Protein Phosphatases

Arino J. (ed.), Alexander D. (ed.)

This book provides an up-to-date and comprehensive overview of protein phosphatase research, a rapidly evolving field with increasing importance in our understanding of the molecular basis of cell biology and of pathological processes. The book covers dephosphorylation processes involving serine/threonine, as well as tyrosine and histidine residues, and aims to be a useful resource for both the advanced reader as well as the newcomer to the field. It is also valuable for those working in the pharmaceutical and Biotech industries.

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Table of contents :
32.pdf……Page 0
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 ([