Preface
Reversible phosphorylation affects proteins in all compartments of a
cell and is crucial for life and death. It is therefore not surprising that this
topic has been covered three times already in the Method s in Enzymology
series: (1) in 1983 in Volume 99 on protein kinases, and (2)/(3) in 1991 in
Volumes 200 and 201 (Part A and Part B) on protein phosphorylation.
Much has happened since the last volume on protein phosphorylation
was published in Methods in Enzymology. Recognition of the fundamental
importance of reversible protein phosphorylation in cellular processes
culminated in the awarding of the 1992 Nobel Prize for Physiology or
Medicine to Edmond Fischer and Edwin Krebs for their discovery of
the regulation of phosphorylase by phosphorylation in the early 1950s.
In the year 2000, Paul Greengard, Eric Kandel, and Arvid Carlson received
the Nobel Prize for their findings concerning signal transduction—including
phosphorylation—in the nervous system. Finally, Timothy Hunt, Leland
Hartwell, and Paul Nurse were awarded the Nobel Prize in 2001 for their
achievements in studying cell cycle control (discovery of cyclins and cyclin-
dependent kinases).
This volume deals with protein phosphatases exclusively. The
phosphatases had been overlooked for a long time. However, it is obvious
by now that they are as equally important as the kinases, and the field is
expanding rapidly. A major development over the past 10 years has been the
realization that serine/threonine phosphatases acquire specificity and are
controlled by association with targeting polypeptides that not only localize
the enzymes to different cellular compartments, but also impart regulation
and substrate recognition. To date, many phosphatase-binding proteins
(including subunits, anchoring proteins, and regulatory proteins) have been
identified. This applies for most of the beloved oldies such as type-1, type-
2A, etc., that continue to be hot spots in science. Protein tyrosine
phosphatases that emerged much later also advanced to become major
targets for further research in recent years. They have revealed an enormous

structural diversity, and the ability of some of the protein tyrosine
phosphatases also to act as a lipid phosphatase is most stunning. A novelty
is the third class of phosphatases acting on labile, N -phosphorylated amino
acids such as histidine: The first vertebrate histidine phosphatase has been
discovered.
Once more, technology and methodol ogy have greatly advanced over
the past years. Overexpression on the one side and downregulation or even
xiii
knock out on the other side are just a few examples of what has emerged and
what has been used in the field of protein phosphatases as represented in this
volume.
It is our pleasure to thank the authors for their cooperation, Drs. M. I.
Simon and J. N. Ableson (the editors-in-chief) for their confidence, and the
publisher for their generous support, thus making this project possible.
S
USANNE KLUMPP
JOSEF KRIEGLSTEIN
xiv PREFACE
Table of Contents
CONTRIBUTORS TO VOLUME 366 ix
P
REFACE xiii
V
OLUMES IN SERIES xv
F
OREWORD BY PHILIP COHEN xlv
Section I. Determination, Detection, and Localization
1. Monitoring of PP2A and PP2C by
Phosphothreonyl Peptide Substrates
ARIANNA DONELLA-DEANA,

MARCO BOSCHETTI, AND
LORENZO A. PINNA
3
2. Determination of Mammalian Glycogen
Synthase Phosphatase Activity
A
NNA A. DEPAOLI-ROACH,
PIER GIUSEPPE VILARDO,
J
ONG-HWA KIM,
NIRMALA MAVILA,
BHARGAVI VEMURI, AND
PETER J. ROACH
17
3. Measuring Protein Phosphatase Activity with
Physiological Substrates
B
O ZHOU AND
ZHONG-YIN ZHANG
34
4. PTEN and Myotubularins: Families of
Phosphoinositide Phosphatases
G
REGORY S. TAYLOR AND
JACK E. DIXON
43
5. Detection of Protein Histidine Phosphatase in
Vertebrates
S
USANNE KLUMPP,

J
AN HERMESMEIER, AND
JOSEF KRIEGLSTEIN
56
6. Advances in Procedures for the Detection and
Localization of Inositol Phospholipid Signals
in Cells, Tissues, and Enzyme Assays
C. P
ETER DOWNES,
ALEXANDER GRAY,
S
TEPHEN A. WATT, AND
JOHN M. LUCOCQ
64
7. Mixed Peptide Sequencing and the FASTF/
FASTS Algorithms
R
UPA RAY AND
TIMOTHY A. J. HAYSTEAD
84
8. Phosphoproteome Analysis in Yeast R
UPA RAY AND
TIMOTHY A. J. HAYSTEAD
95
9. cDNA Microarray Analysis Reveals an
Overexpression of the Dual-Specificity
MAPK Phosphatase PYST2 in Acute
Leukemia
O
RLEV LEVY-NISSENBAUM,

O
RIT SAGI-ASSIF,PIA RAANANI,
A
BRAHAM AVIGDOR,
ISAAC BEN-BASSAT, AND
ISAAC P. WITZ
103
v
10. Protein Phosphatase Translocation in
RBL-2H3 Cells
ALISTAIR T. R. SIM,JEFF HOLST,
AND RUSSELL I. LUDOWYKE
113
11. Whole Mount Analysis of Mammary Gland
Structure in PTP Epsilon Transgenic Mice
Z
OHAR TIRAN AND ARI ELSON 124
Section II. Interacting Proteins and Subunits
12. Assay of Protein Phosphatase 1 Complexes PATRICIA T. W. COHEN,
GARETH J. BROWNE,
MIRELA DELIBEGOVIC, AND
SHONAGH MUNRO
135
13. Validation of Interactions with Protein
Phosphatase-1
A
LEYDE VAN EYNDE
AND
MATHIEU BOLLEN
144

14. Analysis of Specific Interactions of Native
Protein Phosphatase 1 Isoforms with
Targeting Subunits
R
OGER J. COLBRAN,
L
EIGH C. CARMODY,
PATRICIA A. BAUMAN,
BRIAN E. WADZINSKI,
AND MARTHA A. BASS
156
15. Using the Ras Recruitment System to
Identify PP2A–B55-Interacting Proteins
H
AIM M. BARR,
R
AKEFET SHARF, AND
TAMAR KLEINBERGER
175
16. Altering the Holoenzyme Composition and
Substrate Specificity of Protein Phosphatase
2A
T
HOMAS FELLNER,
P
ATRICK PIRIBAUER,
AND EGON OGRIS
187
17. The Application of Fluorescence Resonance
Energy Transfer to the Investigation of

Phosphatases
J
A
´
NOS SZO
¨
LLO
00
SI AND
DENIS R. ALEXANDER 203
18. Receptor Protein-Tyrosine Phosphatase
Dimerization
J
EROEN DEN HERTOG,
T
HEA VAN DER WIJK,
L
EON G. J. TERTOOLEN, AND
CHRISTOPHE BLANCHETOT
224
Section III. Inhibition, Stimulation, and Modulation of Activity
19. Phosphoprotein Inhibitors of Protein
Phosphatase-1
MASUMI ETO,CRAIG LEACH,
N
IKOLAOS A. TOUNTAS, AND
DAVID L. BRAUTIGAN
243
20. Combinatorial Chemistry and Peptide
Library Methods to Characterize

Protein Phosphatases
S
TEFAN W. VETTER AND
ZHONG-YIN ZHANG 260
21. Activity of PP2C is Increased by
Divalent Cations and Lipophilic Compounds
Depending on the Substrate
J
OSEF KRIEGLSTEIN,
DAGMAR SELKE,
ALEXANDER MAAßEN, AND
SUSANNE KLUMPP
282
vi TABLE OF CONTENTS
22. Regulation of Calcineurin by Oxidative Stress MANIK C. GHOSH,
X
UTONG WANG,SHIPENG LI,
AND CLAUDE KLEE
289
23. Analysis of the Regulation of Protein
Tyrosine Phosphatases in Vivo by Reversible
Oxidation
T
ZU-CHING MENG AND
NICHOLAS K. TONKS 304
Section IV. Expression Systems
24. Preparation and Characterization of Recom-
binant Protein Phosphatase 1
TAKUO WATANABE,
EDGAR F. DA CRUZ E SILVA,

H
SIEN-BIN HUANG,
N
ATALIA STARKOVA,
Y
OUNG-GUEN KWON,
ATSUKO HORIUCHI,
P
AUL GREENGARD, AND
ANGUS C. NAIRN
321
25. An Inducible System to Study the Growth
Arrest Properties of Protein Phosphatase 2C
P
AULA OFEK,
DANIELLA BEN-MEIR,
AND SARA LAVI
338
26. Use of Tetracycline-Regulatable Promoters for
Functional Analysis of Protein Phosphatases
in Yeast
J
OAQUI
´
N ARIN
˜
O AND
ENRIC HERRERO 347
Section V. Knockdown and Knockout Technologies
27. Analysis of Protein Phosphatase Function in

Drosophila Cells Using RNA Interference
ADAM M. SILVERSTEIN AND
MARC C. MUMBY
361
28. Regulating the Expression of Protein
Phosphatase Type 5
T
ERESA A. GOLDEN AND
RICHARD E. HONKANEN
372
29. Transgenic and Knockout Models of PP2A J
U
¨
RGEN GO
¨
TZ AND
ANDREAS SCHILD
390
30. Saccharomyces Gene Deletion Project:
Applications and Use in the Study of
Protein Kinases and Phosphatases
W
AYNE A. WILSON AND
PETER J. ROACH 403
A
UTHOR INDEX 419
S
UBJECT INDEX 443
TABLE OF CONTENTS vii
Contributors to Volume 366

Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
DENIS R. ALEXANDER (17), Laboratory of
Lymphocyte Signalling and Development,
Molecular Immunology Programme, The Bab-
raham Institute, Cambridge CB2 4AT, UK
J
OAQUI
´
N ARIN
˜
O (26), Departament de Bioquı
´
-
mica i Biologia Molecular, Universitat
Auto
`
noma de Barcelona Bellaterra, Barce-
lona, 08193, Spain
A
BRAHAM AVIGDOR (9), Institute of Hematol-
ogy, The Chaim Sheba Medical Center,
Tel-Hashomer, Israel, and Sackler School
of Medicine, Tel-Aviv University, Tel-Aviv,
69978, Israel
H
AIM M. BARR (15), Gonda Center for
Molecular Biology, B. Rappaport Faculty
of Medicine, Technion-Israel Institute of
Technology, P.O.Box 9649, Bat-Galim,

Haifa, 31096, Israel
M
ARTHA A. BASS (14), Room 702, Light Hall,
Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
P
ATRICIA A. BAUMAN (14), Room 702, Light
Hall, Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
I
SAAC BEN-BASSAT (9), Institute of Hematol-
ogy, The Chaim Sheba Medical Center, Tel-
Hashomer, Israel, and Sackler School of
Medicine, Tel-Aviv University, Tel-Aviv,
69978, Israel
D
ANIELLA BEN-MEIR (25), Department of Cell
Research and Immunology, Tel Aviv Uni-
versity, Tel Aviv, 69978, Israel
C
HRISTOPHE BLANCHETOT (18), McGill Uni-
versity, McIntyre Medical Sciences Build-
ing, 3655 Drummond Street, Montreal,
Quebec, H3G 1Y6, Canada
M
ATHIEU BOLLEN (13), Afdeling Biochemie,
Faculteit Geneeskunde, Katholieke
Universiteit Leuven, Herestraat 49, B-3000
Leuven, Belgium
M

ARCO BOSCHETTI (1), Dipartimento di
Chimica Biologica, University of Padova,
Viale G. Colombo 3, Padova 35121, Italy
D
AVID L. BRAUTIGAN (19), Center for Cell
Signaling and Department of Microbiology,
University of Virginia, School of Medicine,
Charlottesville, Virginia 22908
G
ARETH J. BROWNE (12), Medical Research
Council Protein Phosphorylation Unit,
School of Life Sciences, MSI/WTB
Complex, University of Dundee, Dow
Street, Dundee DD1 5EH, Scotland, UK
L
EIGH C. CARMODY (14), Room 702, Light
Hall, Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
P
ATRICIA T. W. COHEN (12), MRC Protein
Phosphorylation Unit, School of Life
Sciences, MSI/WTB Complex, University
of Dundee, Dow Street, Dundee DD1 5EH,
Scotland, UK
P
HILIP COHEN (Foreword), MRC Protein
Phosphorylation Unit, School of Life
Sciences, MSI/WTB Complex, University
of Dundee, Dow Street, Dundee DD1 5EH,
Scotland, UK

R
OGER J. COLBRAN (14), Room 702, Light
Hall, Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
E
DGAR F. DA CRUZ E SILVA (24), Centro de
Biologia Celular, Universidade de Aveiro,
3810-193 Aveiro, Portugal
M
IRELA DELIBEGOVIC (12), Medical Research
Council Protein Phosphorylation Unit,
School of Life Sciences, MSI/WTB Com-
plex, University of Dundee, Dow Street,
Dundee DD1 5EH, Scotland, UK
J
EROEN DEN HERTOG (18), Hubrecht Labora-
tory, Netherlands Institute for Developmen-
tal Biology, Uppsalalaan 8, Utrecht, 3584
CT, The Netherlands
ix
ANNA A. DEPAOLI-ROACH (2), Department of
Biochemistry and Molecular Biology, and
Center for Diabetes Research, Indiana
University School of Medicine, 635 Barnhill
Dr., Indianapolis, Indiana 46202
J
ACK E. DIXON (4), Departments of Pharma-
cology, Cellular and Molecular Medicine,
Chemistry and Biochemistry, University
of California, Basic Science Building

3092A-M/C 0636, 9500 Gilman Drive,
La Jolla, California 92093-0636
A
RIANNA DONELLA-DEANA (1), Dipartimento
di Chimica Biologica, University of Padova,
Viale G. Colombo 3, Padova 35121, Italy
C. P
ETER DOWNES (6), School of Life Sciences,
MSI/WTB Complex, Dow Street, Univer-
sity of Dundee, Dundee DD1 5EH, UK
A
RI ELSON (11), Department of Molecular
Genetics, The Weizmann Institute of
Science, Rehovot, 76100, Israel
M
ASUMI ETO (19), Center for Cell Signaling
and Department of Molecular Physiology
and Biological Physics, University of Virgi-
nia, School of Medicine, Charlottesville,
Virginia 22908
T
HOMAS FELLNER (16), Institute of Medical
Biochemistry, Division of Molecular Bio-
logy, ViennaBiocenter, Universityof Vienna,
Dr. Bohr-Gasse 9, Vienna A-1030, Austria
M
ANIK C. GHOSH (22), Cell Biology and
Metabolism Branch, National Institute of
Child Health and Human Development,
NIH, Bethesda, Maryland 20892

T
ERESA A. GOLDEN (28), Department of
Biochemistry & Molecular Biology, College
of Medicine, University of South Alabama,
Mobile, Alabama 36688
J
U
¨
RGEN GO
¨
TZ (29), Division of Psychiatry
Research, University of Zurich, AugustForel
Str 1, Zurich CH 8008, Zu
¨
rich, Switzerland
A
LEXANDER GRAY (6), School of Life
Sciences, MSI/WTB Complex, Dow Street,
University of Dundee,Dundee DD1 5EH,UK
P
AUL GREENGARD (24), Laboratory of
Molecular and Cellular Neuroscience,
The Rockefeller University, 1230 York
Avenue, New York, New York 10021
T
IMOTHY A. J. HAYSTEAD (7,8), Chief Scien-
tific Founder, Serenex Inc., Durham, North
Carolina 27710
J
AN HERMESMEIER (5) Institut fu

¨
r Pharma-
zeutische & Medizinische Chemie, Westfa
¨
-
lische Wilhelms-Universita
¨
t, Hittorfstr.
58-62, 48149 Mu
¨
nster, Germany
E
NRIC HERRERO (26), Departament de Cie
`
n-
cies Me
`
diques Ba
`
siques, Universitat de
Lleida, Lleida, 25198, Spain
J
EFF HOLST (10), Centre for Immunology, St.
Vincent’s Hospital, University of New
South Wales, Sydney, New South Wales,
Australia
R
ICHARD E. HONKANEN (28), Department of
Biochemistry and Molecular Biology,
MSB 2198, University of South Alabama,

Mobile, Alabama 36688
A
TSUKO HORIUCHI (24) Laboratory of
Molecular and Cellular Neuroscience,
The Rockefeller University, 1230 York
Avenue, New York, New York 10021
H
SIEN-BIN HUANG (24), Institute of Molecular
Biology, National Chung Cheng University,
Chia-Yi 621, Taiwan, ROC
J
ONG-HWA KIM (2), Department of Biochem-
istry and Molecular Biology, and Center for
Diabetes Research, Indiana University
School of Medicine, 635 Barnhill Dr.,
Indianapolis, Indiana 46202
C
LAUDE KLEE (22), Laboratory of Bio-
chemistry, National Cancer Institute,
National Institutes of Health, Bethesda,
Maryland 20892
T
AMAR KLEINBERGER (15), Gonda Center for
Molecular Biology, B. Rappaport Faculty
of Medicine, Technion-Israel Institute of
Technology, P.O.Box 9649, Bat-Galim,
Haifa, 31096, Israel
S
USANNE KLUMPP (5,21) Institut fu
¨

r
Pharmazeutische & Medizinische Chemie,
Westfa
¨
lische Wilhelms-Universita
¨
t, Hittorf-
strasse 58-62, 48149 Mu
¨
nster, Germany
J
OSEF KRIEGLSTEIN (5,21) Institut fu
¨
r
Pharmakologie und Toxikologie, Philipps-
Universita
¨
t, Ketzerbach 63, 35032
Marburg, Germany
x CONTRIBUTORS
YOUNG-GUEN KWON (24), Department of
Biochemistry, College of Natural Sciences,
School of Medicine, Kangwon National
University, Chunchon, Kangwon-Do,
200-701, Korea
S
ARA LAVI (25), Department of Cell Research
and Immunology, Tel Aviv University, Tel
Aviv, 69978, Israel
C

RAIG LEACH (19), Center for Cell Signaling
and Department of Microbiology, Univer-
sity of Virginia, School of Medicine,
Charlottesville, Virginia 22908
O
RLEV LEVY-NISSENBAUM (9), Department of
Cell Research and Immunology, George S.
Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv, 69978, Israel
S
HIPENG LI (22), Laboratory of Biochemistry,
National Cancer Institute National Insti-
tutes of Health, Bethesda, Maryland 20892
J
OHN M. LUCOCQ (6), School of Life Sciences,
MSI/WTB Complex, Dow Street, Univer-
sity of Dundee, Dundee DD1 5EH, UK
R
USSELL I. LUDOWYKE (10), Proteome Sys-
tems Ltd, 1/35-41 Waterloo Road, North
Ryde, NSW 2113, Australia
A
LEXANDER MAAßEN (21), Institut fu
¨
r
Pharmazeutische & Medizinische Chemie,
Westfa
¨
lische Wilhelms-Universita
¨

t, Hit-
torfstr. 58-62, 48149 Mu
¨
nster, Germany
N
IRMALA MAVILA (2), Department of Bio-
chemistry and Molecular Biology, and
Center for Diabetes Research, Indiana
University School of Medicine, 635 Barnhill
Dr., Indianapolis, Illinois 46202
T
ZU-CHING MENG (23), Cold Spring Harbor
Laboratory, 1 Bungtown Road, Cold Spring
Harbor, New York 11724
M
ARC C. MUMBY (27), Department of Phar-
macology, University of Texas Southwes-
tern Medical Center, Dallas, Texas
75390-9041
S
HONAGH MUNRO (12), Medical Research
Council Protein Phosphorylation Unit,
School of Life Sciences MSI/WTB Com-
plex, University of Dundee, Dow Street,
Dundee DD1 5EH, Scotland, UK
A
NGUS C. NAIRN (24), Department of Psy-
chiatry, Yale University School of Medi-
cine, New Haven, Connecticut 06508
P

AULA OFEK (25), Department of Cell
Research and Immunology, Tel Aviv Uni-
versity, Tel Aviv, 69978, Israel
E
GON OGRIS (16), Institute of Medical Bio-
chemistry, Division of Molecular Biology,
Vienna Biocenter, University of Vienna, Dr.
Bohr-Gasse 9, Vienna A-1030, Austria
L
ORENZO A. PINNA (1), Dipartimento di
Chimica Biologica, University of Padova,
Viale G. Colombo 3, Padova 35121, Italy
P
ATRICK PIRIBAUER (16), Institute of Medical
Biochemistry, Division of Molecular Biol-
ogy, Vienna Biocenter, University of Vienna,
Dr. Bohr-Gasse 9, Vienna A-1030, Austria
P
IA RAANANI (9), Institute of Hematology,
The Chaim Sheba Medical Center,
Tel-Hashomer, Israel, and Sackler School
of Medicine, Tel-Aviv University, Tel-Aviv,
69978, Israel
R
UPA RAY (7,8), Department of Pharmacol-
ogy and Cancer Biology, Duke University,
Research Drive, C118 LSRC, Durham,
North Carolina 27710-3686
P
ETER J. ROACH (2,30), Department of Bio-

chemistry and Molecular Biology, and
Center for Diabetes Research, Indiana
University School of Medicine, 635 Barnhill
Dr., Indianapolis, Indiana 46202
O
RIT SAGI-ASSIF (9) Department of Cell
Research and Immunology, George S.
Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv, 69978, Israel
A
NDREAS SCHILD (29), Division of Psychiatry
Research, University of Zurich, August
Forel Str 1, Zurich CH 8008, Zu
¨
rich,
Switzerland
R
AKEFET SHARF (15), Gonda Center for
Molecular Biology, B. Rappaport Faculty
of Medicine, Technion-Israel Institute of
Technology, P.O. Box 9649, Bat-Galim,
Haifa, 31096, Israel
D
AGMAR SELKE (21), Institut fu
¨
r Pharmazeu-
tische & Medizinische Chemie, Westfa
¨
lische
Wilhelms-Universita

¨
t, Hittorfstr. 58-62,
48149 Mu
¨
nster, Germany
CONTRIBUTORS xi
ADAM M. SILVERSTEIN (27), Department of
Pharmacology, University of Texas South-
western Medical Center, Dallas, Texas
75390-9041
A
LISTAIR T. R. SIM (10), School of Biomedical
Sciences, University of Newcastle and
Clinical Neuroscience Program, Hunter
Medical Research Institute, Callaghan,
NSW 2308, Australia
N
ATALIA STARKOVA (24), Laboratory of
Molecular and Cellular Neuroscience, The
Rockefeller University, 1230 York Avenue,
New York, New York 10021
J
A
´
NOS SZO
¨
LLO
00
SI (17), Department of Biophy-
sics and Cell Biology, Faculty of Medicine,

Medical and Health Science Center, Uni-
versity of Debrecen, Debrecen H-4012,
Hungary
G
REGORY S. TAYLOR (4), Departments of
Pharmacology, Cellular and Molecular
Medicine, Chemistry and Biochemistry,
University of California, Basic Science
Building 3092A-M/C 0636, 9500 Gilman
Drive, La Jolla, California 92093-0636
L
EON G. J. TERTOOLEN (18), Hubrecht
Laboratory, Netherlands Institute for
Developmental Biology, Uppsalalaan 8,
Utrecht, 3584 CT, The Netherlands
Z
OHAR TIRAN (11), Department of Molecular
Genetics, The Weizmann Institute of
Science, Rehovot, 76100, Israel
N
ICHOLAS K. TONKS (23), Cold Spring Harbor
Laboratory, 1 Bungtown Road, Cold Spring
Harbor, New York 11724
N
IKOLAOS A. TOUNTAS (19), Center for Cell
Signaling and Department of Microbiology,
University of Virginia, School of Medicine,
Charlottesville, Virginia 22908
T
HEA VAN DER WIJK (18), Hubrecht Labora-

tory, Netherlands Institute for Developmen-
tal Biology, Uppsalalaan 8, Utrecht, 3584
CT, The Netherlands
A
LEYDE VAN EYNDE (13), Afdeling Bio-
chemie, Faculteit Geneeskunde, Katholieke
Universiteit Leuven, Herestraat 49, B-3000
Leuven, Belgium
B
HARGAVI VEMURI (2), Department of Bio-
chemistry and Molecular Biology, and
Center for Diabetes Research, Indiana
University School of Medicine, 635 Barnhill
Dr., Indianapolis, Indiana 46202
S
TEFAN W. VETTER (20), Department of
Molecular Biology, The Scripps Research
Institute, 10550 North Torrey Pines Blvd,
La Jolla, California 92037
P
IER GIUSEPPE VILARDO (2), Department of
Biochemistry and Molecular Biology, and
Center for Diabetes Research, Indiana
University School of Medicine, 635 Barnhill
Dr., Indianapolis, Indiana 46202
B
RIAN E. WADZINSKI (14), Room 702 Light
Hall, Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
X

UTONG WANG (22), Board of Governors,
Federal Reserve System, 20th & C Street,
Washington, District of Columbia 20551
T
AKUO WATANABE (24), Department of Bio-
chemistry and Molecular Vascular Biology,
Kanazawa University Graduate School of
Medical Science, 13-1, Takara-machi,
Kanazawa 920-8640, Japan
S
TEPHEN A. WATT (6), School of Life
Sciences, MSI/WTB Complex, Dow Street,
University of Dundee, Dundee DD1 5EH,
UK
W
AYNE A. WILSON (30), Department of
Biochemistry and Molecular Biology, and
Center for Diabetes Research, Indiana
University School of Medicine, Indiana-
polis, Indiana 46202
I
SAAC P. WITZ (9), Department of Cell
Research and Immunology, George S.
Wise Faculty of Life Sciences, and The
Ela Kodesz Institute for Research on
Cancer Development and Prevention, Tel
Aviv University, Tel Aviv, 69978, Israel
Z
HONG-YIN ZHANG (3,20), Department of
Molecular Pharmacology, Albert Einstein

College of Medicine, 1300 Morris Park
Avenue, Bronx, New York 10461
B
O ZHOU (3), Department of Molecular
Pharmacology, Albert Einstein College of
Medicine, 1300 Morris Park Avenue,
Bronx, New York 10461
xii CONTRIBUTORS
Foreword
The Past and Future of Protein Phosphatase Research
I have had relatively little involvement with protein phosphatase s
over the past 10 years. Nevertheless, as it is still a subject that is dear to my
heart, I was delighted to accept the Editors’ invitation to write this
foreword. It has given me an opportunity to take a nostalgic look back at
the early days of the field when protein phosphatases were my major
preoccupation, as well as to consider what the future may hold. The first
part is therefore a personal account of the events that led my laboratory to
classify the serine/threonine protein phosphatases 20 years ago and an
outline of the discoveries I have been most fascinated by over the past
20 years. A more detailed historical account containing the original
references can be found elsewhere.
1
The second half of this foreword contains
a few thoughts about how the field of protei n phosphatases might develop.
The Past
The study of protein phosphatases, like that of protein phosphoryla-
tion, originated from studies of the control of glycogen metabolism. Over
60 years ago, Car l and Gerty Cori found that glycogen phosphorylase
could be isolated in two forms termed phosphorylase b and phosphorylase
a. Since the b-form required 5

0
AMP for activity but the a-form was almost
fully acti ve without AMP, they reasoned (incorrectly) that the a-form must
contain tightly bound 5
0
-AMP, and that the a to b converting enzyme they
had discovered, must catalyze the release of 5
0
AMP from the a-form. It was
only many years later, with the discovery by Edmond Fischer and Edwin
Krebs that the conversion of phosphorylase b to a involved a
phosphorylation event, that it became obvious that the a to b converting
enzyme was a pro tein phosphatase.
My own involvement with protein phosphatases began in 1972 when my
first graduate student, John Antoniw, began to isolate the protein
phosphatase that dephosphorylated and inactivated phosphorylase kinase
in skeletal muscle, the enzyme that converts phosphorylase b to a.
Surprisingly, John resolved the activity into two enzymes, one of which
dephosphorylated the -subunit of the enzyme much faster than the
1
P. Cohen, Bioessays 16, 583–588 (1994).
xlv
-subunit, the other being highly specific for the -subunit. Subsequently,
John showed that the phosphatase specific for the -subunit was also the
major phosphatase activity in skeletal muscle acting on glycogen synthase
and glycogen phosphorylase and was therefore the original a to b converting
enzyme. He and a postdoctoral fellow, Gillian Nimmo, also showed that
this phosphatase was inhibited by two heat-stable proteins termed inhibitor
1 and inhibitor 2 that Walter Glinsmann had identified in 1976. The
phosphatase was therefore renamed protein phosphatase-1 (PP1) to reflect

the fact that it had a broad specificity and tissue distribution, and was likely
to dephosphorylate many regulatory proteins in vivo.
The phosphatase that dephosphorylated the -subunit was insensitive
to inhibitors 1 and 2 and initially termed protein phosphatase 2 (PP2).
It was only several years later that another graduate student, Lex Stewart,
discovered that it was dependent on calcium ions and calmodulin for activity
and, in collaboration with Claude Klee, that it was identical to calcineurin,
a major calmodulin-binding protein in the brain of previously unknown
function.
Studies by a postdoctoral fellow, Tom Ingebritsen, and a graduate
student, Gordon Foulkes, identified a protein phosphatase in the liver that,
like PP2 from muscle, dephosphorylated the -subunit of phosphorylase
kinase much faster than the -subunit and was insensitive to inhibitors 1
and 2. However, in contrast to PP2 from muscle, the hepatic protein
phosphatase had a much higher activity towards phosphorylase. It was
therefore termed PP2A to distinguish it from the muscle PP2, which was
renamed PP2B. A Mg
2 þ
-dependent protein phosphatase that had been
identified in several laboratories and purified to homogeneity by
Shigeru Tsuiki in 1981 was also found to dephosphorylate the -subunit
preferentially, was insensitive to inhibitors 1 and 2, and we therefore termed
it PP2C.
As a result of further investigations by Tom Ingebritsen, Gordon
Foulkes and others in the laboratory, it became obv ious that PP1, PP2A,
PP2B and PP2C accounted for most, if not all, of the serine/threonine-
specific protein phosphatases towards many regulatory proteins involved in
controlling a variety of cellular processes. Although the publication of our
ideas in 1983 was initially greeted with skepticism in some quarters, they
came to be accepted because the simple methods introduced to distinguish

each protein serine/threonine phosphatase enabled other investigators to
rapidly classify the phosphatase that they had been studying.
Over the past 20 years, there has been huge progress in understanding
the structure, substrate specificity and regulation of these and many other
protein phosphatases. One crucially important advance was the isolation
and characterization of the first protein tyrosine phosphatase (PTPase) by
xlvi FOREWORD
one of my former graduate students Nick Tonks, when he joined Edmond
Fischer as a postdoctoral fellow. The PTPase subfamily encompasses many
enzymes, including transmembrane receptor-like molecules whose activities
are likely to be modulated by as yet unidentified ligands, and ‘‘dual
specificity phosphatases that play key roles in the regulation of MAP kinase
cascades and the cell division cycle.’’ The amino acid sequence of every
human protein phosphatase is probably now known and structures of
members of each subclass have been solved at atomic resolution by X-ray
crystallography. We now know that the isoforms of PP1, PP2A and PP2B
are members of the same subfamily (now known as the PPP subfamily),
while the PP2Cs are structurally unrelated (the PPM family). A third
subfamily of protein serine/threonine phosphatases (the FCP family), of
which there are at least five members, was only identified in the late 1990s by
Jack Greenblatt and his colleagues.
2
The amino acid sequences of the FCP
phosphatases are unrelated to either the PPP or PPM subfamilies or to the
PTPases. Although Harry Matthews has shown that members of the PPP
and PPM subfamilies dephosphorylate histidine residues in proteins very
efficiently, the first eukaryotic protein histidine phosphatase that is unable to
dephosphorylate serine and threonine residues was recently identified by
Susanne Klumpp and her colleagues.
3

The structure of this protein histidine
phosphatase is also unrelated to other protein phosphatases. Thus there are
at least five structurally distinct subfamilies of protein phosphatases in
eukaryotic cell, or more if there are separate phosphatases that
dephosphorylate lysine and arginine residues in proteins.
We now appreciate that protein kinases and protein phosphatases do
not find their substrates by simple diffusion, but are directed to specific
locations by targeting subunits and/or by the presence of ‘‘docking’’ sites
located on the phosphatases and their substrates. This has become an
enormous area of research in which the protein phosphatases can be said to
have paved the way, starting with the finding in 1985 by Peter Stralfors, a
postdoctoral fellow in my laboratory, that PP1 is directed to glycogen by a
specific glycogen-targeting subunit.
A surprising development at the time was the discovery of connections
between protein phosphatases and human diseas e, starting with the report
by Akira Takai in 1987 that okadaic acid was a potent inhibitor of PP1 and
PP2A. This complex polyketal is a major toxic component associated with
diarrhetic seafood poisoning, which has poisoned thousands of people and
2
J. Archambault, G. Pan, G. K. Dahmus, M. Cartier, N. F. Marshall, S. Zhang, M. E. Dahmus,
and J. Greenblatt, J. Biol. Chem. 273, 27593–27601 (1998).
3
S. Klumpp, J. Hermesmeier, D. Selke, R. Baumeister, R. Kellner, and J. Krieglstein, J. Cereb.
Blood Flow Metab. 22, 1420–1424 (2002).
FOREWORD xlvii
damaged the shellfish industries of many countries. M icrocystins were
subsequently found to be even more potent inhibitors of PP1 and PP2A.
These cyclic heptapeptides are liver toxins produced by blue-green algae and
the most potent liver carcinogens known to man. A plethora of other
naturally occurring inhibitors of PP1 and PP2A have been identified

subsequently.
A further interesting connection between protein phosphatases and
cancer was made by Gernot Walter, who found that the small T-antigens of
simian virus 40 and polyomavirus, which enhance the transformation of
mammalian cells, form complexes with a particular species of PP2A. Marc
Mumby showed that this decreases their activity towards some substrates,
explaining their oncogenicity.
No less remarkable was the discovery by Stuart Schreiber in 1991 that
cyclosporin and FK506, when bound to immunophilins, are potent and
specific inhibitors of PP2B. These immunosuppressants, which have
permitted the widespread use of organ transplantation, block the
production of interleukin-2 and hence the proliferation of T cells.
Remarkable connections between PTPases and disease were also made
by Jack Dixon in the early 1990s. He showed that bacteria of the genus
Yersinia produce a PTPase which is a virulence factor for several diseases,
including pseudotuberculosis, a range of gastrointestinal syndromes and the
bubonic plague. The last mentioned has been responsible for several
pandemics over the past millennium, the most recent being the ‘‘Black
Death’’ which killed no less than a quarter of the population of Europe in
the 17th century.
The Future
So what does the future of protein phosphatase research hold and what
are the key issues that still need to be resolved? One of the most challenging
problems in the postgenomic era will be to identify all the physiological
substrates of each protein phosphatase. This is a formidable task bearing in
mind that about a third of the 30,000 proteins encoded by the human
genome are phosphorylated frequently at multiple sites. Since about 150
protein phosphatases are encoded by the human genome, one can calculate
that ‘‘on average’’ a protein phosphatase must have 60–70 substrates in vivo.
However, this is an underestimate since many substrates, like phosphorylase

kinase, are dephosphorylated by two or more phosphatases. Clearly,
powerful new methodologies will be ne eded to tackle this problem, such as
the substrate trapping mutants of PTPases developed by Nick Tonks, which
are described in one of the chapters of this volume.
xlviii FOREWORD
Disruption or mutation of the genes encoding some protein
phosphatases has been useful in defining some of the physiological
processes that they regulate. For example, Mitsuhiro Yanagida showed that
the mutation of PP1 in yeast caused chromosome separat ion, while Tricia
Cohen showed that the disruption of PP4 in Drosophila caused defective
nucleation of microtubules from centrosomes. However, inactivating a
protein phosphatase catalytic subunit may frequently not be that helpful in
identifying the direct substrates of a protein phosphatase in vivo. One reason
for this is that many protein kinases are activated by phosphorylation.
Therefore suppressing the activity of a phosphatase, whether by gene
disruption, RNAi or the use of a specific inhibitor, will activate many
protein kinases and therefore lead to the hyperphosphorylation of proteins
that are not themselves direct substrates of the phosphatase under
investigation. A more effective approach may therefore be to prevent the
expression of a particular targeting or regulatory subunit. For example,
Anna de Paoli-Roach and Tricia Cohen have both generated mice that do
not express the gene encoding the G
M
glycogen-targeting subunit of PP1,
providing genetic evidence that the PP1–G
M
complex is indeed the major
phosphatase acting on glycogen synthase and glycogen phosphorylase
in muscle. Extending this approach to other targeting subunits will
undoubtedly aid the identification of other substrates of PP1, although

‘‘knock-in’’ mutations expressing a modified targeting subunit in which the
PP1-interacting ‘‘RVXF’’ motif is disabled may be even better.
Methodological problems associated with studying PP1-targeting subunits
complexes are addressed in Section II of this volume.
Many naturally occurring inhibitors of the PPP subfamily have been
identified and exploited to demonstrate that a number of physiological
processes are regulated by reversible phosphorylation. However, their
usefulness is limited, because they all inhibit several PPP family members.
Despite the caveats outlined earlier, far more specific inhibitors would be
valuable. The recent report from Kunimi Kikuchi and his colleagues that
tautomycetin is a relatively specific inhibitor of PP1 may therefore be a
significant development.
4
Finally, what are the chances that specific inhibitors of protein
phosphatases will be developed for the treatment of disease? Enthusiasts of
this idea received a major boost five years ago when Michel Trembly
reported that mice deficient in PTP1B were not only hypersensitive to
insulin, but also did not gain weight when fed on a high carbohydrate, high
fat diet. For this reason, many pharmaceutical companies are attempting to
4
S. Mitsuhashi, N. Matsuura, M. Ubukata, H. Oikawa, H. Shima, and K. Kikuchi, Biochem.
Biophys. Res. Commun. 287, 328–331 (2001).
FOREWORD xlix
develop drugs that target this enzyme, which may be useful in the treatment
of diabetes and obesity. Some relatively specific inhibitors have already been
generated but, to my knowledge, no PTP1B inhibitor has yet entered human
clinical trials. However, there are other protein phosphatases that are
attractive drug targets, such as CD45, a PTPase expressed in cells of the
immune system that is essential for T-cell activation. Inhibitors of CD45
could be effective immunosuppressants.

However, simply inhibiting the catalytic subunit of most protein
phosphatases will be toxic or oncogenic. The future of drug discovery in this
area may therefore lie in the development of compounds that disrupt the
interaction of a phosphatase with one of its regulatory or targeting subunits,
or the interaction between a targeting subunit and an other regulatory
protein. For example, the form of PP1 associated with hepatic glycogen is
complexed to the liver glycogen-targeting subunit G
L
. The binding of
phosphorylase a to the extreme C-terminus of G
L
prevents PP1 from
dephosphorylating and activating glycogen synthase.
5
A drug that disrupted
the interaction of phosphorylase a with G
L
would be expected to activate
glycogen synthase and hence stimulate the conversion of blood glucose into
hepatic glycogen. Cell permeant inhibitors of the protein kinase GSK3,
which also stimulate the activation of hepatic glycogen synthase, have
recently been shown by Gerald Schulman to normalize blood glucose levels
in animal models of diabetes.
Pharmaceutical companies will tell you that it is extremely difficult (and
therefore unattractive) to develop a drug that disrupts a protein–protein
interaction. However, it was only ten years ago that it was considered
virtually impossible to develop a really specific protein kinase inhibitor, and
yet these enzymes have now become the second most studied family of drug
targets after G protein-coupled receptors. It only takes one success to
overcome a dogma and it may well be that, in the not too distant future,

drugs that target components of protein phosphatases will become a reality.
P
HILIP COHEN
MRC Protein Phosphorylation Unit
University of Dundee
Scotland
5
C. G. Armstrong, M. J. Doherty, and P. T. W. Cohen, Biochem. J. 336, 699–704 (1998).
l FOREWORD
Section I
Determination, Detection, and Localization

[1] Monitoring of PP2A and PP2C by Phosphothreonyl
Peptide Substrates
By ARIANNA DONELLA-DEANA,MARCO BOSCHETTI, and LORENZO A. PINNA
Reversible phosphorylation of Ser, Thr, and Tyr residues occurring
through the concerted action of protein kinases and protein phosphatases
affects about one-third of eukaryotic proteins and represents the most
frequent and general mechanism by which nearly all biological functions are
regulated. The human genome encodes more than 600 protein kinases and
slightly less than 100 protein phosphatases. This figure by itself, in
conjunction with the observation that protein phosphatases operate on a
minority of potential phosphoacceptor residues preselected by protein
kinases, would indicate that the specificity of phosphatases needs to be less
stringent than that of protein kinases. This is especially true of site
specificity, i.e., the ability to recognize consensus sequences defined by local
structural features surrounding the phosphoacceptor aminoacid. This is a
prominent feature of most protein kinases, with special reference of Ser/Thr
specific ones, and has been exploited for the design of hundreds of specific
peptide substrates. These proved useful for the selective monitoring of

individual kinases or classes of kinases, whose structural features
responsible for site specificity in many instances have been disclosed by
mutational and structural analyzes (for a comp ilation of consensus
sequences and peptide substrates of kinases see Pinna and Ruzzene
1
and
Ruzzene and Pinna
2
). In contrast many attempts to define consensus
sequences recognized by individual protein phosphatases were less
rewarding and consequently the development of phosphopeptides useful
for the specific assay of different categories of protein phosphatases proved
rather unsuccessful
3
consistent with the notion that residues surrounding the
phosphoaminoacid may play a marginal role in substrate recognition, which
is mainly mediated by targeting subunits and/or struc tural modules outside
the catalytic core of the phosphatase.
4–7
In a way this was expectable if it is
1
L. A. Pinna and M. Ruzzene, Biochim. Biophys. Acta 131, 191 (1996).
2
M. Ruzzene and L. A. Pinna, in ‘‘Protein Phosphorylation: A Practical Approach’’ (G. Hardy,
ed.), 2nd Ed., p. 221. Oxford University Press, Oxford, UK, 1999.
3
L. A. Pinna and A. Donella-Deana, Biochim. Biophys. Acta 1222, 415 (1994).
4
M. J. Hubbard and P. Cohen, Trends Biochem. Sci. 18, 172 (1993).
5

F. Shibasaki, E. R. Price, D. Milan, and F. McKeon, Nature 382, 370 (1996).
6
H. Song, N. Hanlon, N. R. Brown, N. E. M. Noble, L. N. Johnson, and D. Barford, Mol. Cell
7, 615 (2001).
7
N. K. Tonks and B. G. Neel, Curr. Opin. Cell Biol. 13, 182 (2001).
Copyright ß 2003, Elsevier Inc.
All rights reserved.
METHODS IN ENZYMOLOGY, VOL. 366 0076-6879/2003 $35.00
[1] DEPHOSPHORYLATION OF PHOSPHOTHREONINE PEPTIDES 3
considered that an individual phosphatase is committed to the dephos-
phorylation of a variety of proteins previously phosphorylated by kinases
that recognize different consensus sequences. Calcineurin, e.g., is implicated
in the dephosphorylation of several transcription factors, whose phos-
phoacceptor sites are very variable for being phosphorylated by different
kinases. Likewise it would be hard to figure out any common denominator
around the phosphorylated residues which are affected by PP2A.
8
A telling
example is also provided by two physiological targets of PP2C,
hydroxymethylglutaryl Coenzyme A (HMG-CoA) reductase
9
and BAD
(Klumpp and Krieglstein, personal communication) whose dephosphory-
lated residues display sequences (HNRpSKIN and PAGpTEED, respec-
tively) having nothing in common.
The circumstance that phosphopeptides are scarcely selective substrates
does not hamper their usage for handy and sensitive assays of protein
phosphatase activities whenever specificity is not a priority requirement.
This concept especially applies to Tyr specific protein phosphatase s (PTPs),

whose activity is in fact often monitored and scrutinized with the aid of
phosphopeptides displaying kinetic parameters comparable to those of
protein substrates.
3
A sample of outstanding phosphopeptide substrates
of PTPs is provided in Table I with their K
cat
/K
m
ratio values and pertinent
references. Clearly the successful usage of phosphopeptides as tools for
monitoring and characterizing PTPs is due to a con comitance of properties
which are not shared by Ser/Thr specific protein phosphatases (PPs). First,
PTPs are extremely proficient enzymes with K
cat
/K
m
ratios approaching in
some cases the efficiency limited by diffusion events, a circumstance that
makes easily applicable nonradioactive methods for monitoring their
activity. Second, the dephosphorylation of tyrosyl residues can be
monitored by spectrophotometric methods (taking advantage of the
different absorption spectra of phosphorylated vs unphosphorylated
tyrosine) and specific immunochemical assays (based on fluorescent anti-
phosphotyrosine antibodies) which are not applicable to P-Ser and P-Thr
residues. Third, the promiscuity of PTPs is such that they are able to
dephosphorylate even free phosphotyrosine
10
and related compounds if
these are added at appropriate concentration; thus a broadly employed

PTPs substrate is p-nitrophenyl phosphate (pNPP) whose dephosphoryla-
tion correlates with a change in color which can be followed spectro-
photometrically.
8
T. A. Millward, S. Zolnierowicz, and B. A. Hemmings, Trends Biochem. Sci. 24, 186 (1999).
9
Y. P. Ching, T. Kobayashi, S. Tamura, and D. G. Hardie, FEBS Lett. 411, 265 (1997).
10
Z. Zhao, N. F. Zander, D. A. Malencik, S. R. Anderson, and E. H. Fischer, Anal. Biochem.
202, 361 (1992).
4 DETERMINATION, DETECTION, AND LOCALIZATION [1]
By sharp contrast to PTPs, Ser/Thr PPs, whose catalytic efficiency is on
the average much lower than that of PTPs (see Table I), are generally
assayed by means of phosphoradiolabeled protein substrates. Commonly
used are phosphorylase-a and histones (for the assay of PP1 and PP2A), RII
(regulatory subunit of cAMP-dependent protein kinase) and dopamine-and-
cAMP-regulated phosphoprotein (for assaying calcineurin/PP2B) and
casein phosphorylated by PKA (cAMP-dependent protein kinase) (for the
assay of PP2C). Some PPs, notably calcineurin (which also dephos-
phorylates phosphotyrosyl residues) and, under special circumstances,
PP2A, display significant activity toward pNPP. Unless the phosphatase is
highly purified, however, pNPP cannot be exploited for the reliable
monitoring of its activity owing to the much higher pNPP-ase activity of
PTPs and nonspecific phosphatases which might contaminate the sample. It
should be noted on the other hand that PP1 is nearly inactive on
phosphopeptides,
3,11–13
while calcineurin needs quite large phosphopeptides
TABLE I
P

HOSPHOPEPTIDES USED FOR SENSITIVE ASSAYS OF PROTEIN TYROSINE PHOSPHATASES
K
cat
/K
m
Phosphopeptide PPs (S
À1
Á M
À1
) Â 10
7
Ref.
AFLEDFFTSTEPQpYQPGENL TC-PTP 3.70 a
NIDGDEVNpYEE TC-PTP 4.42 a
DADEpYLIPQQG PTPU323 2.88 b
Yersinia PTP 2.22 b
TAEPDpYGALYE HPTPb 5.71 c
Ac-ELEFpYMDE-NH
2
PTP1B 2.20 d
RRApTVA
f
PP2A 0.02 e
a
M. Ruzzene, A. Donella-Deana, O. Marin, J. W. Perich, P. Ruzza, G. Borin, A. Calderan,
and L. A. Pinna, Eur. J. Biochem. 211, 289 (1993).
b
J. Zhang, Z. Zhang, K. Brew, and E. Y. Lee, Biochemistry 35, 6276 (1996).
c
H. Cho, R. Krishnaraj, M. Itoh, E. Kitas, W. Bannwarth, H. Saito, and C. T. Walsh,

Protein Sci. 2, 977 (1993).
d
S. W. Vetter, Y. F. Keng, D. S. Lawrence, and Z. Y. Zhang, J. Biol. Chem. 275, 2265 (2000).
e
P. Agostinis J. Goris, E. Waelkens, L. A. Pinna, F. Marchiori, and W. Merlevede, J. Biol.
Chem. 262, 1060 (1987).
f
For sake of comparison the K
cat
/K
m
ratio of one of the best peptide substrates for Ser/Thr
PPs is also reported.
11
S. J. McNall and E. H. Fischer, J. Biol. Chem. 263, 1893 (1988).
12
P. Agostinis, J. Goris, L. A. Pinna, F. Marchiori, J. W. Perich, H. E. Meyer, and
W. Merlevede, Eur. J. Biochem. 189, 235 (1990).
13
P. Agostinis, J. Goris, E. Waelkens, L. A. Pinna, F. Marchiori, and W. Merlevede, J. Biol.
Chem. 262, 1060 (1987).
[1] DEPHOSPHORYLATION OF PHOSPHOTHREONINE PEPTIDES 5
(>20 residues long) in order to display an activity comparable to that
observed with its protein substrates.
14
At variance with PP1 and calcineurin,
PP2A and PP2C do dephosphorylate a variety of short phosphopeptides
with efficiencies comparable to protein substrates routinely used for
monitoring their activity.
12,15

Some of these peptides were subjected to
structural modifications with the aim to disclose local structural features
eventually acting as positive or negative determinants, irrespective of their
actual physiological relevance. An unexpected outcome of these studies was
that in general the replacement of phosphothreonine for phosphoserine
dramatically improved dephosphorylation efficiency by PP2A, PP2C, and
PP1. In particular the phosphorylated form of the peptide RR ATVA, i.e.,
the threonyl derivative of the ‘‘kemptide’’ (RRASVA), reproducing the
phosphoacceptor site of pyruvate kinase, a substrate of PP2A,
16
turned
out to be dephosphorylated much more readily than its phosphoseryl
counterpart and also faster than the protein substrates commonly employed
for monitoring PP2A and PPC, phosphorylase-a and casein phospho-
radiolabeled by PKA, respectively (see Table II). A quite high P-Thr vs P-
Ser dephosphorylation rate was also observed with PP1; in this case however
the dephosphorylation rate of the peptides is negligible as compared to that
of phosphorylase-a. Calcineurin does not display any marked preference for
the phosphothreonyl kemptide whose dephosphorylation rate is similar to
that of its phosphoseryl derivative, both being much slower than that of the
protein substrate RII. By sharp contrast, as also reported in Table II,a
number of nonspecific phosphatases, either acidic or alkaline, invariably
display a remarkable preference for the phosphoseryl kemptide. Collectively
taken these data show that the phosphothreonyl kemptide, RRApTVA,
represents a first choice substrate for the assay of PP2A and PP2C and, in
combination with its pS counterpart, it also provides a valuable tool
for discriminating between phosphatase activities accounted for either by
bona fide protein phosphatases of the PP2A and PP2C classes or by
nonspecific alkaline/acidic phosphatases. The general applicability of these
criteria was validated by showing that the marked preference for pT vs pS

peptides is not restricted to the catalytic subunit of PP2A but is also
shared by all its oligomeric forms.
12,13
Likewise the pT-kemptide is by far
preferred over its pS congener by both the alpha and beta isoforms of
animal PP2C as well as by PP2C from the protozoan Paramecium
14
A. Donella-Deana, M. H. Krinks, C. Klee, M. Ruzzene, and L. A. Pinna, Eur. J. Biochem.
219, 109 (1994).
15
A. Donella-Deana, C. H. Mac Gowan, P. Cohen, F. Marchiori, H. E. Meyer, and L. A. Pinna,
Biochim. Biophys. Acta 1051, 199 (1990).
16
M. Nishimura and K. Uyeda, J. Biol. Chem. 270, 26341 (1995).
6 DETERMINATION, DETECTION, AND LOCALIZATION [1]
tetraurelia
17
and by SpoIIE, a bacterial protein phosphatase related to the
PP2C class of eukaryotic protein phosphatases.
18
Interestingly, moreover,
preferential dephosphorylation of P-Thr residues is not restricted to peptide
substrates, since PP2C specifically dephosphorylated only pT residues
present in a sample of
32
P-casein evenly radiolabeled at Se r and Thr
residues,
15
and a mutant of HMG-CoA reductase with the phosphorylatable
serine replaced by threonine, once phosphorylated by AMP-activated

protein Kinase (AMPK) was dephosphorylated by PP2A much more readily
than the phospho-wild type protei n.
9
This does not necessarily mean that
preferential P-Thr dep hosphorylation reflects a physiological situation.
As a matter of fact P-Ser is by far predominant over P-Thr in naturally
TABLE II
P
REFERENTIAL DEPHOSPHORYLATION OF PHOSPHOTHREONYL VS PHOSPHOSERYL
PEPTIDE BY SOME CLASSES OF PPs
Relative activity
a
Dephosphorylation ratio
RRApTVA RRApSVA (pT/pS)
Protein phosphatases
PP1 2.0 0.1 20.02
PP2A 712.1 11.0 64.69
PP2B (calcineurin) 1.6 0.5 3.20
PP2C 253.2 8.8 28.71
Alkaline phosphatases
S. cerevisiae 0.0 72.4 0.00
Intestinal 40.2 326.3 0.12
E. coli 0.0 563.9 0.00
Acid phosphatases
S. cerevisiae 2.4 51.4 0.04
Prostatic 32.4 205.8 0.15
Potato 0.4 5.1 0.08
Wheat germ 1.4 8.6 0.16
a
Expressed as percent of the dephosphorylation rate of the protein substrates phosphorylase

a, RII and phosphocasein in the case of protein phosphatases PP1/PP2A, PP2B and PP2C,
respectively, and of pNPP in the case of alkaline and acid phosphatases. Drawn from A.
Donella-Deana, M. H. Krinks, C. Klee, M. Ruzzene, and L. A. Pinna, Eur. J. Biochem. 219, 109
(1994), and A. Donella-Deana, H. E. Meyer, and L. A. Pinna, Biochim. Biophys. Acta 1094, 130
(1991), respectively.
17
S. Klumpp, C. Hanke, A. Donella-Deana, A. Beyer, R. Kellner, L. A. Pinna, and J. E. Schultz,
J. Biol. Chem. 269, 32774 (1994).
18
E. Adler, A. Donella-Deana, F. Arigoni, L. A. Pinna, and P. Stragier, Mol. Microbiol. 23,57
(1997).
[1] DEPHOSPHORYLATION OF PHOSPHOTHREONINE PEPTIDES 7
occurring phosphoproteins
19
and, to the best of our knowledge there is
no in vivo data supporting the view that the latter are turning over more
rapidly than the former. However, it may be pertinent to note that
phosphothreonine is frequently found at sites which have been reported to be
affected by PP2C, notably the regulatory phosphothreonines found
in the glycine rich loop of cyclin-dependent kinases (CDKs)
20
and at the
activation loop of MAP kinases
21,22
and of AMPK
23
and the aforemen-
tioned T-117 of the BAD protein. This may suggest that PP2C has been
adopted as a specialized ‘‘phosphothreonyl phosphatase.’’ On the other
hand, the preference of PP2C for P-Thr vs P-Ser residues is also deeply

influenced by the surrounding aminoacids. In fact the replacement of pT for
pS in the context of the HMG-CoA reductase phosphosite (HNRpSKINL)
which accelerates >10 fold the rate of dephosphorylation by PP2A, has
only a modest effect on dephosphorylation by PP2C.
9
Likewise the
phosphopeptide RRREEpTEEE, which is a n excellent substrate of PP2A
by virtue of its phosphothreonyl residue, is almost unaffected by PP2C.
15
This provides a tool for discriminating PP2A from PP2C activities using
peptide substrates.
The structural features underlying the striking preference of PP2A and
PP2C for phosphothreonyl over phosphoseryl residues are still unknown,
since structural data of complexes between Ser/Thr protein phosphatases
and peptide substrates are not available. Kinetic data show that with
PP2A and, to a lesser extent, with PP2C the be neficial effect of
phosphothreonine is mostly accounted for by increased V
max
, whereas the
K
m
values of the pT and pS peptides are similar (see Table III), suggesting
that the methyl group of threonine accelerates the catalytic event rather than
increasing the binding affinity. Paradoxically the opposite might be
expectable assuming a catalytic mechanism based on protonation of the
leaving hydroxyaminoacid side chain, since the methyl group is a better
electron donor than an hydrogen atom. This would make the P–O ester
bond of phosphothreonine less polari zed toward the oxygen which
would therefore be less susceptible to protonation as compared to the
oxygen of phosphoserine. Consequently phosphothreonyl residues, on the

average, are more acid stable than phosphoserine ones.
24
The reason why
19
T. Mustelin, ‘‘Src Family Tyrosine Kinases in Leukocytes.’’ MBIU, R.G. Landes Co, Austin,
TX, 1994.
20
A. Cheng, P. Kaldis, and M. J. Solomon, J. Biol. Chem. 275, 34744 (2000).
21
C. C. Fjeld and J. M. Denu, J. Biol. Chem. 274, 20336 (1999).
22
J. Warmka, J. Hanneman, J. I. Lee, D. Amin, and I. Ota, Mol. Cell Biol. 21, 51 (2001).
23
A. E. Marley, J. E. Sullivan, D. Carling, W. M. Abbott, G. J. Smith, I. W. Taylor, F. Carey,
and R. K. Beri, Biochem. J. 320, 801 (1996).
24
D. B. Bylund and T. S. Huang, Anal. Biochem. 73, 477 (1976).
8 DETERMINATION, DETECTION, AND LOCALIZATION [1]
they conversely are much proner to cleavage by some classes of protein
phosphatases, notably 2A and 2C, remains unexplained. One possibility
would be that the methyl group becomes a steric hindrance once
the phosphoester bond has been cleaved, thus accelerating the release of
the dephosphorylated product, assuming this is the rate limiting step in the
overall catalytic reaction. This may apply to PP2A, whose mechanism of
catalysis is unknown, while in the case of PP2C the rate limiting step seems
to be the release of the phosphate.
21
To sum up, while the mechanistic features underlying preferential
dephosphorylation of phosphothreonyl residues by PP2A and PP2C, and,
even more the possible physiological significance of such a selection are still

enigmatic, the practical usefulness of phosphothreonyl peptides for assaying
their activity is self evident and it may deserve more attention than it has
been given in the past.
Experimental Procedures
Assay with Radioactive Phosphopeptides
1a Preparation of Radiolabeled Phosphopeptides
Solutions
Solution A: A buffer, suitable for the specific kinase, containing
10 mM unlabeled ATP and [
32
P]ATP or [
33
P]ATP (Amersham
Pharmacia Biotech) to reach the specific activity of 2000 cpm/pmol,
10 mM MgCl
2
or MnCl
2
,1mM peptide, the kinase (in the case of
TABLE III
K
INETIC CONSTANTS FOR THE ENZYMATIC DEPHOSPHORYLATION
OF
pT AND pS KEMPTIDES BY PP2A AND PP2C
PP2A
a
PP2C
b
V
max

c
K
m
d
V
max
c
K
m
d
RRApTVA 1250 16 2400 1.0
RRApSVA 131 16 416 2.5
a
P. Agostinis, J. Goris, L. A. Pinna, F. Marchiori, J. W. Perich, H. E.
Meyer, and W. Merlevede, Eur. J. Biochem. 189, 235 (1990).
b
S. Klumpp, C. Hanke, A. Donella-Deana, A. Beyer, R. Kellner, L. A.
Pinna, and J. E. Schultz, J. Biol. Chem. 269, 32774 (1994).
c
Expressed as nmol/min/mg.
d
Expressed as M.
[1] DEPHOSPHORYLATION OF PHOSPHOTHREONINE PEPTIDES 9
kemptides the catalytic subunit of PKA) and the appropriate cofactors/
activators.
Solution B: 30% (v : v) acetic acid.
Procedure
1. The standard peptide phosphorylation is carried out in 200 l (or
more depending on the amount of phosphopep tide required) of
solution A.

2. The reaction is stopped by addition of 85 l of 100% (v : v) acetic
acid plus 215 l of 30% (v : v) acetic acid to reach a 0.5 ml volume
and the acetic acid concentration of 30% (v : v).
3. A number of Pasteur pipettes, corresponding to the numb er of
phosphorylated peptides are prepared. Each pipette is plugged with
glass wool and held by a suitable rack. AG 1-X8 resin (0.5 ml) is
poured onto each pipette and the resin is extensively washed with
30% (v : v) acetic acid.
4. Each sample is loaded on a pipette and the flow through fraction is
collected. The resin is washed three times with 1 ml of 30% (v : v)
acetic acid and the eluate is collected in the same test tube as the flow
through to a total volume of 3.5 ml.
5. 20 l of each sample is counted by scintillation counter to estimate
the degree of peptide phosphorylation and to calculate the con-
centration of the phosphopeptide.
6. The pipett e eluate, containing a mixture of the unphosphorylated
and the phosphorylated peptide, is supplemented with an equal
volume of water to allow its freezing at À80

C, lyophilized,
resuspended with water and lyophilized three times to remove traces
of acetic acid.
7. The lyophilized phosphopeptide is redissolved at the desired
concentration in water, or in buffer at the appropriate pH.
8. 2 l of the resuspended phosphopeptide are counted in a scintillation
counter and the concentration (pmol) of the phosphopeptide is
calculated by dividing the cpm of the radioactive peptide by the
specific radioactivity of the [
32
P]ATP or [

33
P]ATP (cpm/pmol)
used for the phosphorylation reaction.
Generally it is not necessary to separate the phosphorylated peptide
from its nonphosphorylated counterpart since it has been shown that even
large excess of the latter does not appreciably affect the kinetic parameters
of protein phosphatases, either Ser/Thr or Tyr specific. If the mixture of
phosphorylated and nonphosphorylated peptides is going to be used in the
subsequent phosphatase assay it is especially important to know exactly the
10 DETERMINATION, DETECTION, AND LOCALIZATION [1]