Regulators and Effectors of Small GTPases: Ras Family (eBook)

Cover Page 1
Table of Contents 6
Contributors to Volume 407 12
Preface 20
Volumes in Series 22
Chapter 1: Ras Superfamily and Interacting Proteins Database 45
Introduction 45
A Database of Ras Superfamily Proteins and Their Regulators 46
References 52
Chapter 2: Real-Time In Vitro Measurement of Intrinsic and Ras GAP-Mediated GTP Hydrolysis 53
Introduction 54
Experimental Procedures 56
Data Analysis 64
Concluding Remarks 65
References 65
Chapter 3: Schwann Cell Preparation from Single Mouse Embryos: Analyses of Neurofibromin Function in Schwann Cells 66
Introduction 67
Materials and Methods 69
Characterization of Phenotypes of Schwann Cells Derived from Wild-Type and Nf1 Mutant Mice 73
Summary 75
Acknowledgments 75
References 75
Chapter 4: Regulation of the Nucleotide State of Oncogenic Ras Proteins by Nucleoside Diphosphate Kinase 77
Introduction 77
Methods 79
Establishing a "Medium-Throughput" GTPase Activity Assay 81
Detection of Protein-Bound Nucleotides by 96-Well Vacuum Filtration 81
Detection of Protein-Bound Nucleotides by Thin-Layer Chromatography 81
Characterization of a Bacterial Lysate Component with GTPase-Stimulating Activity 82
Purification and Identification of the Protein Responsible for Lysate Activity 83
NDK Is the E. coli Protein Responsible for Inactivation of Oncogenic Ras 84
GTPase-Inactivating Activity of NDK Is Specific to Mutant Ras Enzymes 84
Nucleotides Do Not Detectably Dissociate from Ras During the Reaction with NDK 85
NDK Catalyzes the Reverse Reaction: Conversion of Ras-GDP to Ras-GTP 86
Conclusions 88
Acknowledgments 89
References 89
Chapter 5: Measurements of TSC2 GAP Activity Toward Rheb 90
Introduction 90
In Vitro Assay of TSC2 GAP Stimulated Rheb GTP Hydrolysis 91
TSC2 GAP-Stimulated Rheb GTP Hydrolysis In Vivo 94
TSC2 Inhibits the Phosphorylation of Thr-389 of S6K1 96
Conclusion and Discussion 97
References 98
Chapter 6: Characterization of AND-34 Function and Signaling 99
Overview 99
Retrovirus-Mediated Analysis of AND-34-Induced Cdc42 Activation in Lymphoid Cell Lines 100
AND-34-Induced Cdc42 and Rac Activation in MCF-7 Cells 102
PAK1 and Akt Kinase Assays 103
Measurement of R-Ras GTP Levels 104
Acknowledgments 106
References 106
Chapter 7: Studying the Spatial and Temporal Regulation of Ras GTPase-Activating Proteins 108
Introduction 108
Methods 111
Conclusion 124
Acknowledgments 124
References 124
Chapter 8: Activation of Ras Proteins by Ras Guanine Nucleotide Releasing Protein Family Members 126
Introduction 126
Ras Guanine Nucleotide Releasing Proteins (RasGRPs) 127
Analysis of RasGRP Protein Activity in Cells 132
Concluding Remarks 140
References 140
Chapter 9: Ras and Rap1 Activation of PLCepsiv Lipase Activity 143
Introduction 143
Methods 145
References 150
Chapter 10: Specificity and Expression of RalGPS as RalGEFs 152
Background 152
Expression of RalGPS1A/B in 293T Cells 153
Production and Purification of GST Fusion Proteins 153
Ras Protein Binding Assay 154
SH3 Binding Assays 155
In Vivo Exchange Assay 156
Subcellular Fractionation 157
Binding of RalGPS PH Domain to Phospholipids 157
References 158
Chapter 11: Biochemical and Biological Analyses of Rgr RalGEF Oncogene 159
Introduction 159
Characterization of the Rabbit Rgr Oncogene 161
Molecular Analysis of the Human Rgr Oncogene 168
Summary 170
Acknowledgments 171
References 171
Chapter 12: Analysis of Ras Activation in Living Cells with GFP-RBD 172
Introduction 173
Cloning 178
Cell Culture and Transfection 179
Imaging and Stimulation 181
Post-acquisition Image Analysis 182
Bystander FRET 183
Conclusions 186
Acknowledgments 186
References 187
Chapter 13: Genetic and Pharmacologic Analyses of the Role of Icmt in Ras Membrane Association and Function 188
Introduction 188
Methods 190
Experimental Results 193
Pharmacologic Inhibition of Icmt 199
Conclusion and Future Directions 200
Acknowledgments 200
References 201
Chapter 14: Characterization of the Activation of the Rap-Specific Exchange Factor Epac by Cyclic Nucleotides 203
Introduction 204
General Overview 205
Technical Procedure 208
Conclusion 211
Appendix 211
Acknowledgments 216
References 217
Chapter 15: Biochemistry of the Rap-Specific Guanine Nucleotide Exchange Factors PDZ-GEF1 and -2 218
Introduction 218
In Vitro Analysis of PDZ-GEF Regulation 220
In Vivo Analysis of PDZ-GEF Regulation 227
Discussion 228
Acknowledgments 229
References 229
Chapter 16: Characterization of Interactions Between Ras Family GTPases and Their Effectors 231
Introduction 231
In Vitro Interactions with Purified Proteins 232
Interactions in Mammalian Cells 236
Acknowledgments 238
References 238
Chapter 17: Genetic and Pharmacologic Dissection of Ras Effector Utilization in Oncogenesis 239
Introduction 239
Reagents for Assessment of Effector Sufficiency 240
Reagents for Assessment of Effector Necessity 246
Retrovirus, Cell Culture, and Effector Expression Verification Methods 251
Conclusion 257
References 257
Chapter 18: Sequencing Analysis of BRAF Mutations in Human Cancers 262
Introduction 262
Methods 264
References 268
Chapter 19: KSR Regulation of the Raf-MEK-ERK Cascade 268
Introduction 269
Analyzing the ERK Scaffolding Activity of KSR Using the Xenopus laevis Oocyte Meiotic Maturation Assay 270
Analyzing the ERK Scaffolding Activity of KSR in Mammalian Cells 277
Determining KSR Subcellular Localization 278
Acknowledgments 279
References 279
Chapter 20: Ras-Sensitive IMP Modulation of the Raf/MEK/ERK Cascade Through KSR1 281
Introduction 281
Methods 283
Conclusions 290
References 290
Chapter 21: Raf Kinase Inhibitor Protein Regulation of Raf and MAPK Signaling 292
RKIP Functions 292
RKIP Gene Family 294
Methods 295
References 302
Chapter 22: Harnessing RNAi for Analyses of Ras Signaling and Transformation 303
Introduction 303
Protocols 305
References 311
Chapter 23: The Rac Activator Tiam1 and Ras-Induced Oncogenesis 313
Introduction 313
Tiam1 Deficiency and Mouse Tumor Models 315
Acknowledgment 323
References 323
Chapter 24: Phospholipase Cepsi Guanine Nucleotide Exchange Factor Activity and Activation of Rap1 325
Introduction 326
In Vitro Measurement of GEF Activity of PLCepsi 327
Rap1bull1GTP Pull-Down Assay 328
Role of the CDC25 Homology Domain of PLCepsi in Prolonged Increase in Phospholipase Activity of PLCepsi 331
References 333
Chapter 25: Nore1 and RASSF1 Regulation of Cell Proliferation and of the MST1/2 Kinases 334
Introduction 335
Nore1/RASSF1 Polypeptides Inhibit Cell Proliferation and Are Candidate Tumor Suppressors 337
RASSF1A/Nore1A Inhibit G1 Progression Independently of Ras-Like GTPases 339
RASSF1 and Nore1A Participate in Ras-Induced Apoptosis 340
RASSF1A Associates with the Microtubular Apparatus and Controls APC/Cyclosome Activation in Early Mitosis 341
Nore1B/RAPL Mediates Rap1 Regulation of Integrin Activation 342
The Nore1/RASSF Polypeptides All Bind the Class II GC Kinases, MST1 and MST2 342
The MST1/2 Kinases Mediate Ras-Induced Apoptosis 345
Regulation of the MST Kinase Activity by the Nore1/RASSF1 Polypeptides 346
A Phosphopeptide-Specific Antibody Specific for Activated MST1/2 346
Quantitative Assay of MST1/2 Activation In Vitro 347
Estimation of MST Activation In Vivo 348
Effect of NORE1/RASSF1 on MST Activation In Vitro and In Vivo 349
Effect of Small GTPases on the Activity of Recombinant MST1 350
Acknowledgments 350
References 350
Chapter 26: RASSF Family Proteins and Ras Transformation 355
Introduction 355
Ras Binding Assays 356
Biological Assays 358
References 365
Chapter 27: RAS and the RAIN/RasIP1 Effector 366
Introduction 367
Analysis of the RAIN-Ras Interaction 367
Visualization of the RAIN-Ras Interaction In Vivo 372
Endogenous RAIN Expression in Endothelial Cells 374
Concluding Remarks 378
Acknowledgments 378
References 378
Chapter 28: The RIN Family of Ras Effectors 379
Introduction 379
Assays of RIN Protein Function 381
Immunological Reagents for RIN Protein Analysis 384
RIN Family Evolution 384
RIN Genes Have Distinct Patterns of Expression 385
Acknowledgments 387
References 387
Chapter 29: Rap1 Regulation of RIAM and Cell Adhesion 389
Introduction 389
Yeast Two-Hybrid System or Interaction Trap 390
In Vitro Protein-Protein Interaction by Pull-Down Assays 392
Coimmunoprecipitation of Active Rap1 and RIAM after Physiological Stimulation 395
Adhesion Assay to Immobilized Substrate 396
Assessment of Integrin Activation by Activation Epitope Exposure 399
Intracellular Localization of Active Rap1 399
References 401
Chapter 30: Regulation of Cell-Cell Adhesion by Rap1 403
Introduction 403
Cell Culture 404
Rap1 Localization in Epithelial Cells 404
Rap Activation by Cadherin-Mediated Adhesion 410
Determination of Rap Role in Cadherin-Dependent Cell-Cell Contacts 413
References 415
Chapter 31: Effects of Ras Signaling on Gene Expression Analyzed by Customized Microarrays 417
Introduction 417
ROSE 199 and KRAS-Transformed A2/5 Cells as a Model for the Tumorigenic Conversion of Ovarian Surface Epithelium 418
Total RNA Isolation, mRNA Preparation, and cDNA Synthesis 419
Suppression Subtractive Hybridization and Establishment of Subtracted cDNA Libraries 420
Sequence Analysis of Subtracted Libraries and Validation of Differential Expression 422
Oligonucleotide Microarray Design 423
Preparation of Slides and Spotting 423
RNA Labeling and Microarray Hybridization 425
Microarray Evaluation 428
Discussion 428
Acknowledgments 430
References 431
Chapter 32: Protein-Fragment Complementation Assays (PCA) in Small GTPase Research and Drug Discovery 432
Introduction 432
Methods 433
Applications and Examples 436
Conclusions 443
Acknowledgments 444
References 444
Chapter 33: Ras Up-Regulation of Cyclooxygenase-2 445
Introduction 446
Analysis of COX-2 Up-Regulation by Ras Signaling 447
Analysis of PGE2-Mediated Up-Regulation of Ras Signaling 450
Conclusion 453
Acknowledgments 453
References 453
Chapter 34: Regulation of the Expression of Tropomyosins and Actin Cytoskeleton by ras Transformation 454
Introduction 454
The Model System 455
TM mRNAs in Normal and ras-Transformed Fibroblasts 456
Downregulation of TM Proteins by Ras 457
Role of TMs in Regulation of Cytoskeleton and Cell Phenotype 462
Summary 463
References 463
Chapter 35: Regulation of Par-4 by Oncogenic Ras 466
Introduction 466
Analysis of Protein Expression Regulated by Ras 468
Analysis of the Mechanism of Down-Regulation of Par-4 by Oncogenic Ras 473
Analysis of the Functional Significance of Ras-Mediated Down-Regulation of Par-4 479
References 485
Chapter 36: Using Drosophila and Yeast Genetics to Investigate a Role for the Rheb GTPase in Cell Growth 487
Introduction 487
Results and Methods 488
Acknowledgments 496
References 496
Chapter 37: Biochemistry and Biology of ARHI (DIRAS3), an Imprinted Tumor Suppressor Gene Whose Expression Is Lost in Ovarian and Breast Cancers 499
Introduction 499
Imprinted Genes Can Contribute to Human Carcinogenesis 500
ARHI Encodes a 26-kDa GTPase with Homology to Ras 501
ARHI Is Dramatically Downregulated in Most Ovarian and Breast Cancers 504
ARHI Is Monoallelically Expressed and Maternally Imprinted 505
Expression from the Paternal Allele of ARHI Can Be Lost Through LOH, CpG Methylation, and Transcriptional Regulation 505
A Homolog of ARHI Is Not Found in Mice, but Mice Bearing the Human ARHI Transgene Have a Distinct Phenotype 507
Overexpression of ARHI in Cancer Cells Induces Caspase-Independent, Calpain-Dependent Apoptosis, Whereas Physiological Expression of ARHI Induces Autophagy 507
ARHI Regulates Signal Transduction 509
Conclusion 509
Acknowledgment 510
References 510
Chapter 38: Gem Protein Signaling and Regulation 512
Introduction 513
Inhibition of Rho Kinase-Mediated Cytoskeletal Reorganization 514
Redirection of Rho Kinase Substrate Specificity 518
Procedure 520
Inhibition of Voltage-Gated Calcium Channel Activity 523
Regulation of Gem Function by Posttranslational Modification 525
References 526
Chapter 39: Analyses of Rem/RGK Signaling and Biological Activity 528
Introduction 528
Mammalian Expression Vectors for Wild-Type and Mutant Rem Proteins 530
In Vitro Biochemical Assays: Identification of Rem Binding Proteins 531
Interaction with Rem Binding Proteins 533
Analysis of L-Type Calcium Channel Function 536
Acknowledgments 541
References 541
Chapter 40: Analysis of Rit Signaling and Biological Activity 543
Introduction 543
Mammalian Expression Vectors for Wild-Type and Mutant Rit Proteins 544
Evaluation of Rit Effects on Neuronal Signaling and Differentiation 545
Signaling Analyses 548
Nonradioactive Determination of Rit-GTP Levels 553
Yeast Two-Hybrid Analysis of Rit Effector Interactions 555
Acknowledgments 556
References 556
Chapter 41: Characterization of RERG: An Estrogen-Regulated Tumor Suppressor Gene 557
Introduction 557
Molecular Constructs and Mutants of RERG 560
Analyses of RERG Gene Expression 561
Analysis of RERG Protein Subcellular Localization 564
Biochemical Properties and Function 565
RERG as Suppressor of Growth and Tumor Formation 567
Summary 569
References 570
Chapter 42: Inhibition of Transcription Factor NF-kappaB Activation by kappaB-Ras 571
Introduction 571
Materials 574
kappaB-Ras Expression and Purification 574
GTP and GDP Loading 576
In Vitro IkappaB Phosphorylation Inhibition Assay 576
NF-kappaB DNA Binding Inhibition Assay 576
References 578
Chapter 43: Analysis of Rhes Activation State and Effector Function 579
Introduction: The Rhes/Dexras Subfamily 579
Analysis of Rhes Nucleotide Loading 580
Analysis of Rhes Nucleotide Loading by Effector Pull-Down 582
Effect of Rhes on the cAMP Pathway 584
Acknowledgments 586
References 586
Chapter 44: Rheb Activation of mTOR and S6K1 Signaling 586
Introduction 587
Analysis of the Biological Activity of the mTOR Pathway 588
Utilization of Inhibitory Drugs 590
GTPase-Activating Protein (GAP) Assay 591
Forward Scatter FACS Analysis 594
Conclusion 595
Acknowledgments 596
References 596
Chapter 45: Use of Retrovirus Expression of Interfering RNA to Determine the Contribution of Activated K-Ras and Ras Effector Expression to Human Tumor Cell Growth 600
Introduction 600
Identification of Target Sequences and Generation of Retrovirus-Based shRNA Expression Vector Constructs 602
Generation of pSUPER.retro.siRNA Virus for Stable Expression of siRNA to Define the Role of Mutant Ras 604
Consequences of Suppression of K-Ras (12V) Expression 608
Inducible Repression of Mutant K-Ras(12V) Expression 611
Analyses of Ras Effector Function and Oncogenesis 615
Concluding Remarks 617
Acknowledgments 617
References 617
Chapter 46: Using Inhibitors of Prenylation to Block Localization and Transforming Activity 619
Introduction 619
Pharmacological Inhibition 621
Sequestration of Prenylated Small GTPases by RhoGDIs 634
Concluding Remarks 639
References 640
Chapter 47: Sorafenib (BAY 43-9006, Nexavarreg), a Dual-Action Inhibitor That Targets RAF/MEK/ERK Pathway in Tumor Cells and Tyrosine Kinases VEGFR/PDGFR in Tumor Vasculature 641
Introduction 642
Materials and Methods 644
Experimental Results and Discussion 647
Conclusion 653
Acknowledgment 653
References 653
Chapter 48: Yeast Screens for Inhibitors of Ras-Raf Interaction and Characterization of MCP Inhibitors of Ras-Raf Interaction 656
Introduction 656
Developing Yeast Two-Hybrid Strains Permeable for Small Molecular Weight Compounds 659
Developing HTS for Small Molecular Weight Inhibitors of Ras-Raf Interaction in Highly Permeable Yeast Two-Hybrid System 662
Analysis of Selectivity of MCP Compounds by Liquid beta-Galactosidase Yeast Two-Hybrid Assay 664
Preliminary Analysis of HTS Hits with Antifungal Properties 666
Developing a Robust Decision Tree for Compound Selection and Optimization: Summary of Assays Applied 666
References 671
Chapter 49: A Tagging-via-Substrate Technology for Genome-Wide Detection and Identification of Farnesylated Proteins 673
Introduction 673
Chemical Synthesis 675
In Vivo Labeling Proteins with Azido-Farnesyl Substrates 676
Gel Mobility Shift Assay for Ras and Hdj-2 Farnesylated Proteins 676
Reciprocal Immunoprecipitation 678
Global Detection and Affinity Purification of Azido-Farnesylated Proteins 678
Nano-HPLC/Mass Spectrometry for Exhaustive Protein Identification 680
Concluding Remarks 680
Acknowledgments 680
References 680
Chapter 50: A Genetically Defined Normal Human Somatic Cell System to Study Ras Oncogenesis In Vivo and In Vitro 681
Introduction 682
Materials and Methods 684
Creating hTERT T/t-Ag Ras cells 684
Soft Agar Assay 685
Tumorigenic Growth Assay 687
Concluding Remarks 688
References 690
Chapter 51: Analysis of Ras Transformation of Human Thyroid Epithelial Cells 692
Introduction 692
Overview of Biological Results Obtained with the In Vitro Thyroid Model and Effect of Mutant RAS on Thyroid-Specific Differentiation 693
Analysis of Ras Effector Pathways Mediating Mutant Ras-Induced Proliferation of Primary Human Thyroid Epithelial Cells: A Model of Tumor Initiation 695
Differential Response of Human Fibroblasts and Thyrocytes to Mutant Ras Oncoprotein 697
Methods 1: Disaggregation of Thyroid Tissue to Produce Primary Monolayer Cultures of Follicular Epithelium 699
Methods 2: The Stable Expression of Mutant Ras in Normal Thyroid Epithelial Cells Using Retroviral Vectors 700
Summary 702
References 702
Chapter 52: Use of Ras-Transformed Human Ovarian Surface Epithelial Cells as a Model for Studying Ovarian Cancer 704
Introduction 704
Isolation and Culture of Normal Human Ovarian Surface Epithelial Cells 705
Immortalizing Human Ovarian Surface Epithelial Cells with hTERT and SV40 T/t 712
Transforming Immortalized Cells with Ras 713
Protocol for Retroviral Infection 713
Tumorigenicity in Mice 715
Histopathological Analysis of Tumors 716
Other Biochemical Analyses 718
Conclusions 718
Acknowledgment 718
References 718
Chapter 53: Physiological Analysis of Oncogenic K-Ras 720
Introduction 720
Selected Protocols for Generating a Mouse Model of Ductal Pancreatic Cancer 723
Evaluating Endogenous Oncogenic K-ras in Cells 726
References 733
Chapter 54: Use of Conditionally Active Ras Fusion Proteins to Study Epidermal Growth, Differentiation, and Neoplasia 734
Introduction 735
Protocols and Results 737
Concluding Remarks 744
Buffers and Antibodies 745
Acknowledgments 745
References 745
Chapter 55: Pancreatic Duct Epithelial Cell Isolation and Cultivation in Two-Dimensional and Three-Dimensional Culture Systems 747
Introduction 747
Methods 748
Pancreatic Medium 748
Primary Pancreatic Duct Epithelial Cell (PDEC) Isolation 748
Two-Dimensional Cultures 750
Three-Dimensional Cultures on Reconstituted Basement Membrane 751
References 754
Chapter 56: Analyses of RAS Regulation of Eye Development in Drosophila melanogaster 755
Introduction 755
Techniques 756
Acknowledgments 763
References 763
Author Index 767
Subject Index 809