Protein Phosphatases

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Table of Contents......Page 3
Preface......Page 1
Contributors to Volume 353......Page 6
The Past......Page 10
The Future......Page 13
Monitoring of PP2A and PP2C by Phosphothreonyl Peptide Substrates......Page 18
Assay with Radioactive Phosphopeptides......Page 24
Non-Radioactive Assay (Malachite Green)......Page 29
Perspectives......Page 31
Determination of Mammalian Glycogen Synthase Phosphatase Activity......Page 33
Advantages and Disadvantages......Page 38
Buffers and Other Materials......Page 39
Purification of Dephospho-GS......Page 40
Purification of Phospho-GS......Page 43
Determination of GS Activity......Page 45
32P-labeling of Dephospho-GS......Page 46
32P release from......Page 47
Coupled GS Dephosphorylation/Activation......Page 48
Introduction......Page 51
Radioactive Assay......Page 52
Enzyme-Coupled Continuous Spectrophotometric Assay......Page 55
Determining the Activity of a Protein Phosphatase from its Effect on a Kinase Reaction......Page 57
Introduction......Page 61
Materials and Methods......Page 63
Discussion......Page 65
Introduction......Page 67
Materials and Methods......Page 68
Discussion......Page 69
Materials and Methods......Page 71
Discussion......Page 72
Detection of Protein Histidine Phosphatase in Vertebrates......Page 75
Buffers......Page 77
Purification of Protein Histidine Phosphatase......Page 80
Procedure......Page 81
Introduction......Page 84
Measurement of Signaling Lipids as Products in Enzyme Assays and in Cell and Tissue Extracts......Page 87
Formation of PH Domain/Lipid Sensor Complexes......Page 88
Detection of Exogenous Lipids by the Sensor Complex......Page 90
Use of Sensor Complexes to Assay Lipid Metabolizing Enzymes......Page 92
Phosphoinositide Mass Assays......Page 95
Rationale......Page 99
Method and Results......Page 100
Controls......Page 101
Mixed Peptide Sequencing and the FASTF/FASTS Algorithms......Page 105
Mixed Peptide Sequencing......Page 107
Mixed Peptide Sequencing......Page 108
Mass Spectrometry (adapted from Graves and Haystead, 2002)......Page 109
Fast Silver Stain (adapted from Shevchenko et al., 1996)......Page 111
In-Gel Digestion for Silver Stained Gels (adapted from Wilm et al., 1996)......Page 112
Data Analysis and FASTF/FASTS (from Mackey et al., 2002)......Page 114
Conclusions......Page 116
Phosphoproteome Analysis in Yeast......Page 117
Characterizing The Effects of Reg1p on the Yeast Phosphoproteome......Page 118
32P Orthophosphate Labeling......Page 120
Preparation of Yeast Extracts......Page 121
Two-Dimensional Gel Electrophoresis and Mixed Peptide Sequencing......Page 122
Anion-Exchange Chromatography......Page 123
Site-directed Mutagenesis of the PP-1C Binding Site in Reg1p......Page 124
Conclusions......Page 125
Introduction......Page 126
Differential Gene Expression in Leukemic- and Remission Phase Leukocytes from AML Patients......Page 127
Verification of the Microarray Results by RT-PCR......Page 132
Summary......Page 135
Protein Phosphatase Translocation in RBL-2H3 Cells......Page 137
Protocol......Page 139
Sensitization......Page 140
Protocol......Page 141
Protocol......Page 142
Solutions......Page 143
Protocol......Page 144
Fluorescence Microscopy......Page 145
Protocol......Page 146
Protocol......Page 147
Introduction......Page 149
Preparation of Sample for Analysis......Page 154
Comments......Page 156
Introduction......Page 160
Substrates......Page 163
Covalent Coupling of Affinity Purified Antibody to Protein-G-Sepharose......Page 164
Assay Procedure......Page 165
Conclusions......Page 167
Introduction......Page 170
Protein Phosphatase Assays......Page 172
PP1 Overlays......Page 173
GST Pull-downs......Page 175
Microcystin-Sepharose......Page 176
Other Tools to Study the Interaction between PP1 and Associated Polypeptides......Page 177
Protocol 1: PP1 Overlays......Page 178
Protocol 3: GST Pull-downs......Page 179
Protocol 4: Coimmunoprecipitations......Page 180
Protocol 5: Isolation of PP1 complexes with microcystin-sepharose......Page 181
Introduction......Page 183
Generation of PP1 Isoform-specific Antibodies......Page 186
Protocol......Page 187
Results and Discussion......Page 188
Protocol......Page 189
Results and Discussion......Page 190
Far-Western Overlay Assays......Page 191
Protocol......Page 192
Results and Discussion......Page 193
Buffers......Page 194
Protocol......Page 195
Results and Discussion......Page 196
Substrate Preparation ([PP1 Activity Assays......Page 198
Results and Discussion......Page 199
Summary......Page 200
Introduction......Page 203
RRS—principle of method......Page 204
Solutions......Page 206
Construction of the PP2A--B55-RRS Bait......Page 207
Yeast Transformation......Page 208
Testing the PP2A--B55-RRS bait......Page 210
Library Screening (Flow Chart.........Page 211
DNA Plasmid Isolation from Yeast and its Analysis......Page 213
Summary......Page 214
Introduction......Page 216
Primer Design......Page 219
Restriction Digest of the Nonmutated Parental DNA Template and Transformation of the Nicked dsDNA into E. coli......Page 220
Characterization of PP2A Catalytic Subunit Mutants In Vivo......Page 221
Generation of Cell Lines Stably Expressing Epitope-Tagged C Subunit Mutants......Page 222
Preparation of Whole Cell Extracts......Page 223
Coupling Antibodies to Protein-A-Sepharose......Page 224
Preparation of Phosphatase Assays......Page 227
Analysis of Heterotrimeric PP2A Complex Formation......Page 230
Comments......Page 231
Introduction......Page 233
The Theory of FRET......Page 234
FRET Measurements Using Flow Cytometry......Page 237
Method Outline for FRET Measurements Using Flow Cytometry......Page 240
Donor and Acceptor Photobleaching FRET Microscopy......Page 241
Method Outline for Photobleaching FRET Microscopy......Page 242
Intensity Based FRET Microscopy......Page 244
Method Outline for Intensity Based FRET Microscopy......Page 245
Limitations of FRET Studies......Page 246
CD45 and CD45 Isoforms......Page 248
RPTPα......Page 251
Protein Phosphatase 1......Page 252
PTP1B......Page 253
Introduction......Page 255
Chemical Cross-linkers......Page 256
Cross-linking Using bis[sulfosuccinimidyl]-suberate (BS3)......Page 257
Genetic Cross-linking......Page 258
Detection of Enforced Dimers......Page 259
Fluorescence Resonance Energy Transfer......Page 261
Detection of FRET......Page 264
FRET Analysis Hardware......Page 265
CFP and YFP Fusion Proteins......Page 266
Coimmunoprecipitation......Page 267
Coimmunoprecipitation Experiments......Page 268
Concluding Remarks......Page 269
Phosphatase Inhibitory Phosphoproteins......Page 274
Preparation of Recombinant Inhibitor Proteins......Page 277
Preparation of Phospho- and Thiophospho-inhibitor Proteins......Page 278
Assays for Phosphorylation of PP1 Inhibitor Proteins......Page 280
Assay of Phosphorylation by Immunoblotting using Anti-Phosphospecific Antibody . . .......Page 281
Preparation of Antibody Recognizing both Phospho- and Unphospho-inhibitor (Anti Total-inhibitor Protein)......Page 282
Methods......Page 283
Assay of Phosphorylation by Mobility Shift in SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) . . .......Page 284
Assay of Phosphorytlation by Mobility Shift in Glycerol PAGE......Page 286
Assay of Phosphorylation by Mobility Shift in Urea PAGE . . .......Page 287
Assay of Phosphorylation by Mobility Shift in Lutidine PAGE . . .......Page 288
Determination of in vivo Phosphorylation Sites of Endogenous Inhibitor Proteins......Page 289
Methods......Page 290
Combinatorial Chemistry and Peptide Library Methods to Characterize Protein Phosphatases......Page 292
Synthesis of Combinatorial Libraries......Page 293
Synthesis of (phospho)peptide Libraries......Page 296
Fmoc- vs Boc-chemistry......Page 297
Solid Support......Page 298
Amino Acid Derivatives......Page 299
Deprotection/Cleavage......Page 300
N-terminally modified (phospho)peptides......Page 301
Phosphatase Activity......Page 302
Protocol for Continuous Analysis of Phosphotyrosine Peptide Dephosphorylation by Fluorescence Spectrometry......Page 303
Assay......Page 304
Binding of Library Compounds to Protein Phosphatases......Page 305
Deconvolution of Library Screenings......Page 306
Example 1......Page 308
Example 3......Page 310
Example 5......Page 311
Example 8......Page 312
General Advise......Page 313
Introduction......Page 315
Phosphopeptides as Substrates......Page 317
General Remarks......Page 318
Differences in the Effect of Lipophilic Oxidizable Compounds......Page 319
Introduction......Page 323
Reagents......Page 325
Procedure......Page 327
Determination of Calcineurin activity in Crude Tissue Extracts......Page 329
CaM-Dependent Inactivation of Crude Calcineurin......Page 330
Protection of Calcineurin against Inactivation by Superoxide Dismutase......Page 331
Identification of Calcineurin as an (Fe2+--Zn2+) Protein phosphatase.......Page 332
Modified Purification Procedure......Page 333
Identification of the Oxidation State of Iron......Page 335
Concluding Remarks......Page 337
Analysis of the regulation of protein tyrosine phosphatases in vivo by reversible oxidation......Page 339
Mechanism of PTP-Mediated Catalysis......Page 341
General Principles of the Assay......Page 343
Buffers......Page 344
Construction of a Customized Anaerobic Work Station......Page 345
Preparation of Cell Lysates......Page 346
Preparation of a Radioactively-Labeled, Phosphorylated Substrate......Page 347
In-gel PTP Assay......Page 348
Stimulus-Induced Oxidation of PTPs......Page 349
Summary and Perspectives......Page 351
Preparation and Characterization of Recombinant Protein Phosphatase 1......Page 356
Materials......Page 357
Dephosphorylation of . . .......Page 358
Method 1......Page 359
Method 2......Page 360
Method 1......Page 361
Method 2......Page 362
Affinity Chromatography......Page 363
Immunoblotting of Recombinant PP1C......Page 364
Characterization of PP1C Expressed in E. coli using Method 1......Page 365
Characterization of PP1 Expressed in E. coli using Method 2......Page 368
Characterization of PP1 Expressed in Sf9 Cells using Method 1......Page 369
Characterization of PP1 expressed in Sf9 cells using Method 2......Page 371
Introduction......Page 374
Establishment of inducible PP2Calpha Cells......Page 375
Plating Efficiency......Page 376
XTT Assay......Page 377
Establishment of Tet-inducible PP2Calpha-Expressing Cells......Page 378
Characterization of T-Rex-293 PP2Calpha Cells......Page 379
PP2C Induction and Its Effects......Page 381
Introduction......Page 384
Construction and Phenotypic Analysis of a sit4 hal3 Conditional Mutant by Using a One-step Promoter Substitution Cassette and t......Page 385
The Dual Activator-Repressor System Allows Tight Control of Gene Expression......Page 388
Other Constructions Based on the Tet System......Page 390
Substitution of the HAL3 Promoter by a Doxycycline-Regulatable Promoter Amplification of the Substitution Cassette......Page 391
Construction of the sit4Delta tetO:HAL3 Strain......Page 392
Functional Analysis of a sit4 hal3 Conditional Mutant......Page 393
Introduction......Page 398
Identifying Drosophila Protein Phosphatase Homologs and Preparation of cDNA......Page 401
Production of Double Stranded RNA......Page 403
Cytosol Preparation and Western Blotting......Page 404
Reverse Transcription (RT)-PCR......Page 406
Summary......Page 408
Serine/threonine Phosphatase type 5 (PP5)......Page 410
Chemical Composition......Page 412
Experimental Design and Interpretations......Page 415
Screening for a useful antisense molecule......Page 417
Double-stranded RNA interference......Page 422
Conclusions......Page 427
Introduction......Page 429
Classification of Transgenic Approaches......Page 430
PP2A Cα Knockout Mice......Page 431
Animal Models: PP2A Regulatory Subunit Transgenic Mice......Page 433
Animal Models: PP2A Calpha Dominant Negative Mutant Mice......Page 434
The Dom1 Line......Page 435
Animal Models: PP2A PR65 Dominant Negative Mutant Mice......Page 436
Protein Phosphatase Activity Measurements......Page 437
Blastocyst Cultures......Page 438
Advantages and Limitations of the Various Transgenic and Knockout Approaches......Page 439
Conclusion......Page 441
Introduction......Page 443
Equipment Required......Page 445
Initial Steps......Page 446
Genome Wide Screen for Genes Influencing Glycogen Storage......Page 447
Procedure......Page 448
Remarks......Page 450
Rationale for Glycogen Synthase Phosphatase/kinase Assay......Page 452
Assay of Yeast Glycogen Synthase......Page 453
Rationale for Glycogen Phosphorylase Phosphatase Assay......Page 454
Measurement of Endogenous Phosphorylase Phosphatase Activity in Yeast Lysates......Page 455
Remarks......Page 457
Conclusion......Page 458
Author Index......Page 459
Subject Index......Page 483