Small GTPases and Their Regulators, Part B: Rho Family [1st ed.] 0121821579, 9780121821579

Table of contents :
Table of Contents......Page 6
Contributors to Volume 256......Page 10
Preface......Page 13
Section I Expression. Purification. and Posttranslational Modification......Page 14
[ 1] Purification of Recombinant Rho / Rac / G25K from Escherichia coli......Page 16
[2] Purification of Baculovirus-Expressed Cdc42Hs......Page 24
[3] Purification and Properties of Rac2 from Human Leukemia Cells......Page 28
[4] Purification of Rac2 from Human Neutrophils......Page 38
[5] Purification of Rac-GDP Dissociation Inhibitor Complex from Phagocyte Cytosol......Page 46
[6] Purification and Properties of Recombinant Rho-GDP Dissociation Inhibitor......Page 54
[7] Prenylcysteine-Directed Carboxyl Methyltransferase Activity in Human Neutrophil Membranes......Page 62
Section II Guanine Nucleotide Exchange and Hydrolysis......Page 78
[8] Measurement of Intrinsic Nucleotide Exchange and GTP Hydrolysis Rates......Page 80
[9] Guanine Nucleotide Exchange Catalyzed by dbl Oncogene Product......Page 90
[10] Stimulation of Nucleotide Exchange on Ras- andRho-Related Proteins by Small GTP-Binding Protein GDP Dissociation Stimulator......Page 98
[11] Interaction of Ect2 and Dbl with Rho-Related GTPases......Page 103
[13] Purification and GTPase-Activating Protein Activity of Baculovirus Expressed p 190......Page 118
[ 14] GTPase-Activating Protein Activityof n(al)-Chimaerin and Effect of Lipids......Page 127
[15] Characterization of Breakpoint Cluster Region Kinase and SH2-Binding Activities......Page 138
[16] Identification of GTPase-Activating Proteins by Nitrocellulose Overlay Assay......Page 143
[17] Identification of 3BP- 1 in cDNA Expression Libraryby SH3 Domain Screening......Page 153
Section III Cell Expression and in Vitro Analysis......Page 162
[18] Serum Induction of RhoG Expression......Page 164
[19] Microinjection of Epitope-Tagged Rho Family cDNAs and Analysis by Immunolabeling......Page 175
[20] Purification and Assay of Recombinant C3 Transferase......Page 187
[21] In Vitro ADP-Ribosylation of Rho by Bacterial ADP-Ribosyltransferases......Page 197
[22] Preparation of Native and Recombinant Clostridiumbotulinum C3 ADP-Ribosyltransferase and Identification of Rho Proteins by ADP-Ribosylation......Page 209
[23] In Vitro Binding Assay for Interactions of Rho and Rac with GTPase-Activating Proteins and Effectors......Page 220
[24] Purification and Assay of Kinases That Interact with Rac / Cdc42......Page 228
[25] Yeast Two-Hybrid System to Detect Protein-Protein Interactions with Rho GTPases......Page 241
[26] Assay for Rho-Dependent Phosphoinositide 3-Kinase Activity in Platelet Cytosol......Page 254
[27] Neutrophil Phospholipase D: Inhibition by Rho-GDP Dissociation Inhibitor and Stimulation by Small GTPase GDP Dissociation Stimulator......Page 259
[28] Measurement of Rac Translocation from Cytosol to Membranes in Activated Neutrophils......Page 269
[29] Reconstitution of Cell-Free NADPH Oxidase Activity by Purified Components......Page 281
Section IV. Biological Activity......Page 292
[30] Genetic and Biochemical Analysis of Cdc42p Function in Saccharomyces cerevisiae and Schizosaccharomyces pombe......Page 294
[31] Lymphocyte Aggregation Assay and Inhibition by Clostridium botulinum C3 ADP-Ribosyltransferase......Page 303
[32] Inhibition of p21 Rho in Intact Cells by C3 Diphtheria Toxin Chimera Proteins......Page 310
[33] Growth Factor-Induced Actin Reorganization in Swiss 3T3 Cells......Page 319
[34] Microinjection of Rho and Rac into Quiescent Swiss 3T3 Cells......Page 326
[35] Inhibition of Lymphocyte-Mediated Cytotoxicity by Clostridium botulinum C3 Transferase......Page 333
[36] Neutrophil Chemotaxis Assay and Inhibition by C3 ADP-Ribosyltransferase......Page 340
[37] Cell Motility Assay and Inhibition by Rho-GDP Dissociation Inhibitor......Page 349
[38] Cell Transformation by dbl Oncogene......Page 360
[39] Inhibition of Rac Function Using Antisense Oligonucleotides......Page 371
Author Index......Page 380
Subject Index......Page 398