From the Imperial Cancer Research Fund Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital, Dundee DD1 9SY, Scotland, United Kingdom
Received for publication, December 5, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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Mitogen-activated protein (MAP) kinase
phosphatase 1 (MKP-1/CL100) is an inducible nuclear dual specificity
protein phosphatase that can dephosphorylate and inactivate both
mitogen- and stress-activated protein kinases in vitro and
in vivo. However, the molecular mechanism responsible for
the substrate selectivity of MKP-1 is unknown. In addition, it has been
suggested that the signal transducers and activators of transcription 1 (STAT1) transcription factor is a physiological non-MAP kinase
substrate for MKP-1. We have used the yeast two-hybrid assay to
demonstrate that MKP-1 is able to interact selectively with the
extracellular signal-regulated kinase 1/2 (ERK1/2), p38 The mitogen-activated protein
(MAP)1 kinases are key
components of cellular signal transduction pathways, which become
activated in response to a wide variety of external stimuli. They can
be subdivided into at least three classes based on sequence homology and differential activation by agonists (1-3); these include the
growth factor-activated MAP kinases, extracellular signal-regulated kinase 1 (ERK1) and ERK2, and the stress-activated MAP kinases c-Jun
amino-terminal kinase (JNK, SAPK1) and p38 (SAPK2) MAP kinases. In
addition, a number of less well characterized members of the MAP kinase
family have been identified such as BMK1/ERK5, ERK7, and ERK3
(4-6).
MAP kinase pathways relay, amplify, and integrate complex signals in
order to elicit appropriate biological responses. In mammalian cells,
these include cellular proliferation, differentiation, inflammatory
responses, and apoptosis. These responses are associated with
significant alterations in the pattern of cellular gene expression, and
transcription factors are a major target of MAP kinase signaling in vivo (7). To phosphorylate these proteins, activated MAP kinases translocate to the cell nucleus, a process that is generally associated with prolonged activation of the MAP kinase (8). Therefore,
the magnitude and duration of MAP kinase activation are critical
determinants of biological outcome, and regulatory mechanisms governing
the activation of MAP kinase are of key importance in both
physiological and pathological cell functions.
The duration and magnitude of MAP kinase activation can be regulated at
many points within the signal transduction pathway. However, it is now
clear that the MAP kinase itself is a major target for regulation
through the action of specific protein phosphatases. MAP kinase
activation is dependent on the phosphorylation of both the threonine
and tyrosine residues of the TXY motif found within the "activation loop" of the kinase. Since phosphorylation of both
residues is required for activity, dephosphorylation of either residue
is sufficient for inactivation. This can be achieved by protein-tyrosine phosphatases, serine/threonine-specific protein phosphatases, or dual specificity (threonine/tyrosine) protein phosphatases. It is now clear that dual specificity MAP kinase phosphatases (MKPs) play an important role in regulating the activity of MAP kinases (9). Thus far, nine distinct MKPs have been identified
and characterized. These are MKP-1 (CL100) (10-13), PAC1 (14), MKP-2
(hVH2, TYP1) (15-17), hVH3 (B23) (18, 19), hVH5 (M3-6) (20, 21) MKP-3
(Pyst1, rVH6) (22-24), MKP-X (Pyst2, B59) (22, 25, 26), MKP-4 (27),
and MKP-5 (28). MKPs can be divided into two broad classes. The first
group, typified by MKP-1 and PAC1, are nuclear enzymes that are encoded
by growth factor and stress-inducible genes (10, 14). The second group, typified by MKP-3, are predominantly cytosolic enzymes and are not
encoded by immediate early genes (22, 25). Recent work has shown that
certain dual specificity MKPs display marked substrate selectivity for
different MAP kinase isoforms in vitro and in vivo. MKP-3 is ~100-fold more active toward ERK2 than p38 The specific dephosphorylation of ERK2 by MKP-3 is accompanied by the
formation of a tight physical complex between the two proteins (22).
This binding is mediated by the amino-terminal noncatalytic domain of
MKP-3, and removal of this domain abrogates substrate selectivity
in vivo (30). Remarkably, this binding event also results in
the catalytic activation of MKP-3 in vitro as revealed by a
30-fold increase in the ability of the enzyme to hydrolyze the
chromagenic substrate para-nitrophenyl phosphate (pNPP)
(31). Catalytic activation mirrors substrate selectivity in
vivo, since stress activated MAP kinase isoforms were unable either to bind to or increase the catalytic activity of MKP-3. The
closely related enzymes MKP-X (Pyst2) and MKP-4 also undergo catalytic
activation upon binding to ERK2 (25, 31), indicating that this may be a
general mechanism by which all members of this family of MKPs are
regulated. However, MKP-5, a recently characterized MKP, both binds to
and inactivates p38 (SAPK2), but recombinant p38 protein does not
catalytically activate MKP-5 in vitro (28).
The inducible nuclear phosphatase MKP-1 (CL100) was originally
characterized as an ERK2 phosphatase in vitro and in
vivo (11-13). However, it was subsequently demonstrated to have
activity against JNK (SAPK1) and p38 (SAPK2) both in vitro
and in vivo (22, 32, 33), and careful titration of
expression levels in mammalian cells revealed it to be equally
effective in inactivating these three MAP kinase isoforms in
vivo (34). Despite this, nothing is known about the molecular
basis for the substrate selectivity of MKP-1. Wild-type MKP-1 does not
appear to be able to form a physical complex with ERK2 in
vivo (12, 22), and it is not known if MKP-1 is subject to
catalytic activation by mitogen- and stress-activated MAP kinases
in vitro. In addition, the results of experiments using
antisense mRNA to suppress the expression of MKP-1 indicate that a
regulatory tyrosine residue within the signal transducers and
activators of transcription 1 (STAT1) protein is dephosphorylated by
MKP-1 in vivo, thus raising the possibility that there may
be physiological non-MAP kinase substrates for this enzyme (35).
Here we demonstrate that the substrate selectivity of MKP-1 is governed
by its ability to form physical complexes with ERK2, JNK1, and p38 DNA Constructs--
The plasmids pSG5.MKP-1, pSG5.MKP-1CS,
pSG5.p38 Two-hybrid Analysis--
GAL4 DNA binding domain and activation
domain fusion plasmids were transformed into the yeast strains PJ69-2A
and Y187, respectively, according to the manufacturer's instructions
(CLONTECH). Semiquantitative analysis of two-hybrid
interactions was performed using a Protein Expression and Purification--
Wild-type MKP-1 and
MKP-1M were expressed in Escherichia coli
(BL21DE3[pLysS]), purified, and refolded as described previously for
wild-type MKP-1 (10). His-tagged STAT1 Western Analysis--
For detection of Myc epitope-tagged
proteins in COS-1 cells, lysates were separated by SDS-polyacrylamide
gel electrophoresis (10%), transferred to a 0.45-µm polyvinylidene
difluoride membrane (Millipore Corp.), and immunoblotted with the
anti-Myc monoclonal antibody 9E10 as previously described (22). For
detection of endogenous phosphorylated STAT1 Protein Phosphatase Assays--
Phosphatase activities and
catalytic activation of MKP-1 was measured using pNPP hydrolysis at
25 °C as previously described (25). Initial rates of pNPP hydrolysis
were determined using Microplate Manager III, version 1.57 (Bio-Rad).
Kinase Assays--
The activities of Myc (9E10) epitope-tagged
or HA (12CA5) epitope-tagged ERK2, JNK1, p38 Cell Culture, Transfection, and Reporter Assays--
COS-1 cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum and transfected using a standard calcium
phosphate method as previously described (22). Reporter gene assays for
the detection of luciferase and MKP-1 Binds to both Mitogen- and Stress-activated MAP Kinases in
Vivo--
A yeast two-hybrid assay was used to determine the
specificity and selectivity of interactions between MKP-1 and a
comprehensive panel of nine distinct mitogen- and stress-activated MAP
kinase isoforms in vivo. Binding was determined by the
activation of GAL4-dependent ADE2/HIS3/lacZ
reporters. Activation of HIS3 and ADE2 was assessed by selection
on synthetic dropout medium deficient for histidine and adenine.
The strength of the interactions was quantified in a Binding to Mitogen- and Stress-activated MAP Kinases Is Associated
with a Stimulation of MKP-1 Catalytic Activity--
It has been
demonstrated previously for a subfamily of dual specificity MAP kinase
phosphatases, which includes MKP-3 (Pyst1), Pyst2 (MKP-X), and MKP-4,
that binding to ERK2 results in a dramatic increase in the catalytic
activity of these enzymes in vitro (25, 31). Phosphatase
activity of bacterially expressed MKP-1 (2.5 µg) toward pNPP was
investigated to determine whether the selective binding of MKP-1
in vivo to ERK2, JNK1, and p38 MKP-1 Specifically Inactivates ERK2, JNK1, and p38
Taken together, these in vivo and in vitro
findings demonstrate that highly specific interactions between MKP-1
and the MAP kinases ERK2, JNK1, and p38 STAT1 Sevenmaker Mutations in ERK2, JNK1, and p38 ERK2-sevenmaker Does Not Stimulate MKP-1 Phosphatase Activity in
Vitro--
Sevenmaker-like mutations within ERK2, JNK1, and
p38 The NH2 Terminus of MKP-1 Is Required for Interaction
with MAP Kinases--
It has been demonstrated that the noncatalytic
amino-terminal region of MKP-3 is required for interaction with ERK2
(30). To determine whether the corresponding region of MKP-1 is
important for binding to ERK2, JNK1, and p38 Identification of a Selective MAP Kinase Interaction Motif within
MKP-1--
Recent work has identified a common docking site within the
activators, substrates, and regulators of mitogen- and stress-activated MAP kinases (37). For the dual specificity MAP kinase phosphatase MKP-3, this motif is composed of a positively charged amino acid cluster within the noncatalytic amino-terminal domain known to be
critical for ERK2 binding. This is immediately adjacent to a region
found in all dual specificity MKPs, which has significant homology with
the cell cycle regulatory phosphatase Cdc25 (38, 39). A similar
motif was independently identified within the amino-terminal
noncatalytic domain of the tyrosine-specific phosphatase Ptp3p in
S. cerevisiae and is responsible for mediating the binding and inactivation of the yeast Fus3p MAP kinase by this enzyme (40).
Based on sequence homology with Ptp3p and MKP-3, we would predict that
arginine residues at positions 53-55 would be essential for kinase
recognition by MKP-1. To determine whether this motif is important for
binding to ERK, JNK1, and p38 Only JNK1 Is Able to Increase the Phosphatase Activity of
MKP-1M--
For wild-type MKP-1, substrate binding was associated with
marked stimulation of phosphatase activity in vitro. To
investigate whether the MKP-1M mutant retains phosphatase activity and
to determine if this activity could be stimulated specifically by JNK1
binding, recombinant MKP-1M was expressed in bacteria and assayed for
enzymatic activity toward pNPP. MKP-1M exhibited an equivalent level of
phosphatase activity to the wild-type protein, and the ability of JNK1
to cause catalytic activation of the phosphatase was completely
unimpaired (Fig. 10). In contrast and
as predicted by our binding data, MKP-1M did not show any increase in
catalytic activity when incubated with either recombinant ERK2 or
p38 MKP-1M Specifically Inactivates JNK1 Kinase Activity in
Vivo--
Our in vivo and in vitro binding and
activation data strongly suggest that MKP-1M will behave as a
JNK1-specific phosphatase in vivo. To determine whether this
is indeed the case, COS-1 cells were cotransfected with plasmids
encoding Myc (9E10) epitope-tagged MKP-1M and either HA (12CA5)
epitope-tagged ERK2, JNK1, or p38 The prototypic dual specificity MAP kinase phosphatase MKP-1 is
able to dephosphorylate and inactivate both mitogen- and
stress-activated isoforms of MAP kinase in vitro and
in vivo (11-13, 22, 32, 33). However, the full extent of
MKP-1 substrate selectivity and its molecular basis have not been
determined. In an extensive yeast two-hybrid screen, we have
characterized protein-protein interactions between MKP-1 and a
comprehensive panel of mitogen- and stress-activated MAP kinase
isoforms. We find that MKP-1 is only able to interact with a subset of
these enzymes, specifically ERK1, ERK2, JNK1, and p38 For a distinct subfamily of ERK-specific MKPs that includes MKP-3
(Pyst1), MKP-X (Pyst2), and MKP-4 (Pyst3), substrate binding is
associated with an increase in catalytic activity in vitro (25, 31). In contrast, the recently characterized dual specificity phosphatase MKP-5 can interact with and dephosphorylate p38 Our data demonstrate for the first time that catalytic activation of
MKP-1 is mediated by specific protein-protein interactions with ERK2,
JNK1, and p38 The levels of MKP-1 catalytic activation seen here are not comparable
with the 20-30-fold increase in the activity of MKP-3 (Pyst1) and
Pyst2 that results from binding to ERK2 (25, 31). However, we have
observed that the basal activity of recombinant MKP-1 in the absence of
MAP kinase is significantly higher when compared with
MKP-3.2 This may reflect a
fundamental difference in the position of the general acid loop of
MKP-1 in the absence of bound substrate. The crystal structure of the
catalytic domain of MKP-3 (Pyst1) suggests that activation mediated by
ERK2 binding results from the displacement of the general acid
(Asp262) to a more favorable position for catalysis (43).
For MKP-1, the equivalent acidic residue (Asp227) may
already be in a position to contribute significantly to catalytic
activity in the absence of any interaction with MAP kinase. The role of
this aspartate residue in the catalytic activation of MKP-1 is
currently under investigation.
With respect to the substrate selectivity of MKP-1 in vivo,
we find an absolute correlation between substrate binding, catalytic activation, and MAP kinase inactivation. Thus, MKP-1 is able to inhibit
the activities of ERK2, JNK1, and p38 The sevenmaker mutation within ERK2 (D319N) exhibits reduced
sensitivity to inactivation by a number of phosphatases including MKP-1
and MKP-3, both in vitro and in vivo (31, 34,
36). Both JNK1 and p38 The fact that we can still detect catalytic activation by ERK2D319N,
albeit markedly reduced, suggests that other residues within ERK2 may
be important for interaction with MKP-1. This is consistent with the
finding that in p38 MAP kinase other acidic residues close to
Asp316 (Asp313 and Asp315) are
important for interactions with MKK6, MKP-5, and MNK1 (37). Furthermore, a recent report has identified regions of MKP-3 other than
the basic motif as being important for ERK2 binding and catalytic activation (42). We have demonstrated that substrate binding is
critical for catalytic activation; thus, it would be predicted that
both JNKD326N and p38D316N will also be impaired in this assay. This is
indeed the case, since it has recently been reported that a
sevenmaker mutant of p38 MAP kinase also has a reduced ability to activate MKP-1 in vitro (47).
It is clear that the noncatalytic amino-terminal region of both yeast
protein-tyrosine phosphatases and mammalian dual specificity MAP kinase
phosphatases is critical for the binding of MAP kinases (37, 40). We
identified three arginine residues within MKP-1 that would be predicted
to mediate this and found that mutation of these residues abolishes the
ability of MKP-1M to bind to and be activated by ERK1, ERK2, and
p38 Our results strongly suggest that the binding of MKP-1 to JNK1 is
determined by sequences that are distinct from those that mediate
binding of ERK1/2 and p38 In conclusion, we have demonstrated that MKP-1 is able to form physical
complexes with a restricted subset of MAP kinase isoforms and that MAP
kinase binding results in catalytic activation of MKP-1. Both of these
end points show an absolute correlation with the substrate selectivity
of MKP-1, and our quantitative binding and catalytic activation data
would indicate that JNK may be the preferred substrate for MKP-1
in vivo. This is in agreement with previous studies
demonstrating that conditional expression of MKP-1 preferentially
inhibits JNK in U937 cells (51) and more recent data showing that
expression of MKP-1 is able to protect cells against apoptosis mediated
by JNK activation (41, 52). In contrast, both the results of our
previous studies (11) and experiments presented here do not support the
idea that there are physiological non-MAP kinase substrates for MKP-1.
In particular, we can find no evidence to support the recent proposal
that MKP-1 is able to act on the STAT1 transcription factor. Finally,
our identification of distinct determinants within the amino terminus of MKP-1 responsible for binding ERK2/p38, and c-Jun
NH2-terminal kinase (JNK) MAP kinase isoforms. Furthermore,
this binding is accompanied by catalytic activation of recombinant
MKP-1 protein in vitro, and these end points show an
absolute correlation with MKP-1 substrate selectivity in
vivo. In contrast, MKP-1 does not interact with STAT1.
Recombinant STAT1 does not cause catalytic activation of MKP-1; nor
does MKP-1 block tyrosine phosphorylation of STAT1 in vivo.
Both binding and catalytic activation of MKP-1 are abrogated by
mutation of a conserved docking site in ERK2, p38
, and JNK1 MAP
kinases. Within MKP-1, MAP kinase binding is mediated by the amino-terminal noncatalytic domain of the protein. However, mutation of
a conserved cluster of positively charged residues within this domain
abolishes the binding and activation of MKP-1 by ERK2 and p38
but
not JNK1, indicating that there are distinct binding determinants for
these MAP kinase isoforms. We conclude that the substrate selectivity
of MKP-1 is determined by specific protein-protein interactions coupled
with catalytic activation of the phosphatase and that these
interactions are restricted to members of the MAP kinase family of enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(SAPK2a) in vitro (22). Furthermore, this substrate
selectivity is also observed in vivo (22, 29). hVH-5 (M3/6),
another cytosolic MKP, is highly specific for JNK and p38 MAP kinases
(29).
MAP kinase isoforms in vitro and in vivo and that complex formation is accompanied by catalytic activation of MKP-1. In
contrast, STAT1 is unable to bind to MKP-1 or to catalytically activate
the enzyme; nor does overexpression of MKP-1 cause tyrosine dephosphorylation of STAT1 in vivo. Mutation of a conserved
docking site within MAP kinases that is used by activators, regulators, and substrates of these enzymes abrogates both binding and catalytic activation of MKP-1. Within MKP-1, MAP kinase binding is mediated by
the amino-terminal noncatalytic domain of the phosphatase. However,
mutation of a conserved cluster of positively charged residues within
this domain abrogates binding and catalytic activation only by ERK2 and
p38
and not JNK, indicating that MAP kinase interactions may be
mediated by distinct binding sites. We conclude that the substrate
selectivity of MKP-1 is governed by specific protein-protein
interactions coupled with catalytic activation of the phosphatase, and
our results strongly suggest that these interactions are restricted to
the MAP kinase family of proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, pSG5.JNK1, and pSG5.ERK2 containing a single copy of
either the Myc (9E10) epitope tag or the HA (12CA5) epitope
tag have been previously described (22). The IFN-
-responsive
reporter, p(IRF-1.GAS)6tk
[
39]lucter, and the
ptk
[
39]lucter reporter were kindly provided by Dr. S. Goodbourn.
The plasmids (MLVGALELK, which encodes the C-terminal activation domain
of Elk-1 fused to the GAL4 DNA binding domain; G5E4Luc, a
GAL4-dependent luciferase reporter plasmid;
pAG-(1-147), which encodes the DNA binding domain of GAL4
alone; and pJATLac, which encodes
-galactosidase) were kindly
provided by Dr N. Jones. All other plasmid constructs are described
in the Supplemental Material.
-galactosidase assay according to
the manufacturer's instructions (CLONTECH). To
ensure that all protein-kinase interactions were detected, binding
capability was assessed using each kinase isoform expressed as GAL4
DNA-binding domain and activation domain fusions where possible.
and ERK2D319N were expressed
in E. coli and purified on Ni2+-nitrilotriacetic
acid resin according to the manufacturer's instructions (Qiagen).
Protein-containing fractions were pooled and dialyzed overnight at
4 °C against 1× 2 liters DB2 (50 mM Tris, pH
8.0, 150 mM NaCl, 10 mM dithiothreitol, and
50% glycerol).
, immunoblotting was
performed with an anti-phospho-STAT1 (Tyr701) polyclonal
antibody according to the manufacturer's instructions (New England Biolabs).
, p38
, and p38
immunoprecipitated from COS-1 cells using either 9E10 or 12CA5
monoclonal antibodies, respectively, were assayed as described
previously (25).
-galactosidase activities were
performed using the Dual-Light system according to the manufacturer's
instruction (Tropix Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase
assay. Full-length wild-type MKP-1 was shown to specifically interact
with ERK1, ERK2, JNK1, and a kinase-negative form of p38
(Fig.
1A). A very weak interaction was detected with ERK5, while no interaction was seen with ERK3, ERK7,
p38
, or p38
. MKP-1 interacted with ERK1, ERK2, JNK1, and p38
with a rank order of binding strength as follows: JNK1
ERK2 = p38
> ERK1 (Fig. 1B). We consistently find that
ERK1 has a lower binding affinity for MKP-1 when compared with ERK2,
and this is also the case when the ERK-specific phosphatase MKP-3 is
used as bait in these assays (data not shown). The specificity of these
protein-protein interactions was unaltered using a catalytically inactive mutant (Cys258 to Ser) form of MKP-1 (MKP-1CS);
however, the strength of binding was somewhat increased (~2-fold)
(Fig. 1B). Western blot analysis using GAL4-specific
antibodies (CLONTECH) verified expression of each
of the MAP kinase fusion proteins used in the screen (data not
shown).
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Fig. 1.
Analysis of two-hybrid interactions between
MKP-1 and a panel of mitogen- and stress-activated MAP kinase
isoforms. A, pGBKT7.MKP-1 was transformed into PJ69-2A
and mated with Y187 expressing the GAL4 AD fusions pGADT7.ERK1,
pGADT7.ERK2, pGADT7.ERK3, pGADT7.ERK5, pGADT7.ERK7, pGADT7.JNK1,
pGADT7.p38 , and pGADT7.p38
. p38
was expressed in PJ69-2A as a
GAL4 BD fusion (pGBKT7.p38
) and mated with pGADT7.MKP-1 transformed
into Y187. Yeast diploids expressing both BD and AD fusions were
selected on synthetic dropout (SD) medium deficient for leucine
(leu) and tryptophan (trp). Leu/trp positives were restreaked onto
SD
leu/
trp/
his/
ade. Protein-protein interactions were assessed
by growth on this selective medium. B, semiquantitative
analysis of the two-hybrid interactions based on the level of induction
of the
-galactosidase gene was performed for full-length wild-type
(hatched bars) and the catalytically inactive
mutant (MKP-1CS) form of MKP-1 (closed bars).
Assays were performed in triplicate, and the results of a
representative experiment are shown.
was mirrored by catalytic
activation in vitro. The phosphatase activity of MKP-1 was
specifically increased in the presence of 10 µg of recombinant ERK2,
JNK1, and p38
(Fig. 2). ERK5, which
interacted only weakly with MKP-1 in our two-hybrid screen, caused a
small but reproducible increase in activity (~1.6-fold). In contrast,
no significant catalytic activation was observed upon the addition of
recombinant ERK3, p38
, or p38
. The strength of the interactions
between MKP-1 and these kinase isoforms in vivo directly
parallels the level of catalytic activation observed in this in
vitro assay. The phosphatase activity of MKP-1 was only moderately
stimulated by p38
and ERK2 (~2-fold), whereas JNK1, which was
shown to interact most strongly with MKP-1 in vivo, enhanced
activity by up to 5-fold. Incubation of MKP-1 (2.5 µg) with
increasing amounts of these MAP kinases (up to 20 µg) showed that the
activation of MKP-1 was dose-dependent (Fig.
3, A-C) and at least in the
case of p38
was saturable (Fig. 3C). Interestingly, Myc
(9E10) epitope-tagged MKP-1 expressed in mammalian cells and
immunoprecipitated using an anti-9E10 monoclonal antibody also
exhibited phosphatase activity toward pNPP. Furthermore, an identical
pattern of catalytic activation in the rank order JNK1
ERK2 = p38
was observed (Fig. 4). To ensure
that this activity was not mediated by the presence of associated
proteins co-immunoprecipitated with MKP-1, the catalytically inactive
MKP-1CS form was also expressed in COS-1 cells. In contrast to the
wild-type protein, immunoprecipitated MKP-1CS exhibited no measurable
phosphatase activity (Fig. 4).
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Fig. 2.
Catalytic activation of MKP-1 by a panel of
mitogen-and stress-activated MAP kinases. Phosphatase activity was
measured as pNPP hydrolysis at 25 °C monitored by change in optical
density at 405 nm. Catalytic activation is expressed as -fold increase
in the initial rate of pNPP hydrolysis by 2.5 µg of recombinant MKP-1
in the absence or presence of 10 µg of the indicated MAP kinase.
Assays were performed in triplicate, and the results of a
representative experiment are shown.
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Fig. 3.
Catalytic activation of MKP-1 by ERK2, JNK1,
and p38 is dose-dependent.
Time-dependent hydrolysis of pNPP by 2.5 µg of
recombinant MKP-1 at 25 °C was monitored by change in optical
density at 405 nm either alone or in the presence of the indicated
amount of either ERK2 (A), JNK1 (B), or p38
(C). Assays were performed in triplicate, and the results of
representative experiments are shown.
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Fig. 4.
MKP-1 immunoprecipitated from COS-1 cells is
active and undergoes catalytic activation. COS-1 cells were
transfected with 5 µg of a plasmid expression vector encoding either
Myc-tagged wild-type MKP-1 or a catalytically inactive mutant
(MKP-1CS). Following transfection, cells were lysed, and MKP-1 protein
was immunoprecipitated using anti-Myc (9E10) monoclonal antibody.
Immunoprecipitated MKP-1 was then assayed for pNPP hydrolysis either in
the absence or presence of ERK2, JNK1, or p38 as indicated. Assays
were performed in triplicate, and the results of representative
experiments are shown.
in
Vivo--
Since MKP-1 exhibited a restricted ability to both bind to
and be catalytically activated by different mitogen- and
stress-activated MAP kinase isoforms, we set out to determine if these
properties reflect the substrate selectivity of MKP-1 in
vivo. COS-1 cells were cotransfected with plasmids encoding Myc
(9E10) epitope-tagged MKP-1 and HA (12CA5) epitope-tagged ERK2, JNK1,
p38
, p38
, or p38
. Transfected cells were then exposed to an
appropriate stimulus to induce kinase activation, either serum (ERK2)
or anisomycin (JNK1, p38
, p38
, and p38
). MAP kinases were then
immunoprecipitated using an anti-HA (12CA5) monoclonal antibody and
assayed directly for kinase activity using an appropriate substrate,
either myelin basic protein (ERK2 and p38 isoforms) or ATF2 (JNK1). The
data clearly show that expression of MKP-1 specifically inhibits the kinase activity of ERK2, JNK1, and p38
but has no effect upon the
activities of p38
or p38
(Fig. 5).
Furthermore, the resistance of both p38
and p38
to inactivation
by MKP-1 was maintained in titration experiments where even higher
amounts (up to 20 µg) of MKP-1 were transfected and expressed (data
not shown).
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Fig. 5.
MKP-1 exhibits substrate selectivity in
vivo. COS-1 cells were co-transfected with HA-tagged
ERK2, JNK1, p38 , p38
, or p38
MAP kinases (2.5 µg) together
with either empty pSG5 vector (2.5 µg) or pSG5 expression vector
encoding Myc-tagged MKP-1 (2.5 µg). Following 12 h of growth,
cells were either serum-starved overnight and restimulated with 15%
fetal calf serum (ERK2) or treated with 10 µg/ml anisomycin for 30 min (JNK1, p38
, p38
, and p38
). MAP kinases were then
immunoprecipitated using an anti-HA (12CA5) monoclonal antibody, and
immunocomplex assays were performed using myelin basic protein (ERK2,
p38
, p38
, and p38
) or ATF2 (JNK1) as substrates. Radiolabeled
proteins were then analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiography. Equivalent expression of MAP kinases and
expression of MKP-1 was verified by Western blotting using the
appropriate monoclonal antibodies (data not shown).
are associated with
significant stimulation of MKP-1 phosphatase activity. Furthermore,
only those MAP kinase isoforms that are able to bind and cause
catalytic activation of MKP-1 are in vivo substrates for
this dual specificity phosphatase.
Is Not a Substrate for MKP-1--
We have demonstrated
previously that MKP-1 is unable to catalyze the dephosphorylation of a
variety of non-MAP kinase proteins in vitro (11). However, a
recent report that STAT1 tyrosine phosphorylation was specifically
prolonged in the presence of an MKP-1 antisense oligonucleotide
suggested that this transcription factor was also regulated by MKP-1
(35). The nuclear localization and subsequent transcriptional activity
of STAT1 is regulated primarily by tyrosine phosphorylation; thus,
dephosphorylation by a nuclear phosphatase would result in its
inactivation. However, using two-hybrid analysis, we have shown that
MKP-1 is incapable of interacting with STAT1
in vivo
(Fig. 6A). This finding was confirmed by coimmunoprecipitation studies in vitro (data
not shown). Furthermore, recombinant STAT1
(up to 20 µg) was
incapable of increasing the phosphatase activity of MKP-1 toward pNPP
(Fig. 6B), and overexpression of Myc epitope-tagged MKP-1 in
COS-1 cells failed to prevent tyrosine phosphorylation of STAT1
in
response to IFN-
(Fig. 6C). Finally, overexpression of
increasing amounts of MKP-1 (up to 2 µg) in COS-1 cells significantly
reduced the transcriptional activity of the MAP kinase-regulated
transcription factor Elk-1 (Fig. 6D) but did not modulate
the transactivation potential of a STAT1
-dependent
reporter in response to IFN-
(Fig. 6E). In light of these
results, we believe that it is most unlikely that MKP-1 fulfills a role
as a nuclear phosphatase responsible for the dephosphorylation and
subsequent inactivation of STAT1.
View larger version (38K):
[in a new window]
Fig. 6.
STAT1 is not a substrate for MKP-1.
A, semiquantitative analysis of yeast two-hybrid assays
based on the level of induction of the -galactosidase gene was
performed to measure interactions between MKP-1 and either STAT1 or
JNK1. B, phosphatase activity was measured as pNPP
hydrolysis at 25 °C monitored by change in optical density at 405 nm. Catalytic activation is expressed as -fold increase in the initial
rate of pNPP hydrolysis by 2.5 µg of recombinant MKP-1 in the absence
or presence of either 10 µg of recombinant STAT1 or JNK1. Assays were
performed in triplicate, and the results of a representative experiment
are shown. C, COS-1 cells were transfected with 5 µg of
pSG5 expression vector encoding Myc-tagged MKP-1. Cells were then
treated for various times with 1000 IU/ml interferon-
, and cell
lysates were analyzed by Western blotting using antibodies specific for
STAT1 phosphorylated on tyrosine 701 and anti-Myc monoclonal antibody
(9E10). D, COS-1 cells were transiently transfected with
increasing amounts of pSG5.MKP-1 (0.25-2 µg) or pSG5 alone, along
with 2.5 µg of the GAL4-dependent reporter G5E4Luc and
either 2.5 µg of MLVGALELK or 2.5 µg of pAG-(1-147). 1.25 µg of
pJATLac was included in each transfection as a control. The transfected
cells were either untreated or treated for 4 h with 0.1 µg/ml
epidermal growth factor (EGF). GAL4-dependent
luciferase activity was monitored using a luminometer. Elk-1-mediated
expression levels were determined by correcting the luciferase activity
to the
-galactosidase levels (mediated by pJATLac expression) and to
the activity of the GAL4 binding domain plasmid, pAG-(1-147).
E, COS-1 cells were transiently transfected with increasing
amounts of pSG5.MKP-1 (0.25-2 µg) or pSG5 alone, along with either
2.5 µg of an IFN-
-responsive reporter
p(IRF-1.GAS)6tk
[
39]lucter or 2.5 µg of
ptk
[
39]lucter. 1.25 µg of pJATLac was included in each
transfection as a control. The transfected cells were either untreated
or treated for 5 h with 1000 IU/ml IFN-
. GAS reporter-specific
expression levels were determined by correcting the luciferase activity
to the
-galactosidase levels and to the nonspecific activity of the
ptk
[
39]lucter reporter plasmid.
Abrogate Binding to
MKP-1 in Vivo--
The mutant sevenmaker form of ERK2,
ERK2D319N, has been shown to exhibit enhanced kinase activity as a
result of reduced sensitivity to inactivation by several phosphatases
(31, 34, 36). This "docking domain" within the C terminus of ERK2
is common to all members of the mitogen- and stress-activated MAP
kinase family (37). To determine whether this site was important for
the observed interactions between MKP-1 and MAP kinase isoforms,
sevenmaker mutations were introduced into ERK2, JNK1, and
p38
. For p38
, aspartic acid residue 316 was mutated to asparagine
(p38D316N). In the case of JNK1, two docking domain mutants were
created: one in which aspartic acid 326 was replaced by asparagine
(JNKD326N) and another in which three acidic residues within this
domain, aspartic acid 326 and glutamic acid residues 329 and 331, were mutated to asparagine (JNK3N). Two-hybrid analysis showed that in
vivo binding of both wild-type and C258S forms of MKP-1 to ERK2,
p38
, and JNK1 was abrogated by mutation of these conserved acidic
residues. For JNK1, mutation of aspartic acid residue 326 alone was
sufficient to block interaction (Fig.
7A).
View larger version (18K):
[in a new window]
Fig. 7.
The introduction of sevenmaker
mutations into ERK2, JNK1, and p38
abrogates interaction with MKP-1 and impairs catalytic activation
of MKP-1. A, semiquantitative analysis of yeast
two-hybrid assays based on the level of induction of the
-galactosidase gene for interactions between both wild-type MKP-1
(hatched bars) or its inactive mutant MKP-1CS
(closed bars) and either wild-type or
sevenmaker mutants of ERK2, JNK1, and p38
. B,
hydrolysis of pNPP by 2.5 µg of recombinant MKP-1 at 25 °C for
2 h was monitored by change in optical density at 405 nm either
alone or in the presence of the indicated amount of either recombinant
wild-type ERK2 or sevenmaker ERK2D319N. Assays were
performed in triplicate, and the result of a representative experiment
is shown.
abrogated binding of MKP-1 to these kinase isoforms in
vivo. Previous experiments have shown that this mutation within
ERK2 abrogates its ability to bind to and catalytically activate the
dual specificity phosphatase MKP-3 (31), and our experiments
demonstrate that MAP kinase binding is essential for catalytic
activation of MKP-1. To examine the effect of this mutation on the
enzymatic activity of MKP-1 toward pNPP, recombinant MKP-1 (2.5 µg)
was incubated with increasing amounts of either recombinant wild-type
or D319N ERK2. The results clearly show that ERK2-dependent
catalytic activation of MKP-1 was significantly reduced by the presence
of this mutation, with at least 5 times more mutant ERK2 than wild-type
protein required to achieve the same increase in MKP-1 phosphatase
activity (Fig. 7B).
, a deletion mutant of
MKP-1, MKP-1188 (lacking amino acid residues 1-188), was screened in a
two-hybrid assay for its ability to interact with the kinase panel
described previously. The
-galactosidase assay results show that
loss of the noncatalytic NH2 terminus of MKP-1 abrogates binding to ERK1, ERK2, ERK5, JNK1, and p38
in vivo (Fig.
8).
View larger version (26K):
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Fig. 8.
The NH2 terminus of MKP-1 is
required for interaction with MAP kinases. Semiquantitative
analysis of yeast two-hybrid interactions based on the level of
induction of the -galactosidase gene was performed for full-length
wild-type MKP-1 (hatched bars) and a deletion
mutant (MKP-1188) lacking the NH2-terminal noncatalytic
domain of MKP-1 (amino acids 189-367) (closed
bars) in the presence of the indicated MAP kinase.
, a mutant form of MKP-1 was
constructed (MKP-1M) in which these three residues were mutated to
alanine, serine, and alanine, respectively (Fig. 9A). Substrate binding was
then determined by two-hybrid analysis. We find that mutation of these
arginine residues completely abolishes the ability of MKP-1 to
recognize ERK1, ERK2, and p38
(Fig. 9, B and
C). However, to our surprise, the binding of MKP-1 to JNK1 was completely unaffected, suggesting that the binding determinant for
this MAP kinase isoform is distinct from that used by ERKs and
p38
.
View larger version (38K):
[in a new window]
Fig. 9.
Mutations in the NH2-terminal
region of MKP-1 abrogate ERK and p38 but not
JNK1 binding. A, the NH2-terminal region of
MKP-1 contains conserved amino acid residues (shown in
boldface type) present in yeast tyrosine
phosphatases and mammalian MKPs. The Cdc25 homology domain (A box) is
overlined, and the amino acid substitutions introduced into
MKP-1 are indicated. B, pGBKT.MKP-1M was transformed into
PJ69-2A and mated with Y187 expressing the GAL4 AD kinase fusion panel
described in the legend to Fig. 1. p38
was expressed in PJ69-2A as
a GAL4 BD fusion and mated with pGADT7.MKP-1M transformed into Y187.
Yeast diploids were selected onto SD
leu/
trp and then restreaked
onto SD
leu/
trp/
his/
ade as before. C,
semiquantitative analysis of the two-hybrid interactions based on the
level of induction of the
-galactosidase gene was performed for
full-length wild-type MKP-1 (hatched bars) and
the mutant form MKP-1M (closed bars) in the
presence of the indicated MAP kinase. Assays were performed in
triplicate, and the results of a representative experiment are
shown.
.
View larger version (27K):
[in a new window]
Fig. 10.
Catalytic activation of MKP-1M is mediated
specifically by JNK1. Phosphatase activity of wild-type MKP-1 and
MKP-1M was measured as pNPP hydrolysis at 25 °C monitored by change
in optical density at 405 nm. Catalytic activation is expressed as
-fold increase in the initial rate of pNPP hydrolysis by 2.5 µg of
recombinant MKP-1 (hatched bars) and MKP-1M
(closed bars) in the absence or presence of 10 µg of the indicated MAP kinase. Assays were performed in triplicate,
and the results of a representative experiment are shown.
. The kinases were then
immunoprecipitated using an anti-HA (12CA5) monoclonal antibody from
cells treated with either serum (to activate ERK2) or anisomycin (to
activate JNK1 and p38
) and assayed directly for kinase activity
against myelin basic protein (ERK2 and p38
) or ATF2 (JNK1). The
results clearly show that only JNK1-dependent kinase
activity could be abolished by MKP-1M expression; the kinase activities
of ERK2 and p38
are completely unaffected (Fig.
11).
View larger version (44K):
[in a new window]
Fig. 11.
MKP-1M specifically inactivates JNK1
in vivo. COS-1 cells were co-transfected with
HA-tagged ERK2, JNK1, or p38 MAP kinases (2.5 µg) together with
either empty pSG5 vector (2.5 µg) or pSG5 expression vector encoding
Myc-tagged MKP-1M (2.5 µg). Following 12 h of growth, cells were
either serum-starved overnight and restimulated with 15% fetal calf
serum (ERK2) or treated with 10 µg/ml anisomycin for 30 min (JNK1 and
p38
). MAP kinases were then immunoprecipitated using an anti-HA
(12CA5) monoclonal antibody, and immunocomplex assays were performed
using myelin basic protein (ERK2 and p38
) or ATF2 (JNK1) as
substrates. Radiolabeled proteins were then analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. Equivalent
expression of all MAP kinases and expression of MKP-1M was verified by
Western blotting using the appropriate monoclonal antibodies (data not
shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Our finding
that MKP-1 is able to bind most tightly to JNK1 is supported by a
recent study in which wild-type MKP-1 was shown to form a physical
complex with JNK when expressed in the 293T human embryonic kidney cell
line (41). A substrate-trapping mutant of MKP-1 (Cys258
Ser) was also used in this screen and found to interact with this same
subset of MAP kinases, albeit with somewhat higher affinity. This
indicates that such substrate traps do not promote inappropriate binding of dual specificity MKPs to other MAP kinase isoforms and
underlines the utility of such mutants in defining MKP-substrate interactions.
but is
not catalytically activated by this kinase (28). Thus, it is unclear
whether catalytic activation is a general mechanism that determines
substrate selectivity.
. Significantly, the level of catalytic activation by
these kinase isoforms is also consistent with their strength of binding
in vivo. Although catalytic activation of MKP-1 by MAP
kinases is dose-dependent, we find that activation by
p38
MAP kinase saturates at lower concentration than for JNK1. This
is despite the fact that JNK1 binds to MKP-1 more tightly than p38
in vivo. Recent studies of MKP-3 activation by ERK2 have
revealed that multiple regions of the phosphatase are involved in its
recognition by ERK2 (42). Furthermore, certain sites contribute to ERK2
binding but are not essential for MKP-3 activation. As yet, it is
unclear exactly how JNK1 and MKP-1 dock in comparison with complexes
formed between MKP-1 and p38
. However, the results of our
mutagenesis experiments indicate that different binding sites on MKP-1
are involved. Determination of the stoichiometry of binding between
MKP-1 and different MAP kinases and quantitative measurements of
binding affinity are currently under way, and these, together with
studies of the mechanism by which MKP-1 undergoes catalytic activation
(see below), may provide an explanation for our observations.
but not p38
and p38
. The
ability of MKP-1 to discriminate between these p38 MAP kinase isoforms
is particularly striking given that the sequence homology between
p38
, p38
, and p38
is ~60% (44, 45). In our two-hybrid
assays, we did detect a very weak interaction between MKP-1 and ERK5
(Fig. 1B). Furthermore, recombinant ERK5 caused a small but
reproducible increase in the catalytic activity of MKP-1 (~1.6-fold).
Our results are consistent with a recent report that overexpression of
MKP-1 is able to reduce the kinase activity of ERK5 (46).
have conserved aspartic acid residues at
equivalent positions to Asp319 of ERK2, at
Asp326 and Asp316, respectively, and Tanoue
et al. recently demonstrated that mutation of
Asp326 in JNK1 reduced its ability to interact with MKP-5
(37). We have found that mutation of these acidic residues in ERK2,
JNK1, and p38
abrogated binding to MKP-1 in vivo,
suggesting that they represent a conserved docking motif that is
crucial for kinase-phosphatase interactions and thus critical for the
regulation of kinase activity in vivo. Consistent with this,
recombinant ERK2D319N was unable to induce catalytic activation of
MKP-1 in vitro to the same level as wild-type ERK2. Similar
results have also been reported for the catalytic activation of MKP-3
by ERK2D319N (31, 42).
. Furthermore, overexpression of this mutant in COS-1 cells did
not inhibit the activation of either ERK2 or p38
, thus establishing
a critical role for this cluster of basic residues in mediating the
activity of MKP-1 toward both ERK2 and p38
. However, to our
surprise, MKP-1M was still able to bind to and be activated by JNK-1,
and expression of MKP-1M in COS-1 inhibited the kinase activity of
JNK1. To our knowledge MKP-1M is the only dual specificity phosphatase
that is absolutely specific for JNK, since the M3/6 (hVH-5) enzyme also
inactivates p38
(29). Since there are currently no specific JNK
inhibitors available, MKP-1M may be of considerable value in dissecting
the specific role of JNK signaling in mediating a variety of biological end points.
. A motif has been identified within the
transcription factors c-Jun and ATF2 and within the JNK scaffold
protein JIP-1, which acts as a docking site for JNK (48). This
-domain contains an LXL motif located 3-5 amino acids
downstream from a region containing a number of basic residues. Furthermore Net, a MAP kinase-regulated transcription factor, contains
distinct binding domains for ERK/p38 and JNK, and the latter motif
(designated the J box) also contains an LXL element (49).
Mutations within the J box abrogated binding of JNK to Net but had no
effect upon interaction with either ERK2 or p38. Finally, it has been
reported recently that the mutation of an LXL element within
the amino terminus of the JNK-specific phosphatase hVH5 (M3/6) reduced
the ability of this enzyme to dephosphorylate SAPK/JNK in
vivo (50). MKP-1 also contains this LXL motif, and we
are currently examining the effects of mutating this and other residues
close to the ERK/p38 binding site in an attempt to identify the
sequences required for interaction with JNK1.
and JNK imply considerable complexity in the binding and catalytic activation of MKP-1 by MAP
kinases. Future studies will be directed at dissecting these binding
determinants within MKP-1 and determining their role in the regulation
of MAP kinase signaling by this enzyme.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge Iain Goldsmith for
oligonucleotide synthesis and Andy Cassidy for DNA sequencing. We also
thank Ian Sylvester for performing the original p38 and p38
kinase assays, Steve Dowd for generating the MKP-1M mutant, and Chris
Armstrong (MRC Protein Phosphorylation Unit, University of Dundee) for
providing recombinant MAP and SAP kinases. We also thank Dr. Yusen Liu
for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Imperial Cancer Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a listing of plasmid constructs.
Recipient of a postdoctoral fellowship from the Norwegian Research
Council (project 123686/310).
§ To whom correspondence should be addressed. Tel.: 01382 632622; Fax: 01382 669993; E-mail: S.Keyse@icrf.icnet.uk.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M010966200
2 D. N. Slack, O.-M. Seternes, M. Gabrielsen, and S. M. Keyse, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MAP, mitogen-activated protein;
ERK, extracellular signal regulated kinase;
SAPK, stress-activated protein kinase;
JNK, c-Jun
NH2-terminal kinase;
MKP, MAP kinase phosphatase;
pNPP, para-nitrophenyl phosphate;
ATF2, activating transcription
factor 2;
HA, hemagglutinin;
IFN, interferon;
STAT, signal transducers
and activators of transcription;
GAS, -interferon activated
sequence.
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