Protein Phosphatase Protocols

389 Pages·2007·6.83 MB·English

by  Shirish Shenolikar (auth.), Greg Moorhead (eds.)

Protein Phosphatase Protocols
M E T H O D S I N M O L E C U L A R B I O L O G Y™
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M E T H O D S I N M O L E C U L A R B I O L O G Y™
Protein Phosphatase
Protocols
Edited by
Greg Moorhead
Department of Biological Sciences, University of Calgary,
Calgary, AB, Canada
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Protein phosphatase protocols / edited by Greg Moorhead.
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Includes bibliographical references and index.
ISBN-13: 978-1-58829-711-2
ISBN-10: 1-58829-711-X (alk. paper)
1. Phosphoprotein phosphatases—Laboratory manuals.
[DNLM: 1. Phosphoprotein Phosphatase—analysis. 2. Phosphoprotein Phosphatase—isolation &
purification. QU 136 P9671655 2006] I. Moorhead, Greg. II. Series: Methods in molecular biology
(Clifton, N.J.) ; v. 365.
QP609.P56P762 2006
572'.68—dc22 2006007128
Preface
Phosphorylation is recognized as one of the most prevalent and versatile
means to regulate protein function. Alteration in the normal target protein phos-
phorylation state is also established as a major contributor to disease and, as a
consequence the enzymes that add a phosphate group to proteins, the protein
kinases, are considered the second largest drug target of the pharmaceutical
industry. Given the role of protein kinases, it is natural to think then that the
enzymes that remove phosphate from proteins are equally important and thus
are likely contributors to disease when functioning aberrantly. The acceptance
of the protein phosphatases as equal in importance to protein kinases and as
potential drug targets has been slow in coming. Nevertheless, a number of
important research articles over the last few years have dramatically shifted
this paradigm [for instance see, MacKeigan et al. Nat. Cell Biol. 7(6):591–
600]. With a growing interest in protein phosphatase function, Protein Phos-
phatase Protocols represents a timely revisit to phosphatase methodologies.
I have tried to assemble a series of articles covering a broad range of protein
phosphatase methodologies (proteomics, genomics, biochemistry, RNAi and
genetics) with examples from several model organisms, including yeast, Droso-
phila, plant and human cells. By including a variety of approaches across many
organisms, I was able to get contributions from many of the leading and emerg-
ing protein phosphatase researchers from around the world, but at the same
time this meant that many were unable to contribute to this volume. Undoubt-
edly, research by other phosphatase laboratories is referenced within many of
these chapters. I hope that the techniques explained here generate ideas on new
approaches to protein phosphatase research in your laboratory.
Greg Moorhead
v
Contents
Preface ..............................................................................................................v
Contributors .....................................................................................................xi
1 Analysis of Protein Phosphatases:
Toolbox For Unraveling Cell Signaling Networks
Shirish Shenolikar.................................................................................1
2 A Brief Introduction to the Protein Phosphatase Families
Tomas Mustelin.....................................................................................9
3 Small-Molecule Inhibitors of Ser/Thr Protein Phosphatases:
Specificity, Use, and Common Forms of Abuse
Mark Swingle, Li Ni, and Richard E. Honkanen.................................23
4 Synthesis and Use of the Protein Phosphatase Affinity Matrices
Microcystin–Sepharose and Microcystin–Biotin–Sepharose
Greg B. G. Moorhead, Timothy A. J. Haystead
and Carol MacKintosh....................................................................39
5 Utilizing Protein Phosphatase Inhibitors to Define PP2A
as a Regulator of Ataxia-Telangiectasia Mutated (ATM)
Aaron A. Goodarzi, Pauline Douglas, Greg B. G. Moorhead,
and Susan P. Lees-Miller.................................................................47
6 An Automated Fluorescence-Based Method
for Continuous Assay of PP2A Activity
Adam M. Wegner, Jamie L. McConnell, Randy D. Blakely,
and Brian E. Wadzinski...................................................................61
7 An In Vivo Assay to Quantify Stable Protein
Phosphatase 2A (PP2A) Heterotrimeric Species
Matthew S. Gentry, Richard L. Hallberg, and David C. Pallas..........71
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
Ralf Ruediger, Jin Zhou, and Gernot Walter......................................85
9 Isolation and Characterization of PP2A Holoenzymes Containing
FLAG-Tagged B Subunits
Deanna G. Adams and Brian E. Wadzinski......................................101
10 Purification of PP2A Holoenzymes by Sequential
Immunoprecipitation with Anti-Peptide Antibodies
Gernot Walter, Jin Zhou, and Ralf Ruediger....................................113
vii
viii Contents
11 Purification of PP2Ac from Bovine Heart
Hue T. Tran, Tony S. Ferrar, Anne Ulke-Lemée,
and Greg B. G. Moorhead............................................................127
12 Visualization of Intracellular PP1 Targeting Through Transiently
and Stably Expressed Fluorescent Protein Fusions
Laura Trinkle-Mulcahy, Janet Chusainow, Yun Wah Lam,
Sam Swift, and Angus Lamond.....................................................133
13 Yeast Two-Hybrid Screens To Identify
Drosophila PP1-Binding Proteins
Daimark Bennett and Luke Alphey...................................................155
14 Identification of Cellular Protein Phosphatase-1 Regulators
David W. Roadcap, Matthew H. Brush, and Shirish Shenolikar......181
15 Assay for Three-Way Interaction of Protein Phosphatase-1 (Glc7)
with Regulatory Subunits Plus Phosphatase Inhibitor-2
Masumi Etoand David L. Brautigan..................................................197
16 Phosphorylation of the Protein Phosphatase Type 1
Inhibitor Protein CPI-17 by Protein Kinase C
Michael P. Walsh, Marija Susnjar, Jingti Deng,
Cindy Sutherland, Enikó Kiss,
and David P. Wilson.....................................................................209
17 Purification of Smooth Muscle Myosin Phosphatase Using a
Thiophosphorylated Myosin Light-Chain-Affinity Resin
Meredith Borman and Justin MacDonald.........................................225
18 Proteins Interacting with Saccharomyces cerevisiae Type 1
Protein Phosphatase Catalytic Subunit Identified
by Single-Step Affinity Purification and Mass Spectrometry
Edmund P. Walsh, Douglas J. Lamont, Kenneth A. Beattie,
and Michael J. R. Stark.................................................................235
19 Expression of Protein Histidine Phosphatase in Escherichia coli,
Purification, and Determination of Enzyme Activity
Nicole Bäumer, Anette Mäurer, Josef Krieglstein,
and Susanne Klumpp....................................................................247
20 The Use of RNA Interference to Analyze Protein
Phosphatase Function In Mammalian Cells
Iain Fraser, Wei Liu, Robert Rebres, Tamara Roach,
Joelle Zavzavadjian, Leah Santat, Jamie Liu,
Estelle Wall, and Marc Mumby....................................................261
21 Recognition of a PP2C Interaction Motif
in Several Plant Protein Kinases
Niranjan Chakraborty, Masaru Ohta,
and Jian-Kang Zhu........................................................................287
Contents ix
22 Use of Yeast Genetic Tools to Define Biological Roles
of Novel Protein Phosphatases
Joaquín Ariño, Antonio Casamayor, Amparo Ruiz,
Ivan Muñoz, and Maribel Marquina............................................299
23 Targeting of PP2C in Budding Yeast
Irene M. Ota and James Mapes........................................................309
24 Phosphatase Targets in TOR Signaling
Estela Jacinto.....................................................................................323
25 Functional Characterization of the Small CTD Phosphatases
Michele Yeo and Patrick S. Lin.........................................................335
26 Genome-Scale Discovery and Characterization
of Class-Specific Protein Sequences: An Example
Using the Protein Phosphatases of Arabidopsis thaliana
David Kerk........................................................................................347
27 Yeast Substrate-Trapping System for Isolating Substrates
of Protein Tyrosine Phosphatases
Masahide Fukada and Masaharu Noda.............................................371
Index............................................................................................................383
Contributors
DEANNA G. ADAMS• Department of Pharmacology, Vanderbilt University
Medical Center, Nashville, Tennessee, USA
LUKE ALPHEY • Department of Zoology, University of Oxford, Oxford,
United Kingdom
JOAQUÍN ARIÑO • Department of Biochemistry and Molecular Biology,
Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain
NICOLE BÄUMER • Institut für Pharmazeutische und Medizinische Chemie,
Westfälische Wilhelms-Universität, Münster, Germany
KENNETH A. BEATTIE • School of Life Sciences, University of Dundee, United
Kingdom
DAIMARK BENNETT • Department of Zoology, University of Oxford, Oxford,
United Kingdom
RANDY D. BLAKELY • Department of Pharmacology, Vanderbilt University
Medical Center, Nashville, Tennessee, USA
MEREDITH BORMAN• Department of Biochemistry and Molecular Biology,
Smooth Muscle Research Group, University of Calgary, Calgary, AB,
Canada
DAVID L. BRAUTIGAN •Center for Cell Signaling and Department
of Microbiology, University of Virginia School of Medicine,
Charlottesville, Virginia, USA
MATTHEW H. BRUSH • Department of Pharmacology and Cancer Biology,
Duke University Medical Center, Durham, North Carolina, USA
ANTONIO CASAMAYOR • Department of Biochemistry and Molecular Biology,
Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain
NIRANJAN CHAKRABORTY• National Centre for Plant Genome Research
Aruna Asaf Ali Marg JNU Campus, New Delhi, India
JANET CHUSAINOW • Division of Gene Regulation and Expression, School of
Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, United
Kingdom
JINGTI DENG • Smooth Muscle Research Group, Department of Biochemistry
& Molecular Biology, University of Calgary, Calgary, Alberta, Canada
PAULINE DOUGLAS • Southern Alberta Cancer Research Institute and Depart-
ment of Biochemistry & Molecular Biology, University of Calgary,
Calgary, AB, Canada
MASUMI ETO •Department of Physiology, Thomas Jefferson College
of Medicine, Philadelphia, Pennsylvania, USA
xi
xii Contributors
TONY S. FERRAR • Department of Biological Sciences, University of Calgary,
Calgary, Alberta, Canada
IAIN FRASER • AfCS Molecular Biology Laboratory, Division of Biology,
California Institute of Technology, Pasadena, California, USA
MASAHIDE FUKADA • Division of Molecular Neurobiology, National Institute
for Basic Biology, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-
8787, Japan
MATTHEW S. GENTRY • Department of Pharmacology, University of Califor-
nia, San Diego, California, USA
AARON A. GOODARZI• Southern Alberta Cancer Research Institute and
Department of Biochemistry & Molecular Biology, University of Calgary,
Calgary, AB, Canada
RICHARD L. HALLBERG• Department of Biology, Syracuse University,
Syracuse, New York, USA
TIMOTHY A. J. HAYSTEAD • Department of Pharmacology and Cancer
Biology, Duke University Medical Center, Durham, North Carolina, USA
RICHARD E. HONKANEN • Department of Biochemistry and Molecular
Biology, University of South Alabama, Mobile, Alabama, USA
ESTELA JACINTO • Department of Physiology and Biophysics UMDNJ,
Robert Wood Johnson Medical School, Piscataway, New Jersey, USA
ENIKÓ KISS• Smooth Muscle Research Group, Department of Biochemistry
& Molecular Biology, University of Calgary, Calgary, Alberta, Canada
DAVID KERK • Department of Biology, Point Loma Nazarene University,
San Diego, California, USA
SUSANNE KLUMPP • Institut für Pharmazeutische und Medizinische Chemie,
Westfälische Wilhelms-Universität, Münster, Germany
JOSEF KRIEGLSTEIN • Institut für Pharmakologie and Toxikologie, Philipps-
Universität, Marburg, Germany
YUN WAH LAM • Division of Gene Regulation and Expression, School of Life
Sciences, MSI/WTB Complex, University of Dundee, Dundee, United
Kingdom
ANGUS LAMOND • Division of Gene Regulation and Expression, School
of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee,
United Kingdom
DOUGLAS J. LAMONT • School of Life Sciences, University of Dundee, United
Kingdom
SUSAN P. LEES-MILLER • Southern Alberta Cancer Research Institute and
Department of Biochemistry & Molecular Biology, University of Calgary,
Calgary, AB, Canada
PATRICK S. LIN • Division of Oncology, Stanford University School
of Medicine, Stanford, California, USA