2 phosphorylation would be transient and precede significant accumulation of immediate early gene mRNAs in the cytoplasm. Many experiments with cycloheximide and other protein synthesis inhibitors require relatively long exposures that do not mimic the kinetics of EF-2 phosphorylation in response to physiological stimulation. With such long exposures it is even possible to partially replicate, with some protein synthesis inhibitors, the early response to mitogens. This has led to the speculation that EF-2 phosphorylation may play a role in such "reprogramming" of gene expression (e.g., 103). However, caution should be exercised in considering long-term effects of cycloheximide (and other inhibitors like anisomycin) as they may reflect a specific action of the drug on signaling pathways rather than an effect on protein synthesis per se (cf. 119). Another possibility is that EF-2 phosphorylation early in the mitogenic response or at other phases of the cell cycle serves a synchronization function. It seems paradoxical that cells would wish to block elongation, even for a short time, when they need to synthesize more protein on emerging from quiescence or mitosis. However, the situation in the cell at that time is that there are many mRNAs in the cytoplasm that, once the initiation block is removed, would shift onto ribosomes. The consequence of an early elongation block would be that these mRNAs stall at an early stage of polypeptide extension, forcing free ribosomal subunits to engage other, perhaps less efficiently translated, mRNAs. Clearly the result of such a phenomenon would be a diversification of translated messages that may optimize early protein expression. Again an analogy with the effects of cycloheximide can be drawn. As shown in studies largely from the Thach laboratory (e.g., 121; see also 122), the translation of mRNAs with low initiation rates is actually favored by inhibition of elongation because of a reduction in the competition with mRNAs having high initiation rates. A prediction of such a hypothesis is that there would be a lag in the increase of protein synthesis on serum stimulation of quiescent cells. Indeed, such a lag has been found (though not commented on) in at least one study (18). It is interesting to note that distinct lags in the resumption of normal rates of protein synthesis were also seen in those experiments designed to test the reversibility of Ca 2 + depletion on protein synthetic rates. For example, Kumar et al. (37) showed that there was a 5

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216

CALCIUM REGULATION OF PROTEIN SYNTHESIS

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minute lag before resumption of normal protein synthesis on readdition of Ca z+ to Ca 2 +-depleted Ehdich cells, and similar lags were seen earlier (e.g., 22; see Fig. 1). Could such results reflect a transient elongation block following a surge of Ca 2 + into the cytoplasm, or is the lag merely the time it takes for initiation factors to reach the appropriate phosphorylation state prior to a resumption of normal activity? Such questions can be answered only by more careful kinetic analysis of the early phases of such transitions alongside biochemical measurements of initiation and elongation factor phosphorylation. One paradigm that would seem worth investigating further is the possible role of EF-2 phosphorylation in the cell cycle. A number of studies have shown that protein synthetic rates vary throughout the cell cycle. For example, there is a strong suppression of protein synthesis during mitosis that has been proposed to be caused by an initiation block as polysomes decrease, while ribosomal transit times do not change (61; see also 123 for studies in Xenopus embryos). Curiously, there are few studies addressing the molecular mechanism of initiation block in mitosis, though it has been mentioned in passing that elF-2ot phosphorylation does not change (124), and there is a single report of decreased elF-4E phosphorylation in colcemid-arrested cells (125). It is worth noting, however, that in other cell types polysomal profiles are not so drastically affected (126), and it may be that an elongation slowdown comes into play in some cells. In any event, it is conceivable that EF-2 phosphorylation is necessary for some phase of cell cycle traverse. Studies with cycling amnion cells suggested that EF-2 phosphorylation increases slightly in mitosis (127). Data from that study indicate that the ratio of P-EF-2:EF-2 increases from ---0.1 in interphase cells to --~0.25 in cells synchronized by mitotic shake-off. The conclusion that this change may be implicated in altered protein synthetic rates is premature because, as discussed above, it is not known if a 15 percent decline in the availability of functional EF-2 would significantly impact protein synthesis in this cell type. Moreover, neither protein synthesis nor the status of initiation factors was measured in that study and it is uncertain if there was a temporal relationship between increased EF-2 phosphorylation and the presumed decrease in translation. We are currently evaluating the cell cycle dependence of EF-2 phosphorylation in both NIH 3T3 cells and HeLa cells. Our preliminary results do suggest that EF-2 phosphorylation and, surprisingly, CaMK III activity are variable during the cell cycle. It is of interest in this regard to recall that there is substantial evidence that both Ca 2 + and CaM play important roles in the cell cycle (see 128 for review). Ca 2 + transients have been found in some mammalian cells during G1/S and M phases (e.g., 129,130). There is substantial evidence for a regulatory role for Ca 2 + in cell cycle progression in sea urchin embryos (131) but much less consensus on the timing or magnitude of Ca 2 + changes, let alone their significance, in cycling mammalian cells (see 132 for review). Overexpression studies, together with antisense knockouts, have suggested that CaM is involved at several points in the fungal cell cycle (128). It must be borne in mind, however, that there are probably multiple targets for Ca 2 + and CaM control of the cell cycle, and it will be a challenge to sort out a role for EF-2 phosphorylation in this complex network of events.

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217

Given the problems outlined above, alternatives to purely biochemical approaches to investigating the importance of EF-2 phosphorylation in cell function are clearly needed. One method would be to assess the effects of rendering EF-2 nonphosphorylatable in cells. To achieve this, it would be desirable to express a mutated form of EF-2 lacking thr-56 and/or thr-58 in a null background. However, all cells express high levels of EF-2 as it is an essential protein. Naim et al. (120) recently overcame this problem by engineering variant EF-2 molecules that not only lack thr-56 and/or thr-58 but also cannot be ADP-ribosylated by toxin-mediated ADP-ribosylation (G717R mutation). Diphtheria toxin treatment of cells is normally lethal because ADP-ribosylated EF-2 is unable to participate in protein synthesis. Thus it was possible to treat cells that have been transfected with the mutant EF-2s with diphtheria or Pseudomonas A toxin to eliminate the function of the endogenous factor (equivalent to a cellular "knockout" experiment). When this was done with EF-2 (G717R) that was otherwise normal, viable cells resulted. When cells were transfected with EF-2 (T56A or T58A/G717R), viable cells were obtained, but exhibited a slower growth rate. However, cells transfected with the triple mutant (T56A/T58A/G717R) were nonviable. These results suggest that phosphorylation of EF-2 may play an essential role in the life cycle of the cell.

CONCLUSIONS The topic covered by this review is still in flux. As discussed elsewhere in this volume, Ca 2 + is a pleiotropic intracellular messenger with myriad actions on physiological processes. In and of itself, this fact introduces complications in the analysis of any single system because modulation of Ca 2 + levels has multiple effects, and it may be difficult to sort out the direct from the indirect consequences of such manipulations. The field is in the paradoxical position of having a well-characterized phenomenon, i.e., the inhibition of protein synthesis by Ca 2 + depletion of intracellular stores, that lacks a fully described biochemical basis, whereas the physiological function of a well-defined and extremely specific biochemical system, i.e., the phosphorylation of EF-2 by CaMK III is poorly understood. As pointed out above, these two sets of observations are not incompatible. Elongation block by EF-2 phosphorylation is probably a transient event reflecting [Ca 2 +]cyt while the effect of Ca 2 +-store depletion may occur over a much longer time scale and seems independent of prevailing [Ca 2+ ]cyt" It is tempting to speculate that these two effects may act cooperatively to suppress protein synthesis in response to Ca 2 +-mobilizing hormones, but this awaits to be tested. Historical prejudice favors initiation as the most likely site of translational control, but there is now plenty of evidence to suggest that the elongation phase is also regulated. Of course, there is no a priori reason for believing that these two sets of controls are mutually exclusive, though one or the other may predominate under certain physiological conditions or in a temporally sequential manner. Another consideration, touched on only briefly above is the growing evidence for compartmentalization of the translational apparatus within the cell (e.g., 136). It may well be that global measurements of protein

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CALCIUM REGULATION OF PROTEIN SYNTHESIS

synthesis are not sensitive enough to detect changes in individual compartments, and that the regulation of each compartment differs. Future refinements in our ability to measure protein synthesis in these compartments may illuminate the control mechanisms that are prevalent in different parts of the cell.

ACKNOWLEDGMENTS The authors thank Drs. M. A. and C. O. Brostrom and L. S. Jefferson for permission to use figures from their work and Dr. N. Sonenberg for discussion of issues pertaining to elF-4E phosphorylation. Work from the authors' laboratories was supported by USPHS grant GM-42715 (HCP) and the Human Frontiers Program and USPHS grant GM-50402 (ACN).

REFERENCES 1. Hershey JWB. Protein phosphorylation controls translation rates. J Biol Chem 1989;264:2082320826. 2. Proud CG. Protein phosphorylation and translational control. Curr Top Cell Regul 1992;32:243369. 3. Moldave K. Eukaryotic protein synthesis. Annu Rev Biochem 1985;54:1109-1149. 4. Hershey JWB. Translational control in mammalian cells. Annu Rev Biochem 1991;60:717-55. 5. Merrick WC. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev 1992;56: 291-315. 6. Pain VM, Clemens M. Adjustment of translation to special physiological conditions. In: Trachsel H, ed. Translational control in eukaryotes. Boca Raton, FL: CRC Press;293-324. 7. London IM, Levin DH, Matts RL, Thomas NSB, Petryshyn R, Chan J. Regulation of protein synthesis. In: Krebs EG, ed. The enzymes vol XVIII. New York: Academic Press; 1987:359-380. 8. Rowlands AG, Montine KS, Henshaw EC, Panniers R. Physiological stresses inhibit guanine nucleotide exchange factor in Ehrlich cells. Eur J Biochem 1988;175:93-99. 9. Chen J-J, Throop MS, Gehrke L, Kuo I, Pal JK, Brodsky M, London IM. Cloning of the cDNA of the heme-regulated eIF2oLkinase of rabbit reticulocytes. Proc Natl Acad Sci USA 1991;88:77297733. 10. Meurs E, Chong K, Galabru J, Thomas N, Kerr I, Williams B, Hovanessian A. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 1990;62:379-390. 11. Dever TE, Feng L, Weh RC, Cigan AM, Donahue TF, Hinnebusch AG. Phosphorylation of eIF2(x by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell 1992;68:585-596. 12. Hinnebusch AG. Involvement of an initiation factor and protein phosphorylation in translational control of GCN4 mRNA. Trends Biochem Sci 1990;15:148-152. 13. Olmsted EA, O'Brien L, Henshaw EC, Panniers R. Purification and characterization of eIF2(x kinases from Ehrlich ascites tumor cells. J Biol Chem 1993;268:12552-12559. 14. Mellor H, Flowers KM, Kimball SR, Jefferson LS. Cloning and characterization of cDNA encoding rat hemin-sensitive eIF-2a kinase. J Biol Chem 1994;269:10201-10204. 15. Hovanessian A. Interferon-induced and double-standard RNA-activated proteins as key enzymes regulating protein synthesis. In: Ilan J, ed. Translational regulation of gene expression 2. New York: Plenum Press; 1993:163-185. 16. Koromilas AE, Roy S, Barber GN, Katze MG, Sonenberg N. Malignant transformation by a mutant form of the IFN-inducible dsRNA-dependent protein kinase. Science 1992;257:1685-1689. 17. Morley SJ, Thomas G. Intracellular messengers and the control of protein synthesis. Pharmacol Ther 1991;50:291-319.

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18. Montine KS, Henshaw EC. Serum growth factors cause rapid stimulation of protein synthesis and dephosphorylation of eIF-2ot in serum-deprived Ehrlich cells. Biochim Biophys Acta 1989;1014:

282-288. 19. Villereal ML, Byron KL. Calcium signals in growth factor signal transduction. Rev Physiol Biochem Pharmacol 1992;119:68-121. 20. Brostrom CO, Bocckino SB, Brostrom MA. Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells. J Biol Chem 1983;258:14390-14399. 21. Wong WL, Brostrom MA, Brostrom CO. Effects of Ca2+ and ionophore A23187 on protein synthesis in intact rabbit reticulocytes. Int J Biochem 1991;2:605-608. 22. Brostrom CO, Bocckino SB, Brostrom MA, Galuszka EM. Regulation of protein synthesis in isolated hepatocytes by Ca2+-mobilizing hormones. Mol Pharmacol 1986;29:104-111. 23. Menaya J, Parilla R, Ayuso MS. Effect of vasopressin on the regulation of protein synthesis initiation in rat liver. Biochem J 1988;254:773-779. 24. Chin K-V, Cade C, Brostrom MA, Brostrom CO. Regulation of protein synthesis in intact rat liver by Ca2+ mobilizing agents. Int J Biochem 1988;20:1313-1319. 25. Kimball SR, Jefferson LS. Mechanism of inhibition of protein synthesis by vasopressin in rat liver. J Biol Chem 1990;265:16794-16798. 26. Kimball SR, Jefferson LS. Regulation of protein synthesis by modulation of intracellular Ca2 + in rat liver. Am J Physiol 1992;263:E958-964. 27. Thomas AP, Alexander J, Williamson JR. Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes. J Biol Chem 1984;259:5574-5584. 28. Brostrom CO, Chin KV, Wong WL, Cade C, Brostrom MA. Inhibition of translational initiation in eukaryotic cells by Ca2+ ionophore. J Biol Chem 1989;264:1644-1649. regulation of protein synthesis in intact mammalian 29. Brostrom CO, Brostrom MA. Ca2§ cells. Annu Rev Physiol 1990;52:577-590. 30. Takuma T, Kuyatt BL, Baum BJ. Otl-adrenergic inhibition of protein synthesis in rat submandibular gland cells. Am J Physiol 1984;247:G284-289. 31. Putney JW, Bird G St J. The inositol phosphate-Ca 2+ signalling system in non-excitable cells. Endocr Rev 1993;14:610-631. 32. Wong WL, Brostrom MA, Kuznetsov G, Gruitter-Yellen D, Brostrom CO. Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells. Biochem J 1993;289:7179. 33. Preston SF, Berlin RD. An intracellular Ca 2+ store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store. Cell Calcium 1992;13:303-312. 34. Kimball SR, Jefferson LS. Inhibition of microsomal C a 2 + sequestration causes an impairment of initiation of protein synthesis in perfused rat liver. Biochem Biophys Res Commun 1991;177:10821086. 35. Chin K-V, Cade C, Brostrom CO, Galuska EM, Brostrom MA. Calcium-dependent regulation of protein synthesis at translational initiation in eukaryotic cells. J Biol Chem 1987;262:1650916514. 36. Perkins PS, Pandol SJ. CCK-induced changes in polysome structure regulate protein synthesis in pancreas. Biochim Biophys Acta 1992;1136:265-271. 37. Kumar RV, Wolfman A, Panniers R, Henshaw EC. Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells. J Cell Biol 1989;108:2107-2115. 38. Brostrom MA, Cade C, Prostko CR, Gouitter-Yellen D, Brostrom CO. Accommodation of protein synthesis to chronic deprivation of intracellular sequestered calcium. J Biol Chem 1990;265: 20539-20546. 39. Lee AS. Coordinated regulation of a set of genes by glucose and calcium-ionophore in mammalian cells. Trends Biochem Sci 1987;12:20-24. 0. Prostko CR, Brostrom MA, Malara EM, Brostrom CO. Phosphorylation of eIF2ot and inhibition of eIF-213 in GH3 cells by perturbants of early protein processing that induce GRP 78. J Biol Chem 1992;267:16751-16754. 41. Prostko CR, Brostrom MA, Brostrom CO. Reversible phosphorylation of eIF-2ot in response to ER signalling. Mol Cell Biochem 1993;127/8:255-265. 42. Redpath NT, Proud CG. The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor-2. Biochem J 1989;262:69-75. 43. Redpath NT, Proud CG. Activity of protein phosphatases against initiation factor-2 and elongation factor-2. Biochem J 1990;272:175-180.

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1990;54:1034-1039. 113. Brady MJ, Marsh HN, Palfrey HC. Bifurcation of signalling pathways from the NGF receptor. 1994;(submitted). 114. Bagaglio DM, Cheng EHC, Gorelick FS, Mitsui K, Naim AC, Halt WN. Phosphorylation of EF-2 in normal and malignant rat glial cells. Cancer Res 1993;53:2260-2264. 115. Sitikov AS, Simonenko PN, Shestakova EA, Ryazanov AG, Ovchinnikov LP. cAMP-dependent activation of protein synthesis correlates with dephosphorylation of elongation factor 2. FEBS Len 1988;228:327-331. 116. Greenberg ME, Belasco JG. Control of decay of labile protooncogene and cytokine mRNAs. In: Belasco JG, Brawerman G, eds. Control of mRNA stability. New York: Academic Press; 1993: 199-218. 117. Jackson RJ, Standart N. Do the poly (A) tail and 3'UTR control mRNA translation? Cell 1990;62: 15-24. 118. Gay DA, Sisodia SS, Cleveland DW. Autoregulatory control of 13-tubulin mRNA stability is linked to translational elongation. Proc Natl Acad Sci USA 1989;86:5763-5767.

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Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1~

7 Calcium Regulation of Gene Expression L a u r a B. R o s e n , D a v i d D. G i n t y , and M i c h a e l E. G r e e n b e r g Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Calcium is a critical mediator of a wide variety of cellular responses, many of whir involve new gene expression. The concentration of free, ionized calcium in tl cytosol is carefully regulated. Basal calcium in unstimulated cells is approximate 0.1 uM and can rise over 100-fold in response to influx of extracellular calcium (-mM) or mobilization of intracellular calcium stores in the endoplasmic reticulu (ER). Extracellular calcium can enter the cell through a number of different calciu channel types in the plasma membrane, including voltage-activated channels, 1 gand-activated channels, second messenger-activated channels, and calcium r, leased-activated channels (reviewed in 1). Calcium release from internal stores o, curs through ligand-operated channels in the ER membrane, such as the inosit, triphosphate (IP3) receptor. These two sources of calcium can also interact throu~ the process of calcium-induced calcium release (CICR) (reviewed in 2). This intr cate control of cytoplasmic calcium concentration reflects the fact that increas~ cytosolic calcium regulates many cellular responses. These can be divided general] into short-term and long-term responses that are respectively independent and dq pendent on new gene expression. Short-term responses to increased cytosolic calcium include excitation-contra~ tion coupling in skeletal and cardiac muscle and stimulus-secretion coupling in el docrine and neuronal cells. In the nervous system, increased calcium in postsynal tic cells can also lead to modulation of synaptic transmission through effects on ic channel activity. In the short term, the mechanisms underlying these processes il volve posttranslational modification of preexisting factors such as cytoskeletal pr{ teins or ion channel subunits. A common posttranslational modification is phospht rylation, which can be catalyzed by calcium-activated kinases such as calciun calmodulin-dependent kinase II (CaMK II) or calcium/phospholipid-dependent pr~ tein kinase (PKC). In addition, calcium-sensitive adenylate cyclases can lead ! increased production of cAMP and activation of cAMP-dependent protein kinas (PKA), which also regulates short-term responses. However, these responses do nt require new gene expression, at least for their immediate expression. 225

226

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Long-term responses to increased calcium include cell proliferation, differentiation, and the adaptive responses of mature neurons. A number of studies using calcium chelators have shown that calcium is required for cell proliferation, and the release of caged calcium can stimulate cells to progress through the cell cycle. Calcium fluxes are also important in fertilization and embryonic development (2). In the nervous system, electrical activity leading to calcium influx can enhance the survival of developing neurons and lead to their extension of neurites and differentiation. In addition, calcium influx into mature neurons can lead to sustained phenotypic changes known as adaptive responses or neuronal plasticity (reviewed in 3). Long-term processes such as proliferation, differentiation, and neuronal adaptive responses have been shown or are likely to require new gene expression, and a complete understanding of their mechanisms requires studying how gene expression is regulated by calcium.

GENES REGULATED BY INCREASED CYTOSOLIC CALCIUM Many genes are regulated in response to stimulation by extracellular ligands, such as cytokines and growth factors, that lead to the mobilization of intracellular calcium (2). The signaling pathways by which these factors elevate cytosolic calcium have been well studied, although the precise role of this calcium increase in the induction of gene expression is not yet clear. Calcium mobilization occurs in response to activation of the phosphatidyl-inositol signaling pathway by receptor coupling to phospholipase C (PLC)-[3 or-~/isoforms. Activated PLC cleaves phosphatidyl-inositol-biphosphate (PIP2) to produce IP3, which binds to receptors on the ER to mobilize internal calcium stores (2). Experiments using calcium chelators suggest that, in some cases, this mobilization of intracellular calcium may be required for subsequent gene expression. However, these observations may reflect a requirement for basal calcium levels in the cell. The effects of calcium mobilization may be difficult to distinguish from the effects of other signaling pathways that are also activated by the extracellular ligands. In this review, we will focus on studies in which gene expression has been shown to be regulated directly in response to increased calcium in the cell. Intracellular calcium concentrations are carefully maintained at a level that is 10,000 times lower than extracellular calcium. In most cell types, these levels do not increase simply in response to elevated extracellular calcium. However, elevated extracellular calcium can induce gene expression in certain cells. The mechanisms underlying this regulation appear to involve specialized cell surface receptors. For example, extracellular calcium elevation inhibits expression of the parathyroid hormone (PTH) gene in parathyroid cells, and decreased extracellular calcium induces PTH expression (4). Although the mechanism of this response is unknown, it may involve binding of extracellular calcium to a recently cloned plasma membrane calcium sensor that is related to the metabotropic subtype of glutamate receptors (5). Elevated extracellular calcium also induces expression of

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227

keratin proteins in keratinocytes, which correlates with terminal differentiation of these cells (6). This response may reflect signaling that is mediated by calciumbinding adhesion molecules on the cell surface (e.g., see 7). The role of changes in intracellular calcium levels in the induction of gene expression that occurs in response to elevated extracellular calcium is currently unclear. The concentration of cytosolic calcium can be raised experimentally by a number of agents, including: (a) calcium ionophores such as A23187 or ionomycin, which bind calcium and increase its lipid solubility, thereby equilibrating extracellular and intracellular calcium; (b) the microsomal calcium ATPase inhibitor thapsigargin, which depletes ER calcium stores; (c) specific pharmacological agonists of voltagesensitive calcium channels (VSCC), such as Bay K 8644; (d) agonists of the subtype of glutamate receptors that conduct calcium as well as sodium ions (N-methylD-aspartate [NMDA]); or (e) membrane depolarization, which leads to the influx of calcium through VSCC. Membrane depolarization can be achieved using elevated extracellular potassium (KC1), potassium channel blockade, agonists of voltagesensitive Na + channels such as veratridine, or agonists of certain ligand-gated channels such as the nicotinic acetylcholine receptor. In addition, in conjunction with specific ion channel inhibitors, agents that induce seizure activity as well as direct electrical stimulation of neurons can be used to demonstrate calcium regulation of gene expression in vivo. Using these types of agents, a number of different genes have been shown to be regulated directly in response to alterations in intracellular calcium levels (Table I).

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Calcium Regulation of Immediate Early Genes Genes regulated in response to extracellular stimuli can be usefully divided into "immediate early" and late groups, generally reflecting their kinetics of induction and whether new protein synthesis is required for their expression. Immediate early genes (IEGs) are expressed rapidly and transiently in response to many stimuli in many different cell types (reviewed in 8,9). Induction of IEG expression does not require protein synthesis, indicating that posttranslational modification of preexisting proteins is sufficient to activate IEG expression. A number of IEGs encode transcription factors, such as the well-characterized members of the c-fos and c-jun proto-oncogene families, that bind to specific DNA elements in the promoters of target genes and thereby regulate late gene transcription. Calcium regulation of lEG expression was first demonstrated for the c-fos protooncogene in response to membrane depolarization of PC12 cells with elevated extracellular KC1 and with agonists of the nicotinic acetylcholine receptor (AChR), which conducts sodium ions and also leads to membrane depolarization (10,11). Induction of c-fos in response to membrane depolarization was inhibited by the calcium channel antagonists verapamil and nifedipine, indicating that calcium influx was required for the response. Subsequently, a number of lEGs were found to be induced by membrane depolarization of PC12 cells, including junB, zif268 (or

TABLE 1. Examples of mRNAs regulated by changes in intracellular calcium Gene

Cell or tissue

Stimulus

t~

Reference

Oo

lEGs c-fos

fosB c-jun

junB

zif268 (NGFI-A/egr1/krox24/tis8)

PC12 NIH 3T3 Swiss 3T3 HL-60 cortical neurons cerebellar granule neurons dentate gyrus neurons hippocampus dorsal root ganglion neurons hippocampus, cerebral cortex hippocampus dorsal horn, spinal cord suprachiasmatic nucleus cortical neurons NIH 3T3 HL-60 hippocampus hippocampus, cerebral cortex dorsal horn, spinal cord PC12 cortical neurons hippocampus hippocampus, cerebral cortex suprachiasmatic nucleus PC12 HL-60 cortical neurons hippocampus oligodendrocyte precursor cells hippocampus, cerebral cortex dorsal horn, spinal cord suprachiasmatic nucleus

KCI, nicotine thapsigargin A23187, ionomycin ionomycin spontaneous activity, Bay K 8644 glutamate NMDA, kainic acid electrical stimulation electrical stimulation metrazole, picrotoxin kindling sensory stimulation light spontaneous activity, Bay K 8644 thapsigargin ionomycin electrical stimulation metrazole, picrotoxin sensory stimulation KCI spontaneous activity, Bay K 8644 electrical stimulation metrazole, picrotoxin light KCI ionomycin spontaneous activity electrical stimulation glutamate, kainic acid, AMPA metrazole, picrotoxin sensory stimulation light

10,12 13 14 15 16 106 107 18,20,108,109 21 22,23 25 18 102,110 16 13 15 109 23 18 12 16 109 23 112 12 15 16 17,18,19,20,109 113 23 18 110

l

c-myc GM-CSF tis 1,7,8,11,21 tissue plasminogen activator

PC12 dorsal horn, spinal cord cerebral cortex, midbrain, cerebellum HL-60 EL-4 thymoma cells PC12 hippocampal dentate gyrus

candidate plasticity genes

hippocampal dentate gyrus

nur77 (NGFI-B)

KCI dermatome stimulation metrazole A23187 A23187 KCI metrazole, kindling, electrical stimulation kainic acid

12 18 113 114 115 116 117

pulsatile KCI, Bay K 8644 pulsatile KCI, Bay K 8644 pulsatile KCI, Bay K 8644 pulsatile KCI, Bay K 8644 Bay K 8644 Bay K 8644 Bay K 8644 KCI

27 27 27 27 119 120 121 122

118

Hormones glycoprotein oLsubunit FSH 13subunit LH 13subunit prolactin POMC Glucagon

pituitary explant pituitary explant pituitary explant pituitary explant GH4C1 pituitary cells CH3 pituitary cells pituitary cells HIT pancreatic islet cells Neuropeptides

proenkephalin

prodynorphin preprotachykinin VIP cholecystokinin neuropeptide Y galanin

chromaffin cells hippocampus hippocampus embryonic spinal cord adrenal medulla adrenal medullary explants hippocampus superior cervical ganglion superior cervical ganglion hippocampus PC12 adrenal medulla chromaffin cells

nicotine, KCI, veratridine, Bay K 8644 electrical stimulation dentate gyrus lesion, kainic acid spontaneous activity, VSCC agonist splanchnic nerve activity denervation electrical stimulation dentate gyrus lesion denervation denervation dentate gyrus lesion, kainic acid KCI, nicotine splanchnic nerve activity KCI

123-126 29

30 127 128,129 130,131 29 30 132,133 134 30 135 129,135 136 t~ t~ ~D

t~ TABLE 1. Continued Gene

Cell or tissue

Stimulus

Reference

Other neuronal genes tyrosine hydroxylase

nerve growth factor BDNF Trk p75 LNGFR GAP-43 GABAA receptor subunits AMPA receptor subunits NMDA receptor (NR2A) nicotinic AChR Na + channel c=subunit candidate plasticity genes

PC12 chromaffin cells brain, adrenal medulla superior cervical ganglion hippocampal neurons hippocampal neurons cortical neurons MAH sympathoadrenal cells cerebellar Purkinje cells PC12 cerebellar granule neurons cerebellar granule neurons cerebellar granule neurons skeletal myotubes skeletal muscle skeletal myocytes hippocampal dentate gyrus

KCI, veratridine nicotinic agonists electrical stimulation presynaptic activity kainic acid, KCI KCI, veratridine, picrotoxin kainic acid, KCI glutamate, KCI KCI KCI, veratridine, aspartate KCI KCI, NMDA KCI, NMDA KCI, NMDA spontaneous activity denervation spontaneous activity kainic acid

137 138 139 140 141 142 141 105 143 144 145 146 147 32 148,149 150 151 118

ionomycin ionomycin ionomycin A2.3187 A23187

152 152 153 154 155

A23187, thapsigargin A23187 A2.3187

37,156 157 155

A23187 A23187 A23187 A23187, ionomycin, thapsigargin

158 159 160 161

ionomycin

162

Other genes IL-2 IL-2 receptor CD7 IL-3 grp78 (BiP)

grp94 gadd153 I~,~ actin VL30 CFTR Na+/K + ATPase al subunit

T cells T cells T cells mast calls Wgla, LA9 fibroblasts NRK, 293 kidney cells Wg 1a fibroblasts GH3 pituitary cells Wgla, LA9 fibroblasts NRK, 293 kidney cells HeLa H4 hepatoma Rat-1 HT-29, T84 colon carcinoma, bronchial epithelial cells kidney outer medullary tubule

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NGFI-A, a "zinc-finger" domain-containing transcription factor), and nur77 (or NGFI-B, a steroid receptor superfamily member), but notably not c-jun (12). However, c-jun was induced in PC12 cells treated with nerve growth factor (NGF). These results suggested that different extracellular stimuli could lead to expression of different sets of IEGs within a given cell. Because many lEGs encode transcription factors, these distinct patterns of IEG expression might then lead to differential programs of secondary gene expression and distinct cellular responses. Increased cytosolic calcium in NIH 3T3 cells in response to thapsigargin and in Swiss 3T3 or HL-60 cells in response to ionomycin also leads to induction of c-fos and c-jun expression (13-15). In the latter system, intracellular calcium concentrations ([Ca2+]i) were "clamped" at specific levels using ionomycin and various levels of extracellular calcium. Interestingly, the dose response curves of c-fos, c-jun, and zif268 were found to be bell-shaped, with maximal activation occurring at 200-300 nM [Ca2 + ]i but with little induction occurring at concentrations greater than 700 nM [Ca2+ ]i. In addition, a 1-minute transient elevation of cytosolic calcium was sufficient to cause full induction of c-fos mRNA levels by 30 minutes. Because the Fos protein can regulate the expression of other genes, these results suggest that even a transient elevation of cytosolic calcium can have long-term effects on gene expression (15). IEGs are induced by spontaneous synaptic activity in primary cultures of cortical neurons. Basal expression of c-fos, fosB, junB, and zif268 is suppressed by drugs that block action potentials and thereby prevent synaptic release of neurotransmitters. Antagonists of VSCC also lower basal IEG levels, indicating that calcium influx through these channels is involved in transsynaptic regulation of lEG expression (16). Direct electrical stimulation of presynaptic neurons can also induce postsynaptic IEG expression. One of the best cellular models of neuronal plasticity is the long-term potentiation (LTP) of synaptic transmission that is produced in a number of hippocampal synapses in response to high-frequency presynaptic stimulation. Some studies have found that induction of LTP is correlated with induction of an IEG, zif268 (17-19). This induction is blocked by antagonists of the NMDA subtype of glutamate receptors, indicating a need for excitatory glutamatergic neurotransmission. In a number of these experiments, the specific temporal pattern of electrical stimulation has been an important determinant of whether postsynaptic IEG expression was induced, as well as whether a long-term neuronal response (such LTP) was achieved (17-21). Because NMDA receptors conduct calcium current and also lead to membrane depolarization and calcium influx through VSCC, these results suggest that calcium-dependent IEG expression occurs in response to transsynaptic stimulation. In addition, the temporal pattern of elevated cytosolic calcium may play a critical role in regulation of IEG expression (21). lEGs have also been shown to be induced by electrical activity in the intact nervous system. Induction of generalized seizures with metrazol or picrotoxin leads to expression of c-fos, c-jun, junB, and zif268 in the hippocampal dentate gyrus and various parts of the cerebral cortex (22,23). Because these agents act to block inhibitory neurotransmission mediated by GABA receptor chloride channels, the lEG

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induction is regarded as the result of excitatory synaptic activation that is likely to involve an increase in postsynaptic calcium. Expression of c-fos is also stimulated by electrically-induced seizure activity that leads to a permanent increase in the excitability of the brain (kindling). This result links IEG expression with long-term neuronal plasticity that is associated with seizure activity (24,25).

Calcium Regulation of Late Genes Late response genes are expressed with delayed kinetics and are generally tissuespecific, often representing effector proteins that determine the differentiated phenotype of a cell. Examples of genes in this class include hormones of endocrine secretory cells, neuropeptides and ion channels of differentiated neurons, cytokines of activated leukocytes, and metabolic enzymes of liver and fat cells. Induction of late gene expression is generally defined as being dependent on new protein synthesis, and in many cases these genes are regulated by transcription factors that are products of the lEG class. However, some tissue-specific genes, such as the opioid precursor proenkephalin, are expressed with delayed kinetics but are regulated by both lEG transcription factors and by protein synthesis-independent mechanisms. Thus, although the distinction between immediate-early and late programs of gene expression is useful in deciphering mechanisms of activation, the boundary between these two groups can be blurred. In the endocrine system, gene expression in a target organ is often regulated by trophic factors released from another gland, many of which act to mobilize intracellular calcium (reviewed in 26). In some cases, increased cytosolic calcium can mimic the effect of these trophic factors on activation of target gene expression. For example, expression of the genes encoding the et and 13 subunits of the pituitary gonadotropin genes is induced by secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus and requires mobilization of intracellular calcium in target pituitary cells. Gonadotropin gene expression can also be induced by administering KC1 or VSCC agonists to pituitary explants. This effect can be achieved only if these agents are administered in a pulsatile fashion; continuous infusion does not induce expression of the pituitary genes (27). These results suggest that the hypothalamic-releasing factors are activating gene expression by inducing calcium oscillations in target pituitary cells. As with c-fos expression in neurons, this finding also illustrates the importance of temporal signals in calcium regulation of gene expression. In the nervous system, calcium regulation of late gene expression can play a role in neuronal adaptive responses. Excitatory neuronal activity leading to membrane depolarization and increased cytosolic calcium can regulate the expression of neuropeptide genes and enzymes involved in neurotransmitter biosynthesis. This regulation changes the stores of neuropeptides or neurotransmitters that a neuron can secrete and thus represents a form of activity-dependent neuronal plasticity (28). A well-studied example of this type of regulation is the neuropeptide proenkephalin, a

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precursor of the endogenous opioid enkephalin. Enkephalin peptides modulate diverse neuronal pathways involved in motivation, reward, and analgesia, which are also affected by narcotic drugs. Proenkephalin expression in the hippocampus is regulated in response to high-frequency stimulation, which can produce LTP, as well as in response to seizure activity produced by kainic acid or hilus lesion (29,30). The increased proenkephalin synthesis seen under these conditions is countered by a decrease in the expression of another opioid precursor, prodynorphin. Since proenkephalin appears to have excitatory effects on hippocampal neurons, whereas prodynorphin appears to be inhibitory, it was suggested that this transsynaptic regulation of opioid gene expression may lead to an overall enhancement of hippocampal excitability (29). This differential calcium regulation of gene expression could thereby underlie long-term neuronal adaptive responses such as LTP. Other neuronal late response genes regulated by calcium include neurotrophic factors and their receptors as well as ion channels, all of which may be important for defining specific characteristics of a differentiated phenotype. For example, developing cerebellar granule neurons differentiate in response to afferent innervation from cerebellar mossy fibers. This activity-dependent differentiation can be mimicked in vitro by culturing the neurons in the presence of elevated KC1 or NMDA (31). The enhanced survival and neuronal differentiation seen under these conditions appears to be dependent on calcium influx through VSCC or glutamate receptors. Treatment with either KC1 or NMDA also leads to an increase in functional NMDA receptors in cerebellular granule neurons. A recent study demonstrates that this increased NMDA receptor activity is due to a calcium-dependent increase in the mRNA of a specific subunit of the NMDA receptor, NR2A (32). Hetero-oligomerization with NR2A increases the sensitivity of the NR1 subunit of the NMDA receptor to stimulation with NMDA. These studies suggest that calcium-dependent gene expression is likely to play a role in neuronal survival and differentiation in vivo. There are a number of other genes whose expression has been shown to be regulated by alterations in intracellular calcium concentration. Many cytokines that are induced in activated Iymphocytes in response to binding of antigen require the mobilization of intracellular calcium for their expression (reviewed in 33-35). Some of these genes can be induced with ionophore alone (although generally to a submaximal extent). Another notable example is the set of "glucose-regulated proteins" (GRPs), which are expressed in various cell types upon depletion of glucose in the extracellular medium. The grp genes can also be induced by calcium ionophores (reviewed in 36). The best characterized member of this family is GRP78, also identified as BiP, a molecular "chaperone" that binds transiently to proteins in the ER to assist their folding, assembly, and transport. Because protein folding and association of ER proteins with GRP78/BiP are calcium-dependent processes, depletion of calcium from the ER might be expected to produce malfolding of proteins. This would increase the requirement for chaperonins and create a need for new grp78 gene expression. Experiments with calcium chelators have suggested that ionophore induction of the grp genes does not actually require an increase in cytosolic calcium levels but rather is stimulated in response to the depletion of ER

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calcium stores (37). Thus, altering calcium homeostasis will likely activate gene expression by a variety of mechanisms. Determining these mechanisms may begin to account for the great variety of calcium-dependent long-term cellular responses.

MECHANISMS OF GENE REGULATION BY CALCIUM The most extensively studied mechanism by which calcium regulates gene expression is by affecting the level of transcription initiation. However, calcium also regulates gene expression at other levels. These mechanisms include effects on the elongation of nascent mRNA transcripts, mRNA stability, and the translation of mRNA into protein. For example, the c-fos gene is regulated by calcium at the level of both transcriptional initiation and elongation. Using nuclear run-off transcription analysis, it has been shown that c-fos transcription is already initiated to some extent in unstimulated cells, but elongation of the nascent transcripts is blocked within the first intron. This block can be inducibly released by a calcium-dependent mechanism in response to a number of agents, including the calcium ionophore A23187 and KC1 (38,39). Together with increased transcriptional initiation, removal of the elongation block increases c-fos mRNA levels. Calcium can also inhibit the overall rate of translational elongation in the cell by leading to phosphorylation of the eukaryotic translation elongation factor eEF-2 via a calcium/calmOdulin-dependent kinase (CaMK HI or eEF-2 kinase) (reviewed in 40). This effect has been suggested to inhibit protein synthesis during mitosis, when cytosolic calcium is elevated. It has also been speculated that making protein synthesis a ratelimiting step for gene expression could lead to the preferential translation of certain mRNAs that have a higher affinity for limiting elongation factors. The mechanisms by which increased cytosolic calcium regulates gene transcription have been studied in detail for only a handful of genes. A useful system for studying regulation of gene expression by calcium has been the rat pheochromocytoma cell line PC12 (41). PC12 cells have been widely used to study mechanisms of cell growth and differentiation. PC12 cells proliferate in response to epidermal growth factor (EGF) and differentiate into sympathetic-like neurons upon exposure to NGF. However, EGF and NGF activate similar sets of lEGs, and the specific early signals that lead to their distinct programs of late gene expression and biological responses remain to be determined. PC 12 cells respond to membrane depolarization with an influx of extracellular calcium, primarily through L-type VSCC, which also leads to the induction of lEG expression. However, a detailed comparison of lEG expression patterns showed that membrane depolarization and growth factors induce overlapping but distinct sets of lEGs. This result indicated that the mechanisms of gene induction by calcium are at least partly distinct from those mediating the neurotrophin and growth factor responses (12). One of the best studied calcium-inducible genes is the c-fos proto-oncogene. The mechanisms underlying c-fos induction by calcium were first addressed by mapping the cis-acting DNA regulatory sequences involved in this response. In transient

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transfection analysis, deletion mutants revealed two elements important for mediating induction of c-fos transcription in PC12 cells (Fig. 1). A 5' element, located at - 3 0 0 relative to the transcription start site, was found to be required for NGF induction of c-fos, but deletion of this element did not significantly reduce the ability of the c-fos promoter to respond to calcium (42). A 3' element, located at - 6 0 relative to the transcription start site, was found to be required for calcium induction of c-fos and was therefore termed the calcium response element (CARE). The ability of calcium but not NGF to activate c-fos transcription through the isolated CaRE indicates that the mechanisms of c-fos induction in response to growth factors and calcium are distinct at the level of the promoter (42).

Calcium Regulation of c-fos Through the Ca/CRE The CaRE contains within it a five-base pair match with the consensus cAMP response element (CRE) that was first identified in the promoters of neuropeptide genes (43; reviewed in 44). The mechanism by which the CRE mediates transcriptional activation in response to elevated cAMP has been well studied. The first factor found to bind this element was termed the CRE-binding protein (CREB) (45,46). CREB is a member of a large family of "bZIP" transcription factors, which contain "leucine zipper" (LZ) dimerization motifs and adjacent basic regions that contact the DNA. CREB is phosphorylated in vitro on serine 133 by the cAMPdependent protein kinase (PKA) (47). PKA can be stimulated in cells by treatment with agents such as forskolin, which directly activates adenylate cyclase to increase production of cAMP. Forskolin treatment of PC12 cells leads to phosphorylation of

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CREB on serine 133 and the transcriptional activation of a CRE-containing reporter gene (47). Furthermore, cotransfection of expression vectors for CREB and the catalytic subunit of PKA are sufficient to activate transcription of a CRE-reporter gene, and mutation of CREB serine 133 blocks this effect (47). Thus, agents that increase intracellular cAMP levels and PKA activity induce CREB phosphorylation on serine 133 and thereby activate transcription through the CRE (Fig. 2) When cloned upstream of a minimal c-fos promoter (42 base pairs 5' of the transcription start site) or a heterologous reporter gene, the Ca/CRE is sufficient to mediate transcriptional activation in response to both cAMP and calcium (48-50). Fine mutational analysis of the c-fos Ca/CRE demonstrated that the sequences required for calcium inducibility through this element were identical with those required for induction by cAMP. This result suggested that the calcium and cAMP pathways utilize the same or highly related transcription factors to activate c-fos

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transcription through the Ca/CRE (50). Gel mobility shift analysis revealed that the c-fos Ca/CRE binds constitutively to a nuclear factor. In vitro translated CREB also binds the Ca/CRE, and in studies using Ca/CRE point mutants, the affinity of CREB binding correlated directly with the ability of the Ca/CRE mutants to activate c-fos transcription. The possibility that CREB was a calcium-regulated transcription factor was tested by examining the phosphorylation of CREB in PC12 cells treated with membrane depolarizing agents or forskolin. Both stimuli led to CREB phosphorylation on serine 133 (50). These results indicate that calcium- and cAMPsignaling pathways converge at the level of CREB phosphorylation on serine 133 to activate c-fos transcription (Fig. 2). To assess the importance of serine 133 phosphorylation in vivo, it was necessary to use an approach that allows the introduction of modified forms of CREB into a cell such that the properties of the exogenously introduced CREB can be distinguished from endogenous CREB family members. This was accomplished by fusing CREB to the DNA-binding domain of the yeast bipartite transcription factor GAL4 and assessing the ability of this GAL4-CREB fusion protein to activate transcription of a reporter gene that contained a GAL4 DNA-binding site within its regulatory region. Upon transfection into PC12 cells, the GAL4-CREB protein was sufficient to mediate transcriptional activation of the cotransfected reporter gene in response to both increased calcium and cAMP (51). To control for the possibility that the fusion protein could still heterodimerize with endogenous CREB family members, the LZ dimerization motif of CREB was deleted. Although reduced in activity, this GAL4-CREBALZ mutant was still able to confer a transcriptional response. However, a mutation of serine 133 to alanine in this construct decreased transcriptional activation in response to calcium and cAMP by 80 percent (51). These experiments demonstrate that CREB is sufficient to mediate the transcriptional activation of c-fos in response to both calcium and cAMP in vivo and that phosphorylation of CREB on serine 133 is critical for this response. Membrane depolarization of PC12 cells does not lead to an increase in cAMP levels, suggesting that calcium induces CREB phosphorylation by a distinct signaling pathway (50). This possibility was confirmed using a PKA-deficient PC12 cell line, in which an overexpressed mutant form of the PKA regulatory subunit acts as a dominant inhibitor of wildtype PKA (52). Membrane depolarization was still able to induce CREB phosphorylation on serine 133 in these cells, indicating that calcium can use a PKA-independent signaling pathway to phosphorylate CREB (39). However, membrane depolarization did not effectively induce c-fos transcription in the PKA-deficient cells, suggesting that there is a requirement for basal PKA activity to induce c-fos transcription (53). Taken together, these results demonstrate that serine 133 phosphorylation, although necessary, is not sufficient to activate c-fos transcription in response to calcium. A number of close relatives of CREB have been cloned, termed the activating transcription factor (ATF) family (54). These proteins share immunological reactivity and DNA-binding specificity with CREB. Using the GAL4 fusion approach, ATF-1 was also shown to mediate calcium and cAMP induction of gene expression

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in PC12 cells (55). Although the response of ATF-1 to calcium signaling was equal to that of CREB, its response to cAMP signals was significantly reduced. This difference may provide a mechanism by which cells distinguish between calcium and cAMP signaling (55). Serine 133 is contained within the kinase-inducible domain (KID) of CREB, which separates two glutamine-rich domains (Q1 and Q2) (56). The Q2 domain alone is sufficient to activate transcription in vitro, and it has recently been shown to interact with a component of the basal transcription machinery that is associated with RNA polymerase II (57). The TFIID fraction of basal transcription factors consists of the TATA binding protein (TBP) and a number of TBP-associated factors (TAFs). The 110-kDa TAF (TAFII110) binds to the CREB Q2 domain both in vitro and in the yeast two-hybrid protein interaction system (57). This interaction of the CREB Q2 region with TAFII110 may stabilize association of RNA polymerase II with the promoter and thereby promote formation of transcription initiation complexes. Interestingly, the sequences in Q2 required for this interaction are not present in members of the CREB/ATF family that act as transcriptional repressors (CREMs) (56). It is not yet clear how phosphorylation of serine 133 in the KID enhances the ability of CREB to activate transcription. Although the KID and serine 133 are clearly required for calcium and cAMP induction of CREB transactivation potential, the KID is not sufficient to activate transcription if targeted alone to the DNA. Rather, the KID functions synergistically with the adjacent Q2 region to activate transcription (58,59). CREB phosphorylated on serine 133 has recently been shown to bind to a nuclear factor termed the CREB-binding protein (CBP) (60). CBP binds specifically to the region of CREB containing the inducibly phosphorylated serine 133. CBP fused to a DNA-binding domain is sufficient to act as a transcriptional activator, and its transactivating ability is enhanced by unknown mechanisms in response to PKA activation. It has been postulated that CBP may act as a transcriptional coactivator by forming a bridge between the CREB enhancer and the basal transcription machinery (60). In this way, calcium- or cAMP-induced CREB phosphorylation and subsequent CBP binding may allow protein-protein interactions that stabilize the binding of RNA polymerase II to the DNA to promote the initiation of transcription. Because membrane depolarization of PC12 cells does not elevate cAMP levels and can still lead to serine 133 phosphorylation in the PKA-deficient PC12 line, a distinct calcium-induced CREB kinase must exist. This kinase would have to satisfy three requirements: the ability to phosphorylate CREB on serine 133; transient induction in response to calcium influx in PC12 cells; and nuclear localization, since CREB is exclusively found in the nucleus. Some evidence suggests that the CaMKs may mediate calcium-induced phosphorylation and activation of CREB. In vitro phosphorylation assays and two-dimensional tryptic phosphopeptide analyses have shown that CaMKs I, II, and IV can all phosphorylate CREB in vitro on serine 133 (51,61,62). Membrane depolarization of PC12 cells activates CaMK II with kinetics that are consistent with it mediating serine 133 phosphorylation (62, 63), and CaMK IV has been shown to be localized to the nucleus (64). However, a novel

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CREB kinase activity has recently been described that phosphorylates serine 133 is inducibly activated in response to membrane depolarization and is present in both cytoplasmic and nuclear extracts of PC12 cells (65; J. Xing, D. D. G., and M. E. G., unpublished data). Thus, the identity of the calcium-inducible CREB kinase(s) and signaling pathway(s) that mediate c-fos transcription through the Ca/ CRE in vivo remains to be determined.

Calcium Regulation of c-fos Through the SRE Although the isolated Ca/CRE was sufficient to confer calcium inducibility on a minimal c-fos promoter, an internal deletion of the Ca/CRE from the full-length c-fos promoter did not abolish the ability of calcium to induce transcription. This result clearly indicated that other elements in the c-fos promoter are calciumresponsive (42). Subsequent studies have shown that one additional calcium response element in the c-fos promoter is the serum response element (SRE) (62,66,67). The SRE, located at - 3 0 0 relative to the transcription start site, was first identified as being involved in serum induction of c-fos in quiescent fibroblasts and is critical for c-fos induction in response to growth factors, phorbol esters, and NGF (68,69). Recently, it has been shown that an isolated SRE cloned upstream of a minimal c-fos promoter can also mediate c-fos induction in response to membrane depolarization and glutamate by mechanisms dependent on calcium influx (62,66,67). The SRE consists of an inner "CArG" box, named for the central 10-base pair CC[A/T]6GG sequence, and flanking palindromic arms. These outer sequences contain dyad symmetry, so the SRE is also referred to as a dyad symmetry element (DSE). Fine mutational analysis of the SRE indicates that growth factors regulate c-fos transcription through the CArG box and sequences in the 5' palindromic arm, whereas calcium induction involves the CArG box and sequences in the 3' palindromic arm (62). This result suggests that the mechanism of calcium induction through the SRE is distinct from that of growth factors at the level of the DNAbinding proteins involved. DNA mobility shift and methylation interference assays identified a nuclear factor, termed the serum response factor (SRF), that specifically binds the SRE (70). Point mutational analysis of the SRE demonstrated that the affinity of SRF binding correlates with the ability of the SRE to mediate serum induction of c-fos transcription (71,72). SRF binds as a dimer to the CArG box of the SRE, and the CArG box alone is sufficient to mediate serum induction of transcription. However, the flanking palindromic arms increase the affinity of SRF for the SRE (73). Another transcription factor termed p62 TcF forms a ternary complex with SRF and the SRE (reviewed in 74). p62 TcF contains a DNA binding domain similar to that present in the ets family of transcription factors and is therefore also known as Elk (ets-like). p62TCF/Elk is inducibly phosphorylated on a number of C-terminal sites in cells exposed to serum, growth factors, or phorbol esters. Some of these phosphorylation events mediate SRE-dependent c-fos transcription.

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A signaling pathway by which growth factors stimulate Elk phosphorylation and c-fos transcription through the SRE has recently been pieced together (Fig. 3). This pathway is highly conserved over evolution and is activated in a variety of cell types in response to many different extracellular signals. For these reasons, it probably represents a major point of convergence for signals emanating from many different sources and may be a means by which such diverse information is integrated. Some aspects of this signaling pathway have recently been shown to be activated by elevated intracellular calcium in PC12 cells (75). In the well-studied case of growth factors or neurotrophins, the pathway begins

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with ligand binding to cell surface receptor tyrosine kinases, which causes the receptors to dimerize and autophosphorylate in trans (reviewed in 76,77). Phosphorylated tyrosines on the receptors provide docking sites for "adaptor" proteins such as GRB2 and SHC that mediate receptor coupling to the small G protein Ras (reviewed in 78,79). GRB-2 is bound to a guanine nucleotide exchange factor (GRF) that catalyzes the exchange of GDP for GTP on the small G protein Ras. Binding of GTP activates Ras, which then binds the serine/threonine kinase Raf-1 to initiate a kinase cascade, which ultimately leads to phosphorylation and activation of transcription factors in the nucleus (reviewed in 80). Activated Raf phosphorylates and activates the MAPK/ERK kinase (MEK), which then phosphorylates and activates its target MAP kinase (MAPK, also known as ERK). Activated MAPK phosphorylates a number of cytoplasmic and nuclear proteins, including the SRF-associated transcription factor p62XCF/Elk (reviewed in 74,81-83). Recently, it has been shown that calcium influx through VSCC in PC12 cells is sufficient to activate Ras, MEK, and MAPK (75). Calcium influx is therefore capable of initiating a signaling pathway previously associated with growth factors and NGF. The mechanism by which calcium influx leads to Ras activation is not yet clear. In addition, it is not known whether calcium activation of this pathway leads to induction of gene expression. For example, the Ras/MAPK pathway does not appear to be required for calcium activation of c-fos transcription through the SRE (84). Nevertheless, calcium activation of the Ras/MAPK pathway provides another signaling mechanism by which calcium may regulate gene expression. An additional mechanism by which calcium could affect SRE-mediated c-fos transcription is by modification of SRF itself. Using two-dimensional tryptic phosphopeptide mapping, SRF has been shown to be inducibly phosphorylated on serine 103 in response to serum stimulation of fibroblasts (85). An antibody specific for SRF phosphorylated at serine 103 detects this inducible phosphorylation event in PC12 cells (84). This effect may be calcium-specific, since it occurs in response to membrane depolarization and ionomycin but not in response to NGF or EGF. SRF serine 103 is phosphorylated in vitro by CaMK II and CaMK IV (62). In addition, CaMK II activity is stimulated with kinetics that parallel the inducible phosphorylation of SRF on serine 103 in vivo, raising the possibility that CaMK II or a related kinase may catalyze this event (62). Phosphorylation of serine 103 enhances the affinity of SRF binding to the SRE (85). However, this modification has not yet been shown to enhance transcriptional activation in transfection assays, in which altered DNA-binding sites were used to distinguish responses mediated by the transfected SRF proteins from those mediated by endogenous SRF (84). It is possible that the failure to see a difference in transcriptional activation between wild-type and serine 103-mutated SRF proteins in these assays was due to the changes introduced in the SRE. Since SRF phosphorylation at serine 103 affects DNA binding, the native SRE may be necessary to see an effect of this event on transcriptional activation. Thus, calcium-dependent phosphorylation and increased DNA binding of SRF remains a potential mechanism for mediating calcium induction of c-fos transcription through the SRE.

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Calcium Regulation of Gene Expression Through Other Elements

The sequences flanking the SRE contain consensus binding sites for a number of other transcription factors, some of which have been shown to bind the element in vitro. Fine mutational analysis of the SRE revealed that sequences in the 3' palindromic arm contribute to transcriptional activation in response to calcium signals in PC12 cells, whereas this region could be mutated without effect on the NGF response (62). One factor that binds to this 3' region is the CCAAT/enhancer binding protein C/EBPI3 (also called NF-IL-6) (86). The C/EBPs are members of the bZ1P family of transcription factors and were originally characterized as sequence-specific DNA-binding proteins that regulated transcription of genes involved in the differentiation of liver and fat cells (reviewed in 87). More recently, experiments have shown that C/EBPI3 can mediate calcium induction of gene expression (88). A role for C/EBP[3 in c-fos regulation was first found in response to elevated cAMP. In PC12 cells, C/EBPI3 in the cytoplasm is inducibly phosphorylated upon exposure of cells to forskolin and then translocates to the nucleus, where it binds to the c-fos SRE. In transient cotransfection assays of NIH 3T3 cells, C/EBP[3 can activate transcription of a c-fos reporter gene in an SRE-dependent manner (89). C/EBPI3 has also been shown to regulate the expression of phosphoenolpyruvate carboxykinase (PEPCK) in response to elevation of cAMP. In this case, C/EBP[3 acts primarily through binding a CRE in the PEPCK promoter (90). In contrast to these in vivo studies, in vitro experiments suggest that C/EBPI3 is not efficiently phosphorylated by PKA but that it is phosphorylated by CaMK II (88). Taken together, these results suggest that the C/EBPI3 responses detected in vivo may be only indirectly related to the elevation of cAMP. The involvement of C/EBP in calcium regulation of transcriptional activation has been demonstrated in transient transfection assays of pituitary G/C cells. Transfection of a constitutively active mutant of CaMK U stimulated transcription of a reporter gene containing a C/EBP site in its regulatory region (91), and cotransfection of C/EBPI3 increased this response six-fold (88). In addition, treatment of G/C cells with A23187 leads to phosphorylation of C/EBPI3 within its LZ domain. This site was determined to be serine 276, which was also phosphorylated by CaMK II in vivo. Mutation of serine 276 blocked the ability of CaMK II to enhance C/EBPI3mediated transcription (88). The mechanism by which serine 276 phosphorylation regulates C/EBPI3 transactivation potential is unclear. Serine 276 phosphorylation does not affect the ability of C/EBPI3 to form homodimers or to bind DNA, and in G/C cells it does not appear to regulate nuclear localization. One possibility suggested is that serine 276 phosphorylation alters the conformation of the C/EBPI3 LZ, thereby affecting its ability to heterodimerize with another LZ-containing protein. Because there are C/EBPI3 family members that lack the DNA-binding basic region, regulation of heterodimerization may affect the ability of C/EBPI3 complexes to bind DNA (88). These results suggest that increased cytosolic calcium induces the phosphorylation of C/EBPI3 and increases its ability to activate transcription.

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Other transcription factors also bind to the 3' region of the SRE and may be involved in calcium regulation of c-fos transcription. Two of these, El2 and E47, are members of the basic-helix-loop-helix (bHLH) family of transcription factors that are important in cellular differentiation (reviewed in 92). The basic domain is required for DNA binding and the HLH domain for dimerization of these factors. The members of the bHLH family can homodimerize and/or heterodimerize, and these different interactions regulate their specific DNA-binding properties. El2 homodimers bind to a DNA sequence termed the E-box. Recently, El2 and related bHLH proteins were found to be directly regulated by calcium/calmodulin (Ca2+/ CaM) binding and were therefore designated ECa proteins (E-box and calciumbinding). Addition of Ca 2+/CaM specifically inhibited binding of ECa proteins to an E-box in a DNA mobility shift assay, and Ca 2+/CAM was found to bind the bHLH domain directly in glutaraldehyde cross-linking experiments (93). Of the bHLH proteins tested, those that bound Ca 2+/CaM were all members of the class A bHLH factors, which are widely expressed in different cell types. Class A bHLH proteins heterodimerize with members of class B, which are more cell-type specific (94). Because A/A homodimers were more sensitive to Ca 2+/CAM inhibition than A/B heterodimers, it was suggested that calcium may increase the relative binding activity of A/B heterodimers (93). Since the class B bHLH proteins are important in determining cell-type specificity, these results suggest a mechanism by which elevated intracellular calcium may regulate cellular differentiation. Other factors that bind to sequences in the 3' region of the SRE are the Fos and Jun family members that comprise the AP-1 transcription factor complex. The c-fos AP-1 sequence (FAP site) (see Fig. 1), as well as the consensus AP-1 site (TGACTCA), is similar to the consensus CRE (TGACGTCA) and can function as a cAMP-inducible element (49), which reflects the ability of some AP-1 sites to bind both Fos/Jun family members as well as CREB/ATF family members. Another such element is found in the promoter of the opioid precursor proenkephalin (reviewed in 95). This sequence, termed ENK-CRE2, mediates transcriptional activation of proenkephalin in response to both calcium and cAMP (96). The expression of both Fos and Jun family members can be induced by calcium (12), and preexisting Jun proteins can be posttranslationally modified in response to activation of PKC, resulting in an increase in their DNA-binding affinity and transactivation potential (97). Increased binding of Fos and Jun proteins to ENK-CRE2 is observed in the hippocampus upon induction of seizure activity, which correlates with the induction of proenkephalin expression (98). These findings suggest a role for Fos and Jun proteins in calcium regulation of gene transcription in vivo. In addition, combinatorial interactions have been described between CREB/ATF family members and Jun family members, as well as between C/EBPI3 and Fos/Jun proteins, which affect the DNA-binding specificity of the heterodimers (87,99). These types of interactions expand the possibilities for fine-tuning gene expression in response to specific extracellular signals and perhaps combinations of signals. Finally, novel cis-acting elements that regulate gene expression in response to increased cytosolic calcium have also been described (100).

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Calcium Regulation of c-fos Transcription In Vivo

The mechanisms and roles of calcium-dependent gene expression in long-term biological responses have also been studied in vivo. One good system for studying these processes is regulation of neuronal activity in the suprachaismatic nucleus (SCN) of the hypothalamus, which comprises the primary pacemaker for the generation of circadian rhythms (reviewed in 101). The SCN pacemaker regulates the periodicity of behavioral and hormonal circadian rhythms. Light/dark cycles in the environment synchronize, or entrain, the SCN pacemaker to a period of precisely 24 hours. Light activates retinal ganglion neurons, a subset of which sends glutamatergic projections directly to SCN neurons. The integrity of these projections is critical for photic entrainment of the SCN pacemaker. Hamsters placed in a constant dark environment maintain a natural circadian rhythm of running-wheel activity that includes periods of subjective day, when the animals rest, and subjective night, when the animals are active. Hamsters exposed to light during their subjective night undergo a phase shift in their circadian rhythm, which does not occur if they are exposed to light during their subjective day (101,102). This difference correlates with the ability of light exposure to induce lEG expression, making this system useful for studying the mechanisms and role of lEG expression in an activity-dependent long-term neuronal adaptive response, the photic entrainment of the SCN pacemaker. The involvement of CREB in the light-induced expression of c-fos in the SCN was tested using an antibody that specifically recognizes the serine 133-phosphorylated, and therefore active, form of CREB (103). PhosphoCREB immunoreactivity was detected in SCN sections of hamsters exposed to light during their subjective night (Fig. 4), whereas no inducible staining was seen if light exposure occurred during subjective day. Thus, CREB is inducibly phosphorylated at the transcriptional regulatory site, serine 133, in response to light-induced synaptic activation of the SCN only at times when this leads to induction of IEG expression and the phase shifting of circadian rhythms (103). These results strongly support a role for CREB in the transsynaptic regulation of gene expression in vivo. Because the light signal is transmitted via excitatory synapses that increase postsynaptic calcium in SCN neurons, these results also suggest that gene expression involved in a neuronal adaptive response can be induced by calcium activation of the transcription factor CREB.

DIFFERENT CALCIUM SOURCES ACTIVATE DISTINCT SIGNALING PATHWAYS As more information is gathered about the transcription factors and signaling pathways involved in calcium-induced gene expression, evidence is accumulating that different modes of calcium entry into the cytoplasm may activate gene expression by distinct mechanisms. These results suggest ways in which calcium influx through different routes of entry into cells might lead to distinct cellular responses.

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FIG. 4. Light induction of CREB serine 133 phosphorylation in the hamster suprachiasmatic nucleus (SCN). An antibody specific for the serine 133-phosphorylated and therefore activated form of CREB (a-PhosphoCREB) detects increased CREB phosphorylation in the SCN of hamsters exposed to light for 5 minutes during their subjective night (circadian time 19), a time when light exposure leads to induction of immediate-early gene expression and the phase shifting of circadian rhythms. This immunostaining is competed by preabsorbing the antibody with the peptide it was raised against (~-PhosphoCREB+ PEP). The level of CREB protein expression did not change in response to light exposure, as immunoreactivity with an antibody that recognizes CREB regardless of its phosphorylation state (~-CREB) was unaffected. These results suggest that light-induced phosphorylation and activation of CREB is a trigger for c-los transcription in SCN neurons. From Ginty et al. (103), with permission.

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In primary cultures of hippocampal neurons transfected with various c-fos promoter constructs, the SRE and CRE were found to have differing abilities to mediate calcium induction of c-fos expression, depending on the mode of calcium entry into the cell (66). The isolated SRE cloned upstream of a minimal c-fos promoter can mediate transcriptional activation in response to both NMDA receptor stimulation, which leads to calcium influx through ionotropic glutamate receptors, and membrane depolarization, which leads to calcium influx through VSCC. However,

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the CRE cloned upstream of a minimal c-fos promoter can activate transcription only in response to calcium influx through VSCC; calcium influx through NMDA receptors has little effect (66). These results suggest that different modes of calcium entry into hippocampal neurons can lead to activation of distinct signal transduction pathways, one targeting the c-fos SRE and the other able to target both the SRE and the CRE. Two distinct calcium signaling pathways have also been identified by pharmacological means in dentate gyrus neurons cultured in vitro (104). In this system, inhibitors of phospholipase A2 and cyclooxygenase blocked NMDA-induced activation of c-fos expression. However, these agents had no effect on activation by the non-NMDA receptor agonist kainic acid, which leads to membrane depolarization and influx of calcium through VSCC. Conversely, the NMDA activation of c-fos was unaffected by the calmodulin antagonist calmidazolium, whereas this agent inhibited c-fos activation in response to kainic acid (104). These studies support the concept that depending on the mode of entry into a cell, calcium may activate different signaling pathways that lead to gene transcription. Different modes of calcium entry can also lead to distinct patterns of gene expression. In primary cultures of cortical neurons, glutamate leads to increased BDNF expression with a rapid and transient time course, whereas membrane depolarization leads to a much more prolonged induction of BDNF (105). This difference correlates with the ability of KC1 to enhance the survival of these neurons in culture, whereas glutamate lacks this effect. Adding a B DNF antibody to the culture medium blocks both basal and KCl-enhanced neuronal survival, suggesting that the prolonged increase in BDNF expression induced by KCI may explain the ability of membrane depolarization to enhance cell survival (105). Taken togther, these findings suggest that distinct signaling pathways can be activated by calcium, depending on its route of entry into the cell. These pathways may be involved in programming alternative long-term cellular responses to different calcium signals.

CONCLUSIONS Calcium is a central regulator of a wide variety of cellular responses, including long-term responses that require new gene expression. Increased cytosolic calcium leads directly to increased expression of many genes, and the signaling pathways that mediate this regulation are becoming increasingly clear. In addition, the observation that calcium can activate distinct signaling pathways depending on its route of entry into the cytoplasm provides a major direction for future research. This specificity of calcium signaling may reflect the absolute level or duration of increased cytosolic calcium in response to various modes of entry. Alternatively, spatial differences in calcium entry could lead to activation differentially localized secondary signaling molecules. It is possible that calcium channels themselves become modified upon calcium influx and then serve as signaling proteins in the manner of growth factor receptors. Determining the specificity involved in signal-

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ing through different calcium channels should help explain how a single, diffusible ion can regulate such diverse cellular processes as proliferation, differentiation, and neuronal adaptive responses.

ACKNOWLEDGMENTS

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Some of the work described in this review was supported by a predoctoral National Research Service Award (NIH MH10169; L. B. R.), the American Heart Association, Massachusetts affiliate (13-446-912; D. D. G.), NIH NS28829 (M. E. G.), an American Cancer Society Faculty Research Award (FRA-379; M. E. G.), and the McKnight Endowment Fund for Neuroscience (M. E. G.)

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is mediated through two distinct regions in the 3'-untranslated region. J Immunol 1993;150:43864394. 116. Kujubu DA, Lira RW, Varnum BC, Herschman HR. Induction of transiently expressed genes in PC-12 pheochromocytoma cells. Oncogene 1987;1:257-262. 117. Qian Z, Gilbert ME, Colicos MA, Kandel ER, Kuhl D. Tissue-plasminogen activator is induced as an immediate-early gene during seizure, kindling, and long-term potentiation. Nature 1993;361: 453-457. 118. Nedivi E, Hevroni D, Naot D, Israeli D, Citri Y. Numerous candidate plasticity-related genes revealed by differential cDNA cloning. Nature 1993;363:718-722. ll9. Hinlde PM, Jackson AE, Thompson TM, Zavacki AM, Coppola DA, Bancroft C. Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium. Mol Endocrinol 1988;2:1132-1138. 120. Day RN, Maurer RA. Pituitary calcium channel modulation and regulation of prolactin gene expression. Mol Endocrinol 1990;4:736-742. 121. Loeffler JP, Kley N, Pittius CW, Hollt V. Calcium ion and cyclic adenosine 3'-,5'-monophosphate regulate proopiomelanocortin messenger ribonucleic acid levels in rat intermediate and anterior pituitary lobes. Endocrinology 1986;119:2840-2847. 122. Schwaninger M, Lux G, Blume R, Oetjen E, Hidaka H, Knepel W. Membrane depolarization and calcium influx induce glucagon gene transcription in pancreatic islet cells through the cyclic AMPresponsive element. J Biol Chem 1992;268:5168-5177. 123. Eiden LE, Giraud P, Dave JR, Hotchkiss AJ, Affolter H-U. Nicotinic receptor stimulation activates enkephalin release and biosynthesis in adrenal chromaffin cells. Nature 1984;312:661-663. 124. Kley N, Loeffler JPh, Pittius CW, Hollt V. Proenkephalin A gene expression in bovine adrenal chromaffin cells is regulated by changes in electrical activity. EMBO J 1986;5:967-970. 125. Kley N, Loeffler J-P, Pittius CW, HoUt V. Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells. J Biol Chem 1987 ;262:4083-4089. 126. Farin C-J, Kley N, Hollt V. Mechanisms involved in the transcriptional activation of proenkephalin gene expression in bovine chromaffin cells. J Biol Chem 1990;265:19116-19121. 127. Agoston DV, Eiden LE, Brenneman DE. Calcium-dependent regulation of the enkephalin phenotype by neuronal activity during early ontogeny. J Neurosci Res 1991;28:140-148. 128. Kanamatsu T, Unsworth CD, Diliberto EJ, Viveros OH, Hong JS. Reflex splanchnic nerve stimulation increases levels of proenkephalin A mRNA and proenkephalin A-related peptides in the rat adrenal medulla. Proc Natl Acad Sci USA 1986;83:9245-9249. 129. Fischer-Colbrie R, Iacangelo A, Eiden LE. Neural and humoral factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla. Proc Natl Acad Sci USA 1988;85:3240-3244. 130. LaGamma EF, White JD, Adler JE, Krause JE, McKelvy IF, Black IB. Depolarization regulates adrenal preproenkephalin mRNA. Proc Natl Acad Sci USA 1985;82:8252-8255. 131. LaGamma EF, White JD, McKelvy JF, Black IB. Second messenger mechanisms governing opiate peptide transmitter regulation in the rat adrenal medulla. Brain Res 1988;441:292-298. 132. Roach A, Adler JE, Black IB. Depolarizing influences regulate preprotachykinin mRNA in sympathetic neurons. Proc Natl Acad Sci USA 1987;84:5078-5081. 133. Rao MS, Sun Y, Vaidyanathan U, Landis SC, Zigmond RE. Regulation of substance P is similar to that of vasoactive intestinal peptide after axotomy or explantation of the rat superior cervical ganglion. J Neurobiol 1993;24:571-580. 134. Zigmond RE, Hyatt-Sachs H, Baldwin C, Qu XM, Sun Y, McKeon TW, Schreiber RC, Vaidyanathaa U. Phenotyptic plasticity in adult sympathetic neurons: changes in neuropeptide expression in organ culture. Proc Natl Acad Sci USA 1992;89:1507-1511. 135. Higuchi H, Iwasa A, Yoshida H, Miki N. Long lasting increase in neuropeptide Y gene expression in rat adrenal gland with reserpine treatment: positive regulation of transsynaptic activation and membrane depolarization. Mol Pharmocol 1990;38:614-623. 136. Rokaeus A, Pruss RM, Eiden LE. Galanin gene expression in chromaffin cells is controlled by calcium and protein kinase signaling pathways. Endocrinology 1990;127:3096-3102. 137. Kilbourne EJ, Sabban EL. Differential effect of membrane depolarization on levels of tyrosine hydroxylase and dopamine [3-hydroxylase mRNAs in PC12 pheochromocytoma cells. Mol Brain Res 1990;8:121-127. 138. Craviso GL, Hemelt VB, Waymire JC. Nicotinic cholinergic regulation of tyrosine hydroxylase gene expression and catecholamine synthesis in isolated bovine adrenal chromaffin cells. J Neurochem 1992;59:2285-2296.

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139. Faucon Biguet N, Rittenhouse AR, Mallet J, Zigmond RE. Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion. Neurosci Lett 1989;104:189-194. 140. Black IB, Chikaraishi DM, Lewis EJ. Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion. Brain Res 1985;339:151-153. 141. Zafra F, Hengerer B, Leibrock J, Thoenen H, Lindholm D. Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors. EMBO J 1990;9:3545-3550. 142. Lu B, Yokoyama M, Dreyfus CF, Black IB. Depolarizing stimuli regulate nerve growth factor gene expression in cultured hippocampal neurons. Proc Natl Acad Sci USA 1991;88:6289-6292. 143. Birren SJ, Verdi JM, Anderson DJ. Membrane depolarization induces pl40~k and NGF responsiveness, but not p75 LN6r~, in MAIl cells. Science 1992;257:395-397. 144. Cohen-Cory S, Elliott RC, Dreyfus CF, Black IB. Depolarizing influences increase low-affinity NGF receptor gene expression in cultured Purkinje neurons. Exp Neurobiol 1993; 119:165-175. 145. Costello B, Meymandi A, Freeman JA. Factors influencing GAP-43 gene expression in PC12 pheochromocytoma cells. J Neurosci 1990; 10:1398-1406. 146. Memo M, Bovolin P, Costa E, Grayson DR. Regulation of ~-aminobutyric acidA receptor subunit expression by activation of N-methyl-D-aspartate-selective glutamate receptors. Mol Pharmacol 1991;39:599-603. 147. Condorelli DF, DellAlbani P, Aronica E, Genazzani AA, Casabona G, Corsaro M, Balazs R, Nicoletti F. Growth conditions differentially regulate the expression of alpha-amino-3-hydroxy-5methylisoxazole-4-propionate (AMPA) receptor subunits in cultured neurons. J Neurochem 1993;61: 133-2139. 148. Klarsfeld A, Changeux J-P. Activity regulates the levels of acetylcholine receptor ot-subunit mRNA in cultured chicken myotubes. Proc Natl Acad Sci USA 1985;82:4558-4562. 149. Klarsfeld A, Laufer R, Fontaine B, Devillers-Thiery A, Dubreuil C, Changeux JP. Regulation of muscle AChR ot subunit gene expression by electrical activity: involvement of protein kinase C and calcium. Neuron 1989;2:1229-1236. 150. Tsay H-J, Schmidt J. Skeletal muscle denervation activates acetylcholine receptor genes. J Cell Biol 1989;108:1523-1526. 151. Offord J, Catterall WA. Electrical activity, cAMP, and cytosolic calcium regulate mRNA encoding sodium channel ot subunits in rat muscle cells. Neuron 1989;2:1447-1452. 152. Chatila T, Silverman L, Miller R, Geha R. Mechanisms of T cell activation by the calcium ionophore ionomycin. J Immunol 1989; 143:1283-1289. 153. Ware RE, Hart MK, Haynes BF. Induction of T cell CD7 gene transcription by nonmitogenic ionomycin-induced transmembrane calcium flux. J Immunol 1991; 147:2787-2794. 154. Wodnar-Filipowicz A, Moroni C. Regulation of interleukin 3 mRNA expression in mast cells occurs at the posttranscriptional level and is mediated by calcium ions. Proc Natl Acad Sci USA 1990;87:777-781. 155. Resendez JE, Attenello JW, Grafsky A, Chang CS, Lee AS. Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes. Mol Cell Biol 1985; 5:1212-1219. 156. Li WW, Alexandre S, Cao X, Lee, AS. Transactivation of the grp78 promoter by calcium depletion. J Biol Chem 1993;268:12003-12009. 157. Prostko CR, Brostrom MA, Galuska-Malara EM, Brostrom CO. Stimulation of GRP78 gene transcription by phorbol ester and cAMP in GH3 pituitary cells. J Biol Chem 1991;266:19790-19795. 158. Bartlett JD, Luethy/D, Carlson SG, Sollott SJ, Holbrook NJ. Calcium ionophore A23187 induces expression of the growth arrest and DNA damage inducible CCAAT/enhancer-binding protein (C/EBP)-related gene, gadd153. J Biol Chem 1992;267:20465-20470. 159. Weinstock RS, Saville CM, Messina JL. Role of cytosolic calcium in regulation of cytoskeletal gene expression by insulin. Am J Physiol 1993;264:E519-E525. 160. Rodland KD, Muldoon LL, Lenormand P, Magun BE. Modulation of RNA expression by intracellular calcium. J Biol Chem 1990;265:11000-11007. 161. Bargon J, Trapnell BC, Chu C-S, Rosenthal ER, Yoshimura K, Guggino WB, Dalemans W, Pavirani A, Lecocq J-P, Crystal RG. Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations. Mol Cell Biol 1992;12:1872-1878. 162. Rayson BM. [Ca2+]i regulates transcription rate of the Na +/K+-ATPase Ot 1 subunit. J Biol Chem 1991 ;266:21335-21338.

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Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1995.

8 Calcium Regulation of Apoptosis D i a n e R. D o w d E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical Center, St. Louis, Missouri 63104

The morphological description of apoptosis or physiological cell death was first reported nearly 50 years ago. Since that time considerable effort has been made to understand the molecular basis of the death process (recently reviewed in 1). A renewed surge of interest has occurred in the past few years with the potential link of apoptosis to disease states such as cancer, autoimmunity, and AIDS. Apoptosis occurs in a variety of systems such as embryonic development, neuronal development, hematopoiesis, and the homeostasis of hormone-dependent tissue. Although the molecular signals triggering apoptosis are as diverse as the cell types they affect, morphologically the death process is virtually identical. The apoptotic cell condenses as it loses water, the chromatin forms dense aggregates on the nuclear membrane, and the plasma membrane pinches off membrane-bound segments called apoptotic bodies. These bodies eventually separate from the cell and are phagocytosed by macrophages without eliciting an immune response. Recent advances in the field of apoptosis implicate the active involvement of a number of different proteins in the apoptotic pathway in either the causation or prevention of apoptotic death. The reported involvement of a number of oncogenes and tumor suppressors has provided insight into the complex signaling pathway to death. However, their roles may be limited to a specific apoptotic system or systems as few, if any, of these play universal roles in all forms of cell death. The ionic balance in a cell may also be a determining factor in the response of the cell to various apoptotic stimuli. For example, the multifunctional Ca 2 + molecule plays many diverse roles in the cell, ranging from cell proliferation to differentiation to death, depending upon other signals which the cell is experiencing. There does not appear to be a single role for the ion in the regulation of apoptosis. Instead, Ca 2+ appears to have multiple roles during the complex pathway to death. This review will concentrate on the putative roles of calcium in the apoptotic death program. Most of the work implicating calcium as a second messenger in apoptosis has been reported using thymocytes as a model; however, other systems will be mentioned when applicable. 255

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CALCIUM FLUXES DURING APOPTOSIS In resting cells, the concentration of intracellular, cytosolic Ca 2+ ([Ca 2 +]i) is maintained at 0.05-0.2 I~M against an extracellular concentration of ---1 mM. Various organelles, such as the endoplasmic reticulum (ER) and mitochondria, also store Ca 2 + at levels higher than the cytoplasmic levels. The maintenance of these gradients is achieved by compartmentalization systems and the action of a number of Ca 2 +-transporting ATPases located in the plasma or organelle membranes. The release of Ca 2 + from intracellular stores or the influx of Ca 2 + from the extracellular media often occurs as part of a signaling pathway where Ca 2 + plays a role as a second messenger in the regulation of many diverse cellular activities (for review see 2). Indeed, [Ca2+]i may play such a signaling role in apoptosis, as several apoptotic systems exhibit death-associated increases in [Ca 2 +]i. In contrast, other apoptotic systems do not exhibit fluxes in [Ca 2 +]i levels, yet apoptosis in these systems appears to be Ca 2 +-dependent (3). Several systems exhibiting Ca 2 + fluxes during the apoptotic death process are listed in Table 1. As is apparent from Table 1, Ca 2 + fluxes are evident in lymphoid-derived cell lines undergoing apoptosis by a wide variety of inducers, thus implicating a central role for calcium in lymphocyte apoptosis. Increases in [Ca 2+ ]i can result from a calcium influx from the extracellular media, from the redistribution of intracellular ion stores, or from a combination of both events. As discussed in the following sections, these processes are responsible for the intracellular calcium fluxes that occur in a wide variety of apoptotic cell systems.

TABLE 1. Apoptotic systems displaying a flux in intracellular calcium

Cell type

Apoptotic inducer

Reference

synovial cells primary thymocytes thymocytes thymocytes thymocytes thymocytes thymocytes thymocytes thymocytes leukemic T-cell line PBL T lymphoma line T lymphoma line T-cell hybridoma lymphoid cells prostate prostatic cancer cells adenocarcinoma cells hepatocytes

cold shock glucocorticoids anti-CD3 antibody calcium ionophore tributyltin X-irradiation *f-irradiation extracellular ATP TCDD Ultraviolet radiation Ultraviolet radiation glucocorticoids thapsigargin anti-CD3 antibody DMBA androgen withdrawal calcium ionophore tumor necrosis factor c= oxidative stress

36 11,12,14 108 11,12,58 30 117 8,9 76 118 4,5 5 18 18 53 119 15,16 17 33 120

TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PBL, peripheral blood lymphocyte; DMBA, 7,12dimethylbenz[a]anthracene.

CALCIUM REGULATION OF APOPTOSIS

Calcium Influx from the Extracellular Media

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Calcium fluxes are associated with various forms of radiation-induced lymphocyte apoptosis. Increases in intracellular Ca 2 + were reported in normal human peripheral blood lymphocytes and in Jurkat, a leukemic T-cell line, exposed to UVB or UVC radiation (4, 5). On the molecular level, UV radiation rapidly induces tyrosine phosphorylation in these cells (5). In cells treated simultaneously with a tyrosine kinase inhibitor and irradiation, phosphorylation and the UV-induced Ca 2 + flux was inhibited, indicating that the Ca 2 + signaling mechanism was dependent upon tyrosine phosphorylation (5). Mechanistically, tyrosine kinases generate Ca 2 + signals via phosphorylation of phospholipase C-y1 (PLC~/1) and associated proteins (6). PLC~/1 cleaves phopatidylinositol 4,5-bisphosphate to produce inositol 1,4,5trisphosphate (IP3) and diacylglycerol. IP3 binds to a membrane receptor to release Ca 2 + from intracellular stores, probably located in the ER (7). Although PLC~/1 was phosphorylated after UV irradiation, the relationship of PLC~/1 phosphorylation and the Ca 2 + signaling pathway to apoptosis has not been elucidated (5). Apoptosis in primary thymocytes subjected to ~/-irradiation involves calcium fluxes as well (8, 9). A two fold increase in [Ca 2+]i was detected 3 hours postirradiation, concurrent with DNA fragmentation (8). The increase in both Ca 2 § and DNA degradation was inhibited by the addition of trolox, a water-soluble form of vitamin E, to the media. The authors propose that as an antioxidant, trolox blocks damage or alterations in membrane functions that would allow for a Ca 2 + flux or Ca 2 + mobilization during apoptosis (8). This hypothesis supports the observation that vitamin E protects calcium translocases which would otherwise be impaired during membrane damage (10). Glucocorticoid treatment induces apoptosis in specific populations of thymocytes in a calcium-dependent manner (11,12). This process is characterized by the inhibition of uridine metabolism, the activation of the apoptosis-associated endonuclease, and subsequent cell death (11,13). The death process could be mimicked by a calcium ionophore, suggesting that glucocorticoids exert their effects in part through calcium-dependent mechanisms (11,13). In support of this hypothesis, McConkey et al. (14) demonstrated an early, gradual, and sustained increase in cytosolic Ca 2 + during treatment of primary rodent thymocytes with the glucocorticoid methylprednisolone (Fig. 1A, filled circles). Ca 2+ levels increased eightfold (from 85 nM to approximately 750 nM) over a 90-min period, and remained constant for at least an additional 30 min (14). The rise in Ca 2 + was dependent upon macromolecular synthesis, as it is blocked by both actinomycin D (filled squares) and cycloheximide (filled triangles). The increase in [Ca 2 +]i preceded DNA fragmentation (Fig. 1B, filled circles), and the chelation of extracellular or intracellular Ca 2 + with ethylene glycol bis (13-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or Quin-2, respectively, inhibited DNA degradation (Fig. 1B). These results suggest that the increase in cytosolic Ca 2 + was due to an influx of Ca 2 + from the extracellular media, and this increase was rate-limiting for activation of the endonuclease. Moreover, treatment of the cells with a calmodulin antagonist inhibited endonuclease activity (Fig.

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1B, filled squares), but not the observed calcium flux, suggesting a role for calmodulin in transmitting the Ca 2+-mediated signal in the cascade to death. The rise in [Ca 2+ ]i was detected in the presence of voltage-dependent and receptor-mediated Ca 2 +-channel blockers, suggesting that the rise is not due to the activation of preexisting channels. To further investigate the mechanism of the calcium increase, the authors prepared cytosol from thymocytes treated for 90 min with methylprednisolone. This cytosolic fraction was incubated with fresh thymocytes and [Ca 2 +]i measured. The "active" cytosol produced a rapid rise in [Ca 2 +]i (Fig. 2A, filled circles) followed later by DNA fragmentation (Fig. 2B, filled circles). These effects were abolished if the cytosol was boiled for 5 min before the incubation (Fig. 2A, open triangles). The authors suggest that glucocorticoid treatment of thymocytes may lead to the synthesis of a potential calcium pore which facilitates the influx of calcium into the cell, thereby initiating the ionic signal for apoptosis (14). A similar apoptosis-associated rise in calcium occurs in several other systems, including apoptosis in the prostate due to androgen withdrawal (15, 16). In contrast to apoptotic thymocytes, ventral prostate involution is blocked by calcium-channel antagonists, suggesting that calcium influx in this system is a cause and not an effect of apoptosis. Later studies supported these findings by showing a calcium-dependent induction of cell death by ionophore treatment of Dunning R-3327 rat prostatic cancer cells (17). A sustained 3 to sixfold elevation in [Ca 2 § was sufficient to induce apoptotic death in this system.

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Redistribution of Intracellular Stores In recent studies, Lam et al. (18,19) measured cytosolic Ca 2 + during glucocorticoid-mediated apoptosis in the murine T-lymphocyte cell lines W7MG1 and WEHI7.2. These lymphoma cells did not exhibit the dramatic influx of extracellular calcium as reported for primary thymocytes (14). Instead, the authors demonstrated that the synthetic glucocorticoid dexamethasone caused a redistribution of intracellular calcium stores. The release of Ca 2 + from internal stores was estimated by incubating the cells in the presence and absence of glucocorticoids. Then the increase in cytosolic Ca 2 + induced by either ionomycin or thapsigargin was measured. Ionomycin treatment releases Ca 2 + from a variety of intracellular stores and also increases Ca 2 + influx from the extracellular media. Thapsigargin increases Ca 2§ release from the ER specifically by inhibiting the ER-associated Ca 2+ATPase. Glucocorticoid treatment induced a significant decrease in ionomycin- and thapsigargin-mobilized Ca 2 + stores (Table 2). The authors proposed the following sequence of events leading to a two-component increase in [Ca 2+ ]i and subsequent cell death (18). Glucocorticoid treatment of W7MG 1 lymphocytes leads to an efflux of calcium from the ER, resulting in a significant depletion of those stores. The initial loss of Ca 2 + from the ER occurs only after a 2-hour lag consistent with the known mechanism of action of glucocorticoids as regulators of gene expression. Then the calcium is pumped out of the cell, an event which is followed by an influx

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100 kDa) in extracts from control and glucocorticoid-treated (apoptotic) thymocytes, but in the apoptotic cells, the endonuclease also exists as a low molecular weight form (---25 kDa). The distinct, inducible, 25-kDa isoform may result from (a) transcriptional induction of the enzyme via the classical steroid receptor pathway; (b) specific stabilization of the nuclease mRNA; (c) specific activation of a constitutively expressed endonuclease via nongenomic mechanisms; or (d) variations in the extraction conditions due to the presence of digested DNA in the apoptotic samples facilitating the release of the endonuclease (73). Whether the activation of the endonuclease is a cause or an effect of apoptosis is still in debate. Some evidence suggests that DNA fragmentation is not a critical event for apoptotic death. For example, although zinc inhibits the endonuclease activity (60,74,75), the metal may not protect thymocytes from either spontaneous or ghcocorticoid-induced apoptosis (75), or from apoptosis resulting from high levels of extracellular ATP (76). Moreover, Ucker et al. (77) isolated a fibroblast clone which underwent cytotoxic T cell lysis but, unlike its parent, failed to exhibit genome digestion. This led the authors to suggest that DNA fragmentation is not essential for cell death triggered by this mechanism. However, because just a few double-strand breaks can be lethal to the cell (69), it is possible that DNA fragmen-

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tation is occurring in these cells, albeit at a level which is not detected by these techniques. Although genome digestion may not be a critical step in apoptosis, DNA fragmentation may allow for greater ease in the budding and separation of the cell and nucleus into apoptotic bodies. The digestion of DNA may also serve to protect the phagocytosing cells against the possibility of gene transfer from the dying cell. EFFECTS ON THE CYTOSKELETON The activation of tissue transglutaminase is only one effect of Ca 2+ on the cytoskeleton. Calcium fluxes can mediate other disrupting effects which lead to membrane bleb formation, a morphological characteristic of apoptosis (78-80). For example: (a) Ca2+-dependent proteases may cleave actin bundles or actin-binding proteins to alter the cellular matrix and reduce cellular integrity; (b) Ca 2+ may induce a modification and rearrangement of the actin network; and (c) Ca 2+ may lead to the fragmentation or depolymerization of actin. In addition to negatively affecting the actin network, Martin and Cotter (81) reported that intracellular calcium fluctuations in HL60 leukemia cells lead to the disruption of microtubules resulting in an apoptotic phenotype. Low levels of the calcium ionophore A23187 (10-6-10 -4 M) caused apoptosis in the cells while higher levels (> 10 -4 M) resuited in a death phenotype characteristic of necrosis. This suggests that there is a precise ionic balance in the cell and the magnitude of its disruption is responsible for causing varied responses such as physiological death (apoptosis) or pathological death (necrosis). Terminally differentiated HL60 cells undergoing spontaneous apoptosis also exhibited a disrupted microtubule network, and microtubule-disrupting agents induced apoptosis in undifferentiated HL60 cells (81). Together, these data suggest that the disruption in the microtubule network is induced by an increase in [Ca2+]i and is an important step in apoptosis and the formation of apoptotic bodies. Later studies demonstrated that A23187 induced apoptosis of HL60 cells in the absence of external Ca 2+, suggesting that the apoptotic mechanism operates independently of Ca 2+ influx (82). The authors did not completely rule out the possibility that the ionophore was causing an efflux of Ca 2+ from intracellular stores which was not detected by their methodology, thereby activating the death pathway. EFFECTS ON GENE EXPRESSION The calcium influx that is induced by A23187 may have many varied effects due to its ability to activate enzymes and disrupt cellular integrity as described above. Intriguingly, lymphocyte death induced by A23187 is blocked by the inhibition of protein synthesis with cycloheximide (58, 83). These results suggest that either the half-life of an essential Ca 2+-dependent protein (such as the endonuclease) is relatively short, or that Ca 2 + fluxes may be required for transcription of genes involved

CALCIUM REGULATION OF APOPTOSIS

269

in the death process. In this regard, by blocking calcium channels during prostate apoptosis, Connor et al. (15) were able to suppress the induction of specific gene expression associated with death and block the regression of the tissue (15). Although the relationship between changes in [Ca2+]i and macromolecular synthesis is not yet defined for systems undergoing apoptosis, our lab has also detected Ca 2+-regulated gene expression during lymphocyte apoptosis induced by a variety of agents, including calcium ionophore and glucocorticoids (manuscript submitted). In the prostatic epithelium, castration initiates a transcriptional cascade including a number of genes such as c-fos, c-myc, and hsp-70 (84). The regulation of c-fos gene expression was reported in a number of apoptotic systems (85), and is linked to fluxes in [Ca2 +]i in other nonapoptotic systems (48,86-89). Specifically, increases in [Ca2+ ]i lead to the activation of a calmodulin-dependent kinase, and this kinase phosphorylates the transcription factor cAMP-response element binding protein (CREB). CREB is directly responsible for the transcriptional induction of the c-fos gene. When castrated rats were treated with the calcium-channel blockers verapamil (Adalat, Procardia) or nifedipine, a significant inhibition of tissue regression and of c-fos induction were reported. Thus, fluxes in [Ca2+]i which occur during castration-induced apoptosis could be potentially involved in the regulation of gene expression (such as c-fos) required for cell death. Since the c-Fos protein is a transcription factor, the increased expression of this protein may be a first step in a complex cascade to cell death

INHIBITION OF APOPTOSIS Bcl-2

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The Bcl-2 oncoprotein was identified recently as an inhibitor of multiple forms of lymphocyte apoptosis (90,91). Subsequently, many reports have emerged linking the protective effects of Bcl-2 with other apoptotic systems, including the IL-3deprived myeloid cell line discussed above (31). The number of apoptotic systems now shown to be inhibited by Bcl-2 expression is steadily increasing. Discussed below are those systems where calcium plays a role in death, and this process is blocked by Bcl-2. In B-cells, Liu et al. (92) reported a correlation between Bcl-2 expression and protection against calcium ionophore-induced apoptosis, i.e., B-cells which express high levels of Bcl-2 were resistant to the induction of apoptosis by calcium, whereas Bcl-2-deficient cells died. Likewise, when Bcl-2-deficient T-lymphocytes were infected with a retrovirus encoding bcl-2, the cells were no longer sensitive to the apoptotic effect of calcium ionophore (93). The Bcl-2-containing T-cells were also resistant to glucocorticoid-mediated apoptosis (93), a calcium-dependent process (11,12). In the neuron-derived cell line PC I2, the stable expression of Bcl-2 inhibits DNA fragmentation and subsequent apoptotic death induced by the calcium ionophore A23187 (94), whereas the wild-type PC12 cells are susceptible to C a 2 + - m e -

270

CALCIUM REGULATION OF AP OPTOSIS

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diated apoptosis. These studies were later extended to demonstrate Bcl-2-mediated protection in other neural cells (95). In the neural cells, Bcl-2 did not affect the intracellular Ca 2 + levels at either the resting concentrations or the peak levels induced by the ionophore, suggesting that the protective effect was distal to the rise in calcium (95). The mechanism whereby Bcl-2 protects cells against Ca 2+-mediated death in any of these cases is unclear at present, although recent studies have begun to address this. While studying glucocorticoid-induced apoptosis in pre-B-leukemia lines, A1nemri et al. (96) reported that Bcl-2 enhances cell survival under conditions of hormone repression of c-myc expression. This may occur due to a Bcl-2-mediated mobilization of Ca 2§ from the mitochondria to the cytoplasm, which in turn may cause the activation of PKC and aid in the enhanced survival in these cells. The activation of PKC is protective in a number of apoptotic systems (discussed below). A report by Lam et al. (19) links Bcl-2 expression to the maintenance of calcium homeostasis in apoptotic lymphocytes. A previous study from this group demonstrated that glucocorticoid treatment of a murine T-lymphoma cell line induced the efflux of calcium from the ER during apoptosis (18). This effect could be mimicked by thapsigargin, an inhibitor of the Ca 2 +-ATPase in the ER. Overexpression of the Bcl-2 oncoprotein in these cells resulted in protection against apoptosis induced by dexamethasone or thapsigargin (19). Bcl-2 did not affect the inhibitory action of thapsigargin on the ER-associated Ca 2§ pump, but did significantly reduce the release of Ca 2§ into the cytosol from the ER. By reducing Ca2 + efflux from this store, the subsequent influx of extracellular Ca 2+ was prevented (18,19). Thus, Bcl-2 may inhibit apoptosis by directly or indirectly regulating intracellular Ca 2 + fluxes which may be responsible for apoptotic signaling.

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Calcium-Binding Proteins A hormone-insensitive prostate cell line was isolated recently which expresses the calcium-binding protein, calbindin, at high levels (97). The overexpression of calbindin in these cells results from the splicing of the calbindin gene to the long terminal repeat of a retroviral-like sequence. The hormone-dependent prostate undergoes extensive apoptosis and involution upon androgen withdrawal by a mechanism which is triggered by an influx of Ca 2 + (15,16). In contrast, the PC3 cells are unaffected by hormone removal, suggesting that the overexpression of calbindin in PC3 cells may contribute to the observed hormone independence by buffeting calcium fluxes in the cell. Lymphocyte apoptosis, a calcium-dependent process, can be blocked by chelating extracellular calcium or by buffeting intracellular calcium fluxes. Moreover, as illustrated in Fig. 5, high-level expression of the calcium-binding protein calbindinD28K in the WEHI7.2 lymphocyte cell line interferes with the apoptotic cascades induced by either glucocorticoids (see Fig. 5, squares) or calcium ionophore (data not shown) (98), possibly by buffeting apoptosis-associated Ca2 + fluxes. Increases

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in cAMP also may lead to apoptosis in some lymphocytes by a mechanism which is Ca 2+-dependent, although large calcium fluxes have not been observed (3). In this regard, DNA fragmentation due to cAMP can be attenuated by the chelation of intracellular calcium with Quin-2. Likewise, expression of calbindin-D28K in the WEHI7.2 cells leads to protection against cAMP-induced cell death induced by forskolin, an activator of adenylyl cyclase (see Fig. 5, circles). These data suggest that calcium is required for apoptosis in lymphocytes and that by buffeting calcium levels in the cell, death can be attenuated. Moreover, calbindin is expressed generally in cells which experience large Ca 2 + fluxes (e.g., intestine, brain, sensory pathways) (99). Thus, calbindin may serve to protect the cells from damage which could result from unbuffered fluxes.

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There is increasing evidence that reduced levels of calcium-binding proteins may be detrimental to some neuronal cells which experience Ca 2+ fluxes. Brain cells particularly susceptible to Ca 2 +-induced damage due to aging, Huntington's disease, Alzheimer's disease, seizure activity, and autoimmune deficiency syndromeassociated dementia express reduced levels of the calcium binding proteins calbindin-D28K or parvalbumin (100-102). Moreover, Scharfman and Schwartzkroin (103) demonstrated that cells containing calcium-binding proteins showed no signs of degeneration during prolonged stimulation (conditions facilitating Ca 2+ influx), in contrast to their calcium-binding protein-negative counterparts. In addition, they were able to inhibit neuronal damage by impaling cells with microelectrodes conmining the calcium chelator BAPTA-2. These results have led to the suggestion that the abnormally low expression of calcium-binding proteins such as calbindin may reduce the buffeting capacity of the cells leading to Ca 2+ fluxes which are now damaging to the neurons (103,104). Calcium-channel blockers also inhibit damage due to Ca 2+ fluxes. For example, neuronal cell death after withdrawal of nerve growth factor (NGF) was inhibited by flunarizine, a voltage-dependent calcium-channel blocker (105). However, the concentration required for inhibiting death was greater than that required for channel blocking. Thus, the authors proposed that the mechanism of action of flunarizine involves the interaction and inhibition of intracellular components, possibly calmodulin, supporting a role for calmodulin in the apoptotic cascade. Recent clinical trials using an L-type Ca 2+ channel-blocking drug were performed in the treatment of Alzheimer's disease (106). The rationale behind this therapy was to slow cholinergic neuronal cell death by blocking Ca 2 + fluxes into the aging cells. The results of the trial suggest that treatment with the L-type Ca 2+ channel blocker slows progressive memory impairment, potentially by preventing the Ca 2+-activated mechanism of neuronal cell death. Treatments focused on the prevention of large calcium fluxes by preventing or buffeting the flux may prove to be beneficial in these neuronal disorders.

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In an attempt to understand the molecular basis of the action of nicotine, Wright et al. (107) demonstrated that nicotine inhibited apoptosis-associated DNA fragmentation in thymocytes treated with the calcium ionophore A23187. This effect did not involve conventional nicotinic cholinergic receptors, and the mechanism of apoptosis repression was not determined. The authors hypothesized that the inhibitory effect on cell death may contribute to the tumorigenicity of tobacco by acting as a tumor-promoting agent. OTHER SIGNALS In the developing thymocyte, a number of molecular signals regulate proliferation and death. It appears that a rise in [Ca2+ ]i is not sufficient to trigger the apopto-

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tic response, but it is the relationship of [Ca2+ ]i to other intracellular signals which determines the response of the cell. In support of this "unbalanced signaling" hypothesis (108,109), apoptotic death of thymocytes induced by glucocorticoids, calcium ionophore, or anti-CD3 antibodies, processes which demonstrate an increase in [Ca2+]i, can be blocked by the coactivation of PKC (83,108). In these cases, activated PKC does not affect the cytosolic Ca 2 + levels, and thus may directly prevent or counteract the Ca 2+-mediated cascade to cell death, presumably through phosphorylation events. Phorbol esters and other tumor promoters inhibit apoptosis in other Ca 2 +-dependent systems as well, including irradiated cells (110), synovial cells exposed to cold shock (36), and mammary adenocarcinoma cells treated with TNF-ot (33). However, contrasting results have been reported by Ojeda et al. (111). In this study, hydrocortisone-induced apoptosis of thymocytes was blocked by the addition of the PKC inhibitor H-7 to the cell suspension. This apparent discrepancy may indicate that a signal may have multiple effects in a cell depending upon the presence of other independent signals. As mentioned previously, glucocorticoids, Ca 2 + ionophore, and anti-CD3 antibody treatment all induce apoptosis in the same population of immature CD4 + CD8 + thymocytes in a calcium-dependent manner. In contrast, mature lymphocytes are often resistant to glucocorticoid or ionophore treatment, and in some cases ionophores can actually block apoptosis due to other agents (112,113). This suggests that in these cells, the signaling pathway to death is developmentally regulated. Alternatively, the apoptotic programming may be functional; however, other protective proteins are expressed which prevent the pathway from being completed. This may be the case for Bcl-2 in the protection of specific populations of lymphocytes (discussed above). In the case of IL-3-dependent myeloid cells, withdrawal of the cytokine induces apoptosis (113,114). Apoptosis was preceded by a calcium drop and was prevented by the concomitant addition of calcium ionophore to the system (114). Calcium ionophore inhibited apoptosis in the IL-3-dependent bone marrow cells by inducing IL-4 expression in a Ca 2+-dependent manner (112). IL-4 production was required for the maintenance of cell viability and thus was considered a survival factor in this system.

APOPTOTIC SYSTEMS DEMONSTRATING NEGATIVE REGULATION BY CALCIUM Although the evidence is strong that calcium plays a major role in some cases of apoptosis, there are other instances where calcium fluxes may have little involvement with the death process (115,116). For example, CEM C7 T cells undergo glucocorticoid-induced apoptosis without a concomitant increase in [Ca2 +]i (115). Moreover, the reduction of calcium in the media appeared to slightly enhance the apoptotic effect of the hormone. These results suggest that a calcium influx is not a requisite early event in apoptosis of CEM C7 cells. However, they do not exclude the possibility of calcium involvement in the initiation or completion of the apopto-

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tic pathway. For example, Ca 2 + could be released from intracellular stores causing subtle changes in [Ca 2 +]i which activate enzymes or mediate intracellular signals (115). Alternatively, apoptosis may occur by a completely separate mechanism with different ion requirements, or there may be shared steps in the pathways which are arrived at by different mechanisms, one being calcium-dependent, the other independent. Thus, apoptotic death and DNA fragmentation may be controlled by multiple intracellular signals. Although a rise in [Ca2+]i often precedes DNA fragmentation, endonuclease activation may occur in the absence of detectable fluxes in Ca 2 +. In these cases the endonuclease involved may not be the Ca 2 +/Mg 2 +-dependent enzyme described in thymocytes. Alternatively, the level of intracellular Ca 2 § may be sufficient to activate the enzyme, or small fluxes in Ca 2 + due to the redistribution of intracellular stores may be the required signal for enzyme activation. For example, Ca 2 + accumulates in the nucleus of TNF-et treated adenocarcinoma cells, potentially triggering events leading to DNA fragmentation (33). CONCLUSIONS This manuscript reviews several putative roles for calcium in the signal transduction mechanism of a variety of distinct apoptotic systems. These various roles are illustrated in Fig. 6 as a general model of calcium-regulated apoptosis. To summarize, altered calcium levels:

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1. Influence the activity of calcium-dependent enzymes such as proteases, lipases, and transglutaminases that participate in the degradation of cellular constituents; 2. May disrupt cytoskeletal and mitochondrial function; 3. Have fundamental consequences in the nucleus where endonucleases are clearly activated but also where transcription of genes that are involved in the apoptotic process may be regulated in a calcium-dependent fashion. Although a significant amount of work focuses on the role of Ca 2 + in the death process, much of the evidence remains indirect and speculative. More work is necessary to establish direct proof that calcium is a causative agent in the apoptotic cascade. A clearer relationship between ionic homeostasis and apoptotic signaling will enable the elucidation of the pathway to death and will provide insight into potential overlap between calcium- and noncalcium-dependent signaling pathways.

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ACKNOWLEDGMENTS

I would like to thank Dr. P. MacDonald for helpful discussions and for the critical reading of the manuscript.

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90. Sentman CL, Shetter JR, Hockenberry D, Kanagawa O, Korsmeyer SJ. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 1991;67:879-888. 91. Strasser A, Harris AW, Cory S. bcl-2 transgene inhibits T cell death and perturbs thymic selfcensorship. Cell 1991;67:889-899. 92. Liu YJ, Mason DY, Johnson GD, et al. Germinal center cells express bcl-2 after activation by signals which prevent their entry into apoptosis. Eur J Immunol 1991;21:1905-1910. 93. Miyashita T, Reed JC. bcl-2 gene transfer increases relative resistance of $49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res 1992;52:5407-5411. 94. Mah SP, Zhong LT, Liu Y, Roghani A, Edwards RH, Bredesen DE. The-protooncogene bcl-2 inhibits apoptosis in PC12 cells. J Neurochem 1993;60:1183-1186. 95. Zhong LT, Sarafian T, Kane DJ, et al. bcl-2 inhibits death of central neural cells induced by multiple agents. Proc Natl Acad Sci USA 1993;90:4533-4537. 96. Alnemri ES, Fernandes TF, Haldar S, Goce CM, Litwack G. Involvement of BCL-2 in glucocorticoid-induced apoptosis of human pre-B-leukemias. Cancer Res 1992;52:491-495. 97. Liu A, Abraham BA. Subtractive cloning of a hybrid human endogenous retrovirus and calbindin gene in the prostate cell line PC3. Cancer Res 1991 ;51:4107-4110. 98. Dowd DR, MacDonald PN, Komm BS, Haussler MR, Miesfeld RL. Stable expression of the calbindin-DEaK cDNA interferes with the apoptotic pathway in lymphocytes. Mol Endocrinol 1992; 6:1843-1848. 99. Christakos S, Gabrielides C, Rhoten WB. Vitamin D-dependent calcium binding proteins: chemistry, distribution, functional considerations, and molecular biology. Endocr Rev 1989; 10:3-26. 100. Suthedand MK, Somerville MJ, Yoong LKK, Bergeron C, Haussler MR, McLachlan DRC. Reduction of vitamin D hormone receptor mRNA levels in Alzheimer as compared to Huntington hippocampus: correlation with calbindin-28k mRNA levels. Mol Brain Res 1992;13:239-250. 101. Seto-Ohshima A, Emson PC, Lawson E, Mountjoy CZ, Carrasco LH. Loss of matrix calciumbinding protein containing neurons in Huntington's disease. Lancet 1988;6:1252-1255. 102. Iacopino AM, Christakos S. Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases. Proc Natl Acad Sci USA 1990; 87:4078-4082. 103. Scharfman HE, Schwartzkroin PA. Protection of dentate hilar cells from prolonged stimulation by intracellular calcium chelation. Science 1989;246:257-260. 104. Mattson MP, Rychlik B, Chu C, Christakos S. Evidence for calcium-reducing and excito-protecfive roles for the calcium-binding protein calbindin-D28k in cultured hippocampal neurons. Neuron 1991;6:41-51. 105. Rich KM, Hollowell JP. Flunarizine protects neurons from death after axotomy and nerve growth factor deprivation. Science 1990;248:1419-1421. 106. Brancormier RJ, Branconnier ME, Walshe TM, McCarthy C, Morse P-A. Blocking the Ca2+-acti vated cytotoxic mechanisms of cholinergic neuronal death: a novel treatment strategy for Alzheimer's disease. Psychopharm Bull 1992;28:175-181. 107. Wright SC, Zhong J, Zheng H, Larrick JW. Nicotine inhibition of apoptosis suggests a role in tumor promotion. FASEB J 1993;7:1045-1051. 108. McConkey DJ, Hartzell P, Amador-Perez JF, Orrenius S, Jondal M. Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex. J Immunol 1989;143: 1801-1806. 109. McConkey DJ, Orrenius S. Cellular signaling in thymocyte apoptosis. In: Tomei LD, Cope FO, eds. Apoptosis: the molecular basis of cell death. Plainview, NY: Cold Spring Harbor Laboratory Press; 1991:227-246 110. Tomei LD, Kanter P, Wenner CE. Inhibition of radiation-induced apoptosis in vitro by tumor promoters. Biochem Biophys Res Commun 1988;155:324-331. 111. Ojeda F, Guarda MI, Maldonado C, Folch H. Protein kinase-C involvement in thymocyte apoptosis induced by hydrocorisone. Cell Immunol 1990;125:535-539. 112. Rodriguez-Tarduchy G, Malde P, L6pez-Rivas A, Collins MKL. Inhibition of apoptosis by calcium ionophores in IL-3 dependent bone marrow cells is dependent upon production of IL-4. J Immunol 1992; 148:1416-1422. 113. Rodriguez-Tarduchy G, Collins M, L6pez-Rivas A. Regulation of apoptosis in interleukin-3-dependent hemopoietic cells by intedeukin-3 and calcium ionophores. EMBO J 1990,9:2997-3002. 114. Magnelli L, Cinelli M, Turchetti A, Chiarugi VP. Apoptosis incution in 32D cells by IL-3 withdrawal is preceded by a drop in the intracellular calcium level. Biochem Biophys Res Commun 1993;194:1394-1397.

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115. Bansal M, Houle AG, Melnykovych G. Dexamethasone-induced killing of neoplastic cells of lymphoid derivation: lack of early calcium involvement. J Cell Physiol 1990;143:105-109. 116. Kelley LL, Koury MJ, Bondurant MC. Regulation of programmed death in erythroid progentor cells by erythropoietin: effects of calcium and of protein and RNA syntheses. J Cell Physio11992; 151:487-496. 117. Zhivotovsky B, Nicotera P, Bellomo G, Hanson K, Orrenuis S. Ca2+ and endonuclease activation in radiation-induced lymphoid cell death Exp Cell Res 1993;207:163-170. 118. McConkey DJ, Hartzell P, Duddy SK, Hakaneson H, Orrenius S. 2,3,7,8 tetrachlorodibenzop-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation. Science 1988; 242:256-259. 119. Burchiel SW, Davis DAP, Ray SD, Archuleta MM, Thilsted JP, Corcoran GB. DMBA-induced cytotoxicity in lymphoid and nonlymphoid organs of B6C3F1 mice: relation of cell death to target cell intracellular calcium and DNA damage. Toxicol Appl Pharmacol 1992; 113:126-132. 120. McConkey DJ, Hartzell P, Nicotera P, WyUie AH, Orrenius S. Stimulation of endogenous endonuclease activity in hepatocytes exposed to oxidative stress. Toxicol Len 1988;42:123-130.

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Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1995.

9 Role of Calcium in T-Lymphocyte Activation M a r i a E. C a r d e n a s * and J o s e p h H e i t m a n t *Departments of Genetics and Pharmacology and tHoward Hughes Medical Institute, Duke University Medical Center, Box 3546, Durham, North Carolina 27710

Activation of T-lymphocytes is required for proper function of the immune system. T-cell activation begins at the membrane of the cell when antigen in complex with MHC is presented to the T-cell receptor (TCR) complex. This ligand-receptor interaction stimulates signal transduction cascades that activate gene expression to promote alterations in physiology, production of lymphokines, and proliferation. During the earliest signaling events, several tyrosine kinases are activated and one or more of these phosphorylates and thereby activates phospholipase C, resulting in the production of the second messengers diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3). Subsequently, IP3 binds to the IP3 receptor and liberates stored calcium ions (Ca 2+) to give rise to an early increase in intracellular Ca 2 + levels. Next, depletion of intracellular Ca 2 § stores signals an influx of Ca 2 § across the plasma membrane to give rise to a larger, sustained increase in intracellular Ca2 +. One target of Ca 2 +, the phosphatase calcineurin, is required to promote nuclear import of the T-cell transcription factor NF-AT, which then associates with Fos and Jun and drives transcription of T-cell activation genes. The immunosuppressants cyclosporin A and FK506 prevent T-cell activation by inhibiting calcineurin. Further studies of T-cell components that regulate and respond to Ca 2 + may provide additional targets for novel immunosuppressants and provide molecular insights into human immunodeficiency disorders. The vertebrate immune system is specialized to recognize, attack, and destroy foreign invaders and tissues. Two different cell types are clonally expanded and activated via specialized families of receptors: B-cells via antigen binding to membrane-bound immunoglobulin and T cells via antigen presentation to the TCR complex. These ligand-receptor interactions stimulate signal transduction cascades that activate gene expression to promote altered cell physiology, proliferation, and cellular activation. The TCR consists of multiple subunits of two different types. The ot and 13 subunits are variable chains that are members of the immunoglobulin superfamily and confer the antigen specificity of the receptor. These chains have very short intra281

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cellular tails and are therefore unlikely to couple to intracellular signaling molecules. In contrast, the CD3 components of the receptor (e, G, xl, and ~) have invariant sequences and large intracellular domains. Thus, these subunits serve to couple the extracellular ligand recognition domain to intracellular signaling machinery. Studies with chimeric molecules reveal that the cytoplasmic domains of either the or the ~ chains can signal (1-3). In addition to the subunits of the TCR, mature T cells also express either of two coreceptors, CD4 or CD8. In conjunction with the TCR, CD4 confers specificity for antigen presentation by MHCII, whereas CD8 confers specificity for MHCI. This ensures that CD4 + helper T cells are activated by cells of the immune system, which express MHCII, and that CD8 + cytotoxic T cells can be activated by virtually any cell in the body that expresses a novel antigen presented by MHCI molecules, which is critical for the elimination of virally infected or neoplastic cells. The earliest biochemical events following antigen presentation to the TCR are the activation of protein tyrosine kinases (recently reviewed in 4). Members of two different tyrosine kinase families have been implicated in T-cell activation cascades: Lck, Fyn, and Yes, members of the Src family, and ZAP70, a member of the syk/ ZAP70 kinase family (5-7). The Lck kinase is physically associated with the cytoplasmic tails of the CD4 and CD8 coreceptors (8). Cross-linking of either CD4 or CD8 serves to activate Lck activity. Lck is also physically associated with the TCR (9). The Fyn kinase is also physically associated with subunits of the TCR, and its activity increases during signaling (10). In contrast, ZAP70 is not associated with either receptor in quiescent T cells, but does become physically associated with the TCR shortly after activation begins (11). At least one chain of the TCR, the ~ chain, becomes tyrosine phosphorylated during T-cell activation. In a surrogate cell system, either Lck or Fyn can phosphorylate the TCR ~ chain. Although ZAP70 does not interact with the nonphosphorylated ~ chain, either Lck or Fyn is sufficient to enable ZAP70 to bind to the ~ chain (12). A likely scenario is that one or both of the Src family kinases Lck and Fyn associate with and are activated by the TCR to tyrosine phosphorylate the TCR ~ subunit, which then interacts with the ZAP70 kinase via the two ZAP70 SH2 domains (13). Thus, this cascade serves to activate two TCR- or CD4/CD8-associated tyrosine kinases, to recruit and activate a third kinase (ZAP70), and to tyrosine phosphorylate at least one chain of the TCR, which can then interact with SH2-containing proteins, including at least ZAP70.

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CALCIUM IS A SECOND MESSENGER DURING T-CELL ACTIVATION

One of the downstream targets of these activated tyrosine kinases is the -y-isoform of phospholipase C, which is known to be activated by the TCR (14). Several lines of evidence argue that this stimulation is via direct tyrosine phosphorylation of phospholipase C. For example, phospholipase C is tyrosine phosphorylated during T-cell activation (15,16), and phosphorylation increases the activity of the enzyme (17). At present, it is not clear which of the three known kinases serves to activate phospholipase C. Activation of phospholipase C initiates the production of two

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second messengers: diacylglycerol, leading to activation of protein kinase C; and IP3, which binds to the IP 3 receptor calcium channel on the ER to liberate Ca 2+ from this intracellular store. This gives rise to the early increase in intracellular Ca 2 + levels during T-cell activation. A number of studies in a variety of different cell types reveal that Ca 2 + itself regulates the activity of the IP3 receptor (18-22). These studies have employed activation of the IP3 receptor by addition of IP3 or release by flash photolysis of caged IP3. The effects of alterations in cellular Ca 2 + concentrations, by microinjection or flash photolysis release of caged Ca 2 +, on the activity of the IP 3 receptor were then monitored. These studies reveal that the activity of the IP3 receptor is stimulated at low concentrations of intracellular Ca 2 + but inhibited at higher concentrations, providing one possible molecular mechanism for the production of osciUating Ca 2 + concentrations evoked in many cells. Ca 2 + could have direct, indirect, or both direct and indirect effects on the IP3 receptor. For example, studies by Zhang and Muallem (22) revealed that Ca 2+ activation of IP3 receptor activity required Mg 2+ and was blocked by inhibitors of calmodulin-dependent protein kinase II, whereas calcium-dependent inhibition of IP3 receptor activity was blocked by cyclosporin A or FK506, which are specific inhibitors of the calciumcalmodulin-regulated protein phosphatase calcineurin (23). Thus, the IP3 receptor could be reciprocally regulated by Ca 2+-dependent phosphorylation and dephosphorylation. MECHANISMS THAT INCREASE AND SUSTAIN CALCIUM LEVELS The production of IP3, activation of the IP3 receptor, and release of ER stores of Ca 2 + can only partially explain the Ca 2 + fluxes that occur during T-cell activation. They are not sufficient to account for both the magnitude and the duration of the sustained increase in intracellular Ca 2 + during T-cell activation. In fact, following the release of Ca 2 + from the ER, calcium channels in the plasma membrane are activated that increase the influx of Ca 2+ into the cell, and it is the activation of these channels that enhances and prolongs increased Ca 2 + levels in the cell. Two mechanisms have been proposed to account for the activation of this Ca 2 + influx. First, IP3 receptors have been discovered to be present on the T-cell plasma membrane. Thus, IP3 could stimulate both Ca 2 + release from the ER and influx across the plasma membrane. An alternative model, the capacitative Ca 2 + entry model, proposes that the emptying of intracellular Ca 2 + stores is coupled, either directly or indirectly, to mechanisms that allow influx of extracellular Ca 2 +. We consider these two models and discuss additional evidence that potassium ion channels are likely to participate in the regulation of Ca 2 + influx across the plasma membrane. Receptors for the second messenger IP3 were first identified on the surface of the ER and serve to regulate Ca 2 + release from this intracellular store. A distinct IP3 receptor present on the plasma membrane of T cells has been identified by several different routes (24-27). First, electrophysiological patch-clamping studies with

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excised portions of the T-cell plasma membrane revealed the presence of an IP3-responsive Ca 2+ conductance in cells stimulated via the TCR (24). Second, antibodies against the IP3 receptor detect a 260-kDa protein that can be surface iodinated in T cells and which, by immunocytochemistry, is on the plasma membrane (26). This IP3 receptor is distinct from the ER form, is modified by sialic acid, and has higher affinity for IP4 compared to IP3 (27). In addition, capping of the TCR results in cocapping of the plasma membrane-localized IP3 receptor; the highest levels of intracellular Ca 2 + underlie this capped portion of the T-cell membrane (26). These findings suggest that the plasma membrane IP3 receptor mediates, or is associated with, the influx of extracellular Ca 2 + that is required for T-cell activation. Thus, in this model, IP3 would play a dual role of liberating Ca 2 + from intracellular stores and mobilizing Ca 2 + influx across the plasma membrane. There are several problems with the view that IP3 also participates in activating the influx phase of Ca 2 + during T-cell activation, although this may be the case in some cell types under some conditions. First, release of intracellular Ca 2 + stores with thapsigargin, an inhibitor of the ER Ca 2 + ATPase, leads to an influx of Ca 2 + in the absence of signaling via the TCR (28-30). Importantly, thapsigargin treatment does not result in IP3 production and thus separates the effects of Ca 2 + depletion and IP3 action. Second, activation of the TCR signaling pathway in thapsigargin-treated cells does not increase intracellular Ca / + above that evoked by thapsigargin alone (31). Lastly, Zweifach and Lewis (32) have shown that the plasma membrane Ca 2 + channels activated by depletion of intracellular Ca 2 + stores differ in electrophysiological properties from previously described IP3-regulated channels. An alternative hypothesis, the capacitative calcium entry model, is more consistent with these observations. In the capacitative calcium entry model, depletion of intracellular Ca 2 + stores stimulates the influx of Ca 2 + across the plasma membrane (33), (reviewed in 3436). Originally, this model suggested that the depletion of intracellular Ca 2 + stores would be signaled to the plasma membrane by a direct physical coupling between components of the ER and the plasma membrane. This view is similar to the model for excitation-contraction coupling in skeletal muscle cells, in which depolarization of the T-tubule membrane causes conformational changes in the dihydropyridine receptor that are physically transmitted to the ryanodine receptor, triggering release of Ca 2 + from the sarcoplasmic reticulum (reviewed in 37). The capacitative calcium entry model has since been modified to speculate that this coupling between depletion of intracellular stores and plasma membrane could be indirectly transmitted via either conformational changes in the cytoskeleton or a diffusible second messenger. Two recent studies provide strong support for a diffusible second messenger coupling Ca 2 +-store depletion to Ca 2 + influx (38,39). Randriamampita and Tsien (38) have identified a novel second messenger, named CIF for calcium-influx factor, that is released from intracellular stores by Ca 2 + depletion in the Jurkat human T-cell line (38). Some CIF was also found to be released to the extracellular medium. CIF stimulated Ca 2 + influx not only in T cells, but also in macrophages,

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astrocytoma cells, and fibroblasts. These findings reveal that CIF is broadly acting and membrane-permeant. Studies of the physical properties of this agent reveal that it is small, with a molecular weight less than 500 Da (based on size exclusion chromatography), and negatively charged (based on retention by anion exchange chromatography). CIF is resistant to heat or protease but sensitive to alkaline phosphatase, suggesting at least one critical phosphate group is present. CIF is sensitive to periodate, but not to iodoacetamide, indicating the presence of either vicinol diols or amino alcohols. Lastly, CIF is resistant to borohydride, excluding the presence of essential aldehydes, ketones, or reducing sugars. The precise structure of CIF remains to be determined. In related studies, Parekh et al. (39) studied Ca 2 + influx in Xenopus oocytes expressing exogenous serotonin receptors which, when bound by ligand, stimulate phospholipase C to generate IP3 and liberate intracellular Ca 2 +. As in T cells, this leads to a stimulation in Ca 2 + influx across the plasma membrane, which Parekh et al. monitored with a patch pipette. To define components of this signaling pathway, a variety of second messengers and inhibitors were tested. Okadaic acid, an inhibitor of protein phosphatase types 1 and 2A, was found to markedly prolong the activity of the calcium ion channels detected, suggesting that either a phosphorylated second messenger or a phosphorylation-activated signaling component are involved. This finding is reminiscent of the findings of Randriamampita and Tsien (38) that CIF contains an essential phosphate group and is negatively charged. Parekh et al. (39) also found that when a portion of the cell membrane was excised, the calcium currents rapidly decayed, whereas when the excised membrane was reintroduced to the cell, the current was restored. Moreover, the channels of the excised membrane could be activated by reintroducing it at any position in the original cell. These findings imply that a diffusible second messenger, present throughout the cell, is responsible for activating the plasma membrane calcium ion channels in response to intracellular Ca 2 + store depletion. Parekh et al. also found that a potassium ion efflux current was also activated by Ca 2 + depletion; the role of K + efflux in Ca 2 + influx will be discussed further below. In addition to the CIF second messenger, additional studies suggest that both Ca 2 +-regulated enzymes and GTP hydrolysis also participate in the signaling pathway coupling intracellular Ca 2 + stores with Ca 2 + influx channels at the plasma membrane. Haverstick and Gray (30) found that Ca 2 + influx following TCR activation or thapsigargin treatment could be blocked with calmodulin inhibitors, including phenothiazines (such as trifluoperazine [Stelazine]) and W7. Moreover, chelation of released intracellular Ca 2 + blocked influx of extracellular calcium (30). These findings implicate one or more calcium-calmodulin-regulated enzymes in this signaling cascade. Although not yet tested in T cells, studies with either mouse lacrimal cells or rat basophilic leukemia cells reveal that nonhydrolyzable GTP analogues such as GTP~/S block the signaling cascade linking intracellular CaE+-store depletion and the plasma membrane influx pathway (40,41). These studies implicate a GTP hydrolysis-dependent step, possibly involving a G protein, in this novel signaling cascade. Further studies will be required to identify the GTP-

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dependent signaling component(s) and to confirm that this GTP-dependent step is also operative in T cells. T-cell activation results in an increase in not only Ca z + influx, but also in the efflux of potassium ions. T cells express both voltage-activated and calcium-activated potassium ion channels (42,43). Recent studies with peptide inhibitors (noxiustoxin or margatoxin) specific for the voltage-regulated potassium ion channel demonstrate that this channel is required for the maximal increase in intracellular Ca 2 + during TCR signaling (44). Inhibition of this potassium ion channel during T-cell stimulation causes membrane depolarization, which reduces Ca z + levels, resulting in decreased production of lymphokines and decreased proliferation (44). The T-cell type n voltage-regulated potassium ion channel has recently been shown to be phosphorylated, suggesting one possible mechanism of regulation (45). Further studies will be required to address the coordinated regulation of Ca 2+ influx and K + efflux.

zyxwvut TARGETS OF CALCIUM IN T CELLS

The identification of one of the principle targets of Ca 2 + in T cells resulted from a convergence of studies on the mechanisms of action of two immunosuppressants, cyclosporin A (CsA) and FK506 (reviewed in 46-49). CsA and FK506 block a common step required for T-cell responses to antigen presentation. Neither CsA nor FK506 impairs early steps activated by the TCR that result in increased intracellular Ca 2 +. Rather, both drugs inhibit an intermediate signal transduction step and prevent activation of transcription factors required for T-cell activation (50,51). CsA and FK506 diffuse across the plasma membrane and associate with intracellular binding proteins. CsA binds to cyclophilins whereas FK506 binds to FK506-binding proteins, or FKBPs (52,53). Cyclophilins and FKBPs represent two different protein families that share no primary sequence homology or tertiary structural identity. Both types of proteins are abundant, ubiquitous, and found in multiple intracellular compartments. In addition, both cyclophilins and FKBPs catalyze an unusual reaction, cis-trans peptidyl-prolyl isomerization, likely to be involved in protein folding in vivo (54). Drug binding inhibits this reaction; however, a variety of different findings demonstrate that CsA, FK506, and rapamycin do not block T-cell activation by inhibiting cyclophilin or FKBP (23,55-57). Instead, the immunophilin-drug complexes bind to and inhibit the activity of other proteins that are required for signal transduction. The target of CsA and FK506 has been identified as a serine-threonine-specific phosphatase, calcineurin (23,58), also known as protein phosphatase 2B (PP2B). Calcineurin is a Ca 2+-regulated protein phosphatase that is abundant in the brain; hence its name is derived from calcium and neuron (59). Calcineurin is a heterodimer composed of a catalytic A subunit and a regulatory B subunit (59). When intracellular Ca 2 + levels increase, Ca 2 + binds to calmodulin and the resulting complex associates with a calmodulin-binding domain in the carboxy-terminal region of

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calcineurin A, resulting in formation of a heterotrimer and activation of calcineurin phosphatase activity (60,61). In the absence of calmodulin-calcium, an autoinhibitory domain in the carboxy-terminal region of calcineurin A binds to and inhibits the active site; calmodulin binding triggers conformational changes that release the autoinliilSitory domain. Peptides derived from the autoinhibitory region inhibit calcineurin activity (62). Interestingly, the calcineurin B regulatory subunit is a calmodulin homologue which shares significant identity with calmodulin. However, calmodulin and calcineurin B cannot substitute for each other and bind to different regions of calcineurin A. The in vivo role of calcineurin B in the function of the calcineurin AB holoenzyme is not yet known. In vitro studies reveal that calcineurin A has little or no activity in the absence of the B subunit, and addition of the B subunit is required for proper folding of the A subunit and reconstitution of calcineurin activity (63). In addition, it is not yet clear how many of the Ca 2+-binding sites of calcineurin B are occupied in the calcineurin AB holoenzyme in resting cells, and it is not clear whether additional Ca 2 § molecules bind to calcineurin B and alter calcineurin activity during intracellular increases in Ca 2 +. The calcineurin B subunit also differs from calmodulin in that it is lipid modified by an N-terminal myristyl group (64,65), which may play a role in membrane association of calcineurin. Lastly, the activity of calcineurin can be stimulated by lipids in vitro via a binding site on calcineurin B (66,67). The immunophilin-immunosuppressant complexes cyclophilin-CsA and FKBP12-FK506 bind to and potently inhibit the activity of bovine brain calcineurin toward phosphopeptide substrates (23,58). In contrast, both protein-drug complexes stimulate the activity of calcineurin against paranitrophenolphosphate (PNPP), a small organic compound that emulates phosphotyrosine (23,68). This finding first suggested that these inhibitors do not compete for the calcineurin active site, and additional studies have since confirmed that the cyclophilin A-CsA complex noncompetitively inhibits calcineurin (69). The current model is that the drug-protein complexes bind to calcineurin and trigger conformational changes that alter the structure of the active site, inhibiting access by phosphopeptides and promoting activity against small molecules that can still enter the active site in the immunophilin-drug-calcineurin complexes. A great deal of structural information is now available about these immunophilinimmunosuppressant-calcineurin complexes. First, we now know that, together, both the immunophilin and the immunosuppressive ligand form a composite surface that interacts with calcineurin. The NMR and X-ray crystal structures are available for cyclophilin-CsA and FKBP12-FK506 (70-78), and site-directed mutagenesis studies have identified residues surrounding the ligand binding pockets that participate in interaction with calcineurin (69,79-81). The precise calcineurin residues that are contacted by either the drug or the immunophilin have not yet been identified. It has been shown that truncation of the carboxy-terminal autoinhibitory and calmodulin-binding domains of calcineurin A does not prevent association of the immunophilin-drug complexes with calcineurin (58,68). Thus calmodulin is not required for binding. The calcineurin B subunit is

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required for cyclophilin-CsA to inhibit calcineurin in vitro (82), and cross-linking studies reveal that FKBP12 and cyclophilin A cross-link to calcineurin B, but not to calcineurin A, in the respective immunophilin-drug-calcineurin complexes (83). These studies reveal a critical role for calcineurin B in the action of CsA and FK506. In contrast, a photocross-linkable form of CsA became cross-linked to only calcineurin A in the cyclophilin A-CsA-calcineurin AB complex, indicating that the drugs are likely to bind between the immunophilin and calcineurin A (84). The resulting model is that the immunophilin-drug complex contacts calcineurin A but that the calcineurin B subunit must also be very near to or in direct contact with the immunophilins. Because the X-ray structure of calmodulin is known and the twodimensional NMR structure of calcineurin B has been recently determined (84a), the complete structure of the immunophilin-drug-calcineurin-AB-calmodulin complex may be attainable. At present the only missing portion is the structure of calcineurin A. Several recent studies indicate that calcineurin is the relevant target for immunosuppressant action in vivo. Although a role for calcineurin in T-cell activation was unknown until the studies implicating calcineurin as the target of CsA and FK506, two early studies examined calcineurin in T cells. First, a protein complex was found to associate with the plasma membrane in a Ca 2+-dependent fashion in either porcine lymphocytes or human lymphoblastoid cell lines (85). This complex was identified as calcineurin based on electrophoretic mobility and Western analysis with calcineurin-specific antisera. Alexander et al. (86) subsequently confirmed that calcineurin was expressed and membrane associated in human T lymphoblasts and Jurkat T-cell lines. Following the identification of calcineurin as the target for CsA and FK506 action in vitro, three studies tested whether calcineurin is a relevant drug target in vivo. In two studies, overexpression of calcineurin A and B subunits or expression of a truncated constitutively active calcineurin A increased the concentration of CsA required to inhibit T-cell activation (87,88). In addition, T cells overexpressing calcineurin were less dependent on intracellular increases in Ca 2+ for activation. In separate studies, Fruman et al. (89) measured the amount of calcineurin activity present in extracts of T cells exposed to immunosuppressants, and found that inhibition of calcineurin activity paralleled the extent to which T-cell activation reporter gene expression was inhibited. Taken together, these studies provide compelling support for models in which calcineurin normally plays a critical role in transducing signals required for T-cell activation. One recent study has addressed the question as to which of the immunophilins are relevant for drug action in vivo (90). Cells are known to express multiple cyclophilins: cyclophilin A is cytoplasmic; cyclophilins B and C are in the ER and secretory pathway; and cyclophilin D is mitochondrial. Similarly, cells express multiple FKBPs; FKBP12 and FKBP59 are cytoplasmic; FKBP13 is in the ER; and FKBP25 is nuclear. Over-expression of either cyclophilin A or cyclophilin B, but not cyclophilin C, renders Jurkat T cells more sensitive to CsA, as monitored by expression of an NF-AT-responsive reporter gene (90). Similarly, overexpression of FKBP12, but not of FBBP13 or FKBP25, conferred increased sensitivity to FK506. These

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findings confirm that cyclophilin A mediates CsA action and FKBP12 mediates FK506 action, but suggest that cyclophilin B (an ER form of cyclophilin) may also participate in CsA action. This is surprising because calcineurin is thought to be exclusively cytoplasmic. Based on their results, Bram et al. (90) suggest that a proportion of calcineurin might be localized to the ER. An alternative possibility not discussed is that CsA might cause cyclophilin B to be mislocalized to the cytoplasm. It is well known that tight binding ligands can prevent protein unfolding events required for mitochondrial protein import. Thus CsA might bind to cyclophilin B in the cytoplasm, prevent transport across the ER membrane, and promote inhibition of calcineurin. Calcineurin is also present in the yeast S. cerevisiae, where one gene encodes the regulatory B subunit and two homologous genes encode two catalytic subunits (65,91-95). In most yeast strains, calcineurin is not required for viability. Calcineurin is required for yeast cell viability under specialized conditions, such as exposure to mating pheromone (65,92,96). It is not yet clear which are the relevant calcineurin substrates in the yeast mating pathway. As in T-cell activation, a rise in intracellular Ca 2 + levels occurs following exposure to mating pheromones in yeast, and this response is necessary to maintain viability (97). Treatment of yeast cells with CsA or FK506 prevents recovery from pheromone-imposed cell cycle arrest (96). Calcineurin is also required for growth of yeast cells in the presence of high levels of extracellular cations (98,99). CsA and FK506 inhibit growth under these conditions; CsA inhibition requires yeast cyclophilin A, and FK506 inhibition requires yeast FKBP12. We have discovered a yeast strain in which calcineurin is required for viability (99). This strain is killed by either CsA or by FK506 in complex with their respective binding proteins, enabling us to select drug-resistant mutants, to genetically dissect these protein-drug-protein complexes, and possibly to identify relevant in vivo calcineurin targets. In contrast, Cunningham and Fink (100) have isolated a Ca 2 + channel mutant yeast strain in which high levels of Ca 2 + are toxic because of inappropriate activation of calcineurin. In this mutant strain, mutation or inhibition of calcineurin restores growth under conditions of high extracellular calcium. These studies suggest that, in addition to responding to Ca 2 + fluxes, calcineurin may also play a role in regulating Ca 2 + fluxes. In summary, genetic studies of calcineurin function in yeast underscore a remarkable conservation in drug action from yeast to T cells (reviewed in 49). In both organisms, both drugs combine with conserved immunophilin proteins to form protein-drug complexes that potently inhibit calcineurin. Similarly, in both yeast and T cells, calcineurin is activated by a Ca 2 + increase and regulates signal transduction pathways mediated by phosphoproteins. Is there any relationship between cyclophilins, FKBPs, and calcineurin? One possibility is that cyclophilin and FKBP12 bind calcineurin only in the presence of an immunosuppressant. Thus, the protein-drug complexes bind, but neither protein nor drug alone associates with or inhibits calcineurin. Alternatively, immunophilins and calcineurin may normally interact. For example, calcineurin and FKBP12 colocalize throughout the vertebrate central nervous system (101), suggesting that the

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proteins may be related. Furthermore, FK506 promotes an FKBP12-FK506-calcineurin complex in yeast (96,99, Cardenas et al., submitted), in the plant Vicia faba (102), and in mammalian lymphocytes (23). Thus, the FKBP12-FK506-calcineurin interaction surfaces have been highly conserved, perhaps because the proteins normally interact. FKBPs and cyclophilins may also play a more direct role in regulating Ca 2+ fluxes in cells. For example, FKBP12 is a modulatory subunit of the ryanodine receptor Ca 2 + channel (103,104). It is not yet known if FKBP12 is also a subunit of the IP3 receptor, which shares significant sequence similarity with the ryanodine receptor. In addition, cyclophilin B is localized to a specialized region of the ER, the calciosome, which may be involved in storage and release of Ca 2 + (105). In complex with Ca 2+, calmodulin is known to regulate the activity of a large number of enzymes. Thus, we expect that, in addition to calcineurin, Ca 2 + might regulate the activity of other calmodulin-dependent enzymes in T cells. In fact, a specific type of calcium-calmodulin-regulated kinase, CaM kinase-Gr (also known as CaM kinase IV), has been recently implicated in T-cell signaling (106). CaM kinase-Gr is known to be expressed in many tissues, including brain and thymus (107), and has recently been found to be differentially expressed in immature thymocytes (double positives) compared to mature thymocytes (single positives) (106). The kinase is also expressed in CD4 § helper T cells but not in other cells of the hematopoietic system, with the exception of EBV-transformed B cells in which the EBV-transforming protein LMP1 transcriptionally activates expression of CaM kinase-Gr (108). CaM kinase-Gr purified from quiescent T cells is inactive, suggesting that the enzyme is under stringent regulation. Activation of Jurkat T cells with antibodies against the CD3 component of the TCR stimulated phosphorylation of the kinase on serine residues, resulting in increased kinase activity. Comparison of in vivo labeling and in vitro two-dimensional phosphopeptide patterns with the purified kinase stimulated with Ca 2+-calmodulin reveals that signaling via the TCR stimulates autophosphorylation of CaM kinase-Gr (106). Although Ca 2+-calmodulin activates the enzyme in vitro, calcium ionophores are not sufficient to activate the enzyme in vivo, suggesting that there is another level of control. The target(s) of CaM kinase-Gr in T cells is as yet unknown.

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Prior to the identification of calcineurin as the target for cyclosporin and FK506 action, earlier studies revealed that these drugs act as a Ca 2 +-dependent signaling event (109) and impair the activity of transcription factors required for the expression of proteins necessary for T-cell activation (50), such as the T-cell growth factor interleukin-2 (IL-2). The most profound effects are observed with the transcription factor NF-AT or nuclear factor of activated T cells. Subsequently. NF-AT was found to be composed of two subunits, one which is T-cell-specific and cytoplasmic in quiescent T cells and a second non-T-cell-specific subunit which is newly synthe-

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sized and transported to the nucleus following T-cell activation (51). With an in vitro transcription system and extract mixing experiments, CsA was shown to block the nuclear import of the cytoplasmic subunit of NF-AT (51). The nuclear subunit of NF-AT is composed of Fos and Jun (110,111). One form of the cytoplasmic subunit of NF-AT has been recently cloned, revealing that it shares some similarity with the rel family of transcriptional regulators (112). The electrophoretic mobility of NF-ATc is increased by treatment with commercially available bovine calcineurin, suggesting that NF-AT is a phosphoprotein substrate for calcineurin (113,114). Importantly, either a peptide inhibitor of calcineurin or the general phosphatase inhibitor pyrophosphate prevent the action of calcineurin preparations on NF-ATc (114). These findings are consistent with models in which the direct target of calcineurin is NF-ATc. However, it is important to bear in mind that it has not yet been demonstrated that NF-AT is a phosphoprotein (by in vivo labeling with 32p, for example), that cyclophilin-CsA or FKBP12-FK506 inhibit the action of calcineurin on NF-AT in vitro, or that NF-AT is the relevant in vivo substrate for calcineurin. To summarize, the resulting model is that calcineurin is a critical Ca 2+ sensor in T cells. In response to antigen presentation, increased intracellular calcium levels result in calcineurin activation via both calmodulin and possibly also calcineurin B. Activated calcineurin then dephosphorylates the cytoplasmic subunit of NF-AT, allowing it to translocate to the nucleus, associate with the nuclear subunit of NFAT (Fos and Jun), and activate transcription. CsA and FK506 block calcineurin activity, preventing nuclear import of the cytoplasmic subunit of NF-AT and inhibiting expression of genes required for T-cell activation. Although most studies of NF-AT function have concentrated on its effects at the IL-2 gene enhancer, CsA and FK506 inhibit the expression of many other lymphokines such as GM-CSF. Recently, Tsuboi et al. (115) showed that the ability of CsA to inhibit GM-CSF transcription was reduced by increased expression of calcineurin A and B or of a constitutively active truncated form of calcineurin A. They also defined a promoter element, CLEO, which contains an AP-1 binding site and in mobility shift assays binds an NF-AT-like factor, which they named NF-CLEO ~/ (115). The relationship of NF-AT to NF-CLEO ~ is not clear at present; these could be identical factors or members of a related family. These findings reveal that calcineurin is required for the activated expression of both IL-2 and GM-CSF through similar mechanisms. In addition to NF-AT, several other transcription factors which enhance IL-2 gene expression are regulated by Ca 2+ and are likely to be, directly or indirectly, regulated by calcineurin. These include Oct-1/OAP (116,117), NF-• (118), and AP-1. The ubiquitous transcription factor Oct-1 cooperates with a T-cell-specific component, OAP, to activate expression of the IL-2 gene via the ARRE-1 element of the IL-2 enhancer (116). OAP has been recently purified and found to be composed of Jun family members, including JunD and c-Jun, but not JunB (117). Both phorbol ester and a calcium ionophore are required to stimulate expression of a reporter construct driven by a multimerized ARRE-1 site (117). These observations suggest that AP-1 can be differentially regulated; in the case of Oct-1/OAP the

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AP-l-related OAP component is Ca 2 +-activated, and in the case of AP-1 alone, Ca 2+ serves to weakly inhibit transcriptional activation (118). Interestingly, although the DNA binding activity of Oct-1/OAP is not reduced in extracts from CsA exposed Jurkat T cells, CsA does block the ability of Oct-1/OAP to activate transcription (117,118). This is in marked contrast to NF-AT, where CsA or FK506 inhibit the appearance of NF-AT-specific DNA binding activity following T-ceU stimulation. These observations suggest that CsA inhibits a calcium-dependent CsA-sensitive posttranslational modification required for Oct-1/OAP to activate transcription. A likely model is that calcineurin dephosphorylates either Oct-1 or OAP. A third transcription factor that binds to and activates the IL-2 enhancer, NF-• is also regulated by Ca2+ and calcineurin (109,118). Expression of an artificial NF-• reporter gene can be stimulated by phorbol ester alone, but the addition of calcium ionophore further stimulates expression. The Ca 2 +-dependent component of NF-• activity is fully inhibitable by either CsA or FK506 (109, 118). In one recent study (118), calcium ionophore-stimulated NF-• B-dependent expression eightfold above the level observed with phorbol ester alone whereas, in other studies, the CsA-sensitive component of NF-• activity has been less pronounced, either twofold (50) or fourfold (109). These observations suggest that calcineurin is required for maximal NF-• activity and that the magnitude of the effect may depend on experimental conditions. How does calcineurin regulate NF-• Frantz et al. (118) present evidence to suggest that calcineurin participates in the inactivation of the NF-• associated protein, I• which normally anchors NF-xB in the cytoplasm and from which NF-• must be released for nuclear import. Previous studies have implicated phosphorylation as one means by which I• can be released from NF-• so it is paradoxical that a phosphatase would augment this process, unless one supposes that calcineurin regulates kinases that inactivate I• Further studies will be required to address this question. Lastly, a recent study has suggested that activation via AP-1 responsive promoter elements is weakly enhanced, about twofold, by calcineurin inhibition (118). However, this effect has not been observed in similar previous studies (50,109). How is it that the immunosuppressants CsA and FK506 suppress the immune system sufficiently to prevent graft rejection but still permit some immune function to occur? One possibility is that some cells escape inhibition and remain functional (P. F. Halloran, personal communication). Variations in the levels of calcineurin or NF-AT between T cells could account for this. In recent studies, Negulescu et al. (119) monitored both intracellular increases in Ca 2 + concentration (using the Ca 2 +-stimulated fluorescence of fura-2) and expression of an NF-AT-regulated reporter gene. Following T-cell receptor stimulation, individual cells varied greatly in the extent to which intracellular Ca 2 + increased. Moreover, although higher levels of intracellular Ca 2 + were associated with reporter gene expression in the entire population, individual cells varied widely in their responsiveness to different Ca 2 + levels. CsA inhibition decreased the percentage of cells responding to Ca 2+, but did not reduce the magnitude of reporter gene expression in those few CsA-treated cells

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which did respond. These studies suggest that T cells vary in their ability to mount and respond to Ca 2 + increases in response to activation, and further suggest that cells do not respond in a graded fashion, but rather respond when a threshold concentration of Ca 2 + has been crossed. CONCLUSIONS We know a great deal about the earliest events that underlie T-cell activation. Thus, all of the subunits of the TCR, CD4, and CD8 are cloned; we know that, together, they serve to recruit and activate three different tyrosine kinases. We know that one or more of these kinases is likely to phosphorylate phospholipase C, resulting in the production of the well studied second messengers DAG and IP3. We know that IP3 initiates the rise in intracellular Ca 2 + via IP3 receptors. We are coming to appreciate the mechanisms that link this early rise in intracellular Ca 2 + to an influx of Ca 2 + across the plasma membrane that increases and prolongs the wave of Ca 2 + signaling. Further study of this interesting coupling between emptying intracellular reserves and events at the plasma membrane promises to reveal a novel second messenger. We now know that one of the events that Ca 2 + regulates is the activation of calcineurin, which may be required to dephosphorylate NF-AT to allow nuclear entry and transcriptional activation. Other transcription factors are likely to be regulated by calcineurin as well. A remarkable series of findings have converged to provide a very detailed view of how the immunosuppressants CsA and FK506 harness two different abundant intracellular proteins to inhibit calcineurin function and block T-cell activation. Outstanding questions that remain are: Which other Ca 2 + dependent events participate in T-cell activation? Is NF-ATc the direct target of calcineurin? Are there other relevant calcineurin targets? and Do the immunophilins normally participate in T-cell activation? Lastly, further studies on the Ca 2 +-dependent signaling events that participate in T-cell activation are likely to provide further insights into signal transduction cascades and may provide both additional targets for novel immunosuppressants and candidates for defects in immunodeficiency disorders. ACKNOWLEDGMENTS

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We thank Sandra Bowling for assistance with manuscript preparation, Joseph Nevins for generous support, and Tony Means for comments. Joseph Heitman is an investigator with the Howard Hughes Medical Institute.

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81. Rosen MK, Yang D, Martin PK, Schreiber SL. Activation of an inactive immunophilin by mutagenesis. J Am Chem Soc 1993;115:821-822. 82. Haddy A, Swanson SK-H, Born TL, Rusnak F. Inhibition of calcineurin by cyclosporin A-cyclophilin requires calcineurin B. FEBS Lett 1992;314:37-40. 83. Li W, Handschumacher RE. Specific interaction of the cyclophilin-cyclosporin complex with the B subunit of calcineurin. J Biol (?hem 1993;268:14040-14044. 84. Ryffel B, Woedy G, Murray M, Eugster H-P, Car B. Binding of active cyclosporins to cyclophilin A and B, complex formation with calcineurin A. Biochem Biophys Res Commun 1993;194:10741083. 84a.Anglister J, Grzesiek S, Wang AC, Ren H, Klee CB, Bax A. ~H, ~3C, 15N Nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B. Biochemistry 1994;33:3540-3547. 85. Chantler PD. Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin. J Cell Biol 1985;101:207-216. 86. Alexander DR, Hexham JM, Crumpton MJ. The association of type 1, type 2A and type 2B phosphatases with the human T lymphocyte plasma membrane. Biochem J 1988;256:885-892. 87. Clipstone NA, Crabtree GR. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 1992;357:695-697. 88. O'Keefe SJ, Tamura J, Kincaid RL, Tocci MJ, O'Neill EA. FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature 1992;357:692-694. 89. Fruman DA, Klee CB, Bierer BE, Burakoff SJ. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK506 and cyclosporin A. Proc Natl Acad Sci USA 1992;89:3686-3690. 90. Bram RJ, Hung DT, Martin PK, Schreiber SL, Crabtree GR. Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location. Mol Cell Biol 1993;13:4760-4769. 91. Liu Y, Ishii S, Tokai M, Tsutsumi H, Ohki O, Akade R, et al. The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B. Mol Gen Genet 1991;227:52-59. 92. Cyert MS, Kunisawa R, Kaim D, Thorner J. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineudn, a calmodulin-regulated phosphoprotein phosphatase. Proc Natl Acad Sci USA 1991;88:7376-7380. 93. Kuno T, Tanaka H, Mukai H, Chang C-D, Hiraga K, Miyakawa T, et al. cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae. Biochem Biophys Res Commun 1991;180: 1159-1163. 94. Ye RR, Bretscher A. Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae. Eur J Biochem 1992;204:713-723. 95. Nakamura T, Tsutsumi H, Mukai H, Kuno T, Miyakawa T. Ca2+/calmodulin-activated protein phosphatase (PP2B)of Saccharomyces cerevisiae. FEBS Lett 1992;309:103-106. 96. Foor F, Parent SA, Morin N, Dahl AM, Ramadan N, Chrebet G, et al. Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from a-factor arrest in yeast. Nature 1992;360: 682-684. 97. Iida H, Yagawa Y, Anraku Y. Essential role for induced Ca2+ influx followed by [Ca2+ ]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. J Biol Chem 1990;265:13391-13399. 98. Nakamura T, Liu Y, Hiram D, Namba H, Harada S-i, Hirokawa T, et al. Protein phosphatase type 2B (calcineufin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. EMBO J 1993;12:4063-4071. 99. Breuder T, Hemenway CS, Movva NR, Cardenas ME, Heitman J. Calcineurin is essential in cyclosporin A and FK506 sensitive yeast strains. Proc Natl Acad Sci USA 1994;91:5372-5376. 100. Cunningham KW, Fink GR. Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J Cell Biol 1994;124: 351-363. 101. Steiner JP, Dawson TM, Fotuhi M, Glatt CE, Snowman AM, Cohen N, et al. High brain densities of the immunophilin FKBP colocalized with calcineurin. Nature 1992;358:584-587. 102. Luan S, Li W, Rusnak F, Assmann SM, Schreiber SL. Immunosuppressants implicate protein phosphatase regulation of K + channels in guard cells. Proc Natl Acad Sci USA 1993;90:22022206. 103. Jayaraman T, Brillantes A-M, Timerman AP, Fleischer S, Erdjument-Bromage H, Tempst P, et al.

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FK506 binding protein associated with the calcium release channel (ryanodine receptor). J Biol Chem 1992;267:9474-9477. 104. Timerman AP, Ogunbumni E, Freund E, Wiederrecht G, Marks AR, Fleischer S. The calcium release channel of sarcoplasmic reticulum is modulated by FK-506-binding protein. J Biol Chem 1993 ;268:22992-22999. 105. Arber S, Krause K-H, Caroni P. s-Cyclophilin is retained intracellularly via a unique COOHterminal sequence and colocalizes with the calcium storage protein calreticulin. J Cell Biol 1992; 116:113-125. 106. Hanissian SH, Frangakis M, Bland MM, Jawahar S, Chatila TA. Expression of a Ca2+/cal modulin-dependent protein kinase, CaM kinase-Gr, in human T lymphocytes. J Biol Chem 1993; 268:20055-20063. 107. Frangakis MV, Chatila T, Wood ER, Sahyoun N. Expression of a neuronal Ca2+/calmodulin dependent protein kinase, CaM kinase-Gr, in rat thymus. J Biol Chem 1991;266:17592-17596. 108. Mosialos G, Hanissian SH, Jawahar S, Vara L, Kieff E, Chatila TA. A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1. J Virol 1994;68:16971705. 109. Mattila PS, Ullman KS, Fiering S, McCutcheon M, Crabtree GR, Herzenberg LA. The actions of cyclosporin A and FKS06 suggest a novel step in the activation of T lymphocytes. EMBO J 1990; 9:4425-4433. 110. Jain J, McCaffrey PG, Valge-Archer VE, Rao A. Nuclear factor of activated T cells contains Fos and Jun. Nature 1992;356:801-804. 111. Northrop JP, Ullman KS, Crabtree GR. Characterization of nuclear and cytoplasmic components of the lymphoid-specific nuclear factor of activated T cells (NF-AT) complex. J Biol Chem 1993; 268:2917-2923. 112. McCaffrey PG, Perrino BA, Sodeding TR, Rao A. NF-ATp, a T lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs. J Biol Chem 1993;268:3747-3752. 113. Jain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JN, Verdine GL, et al. The T-cell transcription factor NFATp is a substrate for calcineurin and interacts with Fos and Jun. Nature 1993; 365:352-355. 114. Jain J, Miner Z, Rao A. Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. J Immunol 1993;151:837-848. 115. Tsuboi A, Masuda ES, Naito Y, Tokumitsu H, Arai K-I, Arai N. Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine dement O. Mol Biol Cell 1994;5:119-128. 116. Ullman KS, Northrop JP, Verweij CL, Crabtree GR. Transmission of signals from the T lymphocyte antigen receptor to the genes responsible for cell proliferation and immune function: the missing rink. Annu Rev Immunol 1990;8:421-452. 117. Ullman KS, Northrop JP, Admon A, Crabtree GR. Jun family members are controlled by a calcium-regulated, cyclosporin A-sensitive signaling pathway in activated T lymphocytes. Gen Dev 1993;7:188-196. 118. Frantz B, Nordby EC, Bren G, Steffan N, Paya CV, Kincaid RL, et al. Calcineurin acts in synergy with PMA to inactivate I• an inhibitor of NF-• EMBO J 1994;13:861-870. 119. Negulescu PA, Shastri N, Cahalan MD. Intracellular calcium dependence of gene expression in single T lymphocytes. Proc Natl Acad Sci USA 1994;91:2873-2877.

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zyx Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1995.

lO

Regulation of the Cell Division Cycle by Inositol Trisphosphate and the Calcium Signaling Pathway Michael Whitaker Department of Physiology, University College London, London WC1E 6BT, UK

Calcium is a ubiquitous and multifunctional cell messenger. We shall discuss the possibility that changes in the intracellular free calcium concentration ([Ca2+]i) may regulate the cell division cycle. There is evidence to support the idea from experiments in a variety of cell types, including plant cells (1). Here, we shall discuss evidence that has accumulated since we last reviewed the subject (2). It has remained true that some of the most convincing evidence in favor of a role for calcium in regulating cell cycle progression has come from experiments on eggs and oocytes. This is in part because these cells are large and easy to manipulate for calcium measurement and microinjection; it is also because these cells are dedicated to cell division to the exclusion of all else. In this they differ from somatic cells that can enter and leave the cell division cycle under the control of growth factors. Calcium is also involved in regulating the growth factor-induced transition from quiescence to proliferation in somatic cells. This is an important area of research that we shall not deal with here. Nonetheless, calcium is probably involved in what could be called intrinsic cell cycle regulation in somatic cells, too, and this we shall discuss. CALCIUM AS A CELL CYCLE REGULATOR The idea that the cell division cycle can be defined by the stages G l, S, G2, and M has and continues to be a very useful way of describing the process of cell division. The idea retains its utility because it has been found that the four stages can be described in biochemical and molecular terms. The simplest (but still surprisingly effective) description of the cell cycle is in terms of a handful of cell cycle kinases (cdks) that are activated during S phase and M phase by association with cyclin subunits. Activating one set of cyclin/cdks causes S-phase to begin and continue. 299

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Activating a second set causes mitosis. Control of the cell division cycle is loosely analogous to other kinase cascades that control various functions in noncycling cells (3). What turns kinases on and off?. In noncycling cells the answer is cell messengers. They are generated through the interaction of ligands with receptors at the cell membranes. There are two schools of thought when it comes to cell cycle kinases. What we might call the protein-only school thinks in terms of protein-protein regulation (4,5). The other school of thought looks to the analogy with kinase control of other cellular functions and suggests that cell messengers may contribute to cell cycle regulation. The two schools also divide along the lines of method. The idea that cell messengers may be important during the cell cycle comes from cell biologists and cell physiologists who observe and manipulate living cells in the microscope, while the protein-only school comprises largely those who use genetics and molecular biology. The division is hardly surprising, since a genetic approach provides the clearest information about proteins with a single function and often provides confusing information about cell signaling genes. Nonetheless, a genetic approach has provided evidence of an essential function for the calcium regulatory protein calmodulin in cell cycle regulation (6). Quite recently, intragenic complementation analysis in yeast has distinguished between calmodulin mutations that block the cell cycle and those that block other cellular processes (7). Protein kinase cascades are regulated by cAMP (3), cGMP (8), and calcium (9); and each of these cell messengers have been suggested to contribute to regulation of the cell division cycle (1,10-13). While there is no reason to dismiss the idea that the cyclic nucleotides may regulate the cell cycle, only for calcium is there a large body of evidence that can be weighed for and against. There are three active areas of research. The first comprises the calcium signal and how the calcium signal is generated. The second consists of events downstream of the calcium signal involving calcium's target, calmodulin, and calmodulin-activated kinase. The third area of research looks at proteins that have already been identified as components of the cell cycle kinase cascade, to see whether they are indirectly calcium-regulated.

CELL CYCLE CALCIUM TRANSIENTS

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The Phosphoinositide Messenger System and Cell Cycle [Ca2+]i Transients

[Ca2 + ]i can increase as a result of influx of calcium across the plasma membrane or as a result of release from internal stores. The internal calcium stores are located in the endoplasmic reticulum (ER), and the ER is located around the cell nucleus just prior to and during mitosis (14), so it is likely that calcium release is the important element in a cell cycle calcium signal. If release from internal calcium stores is important for cell cycle regulation, then we should expect activation of the calciumrek asing phosphoinositide messenger system at key stages during the cell cycle.

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The clearest example of [Ca2 + ]i regulation of a cell cycle checkpoint is at fertilization, where a calcium signal is the necessary and sufficient signal for resumption of the cell cycle, after cell cycle arrest at any of the start, mitosis entry, or mitosis exit checkpoints (2). Here, it is evident that the phosphoinositide messenger 1,4,5 inositol trisphosphate (InsP3) can release calcium and generate the signal that causes the cell division cycle to resume (15-19). The phosphoinositide messenger system has been shown to be stimulated at fertilization in sea urchin eggs (20-23) and in Xenopus oocytes (24,25). It is the [Ca2 +]i transients associated with meiosis during oocyte maturation before fertilization and with mitosis in the early embryonic cell cycles after fertilization whose relevance to cell cycle regulation is less certain (2). The recent discoveries of changes in phosphoinositide messengers coincident with these [Ca2+] i transients reinforces the idea that these transients, though small, are significant. Phosphoinositide lipid turnover increases at the time of first meiosis during maturation (26,27) and at first mitotic cleavage (25) in Xenopus oocytes. InsP3 increases not only at fertilization, but also at germinal vesicle breakdown (GVBD) (27) and at first cleavage in Xenopus oocytes (25). First cleavage in Xenopus is blocked by microinjection of an antiphosphoinositide antibody or by heparin, an InsP3 receptor antagonist (28), as it is by calcium chelators (29,37). In the first two cell division cycles of the sea urchin embryo, there are peaks of InsP3 production (31) that correspond to cell cycle stages where [Ca2+ ]i transients had previously been measured (32). Heparin, the InsP3 antagonist, blocks both the [Ca2+]i transients and entry into mitosis (31). These results suggest that an InsP3-mediated release of calcium from intracellular stores is essential for progression through mitosis (see Fig. 1). Perhaps the most interesting aspect of the activation of the phosphoinositide messenger system in concert with the cell division cycle is the discovery of activation of the phosphoinositide messenger system in the absence of an external agonist. Until now, the production of cell messengers has been thought to be tightly linked to the stimulation of transmembrane receptors at the plasma membrane. The cell cycle InsP3 peaks are a new sort of intracellular signal, generated endogenously by an intracellular mechanism (31). An obvious possibility (and the one that would immediately suggest itself to the protein-only school) might be that InsP3 production is under the control of the mitotic kinase. This is not the case. Preventing activation of the mitotic kinase by blocking the synthesis of its cyclin subunit does not block the InsP3 spikes, nor the cell-cycle synchronized variations in phosphoinositide lipid (31). So the [Ca2 +]i signal is independent of the mitotic kinase; indeed, the evidence points toward [Ca2+]i having a role in activating the kinase. There are no indications of how the InsP3 signal may be generated, but it seems possible that phosphoinositidase C~ (47) is involved. There may be a similar sort of endogenous phosphoinositide messenger production in mammalian cell lines that is linked to cell cycle control. Inhibition of phosphoinositide lipid synthesis causes cells to arrest in G1 just prior to S phase, at a point at which increased turnover of phosphoinositide lipids usually occurs (48). An effect like this in late G1 may be due to the start cell cycle control point. Of

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course, there are well-studied changes in phosphoinositide messengers when growth factors are added to mammalian cell lines, but this is a distinct control mechanism, since it involves the transition from quiescence to proliferation, rather than the regulated passage through the cell cycle restriction points that is a mark of intrinsic cell cycle control (2).

Measuring Cell Cycle Calcium Signals The discovery of the production of InsP3 at key points during the cell cycle has strengthened the idea that [Ca2 +]i is an important element of cell cycle control and counteracts the main defect in the evidence: the frequent lack of detectable [Ca 2+ ]i

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transients during mitosis (2). The defect is also being tested by measuring calcium in different ways. New dextran-conjugated calcium indicator dyes measure [Ca2+ ]i more faithfully because they remain in the cytoplasm and are not taken up into cellular organelles. The frequency of detection of mitotic calcium signals in early sea urchin embryos is much higher when using these dyes (31). Dextran-conjugated dyes reveal three separate [Ca2 +]i peaks during mitosis: the first just before nuclear envelope breakdown (NEB), the second preceding anaphase onset, and the third during cleavage (31). The calcium photoprotein aequorin has also been used to measure a mitotic transient in sea urchin embryos (39), Xenopus embryos (41) and elevated [Ca2 + ]i in the cleavage furrow of the medaka egg (40). Even with modified detection methods, the mitotic [Ca2+]i transients measured in whole single one- or two-cell sea urchin embryos are small, at around 100 nM on average (31). One reason for this may be that the transients are confined to the immediate vicinity of the nucleus (2). Confocal calcium imaging microscopy offers some support for this view, in that very local [Ca2§ increases can be measured near and in the mitotic apparatus during mitosis; the local increases are themselves large, but represent only a small fraction of the total cytoplasmic volume (Wilding and Whitaker, unpublished data). The local changes in [Ca2+ ]i would be small or unnoticed when looking at a signal from the whole embryo. Perhaps the most useful recent study is a comparison of calcium regulation of meiosis and mitosis in mouse oocytes (34), useful because meiosis and mitosis were compared in the same series of experiments by the same investigators and useful also because the findings in this one species seems entirely representative of work by others on meiosis and mitosis across species. As oocytes progress through maturation, they undergo two meiotic divisions and are fertilized. The zygote then begins mitotic cell cycles. Germinal vesicle breakdown of the first meiotic division is not associated with any obvious calcium transient, nor is it blocked by the permeant calcium chelator, BAPTA-AM. In addition, GVBD is independent of extracellular calcium, though the later events of first meiosis are disrupted if external calcium is removed. Second meiotic metaphase in mouse eggs is controlled by the large [Ca2 + ]i transients that accompany fertilization. It is blocked by the calcium chelator B APTA and is not dependent on external calcium. Nuclear envelope breakdown at first mitosis is usually, though not invariably, accompanied by [Ca2+ ]i increases, is blocked by B APTA, and does not depend on external calcium. The pattern in mouse oocytes seems to be a general one (see Fig. 1). Meiotic GVBD is not calcium-dependent in starfish oocytes, either (49), and although others have recorded repetitive [Ca2 +]i transients in maturing mouse oocytes, the transients can be blocked by calcium chelators without preventing GVBD (42). There is one report, though, of BAPTA blocking GVBD in pig oocytes (50). In contrast, the fertilization [Ca2+]i transient is large and is clearly responsible for triggering meiotic progression. This, too, is a general pattern, seen in frog, starfish, and sea urchin (2). The third finding, that mitotic [Ca2+]i transients depend upon internal stores but are intermittently detected, also applies in sea urchin embryos, as does the observation that mitosis (NEB) is blocked by the calcium chelator BAPTA

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(2). We can sum this up by saying that no one would argue over the importance of calcium as a cell cycle signal at fertilization, that the importance of calcium in embryonic mitosis is demonstrated but not beyond debate, and that the evidence for calcium as a cell cycle signal at GVBD is poor at best. CALMODULIN AND CALMODULIN-REGULATED KINASES If [Ca 2 +]i signals are to regulate cell cycle progression, then calcium-binding proteins must also be involved. The most likely candidate is calmodulin (51). There are two ways of trying to find out whether calmodulin is involved in cell cycle control. The first is to alter the expression of the calmodulin gene by overexpression, mutation, or deletion. Manipulating calmodulin expression can cause arrest at specific stages of the cell cycle in yeast, fungi, and in mammalian cells (52). It has to be conceded, though, that calmodulin plays a number of different roles inside the cell, so a simple genetic approach cannot always separate a direct effect on cell cycle control with more indirect effects caused by a more general loss of cell function. In yeast, it has been possible to construct calmodulin mutants by site-directed mutagenesis and to identify 4 complementation groups for 14 such mutants (7). One of the four complementation groups defines a nuclear division defect, implying a possible role in cell cycle control. The more important implication of this study is, however, to illustrate how site-directed mutagenesis can provide a way of mapping the different interactions of a promiscuous protein such as calmodulin (53) that may be useful in more clearly defining its role in cell cycle control. The other way of finding out how calmodulin fits into the picture is to use calmodulin inhibitors. Peptide inhibitors based on a myosin light-chain kinase (MLCK) sequence are potent and specific. The peptide inhibitors have to be microinjected into cells: this is perhaps why results have been reported in eggs and oocytes. The MLCK peptide prevents resumption of the cell cycle after fertilization of Xenopus oocytes (54). Its effects are overcome by adding excess calmodulin or by unregulated calmodulin-activated kinase (55). We have recently found that a similar MLCK peptide prevents NEB in sea urchin embryos (Wilding, T&6k and Whitaker, unpublished data). These data support the idea that calmodulin may regulate mitosis in Xenopus and in sea urchin embryos. Microinjecting peptide inhibitors into mammalian somatic cells is less easy, and different approaches have been used. A nonpeptide calmodulin inhibitor, KN-93, arrests cycling HeLa cells at the G2/M border when added to synchronized cultures during S phase (Patel, Philipova, Hidaka, and Whitaker, unpublished). However, KN-93 blocks unsynchronized cultures in GI (C. Rasmussen, unpublished). The former result is consistent with the observations in sea urchin embryos and with the findings that calcium chelators block the G2/M transition in Swiss 3T3 cells, while artificial [Ca2 + ]i transients can trigger entry in mitosis (56). The latter result implies that calmodulin may also be important at the G1/S boundary. Fertilization in sea urchin eggs represents a cell cycle calcium signal in G 1 (see Fig. 1), and a [Ca2+]i signal and InsP3 peak have also been detected precisely at the G~/S boundary in sea urchin embryos (31,32). Calcium signals have also been detected during the G1/S transi-

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tion in C127 cells (51). In this cell type, transient overexpression of calmodulin accelerates cells past both GI/S and GE/M boundaries, while calmodulin antisense RNA arrested cells in G1, G2, or mitotic metaphase (57). All this suggests that calcium/calmodulin may control cell cycle transitions at G1/S, GE/M, and anaphase in mammalian somatic cells just as in eggs and oocytes (see Fig. 1). Reinforcement of the idea of a calmodulin-dependent G1/S transition comes from experiments on the fungus, A. nidulans. Using conditional calmodulin mutants, it has been shown that loss of calmodulin functions leads to arrest in G2 (80 percent) or in G~/S (20 percent) (52). There is a clear pattern: the restriction points at which calmodulin function is necessary for cell cycle progression coincide with the points at which [Ca2+]i transients have been observed. The pattern seems to apply in mammalian somatic cells as well as in eggs and oocytes. Calmodulin itself regulates a variety of enzymes, but one of its major targets is a protein kinase, CaMK II (9). There is a specific peptide inhibitor of CaMK II that has been shown to delay or block NEB in sea urchin embryos (58,59). The most impressive demonstration that CaMK II regulates mitosis is a series of experiments in Xenopus oocytes at fertilization in which the above peptide inhibitors were used but which, in addition, showed that an unregulated form of the CaMK II was able to bypass the [Ca2 +]i signal and stimulate exit from mitosis directly (19). When expressed constitutively in mammalian cells, on the other hand, the unregulated CaMK II caused arrest in G2. This is a paradoxical result, since the hypothesis is that CaMK II activation triggers mitosis (Fig. 2). However, the mitotic kinase is indeed active in these cells (60), as the hypothesis predicts, so the inhibition of mitosis itself must be due to a secondary CaMK II-induced phosphorylation (51). The CaMK II would normally be only transiently activated (9). Note that experiments where [Ca2 +]i transients are measured (31), where calcium chelators block cell cycle progression (2), and where calmodulin and CaMK II inhibitory peptides have been used all point to calcium-calmodulin-CaMK II regulation at two points during mitosismat NEB and again at anaphase onset. To some it may seem odd that the same signaling pathway should control two dissimilar cell cycle events that follow one another in close succession. It is thus worth mentioning that NEB is accompanied by large structural changes in the nucleus, not least the formation of the mitotic spindle. It has been known for some time that calmodulin is found predominantly at the spindle poles, and it is reasonable to assume that the target specificity of the kinase is governed at least in part by colocalization of kinase and target. Nonetheless, the idea will be the more convincing once CaMK II targets are identified and linked to known cell cycle control proteins. What might these targets be?

TARGETS OF CaMK II AND LINKS WITH KNOWN CELL CYCLE CONTROL PROTEINS In Aspergillus, mitosis is controlled both by the conventional mitotic kinase (cdc2/cyclin) and by a second essential kinase NIMA (61). NIMA may be an equiv-

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alent of the MAP kinase that is activated during meiosis (62) and mitosis (Philipova and Whitaker, unpublished). NIMA is a substrate for CaMK II in vitro (61), so one effect of the premitotic calcium signal may be to activate this kinase. Another target of CaMK II, at least in vitro, is the cell cycle control protein cdc25. Cdc25 is a phosphatase that activates the conventional mitotic kinase (63). After phosphorylation by CaMK II, cdc25 phosphatase activity increases (Patel and Whitaker, un-

R E G U L A T I O N OF THE CELL DIVISION CYCLE

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published). The Aspergillus homologue of cdc25, NIMT, is also a substrate for the CaMK II in vitro and loss of calmodulin in a conditional mutant blocks tyrosine dephosphorylation of p34 edr (51). These observations suggest a control pathway in which [Ca2+]i activates the mitotic kinase by phosphorylation and activation of cdc25 (see Fig. 2). Consistent with this idea is our unpublished observation that mammalian cell lines arrested in G2 by treatment with the calmodulin inhibitor KN-93 lack the phosphorylated cdc25 seen in control cells at this stage of the cell cycle. These data suggest that it may be possible to trace a kinase cascade pathway from the [Ca 2+ ]i signal to NEB and the initiation of mitosis. There is also the suggestion of a link between CaMK II and the cell cycle kinase in the control of anaphase onset and exit from mitosis. The important consequence of the fertilization [Ca 2 +]i transient in Xenopus is the destruction of the cyclin subunit of the mitotic kinase (19). How might this come about? Not, it seems (64) from the destruction of mos, the cytostatic factor, as was originally suggested (65), since the calpain-mediated destruction of mos occurs only after cyclin itself is destroyed (54). Cyclin destruction in these circumstances has been shown very convincingly to be a consequence of activation of the CaMK II. What might the target be? Possibly, it is the proteasome (64) (see Fig. 2), a large proteolytic complex that will destroy ubiquitinated proteins and that is activated at the time of mitosis (66). CONCLUSIONS Upstream of the cell cycle [Ca 2+ ]i transients, the necessary changes in phosphoinositide messengers have been detected. Downstream of the [Ca 2+]i transients, protein targets have begun to be identified. The [Ca 2 +]; transients themselves are being localized to the nucleus. Evidence slowly continues to accumulate that [C a2+ ]i signals may control or modulate cell cycle events.

zyxwvuts ACKNOWLEDGMENTS

Work in the lab is supported by grants from the Royal Society and the Wellcome Trust.

REFERENCES

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8. Murad F, Waldman S, Molina C, Bennet B, Leitman D. Regulation and role of guanylate cyclasecyclic GMP in vascular relaxation. Prog Clin Biol Res 1987;249:65-76. 9. Schulman H. The multifunctional Ca 2+/calmodulin-dependent kinases. Curr Opin Cell Biol 1993;5: 247-253. 10. Ishida K, Yasumasu Y. The periodic changes in cyclic adenosine 3',5'-monophosphate concentration in sea urchin eggs. Biochim Biophys Acta 1982;720:266-273. 11. Shard MA, Rooney DW. Rhythmic cyclic AMP changes in Chlamydomonas cells synchronized by temperature and light cycles in chemostat culture. Cell Biol Int Rep 1985;9:561-567. 12. Kupetz IS, Jeter JR Jr. Cell-cycle-specific activity of cyclic nucleotide phosphodiesterase activity in Physarum polycephalum. Cell Tissue Kinet 1985; 18:159-168. 13. Appel RG. Growth inhibitory activity of atrial natfiuretic factor in rat glomerular mesangial cells. FEBS Lett 1988;238:135-138. 14. Jaffe LA, Terasaki M. Structural changes of the endoplasmic reticulum of sea urchin eggs during fertilization. D ev B iol 1993;156:566-573. 15. Galione A, McDougall A, Busa WB, Willmott N, Gillot I, Whitaker MJ. Redundant mechanisms of calcium-induced calcium release underlie calcium waves during fertilization. Science 1993;261: 348-352. 16. Nuccitelli R, Yim DL, Smart T. The sperm-induced Ca 2§ wave following fertilization of the Xenopus egg requires the production of Ins(1,4,5)P3. Dev Biol 1993;158:200-212. 17. Mi),azaki S, Yuzaki M, Nakada K, Shirakawa H, Nakanishi S, Nakade S, Mikoshiba K. Block of Ca2§ wave and Ca2§ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs. Science 1992;257:251-255. 18. Whitaker MJ, Swann K. Lighting the fuse at fertilization. Development 1993;108:525-542. 19. Lorca T, Cruzalegui FH, Fesquet D, Cavadore J-C, M6ry J, Means AR, Dor6e M. Calmodulindependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs. Nature 1993;366:270-273. 20. Turner PR, Sheetz MP, Jaffe LA. Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature 1984;310:414-415. 21. Kamel LC, Bailey J, Schoenbaum L, Kinsey W. Phosphatidylinositol metabolism during fertilization in the sea urchin egg. Lipids 1985;20:350-356. 22. Ciapa B, Whitaker MJ. Two phases of inositol polyphosphate and diacylglycerol production at fertilization. FEB S Lett 1986;195:347-351. 23. Ciapa B, Borg B, Whitaker MJ. Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs. Development 1992;115:187-195. 24. Larabell C, Nuccitelli R. Inositol lipid hydrolysis contributes to the Ca2§ wave in the activating egg of Xenopus laevis. Dev Biol 1992;153:347-355. 25. Stith BJ, Goalstone M, Silva S, Jaynes C. Inositol 1,4,5-trisphosphate mass changes from fertilization through first cleavage in Xenopus laevis. Mol Biol Cell 1993;4:435-443. 26. Carrasco D, Allende CC, Allende JE. The incorporation of myo-inositol into phosphatidylinositol derivatives is stimulated during hormone-induced meiotic maturation. Exp Cell Res 1990;919:313318. 27. Stith BJ, Jaynes C, Goalstone M, Silva S. Insulin and progesterone increase 32po4-1abelling of phospholipids and inositol 1,4,5-trisphosphate mass in Xenopus oocytes. Cell Calcium 1992;13: 341-352. 28. Han JK, Fukami K, Nuccitelli R. Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability or Ca2§ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres. J Cell Biol 1992;116:147-156. 29. Snow P, Nuccitelli R. Calcium buffer injections delay cleavage in Xenopus laevis blastomeres. J Cell Biol 1993;122:387-394. 30. Miller AL, Fluck RA, McLaughlin JA, Jaffe LF. Calcium buffer injections inhibit cytokinesis in Xenopus eggs. J Cell Sci 1993;106:523-534. 31. Ciapa B, Pesando D, Wilding M, Whitaker MJ. Cell-cycle calcium transients driven by cyclic changes in inositol trisphosphate levels. Nature 1994;368:875-878. 32. Poenie M, Alderton J, Tsien RY, Steinhardt RA. Changes in free calcium with stages of the cell division cycle. Nature 1985;315:147-149. 33. Lee H-C, Aarhus R, Walseth TF. Calcium mobilization by dual receptors during fertilization of the sea urchin egg. Science 1993;261:352-355. 34. Tombes RM, Simerly C, Borisy GG, Schatten G. Meiosis, egg activation and nuclear envelope breakdown are differentially reliant on Ca 2+, while germinal vesicle breakdown is calcium independent. J Cell Biol 1992;177:799-811.

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35. Busa WB, Nuccitelli R. An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog Xenopus laevis. J Cell Biol 1985;100:1325-1329. 36. Igusa Y, Miyazaki S. Effects of altered extracelluar and intracellular calcium concentration on hyperpolarizing responses of hamster egg. J Physiol (Lond) 1983;340:611-632. 37. Steinhardt R, Zucker R. Schatten G. Intracellular calcium release at fertilization in the sea urchin egg. Dev Biol 1977;58:185-196. 38. Swarm K, Whitaker MJ. The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs. J Cell Biol 1986;103:2333-2342. 39. Browne CI, Miller AL, Palazzo RE, Jaffe LF. On the calcium pulse during nuclear envelope breakdown (NEB) in sea urchin eggs. Biol Bull (reports of general scientific meetings) 1992;370-371. 40. Fluck RA, Miller AL, Jaffe LF. Slow calcium waves accompany cytokinesis in medaka fish eggs. J Cell Biol 1991;115:1259-1265. 41. Kubota HY, Yoshimoto Y, Hiramoto Y. Oscillation of intracellular free calcium in cleaving and cleavage-arrested embryos of Xenopus laevis. Dev B iol 1993;160:512-518. 42. Carroll J, Swarm K. Spontaneous calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes. J Biol Chem 1992;267:11196-11201. 43. Kline D. Calcium-dependent events at fertilization in the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis and formation of pronuclei. Dev Biol 1988;126:346361. 44. Swarm K, McCulloh DH, McDougall A, Chambers EL, Whitaker MJ. Sperm-induced currents at fertilization in sea urchin eggs injected with EGTA and neomycin. Dev Biol 1992;151:552-563. 45. Steinhardt RA, Alderton J. Intracellular free calcium rise triggers unclear envelope breakdown in the sea urchin embryo. Nature 1988;332:364-366. 46. Twigg J, Patel R, Whitaker MJ. Translational control of InsP3-induced chromatin condensation during the early cell cycles of sea urchin embryos. Nature 1988;332:366-369. 47. Dennis EA, Rhee SG, Billah MM, Harmun YA. Role of phospholipase in generating second messengers in signal transduction. FASEB J 1991;5:2068-2077. 48. Imoto M, Morii T, Umezawa K. Involvement of phosphatidylinositol synthesis in the regulation of S-phase induction. Exp Cell Res 1994;(in press). 49. Witchell HJ, Steinhardt RA. 1-methyladenine can consistently-induce a fura-detectable transient calcium increase which is neither necessary or sufficient for maturation in oocytes of the starfish Asterina miniata. Dev Biol 1990;141:393-398. 50. Kaufman ML, Homa ST. Defining a role for calcium in the resumption and progression of meiosis in pig oocytes. J Exp Zool 1993;265:69-78. 51. Lu KP, Means AR. Regulation of the cell cycle by calcium and calmodulin. Endocr Rev 1993;14: 40-48. 52. Rasmussen CD, Lu KP, Means RL, Means AR. Calmodulin and cell cycle control. J Physiol (Paris) 1992;86:83-88. 53. T6r6k K, Whitaker MJ. Taking a long cold look at calmodulin's warm embrace. Bioessays 1994;16: 221-224. 54. Lorca T, Galas S, Fesquet D, Devault A, Cavadore J-C, Dor6e M. Degradation of the protooncogene product p39''~ is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a calcium-calmodulin-dependent event. EMBO J 1992;10:2087-2093. 55. Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, M6ry J, Means A, Dor6e M. Calmodulindependent protein kinase II mediates in activation of MPF and CSF upon fertilization of Xenopus eggs. Nature 1993;366:270-273. 56. Kao JP, Alderton JM, Tsien RY, Steinhardt RA. Active involvement of Ca2§ in mitotic progression of Swiss 3T3 fibroblasts. J Cell Biol 1990; 111:183-196. 57. Rasmussen CD, Means AR. Calmodulin is required for cell cycle progression during G~ and mitosis. EMBO J 1989;8:73-82. 58. Baitinger C, Alderton J, Schulman H, Steinhardt RA. Multifunctional Ca/CaM-dependent protein kinase is necessary for nuclear envelope breakdown. J Cell Biol 1990;111:1763-1773. 59. Patel R, Whitaker MJ. Okadaic acid suppresses calcium regulation of mitosis onset in sea urchin embryos. Cell Regul 1991;2:391-402. 60. Planas-Silva MD, Means AR. Expression of a constitutive form of Ca2+/calmodulin-dependent protein kinase II leads to G2 arrest. EMBO J 1992;11:507-517. 61. Lu KP, Osmani SA, Means AR. Properties and regulation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans. J Biol Chem 1993;268:8769-8776. 62. Shibuya EK, Boulton TG, Cobb MH, Ruderman JV. Activiation of p42 MAP kinase and release of oocytes from cell cycle arrest. EMBO J 1992;11:3963-3975.

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63. Hoffman I, Clarke PR, Jesus-Marcote M, Karsenti E, Draetta G. Phosphorylation/amplification of human cdc25 by self amplification of MPF at mitosis. EMBO J 1993;12:53-63. 64. Whitaker MJ. Cell cyclemSharper than a needle. Nature 1993;366:211-212. 65. Watanabe N, Vande Woude GF, Ikawa Y, Sagata N. Specific proteolysis of the c-mos protooncogene product by calpain on fertilization of Xenopus eggs. Nature 1989;342:505-511. 66. Kawahara H, Sawada H, Yokosawa H. The 26S proteasome is activated at two points in the ascidian cell cycle. FEBS Lett 1992;310:119-122.

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zyx zyx Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1995.

11

The Regulation of Calcium in Paramecium Robert D. Hinrichsen, Dean Fraga, and Chris Russell Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104

Cells are able to respond to a large variety of extracellular signals. In many instances the stimulus leads to a transient increase in free intracellular [Ca2+] (1) which is utilized as a second messenger in the regulation of numerous cellular functions (2). The ciliated protozoa, such as Paramecium, have several membraneassociated processes that are regulated by calcium, including the modulation of ion channels, chemotaxis, ciliary beating, mating, and exocytosis. These unicellular organisms are ideal model systems for the study of calcium regulation at the cellular level. The primary feature of the ciliates that makes them such attractive candidates is the ease of genetic analyses and the ability to maintain viable mutants that are defective in various calcium-regulated processes. The cells are amenable to mutagenesis with several strategies to express recessive mutations in a simple and direct manner (3). By using the powerful combination of genetics and biochemistry that are the hallmark of ciliate biology, much has been accomplished in defining the role of calmodulin in the mediation of various calcium signals. This work led to the first demonstration that the two lobes of the calmodulin molecule are involved in regulating different functions in the cell (see below); this observation has been recently confirmed in yeast (4). In addition, experiments with Paramecium have led to the discovery of novel calcium-regulated proteins such as the Ca 2 +-dependent Na + channels and ciliary guanylate cyclase. The combination of several calcium-regulated functions that are easily observable and the ability to genetically dissect these processes has gained the ciliated protozoa a significant position in the field of calcium regulation. This chapter will summarize the work done with Paramecium tetraurelia, with an emphasis on the genetic and biochemical characterization of calcium-dependent cellular functions.

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General Description of the Cell

Paramecium tetraurelia is a ciliated protozoan that has been investigated for over 100 years (5). The cells are approximately 120-150 Ixm in length (Fig. 1), which is large enough to allow for the easy manipulation of the cells, as well as intracellular electrophysiology and microinjection experiments. The cells are covered by 5,0006,000 cilia that are used to propel them through a liquid environment. The cilia beat in metachronal waves to produce a swimming motion, as well as to sweep food particles (primarily bacteria) into the oral apparatus. As with all ciliated protozoa, Paramecium possesses two distinct types of nuclei; two diploid micronuclei that are transcriptionally inactive and involved in the sexual cycle, and a single, highly polyploid macronucleus that is transcriptionally active and responsible for the somatic functions of the cells. The macronucleus develops from a micronuclear precursor and contains up to 1,O00 copies of the genetic material. A form of self-fertilization called autogamy causes the cell to become homozygous at all loci, and this characteristic has been utilized for the convenient identification of recessive mutations. Classical genetic analyses are well established and the genetic characterization of mutants is a routine procedure. The ease of growth of Paramecium and other protozoan ciliates has allowed for the convenient biochemical characterization of many proteins. The fact that they are unicellular eliminates the problem of contamination from numerous cell types that is

FIG. 1. A scanning electron micrograph of Paramecium tetraurelia. The cell is approximately 120 i~m in length. The 5,000-6,000 cilia that cover the surface, and beat in metachronal waves, propel the cells forward or backward. From Hinrichsen and Schultz (47).

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often a problem with multicellular organisms. The cells can be easily fractionated into the various cellular fractions, such as the cilia, cortical membranes, cytoplasm, and nuclei. This enables the investigator to rapidly identify the location of a particular protein.

How Paramecium Handles Calcium

There are at least three major organellar systems that are regulated by calcium in Paramecium: the cilia, the secretory organelles called trichocysts, and several cytoskeletal networks that lie underneath the cell surface (6). In each case the cell must be able to control the influx of calcium to the vicinity of the organelle, as well as to insure a means to eliminate excess calcium. The general processes by which this is thought to occur in Paramecium will be briefly discussed below.

Calcium Channels---Influx The voltage-dependent Ca 2 + channels of the ciliary membrane are the prime method by which calcium enters the cilium of Paramecium and causes the subsequent calcium-dependent behavioral responses. These channels are located primarily in the ciliary membrane and provide the intracellular calcium required for the change in the direction of the ciliary beat (7). There are also calcium channels in the cell body that are opened by mechanical stimulation (8). These channels are primarily located in the anterior end of the cell and result in backward swimming when the cell encounters an object. The voltage-gated channels open upon depolarization of the cells. The resulting influx increases the intracellular [Ca 2 +] from 10 -8 M to 10 -6 M. The channels are closed by a combination of two events. First, the calcium channels in Paramecium tetraurelia have been shown to be inactivated by the calcium that passes through the channels (9). Therefore, prolonged influx of calcium is prevented by the very calcium that enters the cell. Several behavioral mutants have been uncovered that affect this calcium inactivation process (10,11). Second, the channels are inactivated by prolonged depolarization of the cells (12). In other words, if the cells are strongly stimulated over an extended period of time, the channels will inactivate for several minutes before being able to open again. Therefore, the calcium channels of the ciliary membrane cannot be opened continuously, due to a combination of the calcium that is present and the depolarization that ensues when calcium enters the cells. This will be discussed in greater detail in a later section.

Cortical Alveoli--Storage The plasma membrane of Paramecium tetraurelia has a large network of membrane vesicles designated cortical alveoli that reside directly below the plasma

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membrane (13). These vesicles are uninterrupted except in the vicinity of the cilia and trichocysts. It has been suggested that they may be calcium-storage compartments, similar to the muscle sarcoplasmic reticulum (14). This would appear to be reasonable in light of the fact that the cortical alveoli are located near the three primary organelles controlled by calcium, the cilia, trichocysts, and subcortical cytoskeleton. Recent evidence has been presented to indicate this is indeed the function of the alveoli (6). The vesicles were shown to be storage receptacles for large amounts of calcium; also, the membranes of these vesicles have a Ca 2 +-ATPase activity that is regulated by ATP and Mg 2+ . It may be that the alveoli accumulate excess calcium and release it upon demand. However, there has been no evidence to date as to the mechanism by which the calcium is released from the cortical alveoli or for which specific cellular functions it is designated.

Caz+ Pumps--Reequilibrium Several calcium pumps have been identified in the ciliary membrane of Paramecium tetraurelia (15), and these Ca 2 +-ATPase proteins will be described in more detail later. These pumps are presently believed to be the means by which the calcium is removed from the cilium after the calcium channels are opened. In line with this belief, a class of mutants that have difficulty in the extrusion of calcium during an action potential, designated K-shy, may be calcium pump mutants (16,17). Calcium pumps may also be associated with the trichocysts for the removal of calcium which enters during the discharge of the trichocysts (18). Finally, a calcium pump has been identified in the plasma membrane that may be involved in the maintenance of a proper [Ca 2+ ] within the cell (19). In each case, a defined set of calcium pumps appear to be physically associated with an area of calcium influx. It is not known if these are separate pumps or the distribution of the same protein to different areas.

zyxwvuts Calcium-Binding Proteins---Integration

Calmodulin is a well-characterized calcium-binding protein that is ubiquitous throughout the various kingdoms (20). There has been a great deal of research done on the calmodulin from Paramecium, since calmodulin appears to be a major calcium target in the cell. As will be described in later sections, the calmodulin from Paramecium has been shown to be directly involved in a number of cellular processes, including the regulation of ion channels and exocytosis. Besides calmodulin, a number of other calcium-binding proteins have been identified in Paramecium in the cilia (21), cytoskeleton (22), and the cytoplasm (M. Pollack and R. Hinrichsen, unpublished data). While the identity or function of most of these calcium-binding proteins are not known, it is assumed that they are involved in the regulation of various cellular functions. Some of these identified proteins will be discussed in a later section. A great deal of work is required before the roles for these proteins are elucidated.

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GENETICS AND CHARACTERISTICS OF CALCIUM-DEPENDENT PROCESSES IN PARAMECIUM TETRA URELIA

A number of distinct calcium-dependent cellular processes have been identified in Paramecium tetraurelia. There are genetic mutants identified for most of these processes. In this section we discuss the evidence for the involvement of calcium in these various cellular functions and the mutants that are known to affect them. The various mutants are summarized in Table 1. 9

Cell Motility The backward swimming response of Paramecium is directly controlled by the concentration of calcium in the cilia. When Paramecium is stimulated by ions, organic agents, heat, or touch, there is a transient depolarization of the cell membrane. This results in the initiation of a calcium-based action potential regulated by five different ion channels which have been well-described electrophysiologically. Stimulation initiates a transient depolarization that results in the opening of voltagedependent calcium channels; the influx of calcium acts to reverse the cell's ciliary beating and induces backward swimming. The period of time that the cell swims backward is proportional to the duration of the action potential. There are two aspects of this behavioral response which involve calcium. First, the direct action of the calcium upon the ciliary apparatus that affects both the beat duration and frequency and its direction (23-25). Second, calcium is involved in the regulation of the action potential through its activation and inactivation of the ion channels that are involved in its generation (26). These two aspects of the calcium regulation of cell motility will be described separately in the following sections.

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The Control of Direction and Frequency in Ciliary Beating As mentioned previously, the cilia are used to propel the cell forward or backward depending upon the stimulation. For example, the cell must execute a coordi-

TABLE 1. Mutants of calcium-dependent processes in Paramecium tetraurelia

Process Ciliary motility Calcium channels Ca2+-dependent channels Exocytosis Calcium pumps

Phenotype

Atalanta pawn Dancer pantophobiac fast-2 TEA-insensitive pantophobiac nondischarge K-shy

Complementation groups

Ref.

atlA, B, C and D pwA, B, C and D cnrA, B and C Dn cam, pntB cam TeaA and B cam I nd12 ksyA and B

38,37 111 112 10 82 53 113,54 71 60 17

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nated ciliary reversal when it encounters an obstacle or an environment that is undesirable. Conversely, the cell may need to swim faster in order to escape a predator. Thus, two different aspects of ciliary beating must be regulated. The first is the coordination of the reversal of the ciliary beat when a decision has been made to swim backward, and the second is the regulation of the frequency of ciliary beating. Calcium has an important role in both of these properties of ciliary beating. Cilia are composed of an axoneme, an enclosing membrane, and various associated proteins (27). Defining the character and regulation of those associated proteins that modulate ciliary beating has been the focus of much work. Ciliary beating occurs through the regulated interactions among dynein ATPases, axonemal microtubules, a calcium-sensing protein(s), several ion channels, and other unidentified proteins (28). To help identify regulatory proteins and potential pathways, researchers have taken advantage of an in vitro assay for the study of Paramecium swimming behavior. In this assay, cells are gently treated with 0.01 percent Triton-X100 so as to generate holes in the cell membrane. This results in the formation of cell "ghosts" that are depleted of soluble constituents. The axonemal structures of the treated cells are intact and, most importantly, the cilia are capable of beating in the presence of a simple buffer containing 10 - 6 M, the cilia reversed their beating motion and the ghosts swam backward until the calcium concentration was lowered or the ATP exhausted. The method by which calcium reverses ciliary beating does not involve a direct interaction with the basic sliding machinery of the axoneme (30). Interestingly, it may involve some interaction with a dynein-associated Mg 2 +-dependent ATPase activity (31,32), since this activity is stimulated twofold by micromolar calcium but is unaffected by calmodulin inhibitors. The ability to add second messenger molecules to permeabilized cells has been a powerful technique in the identification of potential regulatory pathways involved in the control of the frequency of ciliary beating. Calcium again plays a role in the control of the frequency of ciliary beat in the ghosts; increases in [Ca 2+ ] cause an increase in beat frequency before the direction is altered (25). The addition of cyclic AMP (cAMP) and cyclic GMP (cGMP) also increase ciliary beat frequency at low calcium concentrations and thus increase forward swimming speed two- to threefold, with half-maximal concentrations of near 0.5 uM (33,34). It was also shown that cAMP and cGMP can act as antagonists to calcium-induced backward swimming (35). These results indicated that cAMP is an approximately 500-fold greater antagonist of calcium action than cGMP. The role of cAMP in swimming behavior is supported by in vivo experiments in which cAMP was microinjected into the cells, resulting in increased ciliary beat frequency (36). A number of mutants that affect the ciliary response to calcium have been isolated (37,38). While these mutants, called atalanta, swim forward in a normal fashion (although there is a slight alteration in swimming speed), they are unable to properly

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alter the direction of the ciliary beat upon stimulation. The cells merely spin in place, which indicates that the mutants are unable to fully reverse the direction of the ciliary power stroke. Whereas the normal backward swimming response is also defective in 0.01 percent Triton extracted cells, their electrophysiological response to stimuli is unaffected. This implies that the activity of the various ion channels is normal in the mutants. Thus, a proper calcium signal is produced but not processed effectively. This could be due to a defect in a structural component of the axoneme or in an inability to link the calcium signal with a change in axonemal motion. However, an examination of the axoneme by electron microscopy did not reveal structural defects in any of the mutants. These mutants sort into four complementation groups, suggesting that there are at least four separate components required for the proper translation of the calcium signal to generate a change in ciliary beating. These mutants should be useful for the elucidation of the control of ciliary motility and the role of calcium.

Regulation of lon Channel Activity Ionic Currents of the Behavioral Response As discussed above, the length of time that Paramecium cells swim backward is determined by the length of time that the calcium concentration is elevated. This is controlled by the generation of an action potential across the cell membrane (Fig. 2) and the action of various calcium pumps. The five ion channels that generate this action potential are the voltage-dependent Ca 2+ and K § channels and the calciumdependent Na +, K +, and Mg 2+ channels (26). In addition to these depolarizationand/or calcium-activated channels, Paramecium has a series of hyperpolarizationand/or calcium-induced currents (39,40,41). These are most likely responsible for the regulation of the resting potential in the cells.

A

j !

. . . . . . . . . .

FIG. 2. Action potentials in Paramecium wild-type and pantophobic cells. Membrane potential responses to

B

j

;> E 500 ms

two-step outward-current injections are shown for A: wild-type cells and B: the pantophobiac mutant that lacks the Ca2+-dependent K + current; these demonstrate the change seen with a behavioral mutant. Note the steady dse in the outward K + current in the pantophobiac mutant (asterisks) and the rise in the calcium current (arrows). The cells were bathed in a calcium solution containing 1 mM Ca2+, 1 mM K +, 1 mM HEPES, and 0.01 mM EDTA, pH 7.3. The potentials (Vm) are in response to 0.2-/and 1.0-nA outward currents injected for 1 sec. From Saimi et al. (114).

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When the voltage-dependent Ca 2 + channel is activated there is a temporary rise in the internal concentration of calcium that eventually results in the inactivation of the voltage-dependent calcium channels (9,42). The increasing intracellular calcium also acts to activate the calcium-dependent Na +, K +, and Mg 2+ channels (43-45). These channels become active approximately 100-500 msec after stimulation (46). The calcium-dependent Na + channel acts to keep the membrane depolarized by allowing sodium to enter the cell. The calcium-dependent K + channel acts to repolarize the membrane and thus return the cell to its resting membrane potential. The calcium-activated Mg 2+ channel was first described in Paramecium, but is still relatively poorly understood; it may modulate the action potential indirectly by modifying the K +-based recovery phase (43). In addition, it could directly activate various Mg 2+-dependent enzymes, such as the dynein-associated Mg 2 +-ATPase. This complex regulation of ion channel activity by calcium results in a tightly regulated, calcium-dependent action potential that establishes a defined length of time in which the calcium concentration in the cell is elevated. As discussed previously, this period of elevated calcium in the cell results in the same duration of ciliary reversal. Genetic Mutants of the Ionic Currents

A number of mutants have been isolated that affect swimming behavior by means other than the axonemal proteins of the cilia discussed above (47). Electrophysiological analyses have shown that these mutants affect the various ion currents described above (48). They can be divided into four general categories; (a) mutants that specifically affect one of the depolarization-induced currents; (b) mutants that affect only the hyperpolarization-induced currents; (c) mutants that affect both depolarization- and hyperpolarization-induced currents; and (d) mutants that have indirect effects upon the currents involved in swimming behavior. The fact that some mutants affect more than one class of channel indicates common components are utilized in the regulation of different ion channels in the same cell. The common components could be regulatory proteins, small molecules, or membrane lipid composition. Mutants in each class have some aspect of their calcium regulatory pathways affected. This underscores the central importance of calcium to the swimming behavior of Paramecium. But remarkably, when the influx of calcium is eliminated, as in the pawn mutants, the cells are viable. Thus, the researcher can study calcium regulatory pathways in an organism in which the "null" phenotype is viable. One of the first classes of mutants described was designated pawn. These mutants are unable to generate any calcium influx in response to stimuli. This eliminates the generation of action potentials, and the cells always swim forward. Nonetheless, 0.01 percent Triton-extracted cells will respond to > 1 0 - 6 M Ca 2+ by swimming backward, indicating that the ciliary apparatus is unaffected (49). Interestingly, the four complementation groups of pawn mutants can be restored to wild-type behavior by a transfer of cytoplasm (either by mating or microinjection) (50,51), implying

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that at least four regulatory components are required for proper calcium channel function. Another calcium channel mutant with the opposite phenotype of pawn is called Dancer (10,11); the calcium channels open, or activate, properly in this mutant, but they close, or inactivate, very slowly. This results in excess calcium entering the cell and a subsequent exaggerated backward swimming response. While the gene product for Dancer is not known, it could provide very useful information in the inactivation process of an ion channel. Two of the phenotypes described in Table 1, fast-2 and pantaphobiac A, were shown to map to the same locus even though they affected different ion channel activities (52); fast-2 mutants primarily have a reduced Ca 2 +-dependent Na + current, while pantophobiac mutants primarily lack the Ca 2 +-dependent K + current (53). These mutants have been found to reside within the calmodulin gene; they will be described in detail in a later section. Additionally, the TeaA mutant has an increased Ca 2 +-dependent K + current that probably occurs because of an increased calcium sensitivity of the ion channels (54). This mutation leads to the rapid inactivation of the action potential and a reduced behavioral response. Finally, a group of mutants called Paranoiac have an increased Ca 2 +-dependent Na + current that causes an exaggerated behavioral response to sodium (55,56). Two other genetic mutants have been isolated that also affect these ion channels. First, the restless mutant has an increased hyperpolarization-induced Ca 2 +-dependent K + current (40,57). This mutant loses potassium in a low-potassium solution due to the overactive state of this ion channel. This has a direct effect upon the membrane potential of the cells and ultimately upon the cell's viability. Second, the baA mutant has an alteration in most of the ionic currents in the cell (58) and an aberrant response to Ba 2+. This is the result of an altered phospholipid composition in the ciliary membrane (the sphingolipid s and phosphonolipids are reduced in amount).

zyxwvuts Exocytosis

The plasma membrane of Paramecium is lined with dense, corelike vesicles called trichocysts (18). These contain high concentrations of a variety of related proteins called trichynins, which are small (17-36 kDa), slightly acidic proteins (59). Each trichocyst is docked on the cytoplasmic side of the plasma membrane at a site adjacent to a cilium. They can be triggered to discharge their contents by a variety of means. This explosive discharge results in the rapid expansion and crystallization of the protein contents of the vesicle. Numerous mutants that affect this process have been isolated (18), resulting in a genetic dissection of the steps necessary for the proper formation and discharge of the docked trichocysts. These are summarized in Table 2. Of particular relevance to this discussion is the fact that this process is regulated by calcium, and two mutants have been described that involve calcium-dependent activities. In addition, genetic and biochemical evidence indicates that calmodulin has two potential roles in the

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TABLE 2. Steps in the discharge of trichocysts from Paramecium .,

Biosynthetic step

Ca 2 + or calmodulin involvement

Step-blocking mutants

Morphogenesis

?

Transport Attachment

? Calmodulin

Trigger Membrane Fusion Decondensation

Calmodulin

tl, tam38, ftA, ftB, ptA, pt2, stA, stB, tamA, tamG, ft33 ndA, tam8, tam 10, tam 11 nd2, nd3, nd6, nd7, nd9, nd16, nd17, nd18, cam 1, tam6 nd 12

Ca 2 + (?) Ca 2 §

none none

,,

regulation of trichocyst discharge. These mutants are discussed within the context of a general description of the process below. The exocytosis process may be broken down into two distinct steps. The first is the fusion of the docked vesicle membrane with the plasma membrane. The second is the calcium-dependent expansion of the vesicle matrix. Currently, there is some controversy over the role of calcium in membrane fusion (60,61). However, it has been elegantly demonstrated that the crystallization of the vesicle contents is calcium-dependent (62). It is possible to isolate intact trichocysts that are stable in the presence of millimolar calcium (63). Thus, the structure of the trichocyst itself is impermeable to calcium, and calcium must cross this barrier by either a passive or an active process. Trichocysts can be further fractionated such that the matrix proteins are separated from the vesicle membrane (64), which allows one to study the crystallization process through quantitative biochemical methods. It was found that, contrary to other dense-core vesicle systems, the trichocyst protein matrix is concentrated in the vesicle in the absence of calcium (62). There appears to be a large amount of orthophosphate present (approximately 300 nmol Pi/mg protein), along with the condensed protein. A method of decondensation was proposed in which the calcium (or other ions that precipitate PO4) acts to decondense the trichynins by precipitating the orthophosphate. The role of calcium in the fusion of the trichocyst vesicle with the plasma membrane has been more controversial and is complicated by an inability to fully control internal and external calcium stores. Data have been presented that argue external calcium is involved in exocytosis (60), whereas other data argue that calcium may be necessary only for decondensation of the trichocyst matrix proteins (61). The evidence for the role of calcium in the initiation of trichocyst secretion is based on several pieces of information: the lack of exocytosis in low-calcium solutions ( 100 I~M), the enzyme is inhibited. Furthermore, the guanylyl cyclase is additionally activated by the presence of calmodulin. Therefore, it would appear that there is an important correlation of calcium influx due to an action potential, the presence of calmodulin, and the activation of guanylyl cyclase. Finally, a cGMP-dependent protein kinase has been identified in the cilia of Paramecium (99), along with phosphorylated substrates, which may be the method by which the rise in cGMP is translated into action. While there is no known physiological function for the cGMP and its cGMP-dependent protein kinases, it has been shown that cGMP antagonizes calcium-regulated ciliary reversal in detergent-permeabilized cells. This may imply that cGMP is involved in regulating the direction of the ciliary beat.

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Other Calcium-Dependent Proteins Calcium-Dependent Protein Kinases

Two primary classes of calcium-activated protein kinases have been identified in higher organisms, the Ca 2+/calmodulin-dependent kinases and the Ca 2+/lipid-dependent protein kinases (protein kinase C). However, in Paramecium tetraurelia another class has been identified that is activated solely by calcium (100,101). There are two species of this enzyme, CaPK-1 and -2, that are absolutely dependent on the presence of calcium; calmodulin and lipids have no effect on their activity. A similar enzyme has xecently been cloned from soybeans, and the protein has an EF-hand motif similar to that seen with calmodulin (102). These enzymes are halfmaximally activated by 0.2 }xM free Ca2 +, they bind calcium in calcium overlay blot assays, and they use casein as a substrate (100). The function of this class of

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E

20

0-

Time(s)

331

FIG. 4. The time-dependent increase in cGMP in Paramecium tetraurelia. The increase in [cGMP] in wild-type 51s (o), the mutant pawnA (&), the double mutant pawnAIpawnB (4) and the mutant Dancer (i) after the addition of 3 mM Ba 2 +. The [cGMP] of the cells was determined by radioimmunoassay. Both wild-type and the Dancer mutant show early increases in [cGMP] after stimulation with Ba 2+, whereas the calcium channel mutants (which cannot generate action potentials) do not. From Schultz et al. (97).

protein kinase is unknown at the present time, but it is highly likely that they play an important role in the transduction of the calcium signal during the action potential.

Calcium-Dependent A TPases Ca 2+-ATPases of the plasma membrane play an essential role in the regulation of intracellular calcium levels in all cells. This is particularly the case with the excitable cells of the brain and heart, in which a cellular depolarization increases the intracellular free [Ca2+]; the Ca2+-ATPases return the levels of calcium back to that seen prior to the depolarization. The ATPases found in the heart and brain are also stimulated by calmodulin; the calmodulin binds directly to the ATPase rather than by the indirect means of stimulating a kinase. Several Ca 2+-specific ATPases have been identified in Paramecium tetraurelia, including those isolated from the ciliary membrane, trichocysts, and the cell pellicle. As mentioned earlier, a Ca2+specific ATPase from the alveoli membranes has been isolated that shows a very high affinity for calcium (Km = 0.5 la,M) and is probably involved in the sequestration of intracellular calcium (6). Another Ca 2 +-ATPase, isolated from the deciliation supernatant, has been characterized (103). This enzyme does not contain calmodulin as part of the subunit, but is inhibited by calmodulin antagonists. The Ca 2+-ATPase from the ciliary membrane has also been purified and characterized, and appears to be distinct from the enzyme found in the deciliation supernatant (15). Furthermore, a Ca 2+-ATPase from the plasma membrane has been purified and characterized (19). This enzyme appears to have many of the characteristics of the mammalian enzyme, including the inhibition by vanadate. However, calmodulin did not stimulate the enzyme as it does in many of the mammalian enzymes. The genes for these ATPases have not been isolated to date, but will provide more insight into the relationship of the Paramecium tetraurelia enzymes to those in higher organisms.

zyxwvuts Protein Kinase C

The role of protein kinase C (PKC) in signal transduction has been well studied in the last 10 years (104). Protein kinase C is activated by phospholipid metabolites

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and calcium, but appears to be a large family, and some of the PKC species show different requirements. The classical group of enzymes (cPKC), which are found in numerous cell types, require calcium and diacylglycerol (DAG), and are further enhanced by cis-unsaturated fatty acids and lysoPC. The complex interaction of these different activators results in the stimulation of PKC and the initiation of a series of different signal transduction cascades. While PKC has not been purified from Paramecium tetraurelia, cytoplasmic extracts have been recently shown to contain a PKC activity (105). The injection of the major PKC substrate, MARCKS, into Paramecium has been shown to influence the behavioral response; the application of a stimulator of PKC, the phorbol esters, influenced this reaction. These results indicate the presence of a PKC protein in the cells. Furthermore, the recent isolation of a partial clone of the gene for PKC implies the existence of this enzyme in Paramecium (D. Nelson, personal communication). Further work will be required to elucidate the functional role of PKC in Paramecium tetraurelia.

Adenylyl Cyclase There is a well-established family of adenylyl cyclases in higher organisms that is activated by calcium and calmodulin, while another group of these adenylyl cyclases is activated by G proteins. The roles of cAMP and cAMP-dependent protein kinases are an integral part of many signal transduction pathways. The recently discovered role of cAMP in the regulation of ciliary beating, using detergent-permeabilized cells, solidified the role of cAMP in the motility of Paramecium. Several cAMP-dependent protein kinases have been isolated from the cilia of Paramecium (106), which probably are utilized to transduce the cAMP signal. Adenylyl cyclases have also been isolated from Paramecium tetraurelia; while there is no evidence of activation by G proteins, the role of calcium is still controversial (107, 108). However, the recent discovery that adenylyl cyclase in the cilia of Paramecium tetraurelia also serves as an ion channel for the efflux of K + has dramatically increased the interest in this protein. The recent cloning of portions of two adenylyl cyclase genes from Paramecium (C. Russell and R. Hinrichsen, unpublished data) should assist in the elucidation of the types of adenylyl cyclases present, the role of calcium in their activation, and their ability to function as ion channels. FUTURE STUDIES The genetics of ion channel activity and calcium-dependent process regulation are continuing to produce new insights, and progress in this area can be expected to continue. As more phenotypes are associated with specific biochemical pathways, genetic selections can be more targeted toward those pathways. Hinrichsen and colleagues (86) isolated one calmodulin intragenic suppressor based on the bariumsensitive phenotype of caml (86). The isolation of other intra- and extragenic sup-

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pressors of the wide variety of calcium regulatory mutants should prove to be a useful endeavor and will be assisted by the increasing molecular genetics of Paramecium. The first transformation of Paramecium by exogenous DNA was accomplished by Godiska et al. (109) with the microinjection of DNA into the somatic macronucleus. Kanabroki et al. (110) have utilized this technique to complement the cam2 mutation with the wild-type calmodulin gene. The microinjection method limits the number of possible transformants that can be created, but recent work in the related ciliate organism, Tetrahymena, holds out the possibility of mass transformation by electroporation, which may allow cloning by complementation. Current transformation is only into the somatic macronucleus, but continuing advances may allow germline transformation into the micronucleus and possibly gene replacement. Coupled with the ability to maintain Paramecium mutants defective in many different calcium regulatory pathways, improvements in transformation should promote even more structure-function analyses and use of suppressor analysis to discover the interacting components. Continuing evolution of antisense technology may provide a molecular genetic method of controlling the level of gene expression, allowing in vivo titration of individual components. Recent work has utilized electroporation to generate mass quantities of antisense-treated cells, with a large proportion having a dose-dependent affected behavior (D. Fraga and R. Hinrichsen, unpublished data). Improvements in microscope and photodetector technology may allow time-andlocation resolution of calcium localization in the cell. A former limitation in using fluorescence detection of calcium concentrations has been the rapid time scale of the "Paramecium behavioral response and recovery, as well as the swimming velocity of the motile cell. Technical improvements in imaging hardware and software, and in the immobilization of active, otherwise normal cells (K. Clark and D. Nelson, unpublished data) could provide a time-resolved, in vivo picture of calcium concentrations in the different organelles as the Paramecium performs any one of its many calcium-dependent functions and may demonstrate how calcium mobilization is altered in any of the mutants that may regulate those processes. The continuing evolution of technology and the increasing knowledge of Paramecium signal transduction biology will amplify our understanding of the many calcium regulatory processes in the system. The ability to maintain viable, robust Paramecium mutants defective in pathways analogous to some of the most interesting mammalian calcium-control mechanisms should provide powerful insights in the future.

ACKNOWLEDGMENTS We would like to thank our many colleagues for numerous discussions. This work was supported in part by grants from the National Science Foundation and the National Institutes of Health.

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84. Haiech J, Kilhoffer MC, Lukas TJ, Craig TA, Roberts DM, Watterson DM. Restoration of the calcium binding activity of mutant calmodulins toward normal by the presence of a calmodulin binding structure. J Biol Chem 1991;266:3427-3431. 85. Hinrichsen R, Wilson E, Lukas T, Craig T, Schultz J, Watterson DM. Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection. J Cell Biol 1990;111:2537-2542. 86. Hinrichsen RD, Pollock M, Hennessey T, Russell C. An intragenic suppressor of a calmodulin mutation in Paramecium: genetic and biochemical characterization. Genetics 1991;129:717725. 87. Ling KY, Kung C. Ba 2+ influx measures the duration of membrane excitation in Paramecium. J Exper Biol 1980;84:73-87. 88. Lukas TJ, Wallen-Friedman M, Kung C, Watterson DM. In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and an altered methylation state at Iysine residue 115. Proc Nat Acad Sci USA 1989; 86:7331-7335. 89. Hinrichsen RD, Fraga D, Reed MW. 3'-modified antisense oligodeoxyribonucleotides complementary to calmodulin mRNA alter behavioral responses in Paramecium. Proc Nat Acad Sci USA 1992;89:8601-8605. 90. Saimi Y, Ling KY. Calmodulin activation of calcium-dependent sodium channels in excised membrane patches of Paramecium. Science 1990;249:1441-1444. 91. Preston RR, Wallen-Friedman MA, Saimi Y, Kung C. Calmodulin defects cause the loss of Ca 2 +-dependent K + currents in two pantophobiac mutants of Paramecium tetraurelia. J Membr Biol 1990;115:51-60. 92. Preston RR, Saimi Y, Kung C. Evidence for two K § currents activated upon hyperpolarization of Paramecium tetraurelia. J Membr Biol 1990;115:41-50. 93. Rasmussen CD, Means AR. Increased calmodulin affects cell morphology and mRNA levels of cytoskeletal protein genes. Cell Motil Cytoskeleton 1992;21:45-57. 94. Kincaid R. Calmodulin-dependent protein phosphatases from microorganisms to man. A study in structural conservatism and biological diversity. Adv Second Messenger Phosphoprotein Res 1993; 27:1-23. 95. King MM, Huang CY, Chock PB, Nairn AC, Hemmings HC Jr, Chan KF, Greengard P. Mammalian brain phosphoproteins as substrates for calcineurin. J Biol Chem 1984;259:8080-8083. 96. Schulz S, Yuen PS, Garbers DL. The expanding family of guanylyl cyclases. Trends Pharmacol Sci 1991;12:116-120. 97. Schultz JE, Pohl T, Klumpp S. Voltage-gated Ca z + entry into Paramecium linked to intraciliary increases in cyclic GMP. Nature 1986;322:271-273. 98. Schultz JE, Klumpp S. Calcium/calmodulin-regulated guanylate cyclases in the ciliary membranes from Paramecium and tetrahymena. Adv Cyclic Nucleotide Protein Phosphorylat Res 1984;17: 275-283. 99. Miglietta LA, Nelson DL. A novel cGMP-dependent protein kinase from Paramecium. J Biol Chem 1988;263:16096-16105. 100. Gundersen RE, Nelson DL. A novel Ca 2+-dependent protein kinase from Paramecium tetraurelia. J Biol Chem 1987;262:4602-4609. 101. Son M, Gundersen RE, Nelson DL. A second member of the novel Ca2 + -dependent protein kinase family from Paramecium tetraurelia: purification and characterization. J Biol Chem 1993;268: 5940-5948. 102. Harper JF, Sussman MR, Schaller GE, Putnam-Evans C, Charbonneau H, Harmon AC. A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science 1991 ;252: 951-954. 103. Levin AE, Travis SM, DeVito LD, Park KA, Nelson DL. Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia. J Biol Chem 1989;264:4544-4551. 104. Asaoka Y, Nakamura S, Yoshida K, Nishizuka Y. Protein kinase C, calcium and phospholipid degradation. Trends Biochem Sci 1992; 17:414-417. 105. Hinrichsen RD, Blackshear PJ. Regulation of peptide-calmodulin complexes by protein kinase C in vivo. Proc Nat Acad Sci U S A 1993;90:1585-1589. 106. Hochstrasser M, Nelson DL. Cyclic AMP-dependent protein kinase in Paramecium tetraurelia: its purification and the production of monoclonal antibodies against both subunits. J Biol Chem 1989; 264:14510-14518. 107. Gustin MC, Nelson DL. Regulation of ciliary adenylate cyclase by Ca 2 + in Paramecium. Biochemical J 1987;246:337-345.

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108. Schultz J, Khmpp S. Adenylate cyclase in cilia from Paramecium. Localization and partial characterization. FEBS Len 1983;154:347-350. 109. Godiska R, Aufderheide KJ, Gilley D, Hendrie P, Fitzwater T, Preer LB, Polisky B, et al. Transformation of Paramecium by microinjection of a cloned serotype gene. Proc Nat Acad Sci USA 1987;84:7590-7594. 110. Kanabrocki JA, Saimi Y, Preston RR, Haynes WJ, Kung C. Efficient transformation of cam2, a behavioral mutant of Paramecium tetraurelia, with the calmodulin gene. Proc Nat Acad Sci USA 1991 ;88:10845-10849. 111. Kung C. Genic mutants with altered system of excitation in Paramecium aurelia: II. mutagenesis, screening and genetic analysis of the mutants. Genetics 1971;69:29-45. 112. Takahashi M, Naitoh Y. Behavioural mutants of Paramecium caudatum with defective membranes electrogenesis. Nature 1978;271:656-659. 113. Chang SY, Kung C. Selection and analysis of a mutant Paramecium tetraurelia lacking behavioural response to tetraethylammonium. Genet Res 1976;27:97-107. 114. Saimi Y, Hinrichsen RD, Forte M, Kung C. Mutant analysis shows that the Ca2+-induced K + current shuts off one type of excitation in Paramecium. Proc Nat Acad Sci U S A 1983;80:51125116.

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zyx zyxw Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1995.

12

Calcium in Saccharomyces Cerevisiae Trisha N. Davis

Department of Biochemistry, University of Washington, Seattle, Washington 98195

Two approaches have been taken to identify the functions of Ca 2 + in Saccharomyces cerevisiae. One concentrates on the ion itself and the other focuses on proteins that are regulated by Ca 2 +. Recent advances in loading yeast cells with Ca 2 + indicator dyes has allowed measurement of the intracellular concentration of Ca 2 + under different conditions (1-3). Great successes in studying the proteins regulated by Ca 2+ have yielded exciting new results about their functions in yeast. Protein kinase C (PKC) regulates a kinase cascade that culminates with activation of a yeast homologue to mammalian MAP kinase (4). The PKC pathway is required for cell wall morphogenesis (5). Yeast calcineurin is required for recovery from treatment with mating pheromone (6,7) and is a target for immunosuppressants and the yeast homologues to immunophilins (8). This provides an excellent system for dissecting the functions of calcineurin and immunophilins in signaling. Finally, calmodulin performs at least two essential functions during cell proliferation. First, it is a light chain for a class V unconventional myosin required for polarized growth (9). Second, calmodulin is required for chromosome segregation (10,11) as an essential component of the yeast microtubule organizing center (12). An elegant genetic analysis of calmodulin mutants presages two additional functions (13,14). Two criteria establish that a cellular function is regulated by Ca 2+. First, increases in intracellular Ca 2 + concentrations are necessary for the function to occur. Second, a Ca 2 +-binding protein is required to bind Ca 2 + to perform the function. In fact, these two criteria are difficult to meet, and only the role of calcineurin in recovery from pheromone arrest meets both criteria. This review will discuss each of the cellular processes in which Ca 2 + has been implicated and describe the evidence supporting the role of Ca 2 + as either regulatory or essential. Before launching into the functions performed by Ca 2 +, how yeast cells control the intracellular concentration of Ca 2 + will be reviewed.

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Yeast cells maintain Ca 2 + homeostasis even at concentration gradients of five orders of magnitude. Intracellular concentrations of free Ca 2 + can be measured either in cells loaded with the Ca 2 § indicator indo- 1 at low pH (3) or in cells loaded with the indicator fura-2 by electroporation (1,2). Both methods measure a cytosolic concentration of free Ca 2 § between 100 and 200 nM. This low level of cytosolic Ca 2 + is maintained with external concentrations ranging from 0.1 ttM to 10 mM Ca 2 +. Increasing the extracellular concentration of Ca 2 + to 50 mM increases the cytosolic concentration to only 270 nM (3). The total amount of Ca 2 + i n a yeast cell is much higher than might be suggested from the low cytosolic concentrations. For cells grown in the standard rich medium (0.18 mM [15]), the numbers in the literature vary from 1 x 10-17 mol total Ca 2 + per cell to 5 x 10-17 mol per cell. The difference seems to be in how the cells are washed before determination of total Ca 2 +. The lower numbers come from measurements in which the cells are grown for several generations in medium containing 45Ca2 +, washed with nonradioactive CaC12 and counted ([16], S. Loukin and C. Kung, personal communication, 1994). Washing with nonradioactive Ca 2 + removes any 45Ca2 + bound to the cell wall but must be done rapidly to avoid disturbing readily exchangeable pools. The higher numbers were obtained from cells washed with MgSO4 (17,18), which may not remove all the Ca 2 § bound to the cell wall. In either case, the free cytosolic Ca 2 + represents only 0.006-0.03 percent of the total Ca 2+ (assuming cytosolic volume of 3 • 10 -14 liters per cell [17]). Ca 2 + enters yeast cells through a nonspecific mechanosensitive ion channel in the plasma membrane (19). Electrophysiological analyses of yeast plasma membranes did not detect a specific Ca 2 + channel ([20] and C. Kung, personal communication, 1994). A Ca 2 +-ATPase that pumps Ca 2 + out of the cell across the plasma membrane has not yet been identified. The mitochondrion also does not transport Ca 2+ (21). Instead, four mechanisms for pumping Ca 2 + out of the cytosol have been identified in the other internal membrane systems of yeast. The vacuolar membrane contains a 2H +/Ca 2 + antiport that exploits the proton gradient formed by the vacuolar H + ATPase to drive Ca 2 + import into the vacuole (22,18). The antiport has a low affinity (Km of 100 I~M) for Ca 2 § (22). Its importance in maintaining internal concentrations of Ca 2 + was demonstrated by characterization of mutants defective in the vacuolar H + ATPase. In these mutants, which cannot maintain a pH gradient across the vacuolar membrane, the cytosolic concentration of Ca 2 + is 1 ~M as compared to 100 nM in wild-type cells (2). The mutants are viable even with this increased cytosolic concentration of Ca 2 + but cannot grow in medium containing high concentrations of Ca 2§ (100 mM) (23). Since mutants that cannot use the H+/Ca 2 + antiport to pump Ca 2 + nevertheless can maintain a cytosolic concentration of Ca 2 + of 1 I~M in medium containing 680 I~M Ca 2 + (2), other pumps must exist to regulate Ca 2 § levels. Three such pumps have been identified. The putative Ca 2 + ATPase encoded by the PMR1 gene is a member of the sarco/endoplasmic reticulum (SERCA) family of Ca 2 § pumps (24).

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Unlike many members of this family, Pmrlp is localized in a novel Golgi-like organelle as determined by immunofluorescence and cell fractionation (25). Another Ca 2 + transport activity was found to have similar pharmacological properties to the endomembrane pumps. This activity is found in a membrane fraction with the same buoyant density as the Golgi, but is not Pmrlp because the activity is present even in strains deleted for PMR1 (26). The gene encoding this activity has not yet been isolated. The third pump, encoded by PMC1, is found in vacuolar membranes and is more closely related to the mammalian plasma membrane Ca 2 + ATPases than to the SERCA pumps (16). A fourth pump, Pmr2p, originally reported as a member of the Ca 2 + ATPases, is probably a Na + pump and is required to provide Na + tolerance (27) but not Ca 2 + tolerance (16). Neither Pmrlp nor Pmc l p have unequivocally been shown to pump Ca 2 +. However, substantial evidence suggests that both are involved in maintaining low intracellular concentrations of Ca 2 +. Strains deleted in the PMC1 gene are viable but sequester Ca 2 + into the nonexchangeable pools (believed to be the vacuole) at 20 percent the wild-type levels. The pmclA strains are unable to grow in medium containing high concentrations of Ca 2 +. The inviability is rescued by inactivation of calcineurin. Strains deleted for the regulatory subunit of calcineurin, CNB1 (7) as well as pmclA are insensitive to high extracellular concentrations of Ca 2 + (16). Furthermore. these double delete strains (pmclA, cnblA) sequester Ca 2+ at 90 percent wild-type levels. Thus, calcineurin negatively regulates one of the other Ca 2 + sequestering mechanisms. That a Ca 2 +-/calmOdulin-dependent enzyme negatively regulates a Ca 2 § pump seems surprising. However, this regulation could be part of a positive feedback loop to raise Ca 2 + levels in cells treated with mating pheromone (see below). The Golgi Ca2+-ATPase, Pmrlp, is also not essential for cell viability, but strains deleted for PMR1 show numerous defects in processing of proteins headed for the cell surface (such as pro alpha factor). These defects can largely be overcome by high concentrations of extracellular Ca 2 +. Thus, Pmrlp appears to maintain sufficient Ca 2 + in the Golgi to allow Ca 2+-dependent enzymes to function ([25] and see below). However, Pmrlp presumably substitutes for the vacuolar Ca 2+-ATPase, Pmclp, in maintaining low cytosolic Ca 2 + levels at least under some conditions. Whereas mutants in either pump alone are viable in standard medium, strains carrying deletions of both pumps are dead (16). Finally, a gene encoding a protein that may be a negative regulator of Ca 2 § uptake by the endoplasmic reticulum was identified in a screen for mutants unable to grow in medium containing 100 mM Ca 2 § (28,29). CLS2/CSG2 encodes a protein containing nine putative transmembrane domains and no ATP-binding site. Cells deleted for CLS2/CSG2 accumulate 30-fold more Ca 2 + than wild-type cells in 3 hours. The excess Ca 2 + is exchangeable with the extracellular Ca 2 + and thus different from the large nonexchangeable pool found in wild-type yeast cells (28). Since Cls2p has been localized to the endoplasmic reticulum, this pool may represent the lumen of the ER (29). Mutants in the putative negative regulator of Ca 2§ uptake (Cls2p/Csg2p) are

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killed by high external Ca ~+ . Selection for extragenic suppressor mutations that prevent Ca 2 +-induced death yielded seven complementation groups (30). The gene encoding one of the suppressors apparently encodes serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid biosynthesis (30). The other suppressor mutants, as well as the original csg2 mutant, display altered sphingolipid metabolism. Thus, sphingolipid metabolism in yeast may be regulated by Ca 2 + or involved in Ca 2 + homeostasis. THE ROLE OF Ca 2 + DURING THE CELL CYCLE Yeast cells reproduce by focusing all growth in the daughter cell or bud. Bud emergence, duplication of the microtubule organizing center or spindle pole body, and the initiation of DNA replication all coincide. All three events are dependent on the activation of the Cdc28 protein kinase at the START point in the G 1 phase of the cell cycle. Sketchy data suggest that an increase in the rate of influx of Ca 2 + also occurs at this time (31,32). It is not known if this increase in influx translates into an increase in the cytosolic concentration of Ca 2 +. Nor is it known if this increase is a required signal of subsequent events or a result of the cell wall reshaping that occurs upon bud emergence. More solid evidence for a role of Ca 2+ in bud emergence and spindle pole body duplication is that both processes require Ca 2 +-binding proteins or enzymes regulated by Ca 2+ (see below). There is no evidence that Ca 2+ is required for initiation of DNA replication.

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Bud Emergence Once a mother cell passes START, it reorganizes to choose a bud site and assemble the components required to form a bud. All the materials required to make a new cell are shuttled out to the bud. Bud site assembly is controlled by a group of genes encoding a Rho-type GTPase (Cdc42p) (33), the enzyme that isoprenylates it (subunits are Callp and Ram2p) (34), a guanine nucleotide exchange protein (Cdc24p) and possibly a newly identified GTPase activating protein (Bem3p) (35). Both Callp and Cdc24p may be regulated by Ca 2 +. RAM2 and CALl encode the ct and 13 subunits of the geranylgeranyl transferase I in yeast (34). The enzyme specifically geranylgeranylates proteins that end with the sequence CaaL. In yeast, the two essential targets are Cdc42p required for bud emergence and Rholp (36). The gene encoding the 13 subunit was first identified in a screen for mutant cells that depended on high concentrations (100 mM) of Ca 2 + in the medium for growth at high temperature (15). The gene was given the name CALl for "Ca 2 +-dependent." The same gene was identified in a screen for cell cycle mutants defective in forming a bud and named CDC43 (33). The remediation by Ca 2 + is allele-specific because many of the temperature-sensitive alleles isolated by the latter group are not remediated by Ca 2 +. That Ca 2 + can rescue one of the recessive mutants, call-l, argues Ca 2+ positively regulates Callp. In vitro, the geranylgeranyl transferase is activated by Ca 2 + at 1 mM, which is not physiologi-

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cal, but lower concentrations were not tried (34). In fact, high concentrations of Mg 2 + also activate and may just be providing a low amount of r e q u ~ Ca 2 + (34). The mutant remediated by Ca 2 + may contain a geranylgeranyl transferase that requires more Ca 2 + for activation than the wild-type enzyme. Measuring the minimal concentration of Ca 2 + required to activate the transferase and identifying the mutation in the Ca 2+-remediated mutant are important next steps for def'ming the role of Ca 2 + in geranylgeranylation. Another component required for bud site assembly is a guanine nucleotide dissociation stimulator, Cdc24p (35). CDC24 was identified in the original screen for cell division cycle mutants. Temperature-sensitive mutants in CDC24 arrest as large unbudded cells when incubated at the nonpermissive temperature. The cells continue to grow and the nuclear cycle continues but buds are never made (37). Both genetic and biochemical evidence indicates that Cdc24p acts as the guanine nucleotide dissociation stimulator for the Rho-type GTPase, Cdc42p, during bud site assembly. In vitro, Cdc24p stimulates guanine nucleotide exchange on Cdc42p but not on the related proteins Rholp or Rsrlp (35). Multiple copies of CDC42 efficiently suppress the temperature sensitivity conferred by mutations in CDC24 (38). CDC24 was also identified in a screen for mutants that were sensitive to high concentrations of Ca 2 + in the medium (as opposed to requiting Ca 2 + as in the case of mutants in the geranylgeranyl transferase) (39). A Ca2+-sensitive allele of CDC24 did not alter Ca 2 + uptake nor the amount of Ca 2 + present in cells (23) and thus did not affect Ca 2 + transport. Cdc24p has two putative Ca 2 + binding sites, one related to EF-hands and one related to the Ca 2 +-binding site in et-lactalbumin (40). Neither is a perfect match to the consensuses, and the ability of Cdc24p to bind Ca 2 + directly has not been demonstrated. Two Ca 2 +-sensitive alleles of CDC24 were isolated. The mutation in one maps to the C-terminal region near one Ca 2 +-binding site. The other maps to the region homologous to mammalian DBL (41), which is the region implicated in guanine nucleotide exchange activity. Whether or not guanine nucleotide exchange is regulated by Ca 2 + in Cdc24p is not known. The genetic evidence suggests that Cdc24p is negatively regulated by Ca 2 +. It is not readily obvious how to reconcile evidence indicating that the geranylgeranyl transferase is activated by Ca 2 + and the guanine nucleotide exchange protein is negatively regulated by Ca 2 +. Isoprenylation (42) and presumably guanine nucleotide exchange are both required to make an active form of the Rho-type GTPase Cdc42p, and yet Ca 2 + apparently has opposite effects on these two processes. Perhaps this opposing regulation is important in providing a balanced amount of active Cdc42p at the bud site. A mutational analysis of Cdc42p itself provided evidence that a proper amount of GTP-Cdc42p is crucial to bud formation. Mutations that either activate the GTPase or inactivate the GTPase block bud formation (42).

zyxwvut Spindle Pole Body Duplication

The calcium-binding protein Cdc31p is required for duplication of the microtubule organizing center or spindle pole body. Cdc3 lp has 4 EF-hand Ca 2 +-binding

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sites (43) and is most closely related to caltractin found on the basal body of Chlamydomonas (44). Cdc31p binds Ca 2+, but neither the stoichiometry nor the affinity has been determined (45,46). Complete duplication of the spindle pole body requires the activity of the Cdc28 kinase. However, recent evidence indicates that Cdc3 lp is required during a very early step of duplication, formation of a satellite. Cdc3 l p completes its function and satellite forms even in cells arrested in G1 at START by treatment with mating pheromone (47). Cdc31p is recruited to the spindle pole body by Karlp. The requirement for Karlp during cell growth can be overcome by dominant mutations in Cdc31p (48). These mutations alter Cdc3 l p such that it localizes to the spindle pole body independently of Karlp (46). The interaction between Karlp and Cdc31p does not appear to be Ca 2+-dependent (46) as might be expected if Karlp is required only to bind Cdc3 l p to the spindle pole body, where it interacts with downstream effectors in a Ca 2 +-dependent manner.

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Other Stages of the Cell Cycle Iida and coworkers (49) studied the effects of EGTA and A23187 on yeast cell growth. In the presence of EGTA and A23187, cells display a transient arrest at G1, dependent on the activity of the Ras pathway, which is known to act at G1. Their analysis was done at pH 5.7 (or below) where EGTA has a very low affinity for ions. In fact, a subsequent analysis of their data (50) demonstrated that the ability of cells to grow did not correlate with the concentration of free Ca2+ and instead could be ascribed to toxic effects of A23187 independent of ions. For example, A23187 can also affect the transport of amino acids (51). The results of Iida and coworkers could be explained as the response of yeast cell to starvation for amino acids, a response known to be governed by the Ras pathway. There is little evidence for a role for Ca 2 + in the S, G2, or M phases of the cell cycle. The Ca 2+-dependent mutant in the geranylgeranyl transferase Cal l p does not complete a nuclear cycle (15). This may be a secondary defect stemming from its inability to complete a bud. Calmodulin is required for the function of the microtubule organizing center during mitosis (10-12), but mutant calmodulins completely defective in binding Ca 2+ can still perform the mitotic function (52). CELL WALL INTEGRITY AND PROTEIN KINASE CI In higher eukaryotic cells, one of the cornerstone signaling pathways begins with an extracellular signal, activating phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into diacylglycerol and 1,4,5 inositol triphosphate (IP3). Diacylglycerol is the physiological activator of PKC, whereas IP3 induces release of Ca 2 + from intracellular stores. Yeast contain at least two versions of this pathway (see Table 1). One version has been implicated in cell wall biosynthesis and the other in nutrient response and stress response (see below).

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TABLE 1. Model for signal transduction pathways in yeast" ?

?

9

Stt4p

?

Piklp

Phospholipase C

?

Plcl p

?

Protein kinase C

Pkcl p

Pkc2p

?

?

?

Stress response and nutrient response

Growth

Signal PI 4-kinase

Downstream effectors

Bckl p

?

Mkkl p Mkk2p ipklp ? Function

Cell wall morphogenesis

'Yeast proteins known to perform each of the functions are shown. Description and references are given in text. Also see addendum for Pkc2p.

The yeast gene PKC1 encodes a protein kinase closely related to the et, 13, and isoforms of mammalian PKC (5). Cells depleted of PKC1 gene product uniformly arrest with a small bud and a G2 content of DNA. DNA synthesis continues while bud growth is stopped (5). Further analysis of cells depleted for Pkc l p as well as temperature-sensitive mutants revealed that the defects can be rescued by high osmolarity medium (53,54). Furthermore, temperature-sensitive pkcl mutants lyse and release cellular material into the medium at the nonpermissive temperature (53). Electron micrographs of the mutants at the high temperature show that the buds are blown out at the tip (55). Thus, PKC seems to be required for cell wall construction or strengthening, with the tip of the small growing bud representing the most vulnerable part of a yeast cell. The temperature-sensitive pkcl mutants are remediated by Ca 2 +. Concentrations of Ca 2 + that are below those required to osmotically stabilize the mutants still rescue (53). Ca 2§ cannot remediate the death of a strain deleted for pkcl. Thus, Ca 2 § is not just bypassing the requirement for Pkc lp but strengthening weakened versions of Pkc l p. This is exactly the result expected for an enzyme activated by Ca 2 +. Pkc l p does contain the conserved region believed to be involved in binding Ca 2 + Three different groups are characterizing the enzymatic activity of Pkclp. Using a synthetic peptide as a substrate, two groups did not detect activation by Ca 2 +, phosphatidylserine, diacylglycerol, or phorbol esters (56,57). However, mutational activation of the enzyme by inactivation of the pseudosubstrate sequence yielded nine-fold greater activity than could be detected for the wild-type enzyme. The activity detected with the synthetic peptide is apparently a basal level of activity. In contrast, Ca 2§ phosphatidylserine, and diacylglycerol were required for significant phosphorylation of myelin basic protein or mixed histones (58).

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The only enzyme identified to act upstream of Pkc l p is a phosphatidylinositol-4 kinase (see Table 1) (59). In a screen for mutants that are temperature-sensitive and sensitive to staurosporine (an inhibitor of PKC), Yoshida and coworkers (54) identified new alleles of PKC1. The same screen yielded STT4, which encodes a PI 4-kinase. Strains deleted for STT4 are dead but can be rescued by high osmolarity medium (59), just as the mutants in PKC1 can be rescued. Another enzyme expected to act upstream of PKC is phospholipase C. A phospholipase C has been identified that is specific for phosphoinositides (60-62), but several genetic analyses suggest it is not in the same pathway as Pkc l p (see below). Downstream of Pkc lp is a protein kinase cascade that culminates with activation of a yeast homologue of MAP kinase, Mpklp (see Table 1) (4,63,64). Unlike Pkc lp, not all of the enzymes that act downstream are essential. Deletions of these genes cause cell lysis at high temperature but allow growth at low temperature (4,63,64), whereas deletion of PKC1 causes cell lysis at all temperatures (5). Thus, the Pkclp pathway may bifurcate below Pkc lp, one path leading to Mpklp and the other path as yet unidentified (see Table 1). The targets of the MAP kinase, Mpklp, have not been identified.

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NUTRITIONAL AND STRESS-RELATED RESPONSES AND PHOSPHOLIPASE C

Several reports originally suggested that IP3 and Ca 2 + may be involved in signaling exit from GO or stationary phase. An increase in Ca 2§ influx occurs as starving cells are refed glucose (65). In addition, indirect experiments suggested that in cells shifted to medium containing glucose after glucose starvation, inositol phosphate and IP3 were produced (65). These results implied the presence of a phospholipase C pathway signaling exit from stationary phase. However, recently the inositol metabolites produced after release from stationary were unequivocally identified as extracellular glycerophosphoinositol and glycerophosphoinositol 4,5-bisphosphate rather than intracellular inositol phosphate and IP3 (66). No inositol phosphate or IP3 was detected. Thus, exit from stationary apparently does not involve phospholipase C but perhaps phospholipases A or B. Phospholipase C is important for other nutritional and stress-related responses. Recent identification of the gene (PLC1) encoding a phospholipase C ~ has allowed study of the role of this enzyme in yeast cells (60-62). Plclp is 29 and 51 percent identical to conserved X and Y domains of ~1 phospholipase C and contains one EF-hand Ca 2+-binding site. The enzyme was His6 tagged and partially purified from yeast. Plclp is stimulated 100-fold by 0.5 IxM Ca2 § and hydrolyzes phosphatidylinositol 4,5-bisphosphate at least 40-fold faster than phosphatidylinositol (60). The phenotypes observed in strains deleted for PLC1 depends on the strain background. In some backgrounds, deletion of PLC1 is lethal (62). Other strains lacking Plc l p grow poorly (62), and some plclA strains grow well at low temperature but are dead at high temperature (60). Hick and Thomer (60) found that in addition to

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being temperature-sensitive the plclA stlains are sensitive to high osmotic strength medium and cannot utilize nonfermentable carbon sources. Although these phenotypes point to divergent functions, none suggest a role in glucose response or in exit from stationary. Instead, they suggest a function in stress response and use of nonfermentable carbon sources. Several lines of genetic evidence argue that phospholipase C 1 (Plc lp) is not in the same pathway as PKC1 (Pkc l p). First, mutants in Pkc lp are rescued by high osmolarity (53), whereas mutants in Plclp are sensitive to high osmolarity (60). Extra copies of PKC1 have no effect on the temperature-sensitive phenotype of plcl mutants (60). Hyperactivated Pkc lp does not rescue plcl mutant phenotypes (J. Flick, O. Fields, and J. Thorner, personal communication). Finally, an activated version of a kinase (Bcklp) downstream of Pkclp does not suppress the defects in plcl deletes (62). Another PKC encoded by PKC2 was recently identified (67). Mutants in PKC2 share some phenotypes with mutants in PLC1. Thus, Pkc2p may act downstream of Plc lp. Finally, Payne and Fitzgerald-Hayes identified a mutant in PLC1 in a screen for strains with defects in chromosome segregation (61). The allele they isolated, plcl-1, is also temperature-sensitive and shows a tenfold increase in chromosome segregation. The relationship between chromosome segregation and the defects in nutrient and stress response displayed by strains deleted for PLC1 is unclear.

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PHOSPHATIDYLINOSITOL 4-KINASES AND IP3 Phosphatidylinositol 4-kinase catalyzes the first committed step in biosynthesis of phosphatidylinositol 4,5-bisphosphate. Cleavage of PIP2 by phospholipase C yields diacylglycerol to activate PKC and IP3, which releases Ca 2+ from intracellular stores. Two membrane bound (68,69) and one soluble PI 4-kinase (70) have been identified biochemically in yeast. The gene (PIK1) encoding the soluble PI 4-kinase was isolated based on the sequence of the purified enzyme. The genes encoding the membrane-bound forms are not known. The two PI 4-kinases Piklp and Stt4p do not have identical functions. PIK1 is essential for growth, whether or not osmotic support is provided in the medium (71). S ~ 4 is required only in low osmotic strength medium, but not in medium of high osmotic strength (59) (see Table 1). The two enzymes, both of which synthesize PIP, are required under different conditions. This observation strongly argues that there are distinct pools of PIP. The pool produced by Piklp is required under all conditions, whereas the pool produced by Stt4p is only required in medium containing low osmotic strength. Since mutants in STT4 can be grown under some conditions, identification of the membrane compartment containing the PIP pool produced by Stt4p may be possible. Finally, if PIP2 is involved in signaling then there should be intracellular stores of Ca 2 + sensitive to IP3. Belde and coworkers (72) recently reported that IP3 releases Ca 2 + from vacuolar membrane vesicles without affecting the pH gradient.

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CALCIUM IN SACCHAROMYCES CEREVISIAE Ca 2+ A N D S E C R E T I O N

In vitro assays of protein transport in yeast reveal that Ca 2 + is required for transport of vesicles from the endoplasmic reticulum to the Golgi (73). A detailed analysis of the steps involved in ER to Golgi transport revealed that Ca 2 + is specifically required for the fusion of vesicles with the Golgi but is not required for targeting the vesicles to the Golgi (73). Approximately 100 nM Ca 2 + is required and up to 1 I~M Ca 2§ only inhibits fusion by 20 percent. Thus, vesicle fusion is apparently not regulated by changes in Ca 2 + levels. Early on, there was some confusion about the relationship between Ca 2 + and Yptlp, a member of the Rab family of GTPases. Cells carrying a temperaturesensitive mutation in YPT1 are rescued at the high temperature by increased extracellular Ca 2 § and show an increased rate in the influx of Ca 2 § into the cell (74). These results led to the conclusion that Yptlp was involved in Ca 2 + uptake. However, all the mutants defective in ER to Golgi transport also show an increased rate of Ca 2 + influx, so this seems to be a general characteristic of mutants defective in this early transport step rather than a specific attribute of yptl mutants (74). In vitro, both Yptlp and Ca 2 + are required for transport of vesicles from the ER to the Golgi (75,76). The secretion step requiting Ca 2 + occurs after the step requiring Yptlp. If vesicle transport to the Golgi is allowed to proceed up to the step blocked by EGTA, and then the block released by the addition of Ca 2 +, Yptlp is not required to complete transport (73). Yptlp is required for targeting the vesicles to the Golgi, whereas Ca 2 § is required for vesicle fusion (73). In vitro Ca 2 § cannot rescue transport defects observed in extracts depleted for Yptlp (76) or extracts made from cells carrying mutant yptl proteins (75). Why the yptl mutant is rescued by increased extracellular Ca 2 + in vivo is not known, but the in vitro results suggest the effect is indirect. An additional role for Ca 2+ in secretion is suggested by analysis of strains deleted for PMR1, the gene encoding a Ca 2 + pump in the Golgi. Strains deleted for PMR1 show numerous and diverse secretory defects. In wild-type strains, core glycosylation occurs in the ER, with outer chain mannose residues added in the Golgi. Invertase secreted from pmrlA strains is core-glycosylated, but contains truncated outer chains (25). pmrlA strains secrete multiple forms of unprocessed a-factor similar to that seen in mutants defective in the processing enzyme Kex2p (77). These defects could largely be explained if pmrlA strains have decreased Ca 2 + concentrations in the Golgi. Guanosine diphosphatase, a Golgi enzyme that hydrolyzes GDP (a known inhibitor of mannosyltransferases), requires Ca 2 § for maximal activity. Kex2p, the Golgi enzyme required to process a-factor (77), also requires Ca 2 + (78). Both secretion defects are overcome by increasing extracellular Ca 2 +, perhaps because that drives more Ca 2 + into the Golgi. One defect seen in pmrlA strains is an enigma, given the evidence that Pmrlp is a Golgi Ca 2 § pump (25). Mutants in PMR1 secrete heterologous proteins normally trapped in the cell (24). The trapped proteins are thought to be in the ER because they lack outer chain mannosyls that are added in the Golgi. Thus, changes in the

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Ca z + state of a compartment of the Golgi allows proteins normally trapped in the ER to be secreted. Taken together, these results suggest that maintaining proper Ca 2 + homeostasis in the internal membrane compartments is important for proper secretion.

Ca 2 + A N D M A T I N G P H E R O M O N E

Yeast cells can be stably grown either as haploids or diploids. The haploid cells have two mating types, a or oL, which normally grow by budding. After treatment with the appropriate pheromone, a and et cells enter a developmental pathway that leads to formation of a diploid cell (79). et cells produce a tridecapeptide pheromone, a-factor. Tachikawa and coworkers (80) observed a rapid and transient increase in Ca z + uptake that occurs in the first 10 minutes after treatment of a cells with a-factor (80), but no other evidence for a role for Ca 2 + in response to pheromone has been reported. Furthermore, a similar transient increase was not seen in yeast cells loaded with fura-2 (1). Several lines of evidence indicate that Ca 2 + is involved in recovery from pheromone-induced arrest. Forty minutes after a-factor treatment, a cells begin to accumulate Ca 2 + such that by 120 minutes the a cells have accumulated 30-fold more Ca 2 + than the control et cells, which do not respond to a-factor (32,81). As measured with fura-2, the cytosolic concentration of free Ca 2 + rises two- to tenfold to 200-1000 nM (1). The large accumulation of Ca 2 + is required for a cells to maintain viability in the presence of e~-factor. In medium containing low concentrations of Ca 2 + (0.24 p~M), these increases in Ca 2 + do not occur and the cells are killed by e~-factor (1). Sixty percent of the cells die within 5 hours, and 95 percent in 10 hours, whereas if Ca 2 + is present only 10 percent die in 10 hours. Addition of extracellular Ca 2 + to 10 I~M allows recovery, but 100 ~M is required for full recovery (1). Cells in which the mating pheromone pathway is artificially activated by depletion of G,~ from the cells also require Ca 2+ to survive (1). Two other proteins known to be involved in pheromone recovery, Sst2p and a protease specific for (x-factor, Sstlp, are not involved in the Ca 2 + pathway (1). Information about the role of Ca 2 + in pheromone survival came from the analysis of the Ca z +-/calmodulin-dependent protein phosphatase calcineurin in yeast. Calcineurin contains two subunits, the catalytic A subunit and the regulatory B subunit. In yeast the A subunit is encoded by two genes named CMPllCNA1 and CMP21 CNA2 (6,82). Cells deleted for both genes are viable and lack all detectable calcineurin activity (83). The B subunit is encoded by one gene CNB1 (7,84). Neither the A subunits nor the B subunit are essential for growth. However, they are required for viability in the presence of e~-factor. Strains carrying deletions of both genes encoding A subunits or deletion of the gene encoding the B subunit do not survive pheromone treatment (6,7) (M. Cyert, personal communication). Survival in oL-factor also represents the one identified CaZ+-dependent function for cal-

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modulin. Strains carrying mutant calmodulins defective in binding Ca 2 + do not survive a-factor treatment, presumably because calcineurin is not activated (85). Survival in the presence of a-factor has proven a useful assay for molecules that regulate calcineurin. Both cyclosporin and FK506 prevent pheromone recovery at concentrations of immunosuppressant two to three orders of magnitude below those required to inhibit growth (8). Inhibition of recovery by FK506 requires the FK506binding protein FKBP12, which forms a complex with calcineurin. Inhibition of recovery by cyclosporin A requires cyclophilin (8). Thus, yeast may prove an excellent system to dissect the mechanism of action of the immunosuppressants and their cytosolic receptors (86).

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Ca 2 + AND H O M E O S T A S I S OF O T H E R IONS

Two different observations implicate Ca 2 + in regulating homeostasis of other ions. First, the vacuolar membrane contains a voltage-dependent cation channel detectable by patch clamp techniques. The channel shows little selectivity between K § and Na +. The channel was originally reported to be activated by mM Ca 2 § (87), but under reducing conditions becomes sensitive to I~M Ca 2 + on the cytosolic side of the membrane (88). Second, growth of strains deleted for calcineurin is inhibited by either NaC1 or LiC1 but not by KC1, CaC12, or MgC12 (83,89). In calcineurin mutants shifted to high-salt medium, the cellular content of Na + increases at the same rate as in wild-type cells. In the wild-type cells, Na § levels decrease after 3 hours, whereas in the mutants, Na + content stays high. Further analysis revealed that the mutant has decreased Na + export capacity, which can be rescued by extracellular K + (90). FK506, the inhibitor of calcineurin, causes similar effects. CALMODULIN Calmodulin is essential for growth of yeast cells (91), and the essential functions can be performed by vertebrate calmodulin (92,93). Structural determinants required for calmodulin to function in yeast have been conserved in vertebrate calmodulin. Surprisingly, mutant calmodulins in which the Ca 2+-binding sites are inactivated can still support growth (52). Even mutant vertebrate calmodulin with inactive Ca 2 +-binding sites suffices. These results suggested that calmodulin does not require a high affinity for Ca 2 + to perform its essential functions, which may be Ca 2 +-independent. Two other possibilities are that the mutant calmodulins still bind Ca 2 + and activate Ca2+-dependent targets despite the mutations. Alternatively, the mutations may mimic the conformational change induced by Ca 2 +, and the mutant proteins may thus be constitutively in the Ca 2 +-bound form. These alternatives were originally difficult to test since no Ca 2+-dependent functions for calmodulin or calmodulin-binding proteins were known. The discovery that calcineurin is required for recovery from pheromone arrest (6,7) provided an assay for Ca 2 +/calmodulin.

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All the strains carrying calmodulin mutated in either two or three of the Ca 2 +-binding sites are defective in recovery (85). This result strongly argues that the conformation of the mutant calmodulins are defective in Ca 2 +-dependent activities. Furthermore, their conformation does not mimic Ca 2 +/calmodulin. That the essential functions apparently do not require calmodulin to bind Ca 2 + suggests they may be activities whose importance had not been fully appreciated. One approach to identify these functions was to characterize strains carrying temperature-sensitive mutations in calmodulin. At the nonpermissive temperature, these strains have defects in bud growth, chromosome segregation, and spindle pole body function (10,11,13). Immunolocalization of calmodulin revealed calmodulin concentrates at sites of cell growth (94,95), as expected for a protein involved in bud growth. The next step was to identify the target proteins with which calmodulin must interact to support growth. A screen for mutant cells that could continue to grow but could not form buds identified a yeast homologue (Myo2p) of the class V unconventional myosins known to bind calmodulin (96). Genetic and biochemical analyses of Myo2p provided strong evidence that it is the target of calmodulin at sites of cell growth. Both proteins localize at sites of cell growth (94,97) in patterns that are indistinguishable (9). Mutant calmodulins defective in binding Ca 2 § also localize at sites of cell growth (94). Anticalmodulin antibodies immunoprecipitate Myo2p from yeast crude extracts in the presence of Ca 2 § or EGTA. Direct binding to the IQ sites of Myo2p was demonstrated by a modified gel overlay assay. This interaction also occurs in the presence of Ca 2 + or EGTA (9). The IQ sites are found in all myosins and represent the region where the light chains bind. Finally, mutations in CMD1 show allele-specific synthetic lethality with the myo2-66 temperature-sensitive mutation. Mutations that inactivate the Ca 2 +-binding sites of calmodulin have little or no effect on strains carrying myo2-66, whereas an allele with a mutation outside the Ca 2 +-binding sites dramatically increases the severity of the phenotype conferred by myo2-66 (9). Presumably, Myo2p is involved in moving cellular components to the bud, but how the materials are packaged is unknown. The role of the calmodulin light chains is not clear. Are they regulated, and, if so, by what? Ca 2 + is not an important regulator of binding, since interactions between calmodulin and Myo2p occur in the presence of Ca 2 § or EGTA. Ca 2 + has been implicated as a regulator of p190, the mammalian homologue of Myo2p (98). Since yeast mutants relying on calmodulins defective in binding Ca 2 + grow buds well, calmodulin may not be the Ca 2 + receptor. The calmodulin light chains could be regulated by other factors such as phosphorylation, which regulates the conventional myosin light chains. The second target of calmodulin required for chromosome segregation is the 110kDa component (Nuflp/Spc110p) of the microtubule organizing center or spindle pole body (12,99). Nuflp/Spc110p was identified in two independent screens for calmodulin targets (12). First, dominant suppressors of a calmodulin temperaturesensitive mutant all map to the single gene encoding the 110-kD component. Second, a two-hybrid screen for proteins that interact with a Ca 2 +-binding mutant

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calmodulin yielded the C-terminus of Nuflp/Spc 110p. Direct binding between calmodulin and the 110-kDa component was confmned by a gel overlay assay. Finally calmodulin localizes to the spindle pole body in cells fixed by procedures that enhance visualization of spindle pole body components. By systematically mutagenizing the calmodulin gene such that each phenylalanine was changed to alanine, Ohya and Botstein (13,14) discovered that different calmodulin mutants displayed intragenic complementation. They identified four complementation groups, which may define four different functions of calmodulin. Characterization of representative mutants in each group revealed that one showed G2/M arrest expected for a mutant defective in spindle pole body function (13). Another mutant had a phenotype very similar to myo2-66 mutants. These two complementation groups probably represent calmodulins with defects in interacting with the two targets Nuflp/Spcll0p and Myo2p already identified. An exciting possibility is that the other two complementation groups represent additional unidentified functions for calmodulin. Two Ca 2+-/calmodulin-dependent protein kinases are reported in the literature (100,101). They are not essential for growth. Mutants deleted for the genes encoding both enzymes have no discernible phenotype. A gene encoding another Ca 2 +-/calmodulin-dependent protein kinase was recently identified. It also is not essential for growth, and mutants lacking all three enzymes grow well (M. Melcher and J. Thorner, personal communication).

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Ca 2 + A N D T H E S P I N D L E P O L E B O D Y

Two related Ca 2+-binding proteins, Cdc3 l p and calmodulin, are both components of the spindle pole body (45,46,52). The spindle pole body is a cylindrical multilayered microtubule organizing center in yeast. The central layer or plaque is embedded in the nuclear envelope. Cytoplasmic microtubules emanate from an outer plaque and nuclear microtubules emanate from an inner plaque internal to the nuclear envelope (102). Cdc31p associates with Karlp (46,48), which is on the cytoplasmic side of the nuclear envelope (45,103). Calmodulin associates with Nuflp/Spcll0p ([12] and see previous section), which forms a connecting rod between the central and inner plaque on the nuclear side of the envelope (99,102). Cdc31p is required in G1 for spindle pole body duplication ([47] and see section on Ca 2 + during the cell cycle). Calmodulin is required during mitosis for spindle pole body function (10,11). Thus, two related proteins both act at the spindle pole body, one outside the nucleus during spindle pole body formation, and one inside the nucleus during chromosome segregation. The role of Ca 2 § in each of these functions is not known. In fact, yeast strains carrying mutant calmodulins defective in binding Ca 2§ segregate their chromosomes normally (52). C A N M n 2 + S U B S T I T U T E F O R Ca 2 + ?

Historically, proof that yeast cells actually require Ca 2 + for growth has been difficult to obtain (see [50] for discussion of the difficulties). Ca2 + is a major contami-

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nant in agar and the salts used to make medium for growing yeast. Yeast nitrogen base, which is used to make the common synthetic medium, contains 680 IxM CaC12. YPD, which is the standard rich medium, contains 180 IxM (15). Even when substantial precautions are taken to introduce as little extraneous Ca 2 + as possible, yeast medium contains 0.24 IxM Ca 2 + (49). At that low concentration of Ca 2 +, yeast cells grow well (49). Ideally, one would use a chelator to control Ca 2 + concentrations. Yeast medium is pH 6.5 and becomes more acidic as the yeast cells actively pump H + into the medium. The standard chelator, EGTA, has a very low affinity for ions at acidic pH. S. Loukin and C. Kung (manuscript in preparation) have overcome these difficulties by using B APTA as a chelator, which is less pHsensitive than EGTA. They find that yeast cells can grow well in medium containing 3 x 10-1o free Ca 2 + as long as Mn 2 + is present. Under these conditions the amount of total internal Ca 2 + is 3 percent of the normal level. Perhaps 3 percent of the normal level of internal Ca 2+ is enough to maintain the levels of Ca 2 + required in the cytosol and the membrane compartments. Alternatively, Mn 2 + may substitute for Ca 2 +. At the very least, these results spur all of us working on Ca 2 + in S. cerevisiae to reconsider our results in view of Mn 2 + and not to consider just Ca 2 + and Mg 2 +.

CONCLUSIONS Tremendous progress has been made in delineating the numerous pathways involving proteins regulated by Ca 2 +. The more difficult aspect has been identifying the intracellular or extracellular signals that modulate the activities of these proteins. With the exception of the mating pheromone pathway, neither a signal to initiate the pathway nor an increase in internal Ca 2 + to act as a second messenger has been identified (Table 1). Genetic analyses may identify the initiating signals. As recently developed methods improve for detecting changes in intracellular concentrations of Ca 2 +, we may be able to detect transient increases in Ca 2 + levels.

ACKNOWLEDGMENTS

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I thank Dr. E . G . D . Muller for helpful suggestions on the manuscript. I thank Dr. J. Thorner, Dr. Y. Ohya, Dr. D. Levin, Dr. M. Cyert, and S. Loukin for helpful discussions and communication of unpublished results.

ADDENDUM Levin et al. have recently reported that the PKC2 gene does not exist as a contiguous sequence in the S. cerevisiae genome (104).

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49. Iida H, Sakaguchi S, Yagawa Y, Anraku Y. Cell cycle control by Ca2+ in Saccharomyces cerevisiae. J Biol Chem 1990;265:21216-21222. 50. Youatt J. Calcium and microorganisms. Crit Rev Microbiol 1993;19:83-97. 51. Hovi T, Williams SC, Allison AC. Divalent cation ionophore A23187 forms lipid soluble complexes with leucine and other amino acids. Nature 1975;256:70-72. 52. Geiser JR, van-Tuinen D, Brockerhoff SE, Neff MM, Davis TN. Can calmodulin function without binding calcium? Cell 1991;65;949-959. 53. Levin D, Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle specific osmotic stability defect. J Cell Biol 1992;116:1221-1229. 54. Yoshida S, Ikeda E, Uno I, Mitsuzawa H. Characterization of a staurosporine- and temperaturesensitive mutant, sttl, of Saccharomyces cerevisiae: STT1 is allelic to PKC1. Mol Gen Genet 1992; 231:337-344. 55. Levin DE, Errede B. A multitude of MAP kinase activation pathways. J NIH Res 1993;5:49-52. 56. Antonsson B, Montessuit S, Friedl L, Payton MA, Paravacini G. Characteristics of the Saccharomyces cerevisiae PKC1 gene product. J Biol Chem 1994;269:16821-16828. 57. Watanabe M, Chen C-Y, Levin DE. Saccharomyces cerevisiae PKC1 encodes a PKC homolog with a substrate specificity similar to mammalian PKC. J Biol Chem 1994;269:(in press). 58. Fields FO. Biochemical and molecular genetic analysis of protein kinase C function in the yeast Saccharomyces cerevisiae. University of California at Berkeley: 1991. 59. Yoshida S, Ohya Y, Goebl M, Nakano A, Anraku Y. A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae. J Biol Chem 1994;269:1166-1171. 60. Flick JS, Thorner J. Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae. Mol Cell Biol 1993;13:5861-5876. 61. Payne WE, Fitzgerald-Hayes M. A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation. Mol Cell Biol 1993;13:4351-4364. 62. Yoko-o T, Matsui Y, Yagisawa H, Nojima H, Uno I, Toh-e A. The putative phosphoinositidespecific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth. Proc Natl Acad Sci U S A 1993;90:1804-1808. 63. Lee KS, Levin DE. Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog Mol Cell Biol 1992;12:172-182. 64. Irie K, Takase M, Lee KS, et al. MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C. Mol Cell Biol 1993;13:3076-83. 65. Kaibuchi K, Miyajima A, Arai K, Matsumoto K. Possible involvement of RAS-encoded proteins in glucose-induced inositolphospholipid turnover in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 1986;83:8172-8176. 66. Hawkins PT, Stephens LR, Piggott JR. Analysis of inositol metabolites produced by Saccharomyces cerevisiae in response to glucose stimulation. J Biol Chem 1993;268:3374-3383. 67. Simon AJ, Saville SP, Jamieson L, et al. Characterization of PKC2, a gene encoding a second protein kinase C isotype of Saccharomyces cerevisiae. Curr Biol 1993;3:813-821. 68. Buxeda RJ, Nickels JT, Belunis CJ, Carman GM. Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. J Biol Chem 1991;266:13859-13865. 69. Nickels JT Jr, Buxeda RJ, Carman GM. Purification, characterization, and kinetic analysis of a 55kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. J Biol Chem 1992;267: 16297-16304. 70. Flanagan CA, Thorner J. Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae. J Biol Chem 1992;267:24117-24125. 71. Flanagan CA, Schnieders EA, Emerick AW, Kunisawa R, Admon A, Thorner 1. Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability. Science 1993;262: 1444-1448. 72. Belde PJ, Vossen JH, Borst-Pauwels GW, Theuvenet AP. Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae. FEBS Lett 1993;323:113118. 73. Rexach MF, Schekman RW. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol 1991;114:219-229. 74. Schmitt HD, Puzicha M, Gallwitz D. Study of a temperature-sensitive mutant of the ras-related

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YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium. Cell 1988; 53:635-647. Bacon RA, Salminen A, Ruohola H, Novick P, Ferro-Novick S. The GTP-binding protein Yptl is required for transport in vitro: the Golgi apparatus is defective in yptl mutants. J Cell Biol 1989; 109:1015-1022. Baker D, Wuestehube L, Schekman R, Botstein D, Segev N. GTP-binding Yptl protein and Ca2+ function independently in a cell-free protein transport reaction. Proc Natl Acad Sci USA 1990; 87:355-359. Julius D, Brake A, Blair L, Kunisawa R, Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell 1984;37:1075-1089. Wagner JC, Escher C, Wolf DH. Some characteristics of hormone (pheromone) processing enzymes in yeast [published erratum appears in FEBS Len 1987;221:433]. FEBS Lett 1987;218:3134. Konopka JB, Fields S. The pheromone signal pathway in Saccharomyces cerevisiae. Antonie Van Leeuwenhoek 1992;62:95-108. Tachikawa T, Miyakawa T, Tsughiya E, Fukui S. A rapid and transient increase of cellular Ca2§ in response to mating pheromone in Saccharomyces cerevisiae. Agric Biol Chem 1987;51:12091210. Ohsumi Y, Anraku Y. Specific induction of Ca2+ transport activity in MATa cells of Saccharomyces cerevisiae by a mating pheromone ot factor. J Biol Chem 1985;260:10482-10486. Liu Y, Ishii S, Tokai M, et al. The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B. Mol Gen Genet 1991;227:52-59. Nakamura T, Tsutsumi H, Mukai H, Kuno T, Miyakawa T. Ca 2§ protein phosphatase (PP2B) of Saccharomyces cerevisiae. PP2B activity is not essential for growth. FEBS Lett 1992;309:103-106. Kuno T, Tanaka H, Mukai H, et al. cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae. Biochem Biophys Res Commun 1991;180:1159-1163. Geiser JR. Genetic analysis of the essential calmodulin functions in Saccharomyces cerevsiae. University of Washington: 1993. Rotonda J, Burbaum JJ, Chan HK, Marcy AI, Becker JW. Improved calcineurin inhibition by yeast FKBP12-drug complexes: crystallographic and functional analysis. J Biol Chem 1993;268: 7607-9. Wada Y, Ohsumi Y, Tanifuji M, Kasai M, Anraku Y. Vacuolar ion channel of the yeast, Saccharomyces cerevisiae. J Biol Chem 1987;262:17260-17263. Bertl A, Slayman CL. Cation-selective channels in the vacuolar membrane of Saccharomyces: dependence on calcium, redox state, and voltage. Proc Natl Acad Sci USA 1990;87;78247828. Breuder T, Hemenway CS, Movva NR, Cardenas ME, Heitman J. Calcineurin is essential in cyclosporin A and FK506 sensitive yeast strains. Proc Natl Acad Sci USA. 1994;91:5372-5376. Nakamura T, Liu Y, Hirata D, et al. Protein phosphatase type 2B (calcineurin)-mediated, FK506sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. EMBO J 1993;12:4063-4071. Davis TN, Urdea MS, Masiarz FR, Thomer J. Isolation of the yeast calmodulin gene: Calmodulin is an essential protein. Cell 1986;47:423-431. Davis TN, Thorner J. Vertebrate and yeast calmodulin, despite significant sequence divergence, are functionally interchangeable. Proc Natl Acad Sci USA. 1989;86:7909-7913. Ohya Y, Anraku Y. Functional expression of chicken calmodulin in yeast. Biochem Biophys Res Commun 1989;158:541-547. Brockerhoff SB, Davis TN. Calmodulin concentrates in regions of cell growth in Saccharomyces cerevisiae. J Cell Biol 1992;118:619-629. Sun G-H, Ohya Y, Anraku Y. Yeast calmodulin localizes to sites of cell growth. Protoplasma 1992;166:110-113. Johnston GC, Prendergast JA, Singer RA. The Saccharomyces cerevisiae MY02 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol 1991;113:539-551. Lillie SH, Brown SS. Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smylp, to the same regions of polarized growth in Saccharomyces cerevisiae. J Cell Biol 1994;125:825-842.

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98. Cheney RE, O'Shea MK, Heuser JE, et al. Brain myosin-V is a two-headed unconventional myosin with motor activity [see comments]. Cell 1993;75:13-23. 99. Kilmartin JV, Dyos SL, Kershaw D, Finch JT. A cell cycle-regulated spacer element in the Saccharomyces cerevisiae spindle pole body. J Cell Biol 1993;123:1175-1184. 100. Ohya Y, Kawasaki H, Suzuki K, Londesborough J, Anraku Y. Two yeast genes encoding calmodulin-dependent protein kinases: Isolation, sequencing and bacterial expressions of CMK1 and CMK2. J Biol Chem 1991;266:12784-12794. 101. Pausch MH, Kaim D, Kunisawa R, Admon A, Thorner J. Multiple Ca2§ protein kinase genes in a unicellular eukaryote. EMBO J 1991;10:1511-1522. 102. Rout MP, Kilmartin JV. Components of the yeast spindle and spindle pole body. J Cell Biol 1990;111:1913-1927. 103. Vallen EA, Scherson TY, Roberts T, van Zee K, Rose MD. Asymmetric mitotic segregation of the yeast spindle pole body. Cell 1992;69:505-515.

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zyx zy Advances in Second Messenger and Phosphoprotein Research, Vol. 30, edited by Anthony R. Means Raven Press, Ltd., New York 9 1995.

13

Calcium Regulation of Drosophila Development Kathy Beckingham

Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251

DROSOPHILA IN GENE FUNCTION STUDIES More than half a century of intense genetic analysis has made Drosophila the multicellular organism of choice for studies of gene function in vivo. A by-product of the high level of genetic understanding has been the development of sophisticated molecular/genetic tools that make reintroduction of genes into the genome and cloning of genes identified by mutant phenotypes or unusual expression patterns relatively routine experimental procedures. In addition, methods that will allow substitution of reintroduced, mutated genes for their wild-type chromosomal counterparts have recently been developed (1). The cumulative effect of these various methodologies has been the generation of a system for the study of gene action that is of unparalleled power and scope. To date, however, the central focus for research in Drosophila has been the identification of key regulatory genes that control pattern formation in development, particularly during embryogenesis. Many of the key genes identified have proved to encode transcription factors and thus the emphasis in the more detailed biochemical work that has followed has been on DNA binding-related activities--that is, areas of research in which Ca 2+ regulation has not yet proved to play a major role. Although some of the genes with key roles in pattern formation are proving to be membrane receptor molecules, analysis of the downstream cytoplasmic signaling for these receptors is in its early stages and the contribution, if any, of Ca 2 + ions to regulation in these pathways is, as yet, largely uninvestigated. However, in the one embryonic pattern formation pathway analyzed thus far from this perspective--that of dorsal-ventral axis formation resulting from activation of the receptor Tollm evidence for a role for Ca 2+ has, in fact, been obtained (2). As yet then, the roles of Ca 2 + in Drosophila development, with the notable exception of its roles in phototransduction, are relatively unexplored processes. The small size of the animal and the strong bias toward genetic studies with the whole

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organism have contributed to this state of affairs. Cell culture systems for Drosophila are underdeveloped when compared to mammalian systems, and the Drosophila community contains few cell physiologists specializing in dynamic studies of intracellular events. Thus, for example, no imaging studies of Ca 2 § fluxes during early embryogenesis comparable to those performed for Xenopus and sea urchin embryos have as yet been performed for Drosophila. Our knowledge of the roles of Ca 2+ in Drosophila have so far come from two major approaches. Firstly, specific mutations have proved upon molecular analysis to affect genes whose products are recognizable as having Ca2 + binding properties or Ca 2 +-regulated behavior. Secondly, the genes for the Drosophila equivalents of proteins known to be involved in Ca 2+ regulation in other organisms have been cloned (largely by sequence similarity to mammalian counterparts) and an analysis of their expression and function has been initiated. Thus, the emphasis has been almost exclusively on cloning of genes that function in Ca2 +-regulated pathways and on studies in the whole organism of the expression and mutant phenotypes of these genes. As in vertebrates, inositol triphosphate (IP3) signaling and calmodulin mediation are emerging as major routes for Ca 2 + regulation in Drosophila. At the same time, a significant number of Ca 2 +-regulated proteins specific to neural and muscle tissues that do not appear to act through these two routes have also been identified in Drosophila. This review is therefore organized in the following manner. Firstly, the current status of knowledge on the roles of Drosophila gene products recognized as components of IP3 signaling pathways will be reviewed, followed by a similar overview of genes with roles in calmodulin-mediated functions. Finally, nerve and muscle-specific proteins that act through a Ca 2+-binding role will be considered. INOSITOL PHOSPHATE SIGNALING PATHWAYS

Receptors Linked to IP Pathways

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Currently the only Drosophila membrane receptors known to activate the inositol phosphate (IP) signaling pathway are the rhodopsins of the compound eyes. In fact, phototransduction in Drosophila is perhaps the best characterized IP3 signaling pathway known--mainly as a result of the ability to use mutants to dissect the roles of individual components of the pathway. Invertebrate vision differs significantly from that of vertebrate vision. In both systems, light-induced changes in rhodopsin lead to G-protein activation, but in vertebrates the ultimate result is closure of cation channels so that light produces an atypical hyperpolarization response. In invertebrates, light stimulation of rhodopsin ultimately acts to open photoreceptor cation channels (for review see 3), leading to a more typical depolarization response.

G Proteins Linked to IP Signaling Pathways A specialized G protein appears to be used for the Drosophila IP signaling system involved in photoreception. A G-protein et-subunit (DGq) which is strikingly simi-

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lar to the mammalian ot-subunit that activates phosphoinositide-specific phospholipase C (PI-PLC) has been identified and shown to be specific to the photoreceptor cells (4). A photoreceptor cell-specific G-protein 13-subunit (Gbe) has also been isolated and is thus a good candidate for the 13 partner of DGq in a photoreceptorspecific trimeric G protein (5). This 13 protein is more divergent from counterparts in other species than the DGq ot-subunit, showing only 43 percent identity to the consensus derived for G protein 13 subunits. The gene maps to 76C1 on chromosome 3L. Thus far mutants have not been identified for either DGq or Gbe. A second set of ct/13 subunits that may function in IP3 signaling in tissues other than the eye has also been identified. The expression patterns of the Go ot-subunit gene dgo (6-8) and the 13 subunit gene Gbb (9) overlap strikingly with that of one isoform of PI-PLC, termed plc-21 (see below), suggesting that they may represent subunits of the upstream activator for this particular isozyme. Again, although the chromosome locations of these two genes are known, mutants within the genes have not yet been identified.

zyxwvu PI-PLC Isoforms

At least two Drosophila genes encoding the key initial enzyme of the IP 3 signaling pathway, PI-PLC, have been isolated. The best characterized of these two genes, norpA, is the PI-PLC used in phototransduction and was isolated via a genetic route, using mutations to lead to the gene. Strong norpA mutations have long been known to abolish all light-induced electrical responses in the photoreceptors of the compound eye (hence the name no receptor potential A). The demonstrations that (a) PI-specific phospholipase C activity of Drosophila heads is concentrated in the eyes and absent in norpA mutants and (b) PI-PLC activity shows temperaturedependent inactivation in temperature-sensitive alleles of norpA (9-11) strongly suggested that norpA encodes a form of PI-PLC. Generation of an allele of norpA by transposon tagging allowed the gene to be cloned (12) and provided the definitive proof that norpA does indeed encode a PI-PLC of the PLC 13subtype (13). This discovery has provided compelling evidence for the central role of PI-PLC in signaling in the invertebrate eye (see Fig. 1). The initial intracellular responses to light are clearly generated as a result of the release of diacylglycerol (DAG) and IP3 by the action of the norpA PI-PLC. It is also true that the role of norpA in vision represents the only situation identified to date in which the role of a specific PI-PLC isoform in a particular signaling pathway is known. Within the Drosophila head, the major site of norpA protein expression is the eight photoreceptors of each compound eye ommatidium (14). Ultrastructural localization has shown that the protein is adjacent to the membranes of the light-sensing structures (rhabdomeres) in each photoreceptor (14). Interestingly, however, both more recent expression studies of norpA (15) and detailed studies of the norpA phenotype (16,17) suggest that norpA has additional signaling roles that are unrelated to photodetection. Thus, norpA protein is found outside the headmin the legs and also male, but not female, abdomens. When linked to the observations that

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FIG. 1. The multiple roles of Ca2§ in Drosophila phototransduction. Ten protein components recognized as generating Ca2+ or responding to Ca2+ in the photoreceptors are shown. Light activation of rhodopsin leads, via a specific G protein, to activation of the norpA PLC. IP3 generated in this way causes release of Ca2+ through the IP3 receptor (IP3-R) channels from the stores in the subrhabdomeric cisternae (SRC). This Ca2+, acting directly or indirectly, is believed to open the trpl cation channels in the plasma membrane, leading to large influxes of Ca2+ and Na + and the initial phase (depolarization) of the light response (69). Opening of the more Ca2+specific channel trp is delayed slightly relative to trpl and is speculated to result from emptying of the SRC Ca2+ stores, possibly sensed by trp via direct protein-protein interaction with IP3-R (69). The inaC PKC, after activation by Ca2§ and DAG, plays a role in downregulating the response (i.e., lowering Ca2+ levels) and could act either by closing the IP3-R channels or activating Ca2+ transport back into the SRC. A transmembrane Ca2+-binding protein of the SRC, rdgB, may be a direct phosphorylation target of inaC and could also function as a Ca2§ transporter. The ninaC kinase/calmodulin (CAM) binding protein also plays a role in terminating the light response and may act in either or both of the following ways: (a) to inactivate rhodopsin or the G protein by phosphorylation or (b) to release Ca2+-saturated CaM for binding and inactivation of trp and trpl.

norpA mutants showed reduced feeding responses to some sugars (15) (an indication that the taste receptors of the legs are altered) and that norpA males initiate courtship and mating more slowly than wild-type males (16,17), these findings suggest norpA plays a role in transducing primary sensory information in other pathways besides vision. The presence of norpA in regions of the central nervous system (CNS) including the optic lobes, brain, and thoracic ganglion (15) further suggests that the signaling role of norpA is not limited to processing of primary sensory information. The second Drosophila PI-PLC gene (plc-21) was isolated by reduced stringency screening of cDNA libraries with norpA-derived probes (18). The encoded protein,

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like norpA, is a member of the PLC 13 subfamily and, as for norpA, several transcripts are produced from the gene, with one being head-specific. Analysis of cDNA clones has demonstrated that at least two of these plc-21 transcripts are generated by alternative splicing, yielding variants of the protein that differ by the presence/absence of a seven amino acid insertion. The plc-21 gene is largely expressed in tissues of the CNS, including the optic lobes, central brain, and thoracic ganglion of the adult and the brain lobes of the larva. In contrast to norpA, however, expression is seen in the female abdomenmspecifically in the developing germ cells of the ovary. Although the plc-21 gene has been mapped to region 21C of chromosome 2, no mutations to the gene are known. Thus, unlike norpA, it is not possible as yet to identify specific signaling pathways that require this enzyme. As noted above, however, G-protein et and 13 subunits that may activate this particular isozyme have been identified.

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Inositol 1, 4, 5 Triphosphate Receptor A full-length inositol 1,4,5-triphosphate receptor (IP3-R) cDNA has been cloned from Drosophila using reduced stringency screening with a mouse IP3-R probe (19). A shorter clone encoding part of the C-terminus of the same gene has also been identified by PCR (20). Reduced stringency hybridization to Southern blots suggests that this IP3-R gene is the single gene of the organism encoding an IP3-R protein (20). The protein encoded by the full-length cDNA clone shows very high sequence similarity to the mouse protein and maintains the same overall topology of the protein domains. Thus, the N-terminal region contains the IP3 binding domain and the six membrane-spanning domains which make up the interior of the Ca 2 + channel which are similarly placed in the C-terminus. A noteworthy difference, however, is the absence in the Drosophila protein of the two potential sites for phosphorylation by cAMP-dependent protein kinase seen in the mouse protein. This suggests differences in regulation of IP3-R activity in the two species. The Drosophila protein also shows a 40 amino acid deletion relative to the mouse protein. This same deletion has recently been recognized as one of the splicing variants of the mouse protein, suggesting that alternatively spliced subtypes of the Drosophila protein may also exist. As shown by both Yoshikawa et al. (19) and Hasan and Rosbash (20), a single 10-kb mRNA species predominates at most developmental stages, although a slightly shorter RNA is detected in the early embryo. Thus, any alternative splicing that does occur produces limited variation in transcript size. The IP3 binding studies and expression studies for IP3-R performed by Yoshikawa et al. (19) allow some indications of the developmental stages and tissues in which IP3 signaling is important in Drosophila. They have shown that the gene is expressed throughout development with peaks of expression in embryogenesis and in the midpupal stages. In situ hybridization studies by Hasan and Rosbash (20) demonstrate that in the late embryo the IP3-R expression is exclusively localized in sense organs of the anterior head. Yoshikawa et al. found that expression in all adult body

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parts examined (head, appendages, and abdomen) is significantly higher than expression in any of the earlier developmental stages, with the appendages (antennae, legs, wings, and sensory hairs) showing by far the greatest expression. These results correlate well with their IP3 binding studies which show that the legs and antennae have the highest level of IP3 binding activity of all body parts examined (19). Yoshikawa and co-workers (19) interpret the antennal IP3-R activity as providing evidence for a role for IP3 in olfaction, a view which is supported by more physiological studies in other insects. The high activity in legs is interpreted in terms of a role for IP3-R in invertebrate muscle signal transduction, for which again there is physiological evidence from other organisms. However, given the findings for norpA mutant flies described above it seems possible that some of the IP3-R activity in the legs is involved in sensory signal transduction from sense organs such as the taste receptors. Surprisingly, although IP3 binding activity is clearly present in the eyes and would be expected given the central role of norpA in phototransduction (see above), the studies by Yoshikawa et al. (19) and Hasan and Rosbash (20) both demonstrate that the overall levels of IP3 binding activity/IP3-R expression in the eye are significantly lower than in the antenna. As yet, no mutants in the IP3 receptor have been identified and thus there are no genetic data on the role of the receptor in the eye.

zyxwvuts Protein Kinase C Isoforms

Three Drosophila genes encoding PKC have been identified to date (21,22). A fourth gene (C3-2) with less similarity to the mammalian PKCs has also been isolated, but as yet is not well characterized (23). Interestingly two of the bona fide PKC genes (termed PKC1 and -2) are positioned within 25 kb of one another in region 53E of chromosome 2 (24). The third gene, PKC3, is at a distant location in region 98F of the third chromosome (22). The two genes at 53E are more similar to one another than to the gene at 98F, with PKC1 showing greatest overall similarity to the classical mammalian PKCs (64 percent identity to mammalian PKC et). The proteins encoded by all three genes possess the major structural features seen in the mammalian PKC enzymes, i.e., (a) a pair of cysteine-containing, putative metal binding "fingers" similar to those seen in the steroid hormone receptors; (b) a C-terminal catalytic domain; (c) a central ATP binding site; and (d) an eight amino acid N-terminal pseudosubstrate region. A further indication of the greater similarity of the PKC1 protein to the mammalian enzymes is the fact that the pseudosubstrate region of this enzyme most resembles that of the mammalian counterparts. The more recently discovered mammalian enzymes, ~ and e, lack a region termed C2 which is present in the et, 13, and ~/classes of enzyme and believed to be involved in enzyme regulation. The PKC3 gene is also missing this region, and overall, is most homologous to the mammalian ~ class of enzymes. PKC3 is the only member of the gene family expressed during the embryonic and larval stages and it is thus likely to play a key role in signaling processes before the

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adult stages. The pattern of transcripts produced from the gene changes during development, offering the possibility of stage-specific isoforms. In the adult, the major site of expression of PKC3 is the head and in situ hybridization has localized the transcripts to the cell bodies of neurons in the brain. Similarly, PKC1, whose major site of expression is the adult head, has been shown to be expressed in all the neural cell bodies in the brain. Mutants have not been identified in either the PKC1 or PKC3 gene, and thus a limited amount is known about their functions in vivo. Their expression in the brain suggests a role in CNS signaling, however. In this context, biochemical and genetic studies of the Drosophila learning mutant turnip (25) are particularly intriguing, since they suggest a role for neuronally expressed PKC activity in learning processes. Flies carrying the turnip mutation are deficient in a number of learning paradigms which, when viewed as a whole, suggest a defect in associative learning (26). Heads of turnip mutant flies show very reduced PKC activity and fractionation of PKC isozymes has demonstrated that at least two different PKC enzymes are affected, with Ca z +/phospholipid-activated PKC activity showing a much stronger effect than that seen for a separable phorbol-ester-stimulated PKC activity. The reduced PKC activity is associated with severely reduced PKC autophosphorylation. The turnip mutation maps to a chromosome location (18A5-18D 1-2), which does not correspond to any of the three known PKC genes or the more distantly related C3-2 gene. In addition, gene dosage correlations and the pleiotropic effects of turnip on more than one type of PKC activity further indicate that turnip does not represent a mutation in a structural gene for PKC. The genetic location of turnip does, however, appear to correspond to the locus for a 76-KDa phosphoprotein (pp76) which is itself a substrate for PKC and whose phosphorylation is markedly decreased in the turnip mutant. Although far from proven, these data suggest two interesting possibilities. At the molecular level they indicate that the phosphorylated turnip gene product (assumed to be pp76) in some way back-potentiates PKC activity, particularly Ca 2 +/phospholipid-activated PKC enzymes. At the behavioral level, this work suggests a role for PKC in associative learning behavior. Most molecular studies, including studies of other learning mutants in Drosophila (see below), have identified the adenylyl cyclase/cAMP-dependent kinase pathway as playing the critical role in associative learning, and thus this suggestion of a role for PKC must be evaluated in the context of these previous findings. A noticeable difference between turnip and learning mutants associated with the cAMP pathway such as rutabaga (mutant in adenylyl cyclase) and dunce (mutant in cyclic nucleotide phosphodiesterase) is that the learning defects in turnip show a much greater susceptibility to modification by genetic background. This could suggest a more indirect role for the PKC system in associative learning than that of the cAMP-dependent kinase pathway. The original turnip mutant showed some reduction in adenylyl cyclase and cyclase-coupled receptor binding activity (27), which could indicate that PKC acts via indirect effects on the cAMP system. Interactions of this type between the C- and A-kinase systems have been identified previously in mammalian cells.

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In direct contrast to PKC1 and PKC3, PKC2 is very specifically expressed only in the photoreceptor cells of both the eyes and the ocelli (22). This finding immediately suggested a role for PKC2 in phototransduction and the subsequent demonstration that three phototransduction mutants (two inaC mutants and US 3741) are all mutants in the PKC2 gene (24) has confirmed this hypothesis and permitted detailed functional studies of its role. The two inaC mutants were originally identified as failing to produce the prolonged depolarizing afterpotential (PDA) normally seen in response to cessation of a lengthy light stimulus. The initial depolarization, or inactivation, of the photoreceptors in response to light is normal, however, leading to the name inactivation-no-afterpotential mutants. Like the inaC mutants, the US 3741 mutation was identified as showing a normal inactivating depolarization but as being defective in the repolarization of the photoreceptors after a stimulating flash (24). Thus, these initially recognized properties of PKC2 mutants suggested that PKC2 is not involved in the signal pathway that produces the initial response to light, but rather plays a role in the subsequent inhibition and modulation of that response. In other signaling pathways there is evidence that PKC activated by Ca 2 +/DAG released by the initial action of PI-PLC plays a role in subsequent signal modulation. Later physiological studies uncovered two further defects in the light responses of the inaC mutants (28). Not only does repolarization occur much more slowly, but these mutants also (a) fail to adapt to light stimuli and (b) upon exposure to continuous intense bright light, ultimately lose the ability to maintain any depolarization, with the photoreceptors finally returning to resting polarized state. In contrast, wildtype flies maintain a partially depolarized state during prolonged light exposure. In terms of understanding events at the molecular level, two key discoveries have been the demonstrations that (a) both the depolarization and repolarization aspects of the light response depend upon Ca 2 + ion fluxes into the photoreceptor (29) and (b) that the primary defect in the inaC mutants appears to be a failure to terminate the quantal releases of Ca 2 + that cumulatively produce the depolarization phase of the light response (28). There is good evidence that the initiating Ca 2 + is released from intracellular stores through the IP3-R Ca 2+ channel in response to IP3 generated by the norpA PI-PLC, and thus the current prediction on the role of PKC2 is that it regulates closure of these channels. Most of the physiological defects associated with the mutants such as failure to adapt and failure to repolarize can readily be seen to follow from this proposed molecular defect, but the complete repolarization on exposure to prolonged light is not an immediately obvious prediction of this model. However, it is believed that this phenomenon results from a complete exhaustion of the intracellular Ca 2 + stores which would result in the total inability to respond to light (28). If PKC2 is acting in the manner described above, that is, to modify the IP3-induced release of Ca 2 + from intracellular stores, the critical target of PKC2 is likely to be a signaling component required to produce this Ca 2 + release. It seems unlikely that the initiator of Ca 2 + release, the norpA PLC itself, is the critical PKC2 target, since the defects in quantal Ca 2 + release seen in a temperature-sensitive

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norpA mutant are not equivalent to those seen for inaC mutants (68). Thus, the IP3-R or, alternatively, an as yet unidentified Ca 2 + pump involved in the resequestration of Ca 2 + ions, seem more likely targets. In this context, the interaction of PKC2 mutants with mutants of the rdgB locus is particularly interesting. In the mutation rdgB, the retinal photoreceptor cells are completely normal in dark-reared flies, but undergo degeneration upon exposure to light (30). Flies carrying rdgB are completely protected from this effect if they are also completely lacking (that is, null) for PKC2 function (24). The simplest explanation for these findings is that PKC2 activity effects the functioning of the wildtype version of the rdgB gene, indirectly or directly, via phosphorylation. As described below, the sequence of rdgB indicates that it is an integral membrane protein of the photoreceptors, with both Ca 2 + binding and phosphatidylinositol transfer activities, and some structural similarity to Ca 2 + transporters. The potential roles of inaC and rdgB in phototransduction are shown in Fig. 1.

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PKC Targets As described above, the turnip gene product is a putative PKC target. Although several cloned Drosophila genes have potential PKC phosphorylation sites, the product of the igloo gene, which is a GAP-43-related protein, (see below) is the only Drosophila protein shown definitively to be a PKC target. CALMODULIN SIGNALING

Calmodulin In contrast to other multicellular organisms, Drosophila contains only one geneencoding calmodulin (31,32). The calmodulin encoded by this single gene (32,33) shows a very high level of similarity to vertebrate calmodulin, containing only three conservative amino acid substitutions relative to the mammalian sequence. As for other insects (34), it seems likely that Drosophila calmodulin does not show the posttranslation trimethylation modification of lysine 115 found on all mammalian calmodulins. Uniquely for the Ca 2 + signaling molecules of Drosophila, a significant number of in vitro protein structure/function studies have been performed for Drosophila calmodulin. Thus, the crystal structure for the wild-type protein is known (35), and the first published structure of calmodulin bound to a target peptide binding region was derived for Drosophila calmodulin as opposed to mammalian calmodulin (36). In addition, two series of Ca 2 +-binding site mutants of Drosophila calmodulin have yielded insight into the function of the four individual Ca 2 +-binding sites of calmodulin in (a) overall Ca2+-binding (37,38); (b) Ca2+-induced conformational changes (39-41); and (c) binding of both target peptides (42) and target enzymes (43). A major conclusion to emerge from these studies is that the effects of mutating

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the individual sites within each of the two pairs of Ca 2+-binding sites of the protein are not symmetrical, but rather within each pair a dominant Ca 2+-binding site may be identified. A consideration of all of the studies performed with these mutants has permitted a ranking of the four sites in terms of their overall contributions to the Ca 2 +-induced changes in the protein. Thus, site 4 (the most C-terminal site) has emerged as the most critical site of the protein, followed by site 2, site 3, and finally, site 1. The complete structure of the genomic version of the calmodulin gene is known and thus the exact structure of the transcripts derived from the gene is understood (31-33,44). A single transcription start site is used to generate two mRNAs that differ in size by 200 bp as a result of alternative polyadenylation signal usage. Thus, as for mammals, no tissue-specific isoforms of the protein are produced. Interestingly, the use of the two polyadenylation signals is developmentally regulated, with the more proximal signal, producing a 1.7 kb mRNA, dominating during oogenesis and thus generating the transcripts stored in the mature egg and the more distal signal being used for most of the transcripts produced during embryogenesis and later stages (45). Given the ubiquitous distribution of calmodulin in the differentiated tissues of mammals, the extremely tissue-specific nature of calmodulin mRNA expression during Drosophila embryogenesis is an unexpected finding (45). Although the mature egg contains very high levels of maternally derived calmodulin mRNA, by the time embryogenesis is 25-30 percent completed, these transcripts have largely disappeared. Transcription of the gene is then activated in only two cell lineagesmthe neuroblasts of the CNS and at a later time point, in the equivalent precursors of the peripheral nervous system (PNS). At the end of embryogenesis, the mature neurons of the CNS and PNS are essentially the only cells of the organism that express calmodulin mRNA (45). This discovery, while suggesting a key role for calmodulin in the neural lineages, should not be taken to indicate that calmodulin is not required in other tissues of the embryo. The maternal mRNA present in the mature egg is almost certainly used to generate calmodulin protein, and this could provide sufficient protein for all other developing tissues of the embryo. Beyond the embryonic stages, the expression of the gene continues to be highly regulatedmboth in terms of tissue specificity and developmental stage-specificity (Andruss, B. and Beckingham, K., unpublished data). Expression in the nervous system is interesting in that, as the larval and pupal stages are traversed, periods of intense expression alternate with periods of little or no calmodulin transcription. Within the adult brain and the larval CNS, striking differences in calmodulin mRNA levels can be seen within neighboring cells that, at the anatomical level, appear indistinguishable. The compound eye is a major site of calmodulin transcription, with transcripts concentrated in the photoreceptors. In addition to strong expression in neural cells, a survey of the tissues which show intense calmodulin transcription throughout development reveals a strong correlation with secretory functions. For example, in the larval stages the cardia (a secretory section of the anterior gut) and the salivary glands both show strong ex-

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pression. Similarly, in the adult female, the follicle cells of the ovary (whose function is to secrete the egg shell) show the most intense staining seen within the abdomen. The germ line cells of the ovary also show strong transcription, as might be predicted given the high levels of transcripts found in the mature egg (see above). To date it has not been determined whether, as in mammalian species, the testis is a rich source of calmodulin expression. The recent identification of mutations within the Drosophila calmodulin gene has permitted an analysis of the roles of the protein during development to be initiated (Kovalick, G., Atkinson, R., Heiman, R., and Beckingham, K., unpublished data). Two mutations, both resulting from insertion of transposons (P element DNA) into the gene were initially identified. One of these insertions has been used to generate deletion mutants that lack the transcription start site and, as a result, produce no calmodulin transcripts. These mutants are being used to define the null phenotype--that is, the developmental effects of total loss of calmodulin gene expression. It has been established that calmodulin null embryos do not die during embryogenesis, but rather hatch and die subsequently during the early larval stages. In addition, the differentiation of both the CNS and PNS during embryogenesis appear essentially normal. It must be concluded, therefore, that the specific activation of calmodulin transcription seen in the neural precursors (see above) is not required for the structural formation of the nervous system, but rather has some role in subsequent functioning of the tissue. Current investigations are focused on determining the cause of death in larvae null for calmodulin function. Prior to death the larvae appear sluggish and do not grow at the normal rate. Preliminary evidence suggests they are defective for several neurological functions, most notably chemosensation (Heiman, R. and Beckingham, K., unpublished data). These findings corroborate the suggestion made above that the expression of calmodulin in the embryonic nervous system has a functional rather than a structural role. It is not clear that neurological defects are the ultimate cause of death for calmodulin null larvae, however. It seems more likely that vital role of calmodulin during the larval stages is linked to the tremendous growth phase initiated soon after hatching.

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Adenylyl Cyclase Ca 2 +-/calmodulin-activated adenylyl cyclase represents one of the few examples thus far within Ca 2 + signaling in Drosophila where a mutant phenotype has led to a prediction on the nature of the encoded protein and ultimately to the isolation and cloning of that gene. The mutation rutabaga (rut 1) was originally isolated as part of a large screen to identify mutations affecting learning and memory (46). Prompted by the finding that the learning mutant dunce is defective in cyclic nucleotide phosphodiesterase activity, extracts of rut ~ flies were examined for various aspects of cyclic nucleotide metabolism and shown to be strikingly deficient in Ca2+/cal modulin-activated adenylyl cyclase (46). Gene dosage experiments supported the

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idea that the rut 1 gene encoded a catalytic subunit of the enzyme rather than a regulatory activity. Surprisingly, abdominal rather than head extracts showed greatest loss of cyclase activity, suggesting the presence of multiple forms of the cyclase, with the rutabaga-encoded protein representing a greater fraction of total activity in the body than in the head. Fine mapping of the mutation placed the gene in region 12F5-7 of the X chromosome. The rut gene was finally cloned by two convergent routes. Probes derived from bovine type I and rat type II adenylyl cyclases allowed cloning of four different Drosophila adenylyl cyclase homologues, one of which mapped to the cytological location of rutabaga (47). Simultaneously, a screen to identify 13-galactosidase enhancer trap lines expressed at high levels in brain structures associated with learning and memory (the so-called mushroom bodies) identified seven lines, all of which have proved to be insertions of the 13-galactosidase-expressing transposon into the 5' region of this cyclase gene (48). Several of these insertion lines show effects on learning and memory comparable to those seen with the original rut ~ mutation and at least three of them have proved to be allelic to r u t l ~ t h u s conclusively establishing that rutabaga does indeed encode an adenylyl cyclase gene. The rutabaga protein is very l a r g e ~ a total of 2,249 residues (47). The N-terminal half is about the same size as mammalian type I cyclase, and it is this half of the protein that contains all the similarity to the mammalian enzyme. The same overall arrangement of two sets of six membrane-spanning regions and two proposed catalytic domains is present. The two putative cytoplasmic domains of rutabaga show strong conservation when compared to all known mammalian adenylyl cyclases but are most similar to the bovine type I enzyme (75 percent and 57 percent identity). The original rut ~ mutation was identified and shown to be a substitution of arginine for a conserved glycine in one of the putative cytoplasmic catalytic domains (47). The properties of the encoded cyclase were studied by examining activity after expression in a human cell line under a strong constitutive promoter (47). This allowed several important findings concerning the properties of the enzyme. Firstly, the rut 1 mutation completely inactivates the enzyme. Further, in addition to demonstrating the expected Ca 2 +/calmodulin stimulation, the cyclase was also shown to be capable of G-protein activation. This is an important discovery, since in other systems adenylyl cyclase has been proposed to act as an integrator of signals from distinct signaling pathways during learning and memory processes. Thus, contrary to prior predictions (49), rutabaga may act in this manner in Drosophila. The isolation of several alleles of rut as enhancer trap lines showing preferential expression in the mushroom bodies predicted that the rut gene itself would show strong expression in these structures and this has proved to be the case~as demonstrated by probes for both the RNA and the protein (48). Not surprisingly, the enhancer trap lines which proved to be strong alleles of rut show almost complete loss of this expression. The previous discovery that the cAMP-specific phosphodiesterase encoded by dunce is strongly expressed in the mushroom bodies suggests that these structures play a central role in learning processes. The demonstration that rut is also strongly expressed here strengthens this hypothesis. Interestingly, however, the expression patterns for these two proteins do not overlap completely in the

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mushroom bodies (48). The region containing the dendritic processes of the mushroom body neurons contains high levels of dunce protein and very little rut protein. The roles of these two proteins in regulating cAMP levels may thus show subtle spatial variations. Given that dunce mutations produce high levels of cAMP due to loss of the degradative enzyme and rut mutations produce low levels as a result of loss of the synthetic enzyme, the hypothesis that the learning defects of both mutant types might be corrected in a double rut~dunce mutant has been tested (46). No rescue of learning is detected, however, suggesting that the ability to modulate cAMP levels as opposed to maintaining absolute levels is critical to learning processes. The overall expression of rut in the head has been examined in detail, and other neural structures that express rut at lower levels than the mushroom bodies have been identified (48). The role of rut outside the head has not yet been investigated, however. As discussed above, there is significant cyclase activity in structures outside the head. Clearly, no essential function in the head, thorax, or abdomen is performed by rut, since rut I is a null mutation and yet produces only neurological defects. It is possible that the other adenylyl cyclases shown to be present in the organism (see above) perform more vital functions. The rut gene is structurally complexmcomprising 16 exons spanning over 30 kb and generating two major transcripts of 7.5 and 9.5 kb (47). Whether these encode isoforms of the protein has not yet been established. Given the effects of rut mutations on learning and memory, there is considerable interest in examining the defects in neuronal transmission caused by the mutation. It is technically difficult to study synaptic activity in key brain regions like the mushroom bodies, but on the assumption that rut might affect all synaptic activity, Zhong and Wu (50) have examined the effect of the null mutation on transmission at the larval neuromuscular junction, rut 1 mutants showed reduced facilitation and impaired posttetanic potentiation whereas dunce mutants showed changes consistent with higher transmitter release. Mutants of both loci gave abnormal synaptic responses to direct application of dibutyryl cAMP. These findings establish that through its effect on cAMP metabolism, rut plays a general role in synaptic activity throughout the animal. Interestingly, rut and dunce affect not only the activity of synapses but also their morphology, presumably as an indirect result of the changes in activity (51). Thus the rut ~ mutant produces a slight but statistically significant reduction in the number of synaptic boutons and branches at the nerve termini of the larval neuromuscular junction, with dunce producing more striking changes of the opposite directionm greater numbers of branches and terminal boutons. The effects of dunce were found to be suppressed in the dunce rut double mutant.

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Type II Protein Kinase

The considerable evidence that brain calmodulin-activated protein kinase II (CaM kinase) plays a major role in mammalian learning and memory processes has been a major impetus for isolating the Drosophila equivalent(s) of this enzyme. Cho et al.

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(52) established that Drosophila heads are rich in kinase activity with the expected characteristics of CaM kinase and showed that three isoforms with molecular weights very similar to the rat or, 13 and 131 subunits can be precipitated from head homogenates by an antibody against rat brain CaM kinase. Using PCR to generate a short probe for screening of head cDNA libraries, Cho et al. then proceeded to isolate the full-length cDNA sequence for an a-type CaM kinase subunit. The protein shows very high conservation when compared to the rat ot-subunit (85 percent identical in the N-terminus and 68 percent identical in the C-terminus). All of the essential domains delineated in the rat protein are conserved with the calmodulinbinding regions and nearby inhibitory regions being nearly identical. Similarly, two key threonine residues whose autophosphorylation results in loss of Ca 2 § ulin regulation of the enzyme are also conserved. Genomic DNA Southern blot analysis indicates that this et-subunit is a unique gene, the location of which was shown by in situ hybridization to be region 102E/F on the fourth chromosome. This position is unfortunate from a genetic perspective since the tiny fourth chromosome is intractable to genetic analysis. The demonstration by Cho et al. that this CaM kinase ot-subunit gene produces essentially one size class of transcripts might suggest that a single protein product is derived from the gene. This has proved to be far from the case, however. Using reduced stringency screening with rat CaM kinase et and 13 subunit probes, Ohsaka et al. (53) were able to isolate cDNAs encoding three further isoforms of the protein, each containing one or more additional short (8-22 residues) stretches of amino acids (termed b, c and d), all inserted at the same position within the protein, downstream of the calmodulin-binding regulatory domain. Clones containing insert combinations of b, b + c, and b + d were isolated. The insert position is close to that at which additional residues are detected in the 13 and ~/forms of rat CaM kinase relative to rat CaM kinase or, but nevertheless, it is distinct from that position. The insertion sequences are also quite different from those seen for the rat 13 and ~t insertions. Ohsaka et al. provide evidence that the three CaM kinase isoforms detected in Drosophila heads (see above) correspond to the 490-residue form sequenced by Cho et al. and the 509- and 530-residue sequences identified in their laboratory. The third, 516-residue, isoform they identified appears to be a fairly rare species. From analysis of the genomic version of the gene encoding these isoforms, Ohsaka et al. were able to show that these variants arise by alternative splicing, in some cases involving use of two alternative splice sites for the same exon. PCR studies suggested that even further isoforms might exist, and recent studies by Griffith and Greenspan (54) demonstrate that up to 18 variants of the protein are possible. This additional variation arises from the discovery that (a) insertions seen only together in the Ohsaka clones can exist as separate entities (see above); and (b) an N-terminal glutamic acid residue and a C-terminal alanine are also variably present at the insertion site. The 18 possible CaM kinase variants are shown in Fig. 2. The expression patterns of the individual isoforms will be difficult to delineate, given how subtly some of the variants differ from one another. Using probes that

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