Protein Phosphatase Protocols

By Greg Moorhead

Table of Contents

Preface ………………………………………………………………………………………………..v
Contributors ………………………………………………………………………………………..xi
1 Analysis of Protein Phosphatases:
Toolbox For Unraveling Cell Signaling Networks

2 A Brief Introduction to the Protein Phosphatase Families

3 Small-Molecule Inhibitors of Ser/Thr Protein Phosphatases:
Specificity, Use, and Common Forms of Abuse

4 Synthesis and Use of the Protein Phosphatase Affinity Matrices
Microcystin–Sepharose and Microcystin–Biotin–Sepharose

5 Utilizing Protein Phosphatase Inhibitors to Define PP2A
as a Regulator of Ataxia-Telangiectasia Mutated (ATM)

6 An Automated Fluorescence-Based Method
for Continuous Assay of PP2A Activity

7 An In Vivo Assay to Quantify Stable Protein
Phosphatase 2A (PP2A) Heterotrimeric Species

8 Mutagenesis and Expression of the Scaffolding Aα and Aβ
Subunits of PP2A: Assays for Measuring Defects
in Binding of Cancer-Related Aα and Aβ Mutants
to the Regulatory B and Catalytic C Subunits

9 Isolation and Characterization of PP2A Holoenzymes Containing
FLAG-Tagged B Subunits

10 Purification of PP2A Holoenzymes by Sequential
Immunoprecipitation with Anti-Peptide Antibodies

11 Purification of PP2Ac from Bovine Heart

12 Visualization of Intracellular PP1 Targeting Through Transiently
and Stably Expressed Fluorescent Protein Fusions

13 Yeast Two-Hybrid Screens To Identify
Drosophila PP1-Binding Proteins

14 Identification of Cellular Protein Phosphatase-1 Regulators

15 Assay for Three-Way Interaction of Protein Phosphatase-1 (Glc7)
with Regulatory Subunits Plus Phosphatase Inhibitor-2

16 Phosphorylation of the Protein Phosphatase Type 1
Inhibitor Protein CPI-17 by Protein Kinase C

17 Purification of Smooth Muscle Myosin Phosphatase Using a
Thiophosphorylated Myosin Light-Chain-Affinity Resin

18 Proteins Interacting with Saccharomyces cerevisiae Type 1
Protein Phosphatase Catalytic Subunit Identified
by Single-Step Affinity Purification and Mass Spectrometry

19 Expression of Protein Histidine Phosphatase in Escherichia coli,
Purification, and Determination of Enzyme Activity

20 The Use of RNA Interference to Analyze Protein
Phosphatase Function In Mammalian Cells

21 Recognition of a PP2C Interaction Motif
in Several Plant Protein Kinases

22 Use of Yeast Genetic Tools to Define Biological Roles
of Novel Protein Phosphatases

23 Targeting of PP2C in Budding Yeast

24 Phosphatase Targets in TOR Signaling

25 Functional Characterization of the Small CTD Phosphatases

26 Genome-Scale Discovery and Characterization
of Class-Specific Protein Sequences: An Example
Using the Protein Phosphatases of Arabidopsis thaliana

27 Yeast Substrate-Trapping System for Isolating Substrates
of Protein Tyrosine Phosphatases

Index

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