From the Departments of Biochemistry and
§ Clinical Oncology, Institute of Development, Aging, and
Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku,
Sendai 980-8575, the ¶ Department of Molecular Biology,
Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, and the
Department of Geriatric Dentistry,
Tohoku University School of Dentistry, 4-1 Seiryomachi, Aoba-ku,
Sendai 980-8575, Japan
Received for publication, August 25, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Protein phosphatase 2C (PP2C) is implicated in
the negative regulation of stress-activated protein kinase
cascades in yeast and mammalian cells. In this study, we determined the
role of PP2C Stress-activated protein kinases
(SAPKs)1 are a subfamily of
the mitogen-activated protein kinase (MAPK) superfamily and are highly
conserved from yeast to mammalian cells. SAPKs relay signals in
response to various extracellular stimuli, including environmental stress and inflammatory cytokines. In mammalian cells, two distinct classes of SAPKs have been identified: the c-Jun amino-terminal kinases
(JNKs) (JNK1, JNK2, and JNK3) and the p38 MAPKs (p38 In the absence of signaling, SAPK cascades return to their inactive,
dephosphorylated state, suggesting a possible role for phosphatases in
SAPK regulation. In yeast cells, molecular genetic analysis has
indicated that two distinct protein phosphatase groups, protein
tyrosine phosphatase and protein serine/threonine phosphatase type 2C
(PP2C), act as negative regulators of SAPK pathways (3). In
Schizosaccharomyces pombe, tyrosine phosphatase Pyp2 and the yeast homolog of PP2C (Ptc1 and Ptc3) have been shown to
dephosphorylate and inactivate Spc1, the yeast homolog of SAPK (4,
5).
PP2C is one of four major protein serine/threonine phosphatases (PP1,
PP2A, PP2B, and PP2C) in eukaryotes and is implicated in the regulation
of various cellular functions. To date, at least six distinct PP2C gene
products (2C TAK1 was originally identified as an MKKK that functions in the
transforming growth factor- Materials--
The restriction enzymes and other modifying
enzymes used for DNA manipulation were obtained from Takara (Kyoto,
Japan). Anti-6xHis, anti-Myc, and anti-TAK1 antibodies (Abs) were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-phospho-MKK4 and anti-phospho-MKK3/6 Abs were supplied by New
England Biolabs (Beverly, MA). Anti-hemagglutinin (HA; 12CA5) and
anti-Flag (M2) Abs were purchased from Roche Molecular Biochemicals and Kodak Scientific Imaging Systems, respectively. Anti-PP2C Construction of Expression Plasmids--
Expression plasmids
were constructed by standard procedures. Plasmids that express PP2C,
TAK1, TAB1, MAPKs, MKKs, and MKKKs in mammalian cells were constructed
using cDNAs encoding these proteins (17, 21) under the control of
the CMV promoter. Epitope tags were added to the constructs using
synthesized oligonucleotides. Mutated cDNAs were generated by
polymerase chain reaction. For bacterial expression of proteins,
cDNAs encoding the proteins were subcloned into pGEX (Amersham
Pharmacia Biotech) to generate glutathione
S-transferase (GST) fusion proteins or into pQE31 (Qiagen,
Hilden, Germany) to generate hexahistidine-tagged protein and
affinity-purified by standard procedures. Other expression plasmids
were as described elsewhere (23, 24)
Cell Culture and Transfection--
COS7, 293, and 293IL-1RI (25)
cells were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum. At
50-80% confluency the cells were transfected by the DEAE-dextran
method or using LipofectAMINE (Life Technologies, Inc.). The total
amount of DNA (0.5-2 µg per 35-mm dish) was kept constant by
supplementing with empty vector. The cells were cultured for 24-48 h
after transfection and then harvested.
Kinase and Phosphatase Assays--
Immune complex kinase assays
were performed as follows. The cells were lysed in a buffer containing
20 mM Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM sodium
orthovanadate, 50 mM NaF, 1 mM dithiothreitol,
and 1 mM phenylmethylsulfonyl fluoride, and the lysates
were incubated with appropriate Abs for 1 h at 4 °C. The
resulting immune complexes were recovered with protein G-Sepharose
(Amersham Pharmacia Biotech), washed twice with Tris-buffered saline
(20 mM Tris-HCl, pH 7.5, 150 mM NaCl), twice
with 20 mM Tris-HCl, pH 7.5, and then incubated with or
without appropriate substrates in 25 µl of kinase buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
and 1 mM dithiothreitol) containing 0.5-3 µCi of
[ Western Blot Analysis--
Proteins in the cell lysates and
immunoprecipitates were separated by SDS-PAGE and electroblotted onto
polyvinylidene difluoride membranes. The membranes were incubated with
primary Abs at 4 °C for 16 h and then incubated with
horseradish peroxidase-conjugated secondary Ab at 25 °C for 1 h. The chemiluminescence of each blot was detected with an enhanced
chemiluminescence system (Amersham Pharmacia Biotech).
Coimmunoprecipitation Assay--
Cells were lysed with a buffer
containing 20 mM Tris-HCl, pH 7.5, 5 mM EDTA,
150 mM NaCl, 1% (v/v) Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. The cell lysates were incubated with the
indicated Abs for 1 h at 4 °C. The immunoprecipitated proteins
were washed three times with Tris-buffered saline and submitted to
Western blot analysis.
Reporter Assay--
Cells were transfected with the
AP-1-luciferase reporter plasmid (26). After the transfection, cells
were treated with IL-1 PP2C
We then tested whether PP2C PP2C
To determine whether PP2C
To investigate whether TAK1 is a substrate of PP2C, we examined the
phosphorylation and kinase activity of TAK1 incubated with PP2C
in vitro. Flag-TAK1 and TAB1 were coexpressed in COS7 cells,
and Flag-TAK1 was immunoprecipitated from cell extracts with anti-Flag
Ab. When the immunopurified TAK1 complex was incubated with
[
We then determined whether dephosphorylation of TAK1 by PP2C PP2C
Next, we determined the effect of PP2C PP2C
The observation that the catalytically inactive PP2C
We next examined whether endogenous PP2C The Region of TAK1 Required for Association with
PP2C PP2C Effect of PP2C
It has recently been reported that IL-1 treatment of cells activates
the JNK signaling pathway through activation of TAK1 (21). Therefore,
we examined the ability of PP2C MAPK cascades are intracellular signaling modules composed of
three tiers of sequentially activating protein kinases: MKKK, MKK, and
MAPK (1, 2). Because phosphorylation of these components is essential
for the activation of the MAPK cascades, protein phosphatases may be
expected to play important roles in the regulation of these cascades.
Indeed, we recently demonstrated that two major protein
serine/threonine phosphatases, PP2C We present several lines of evidence suggesting that PP2C Coexpression of TAK1/TAB1 with PP2C TAK1 associates with PP2C The central region of TAK1 is required for its association with PP2C To understand what role PP2C may play in regulating SAPK signaling
pathways, it is important to determine how cellular PP2C activity is
affected by extracellular stimuli. In fission yeast cells, the
expression of Ptc1 is enhanced by hyperosmotic stress (29). In
contrast, expression levels of PP2C Takekawa et al. (14) recently reported that PP2C-1, a major isoform of mammalian PP2C, in the TAK1
signaling pathway, a stress-activated protein kinase cascade that is
activated by interleukin-1, transforming growth factor-
, or stress.
Ectopic expression of PP2C
-1 inhibited the TAK1-mediated
mitogen-activated protein kinase kinase 4-c-Jun amino-terminal
kinase and mitogen-activated protein kinase kinase 6-p38 signaling
pathways. In vitro, PP2C
-1 dephosphorylated and
inactivated TAK1. Coimmunoprecipitation experiments indicated that
PP2C
-1 associates with the central region of TAK1. A
phosphatase-negative mutant of PP2C
-1, PP2C
-1 (R/G), acted as a
dominant negative mutant, inhibiting dephosphorylation of TAK1 by
wild-type PP2C
-1 in vitro. In addition, ectopic
expression of PP2C
-1(R/G) enhanced interleukin-1-induced activation
of an AP-1 reporter gene. Collectively, these results indicate that PP2C
negatively regulates the TAK1 signaling pathway by direct dephosphorylation of TAK1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p38
,
p38
, and p38
) (1, 2). Activation of SAPKs requires phosphorylation at conserved tyrosine and threonine residues in the
catalytic domain. This phosphorylation is mediated by dual specificity
protein kinases, which are the members of the MAPK kinase (MKK) family.
Of these, MKK3 and MKK6 phosphorylate p38, MKK7 phosphorylates JNK, and
MKK4 can phosphorylate either. These MKKs, in turn, are activated by
phosphorylation of conserved serine and threonine residues (1, 2).
Recently, several MKK-activating MKK kinases (MKKKs) have been
identified. Some of these MKKKs are also known to be activated by
phosphorylation, but the details are unclear at present.
, 2C
, 2C
, 2C
, Wip1, and Ca2+/calmodulin-dependent protein kinase
phosphatase) have been found in mammalian cells (6-12). In addition,
two distinct isoforms of the human PP2C
(
-1 and -2) and five
isoforms of the mouse PP2C
(
-1, -2, -3, -4, and -5) have been
identified (13-16). These isoforms are generated in each species as
splicing variants of a single pre-mRNA. We have recently reported
that ectopic expression of mouse PP2C
or PP2C
-1 inhibited the
stress-activated MKK3/6-p38 and MKK4/7-JNK pathways but not the
mitogen-activated MKK1-ERK1 pathway. Thus, negative regulation by
PP2C
and PP2C
-1 is selective for different SAPK pathways (17).
Essentially the same results were obtained in studies of human
PP2C
-1 and -2 in mammalian cells (14). Currently, the in
vivo target molecule(s) of PP2C is unknown, although MKK4, MKK6,
and p38 have been proposed as substrates of PP2C
-2 (14).
signaling pathway (18). TAK1 can
activate both the MKK4-JNK and MKK6-p38 pathways (18). Recent studies
have indicated that TAK1 is also activated by various stimuli,
including environmental stress and inflammatory cytokines, and that it
plays critical roles in various cellular responses (19-22). Studies on
the regulation of TAK1 activity have revealed that a TAK1-binding
protein, TAB1, functions as an activator promoting TAK1
autophosphorylation (21, 23). However, the protein phosphatase(s) responsible for inactivation of TAK1 has not been identified. In this
study, we provide evidence indicating that PP2C
-1 selectively associates with TAK1 and inhibits the TAK1 signaling pathway by direct dephosphorylation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ab was raised in rabbit against an oligopeptide of mouse
PP2C
(RILSAENIPNLPPGGGLAGK). Human interleukin-1
(IL-1
) was
from Roche Molecular Biochemicals. All the other reagents used were
from Wako Pure Chemical (Osaka, Japan).
-32P]ATP (NEG-002A, PerkinElmer Life Sciences)
at 30 °C for 10-30 min. The reactions were stopped by adding
SDS-sample buffer and boiled for 2 min. Protein phosphatase assays were
carried out as follows. COS7 cells seeded onto 10-cm dishes were
cotransfected with Flag-TAK1 and Myc-TAB1 expression plasmids. The
Flag-TAK1-Myc-TAB1 complex was immunoprecipitated from cell
extracts with anti-Flag Ab, and phosphorylation was carried out in
kinase buffer containing [
-32P]ATP at 30 °C for 30 min. After washing three times with 20 mM Tris-HCl, pH 7.5, the immune complex was then incubated with or without recombinant
GST-PP2C
in kinase buffer at 30 °C for the indicated times.
Phosphorylated proteins were separated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), and the radioactivities incorporated into
the proteins were detected with a BAS 2000 image analyzer (Fuji, Tokyo, Japan).
for 6 h. Luciferase activity was
determined with the Luciferase Assay System (Promega).
-actin-
-galactosidase reporter plasmid was cotransfected for
normalizing transfection efficiencies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Inhibits TAK1-induced Signal Transduction--
We have
previously reported that two mouse PP2C isoforms, PP2C
and
PP2C
-1, selectively inhibit stress-activated MKKs (MKK3, MKK4, MKK6,
and MKK7) (17). However, the target molecule(s) of PP2C has not been
identified. Because both the MKK4-JNK and MKK6-p38 signaling pathways
are activated by TAK1 (18), we examined whether expression of PP2C
-1
affects TAK1-induced phosphorylation of MKK4 and MKK6 at their serine
or threonine residues. Coexpression of TAK1 and TAB1 enhanced
phosphorylation of MKK4 or MKK6 when expressed together in COS7 cells
(Fig. 1, A and B).
However, concomitant expression of PP2C
-1 markedly inhibited
TAK1-induced phosphorylation of MKK4 and MKK6.
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Fig. 1.
PP2C inhibits
TAK1-induced signaling pathways. A and B,
expression plasmids for HA-TAK1, HA-TAB1, and His-MKK4 (A)
or His-MKK6 (B) were cotransfected with the indicated
amounts of Myc-PP2C
-1 expression plasmid into COS7 cells. Aliquots
of the lysates were immunoblotted with anti-phospho-MKK4 (A)
or anti-phospho-MKK3/6 (B) Abs (upper panels) or
anti-His Ab (lower panels). C and D,
expression plasmids for Flag-TAK1 and Myc-JNK1 (C) or
Myc-p38
(D) were cotransfected with the indicated amounts
of expression plasmids for HA-PP2C
-1 or HA-PP2C
-1(R/G) into COS7
cells. Myc-JNK1(C) or Myc-p38
(D) was
immunoprecipitated from each cell extract, and immunoprecipitates were
subjected to in vitro kinase assays with bacterially
expressed GST-c-Jun (C) or GST-ATF2 (D),
respectively, as substrate (upper panels). The amounts of
Myc-JNK1 or Myc-p38
in aliquots of the lysates were determined by
immunoblotting with anti-Myc Ab (lower panels).
-1 expression affects TAK1-induced
activation of JNK1 and p38
. Both the JNK1 and p38 kinases expressed
in COS7 cells were activated by the exogenous TAK1. However, these
kinase activities were inhibited when PP2C
-1 was coexpressed (Fig.
1, C and D). In contrast, expression of
PP2C
-1(R/G), a phosphatase-defective mutant containing an Arg-179 to
Gly mutation, had no inhibitory effect on TAK1-induced activation of
JNK1 or p38. These results suggest that PP2C
-1 inhibits the TAK1
signaling pathway at TAK1 or downstream of TAK1, e.g. MKKs
and MAPKs.
Acts upon TAK1--
We have previously shown that TAK1,
when coexpressed with TAB1, is activated by autophosphorylation (23).
TAK1 autophosphorylation can be monitored by decreased mobility on
SDS-PAGE, and this mobility shift was cancelled when Ser-192 of TAK1,
which is the site of autophosphorylation, was mutated to alanine (Ref.
23; also shown in Fig. 2).
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Fig. 2.
PP2C decreases the
levels of TAK1 phosphorylation. Expression plasmids for HA-TAK1
and/or HA-TAB1 were cotransfected with the indicated amounts of
expression plasmids for Myc-PP2C
-1 or Myc-PP2C
-1(R/G) into COS7
cells. Aliquots of the lysates were immunoblotted with anti-HA Ab
(upper panel) or anti-Myc Ab (lower panel).
-1 affects the phosphorylation state of
TAK1, we coexpressed TAK1, TAB1, and PP2C
-1 in COS7 cells. As shown
in Fig. 2, expression of wild-type PP2C
-1, but not PP2C
-1(R/G), caused a substantial decrease in the levels of TAK1 phosphorylation. This result suggests that PP2C
-1 acts upon TAK1 directly.
-32P]ATP, TAK1 became autophosphorylated. This
reaction mixture was next incubated with bacterially produced
GST-PP2C
-1 or GST-PP2C
-1(R/G). TAK1 was found to be
dephosphorylated by GST-PP2C
-1, but not by GST-PP2C
-1(R/G), in a
dose-dependent manner (Fig.
3A). The PP2C
-1-mediated
dephosphorylation reaction was dependent on the presence of
Mg2+ (Fig. 3B).
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Fig. 3.
PP2C
dephosphorylates and inactivates TAK1 in
vitro. A, COS7 cells were cotransfected with
Flag-TAK1 and Myc-TAB1 expression plasmids. The cell lysates were
immunoprecipitated with anti-Flag antibody. The immunoprecipitates were
incubated in the presence of [
-32P]ATP. The
phosphorylated proteins were washed, divided equally, and incubated
with the indicated amounts of recombinant GST-PP2C
-1 or
GST-PP2C
-1(R/G). The proteins in the samples were separated by
SDS-PAGE and analyzed by autoradiography (upper panel) or
Coomassie Brilliant Blue (CBB) staining (lower
panel). B, the immunoprecipitates prepared as described
in A were incubated in the presence of
[
-32P]ATP. The phosphorylated proteins were washed,
divided equally, and incubated with 50 µg/ml recombinant
GST-PP2C
-1 for the indicated times in the presence or absence of
MgCl2. The proteins in the samples were separated by
SDS-PAGE and analyzed by autoradiography (upper panel) or
Coomassie Brilliant Blue staining (lower panel).
C, the immunoprecipitates prepared as described in
A were incubated with recombinant His-MKK6 (1 µg) in the
presence of [
-32P]ATP with or without the indicated
amounts of recombinant GST-PP2C
-1. The proteins in the samples were
separated by SDS-PAGE and analyzed by autoradiography (top
and middle panels) or Coomassie Brilliant Blue staining
(bottom panel).
-1
reduces TAK1 activity. Flag-TAK1 immunoprecipitates were treated with
GST-PP2C
-1 and measured for TAK1 activity in vitro. The presence of PP2C
-1 decreased the ability of TAK1 to phosphorylate itself and MKK6 (Fig. 3C). Thus, PP2C
-1 dephosphorylates
and inactivates TAK1 in vitro. This supports the possibility
that PP2C
-1 negatively regulates the TAK1 signaling pathway by
dephosphorylating TAK1.
Does Not Dephosphorylate MKK6 in Vitro--
Recent studies
have indicated that one of the human PP2C isoforms, PP2C
-2,
dephosphorylates and inactivates MKK4, MKK6, and p38 in
vitro (14). Therefore, we tested whether PP2C
-1 could also
dephosphorylate and inactivate MKK6 in vitro. Bacterially produced MKK6 is activated by autophosphorylation and is able to
phosphorylate p38 in vitro (27). We used this system to
determine the effect of recombinant PP2C
-1 on MKK6 activity. We
found that PP2C
-1 treatment did not affect MKK6 kinase activity
under conditions where it inactivates TAK1 (Fig. 3, A and
C versus Fig.
4A).
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Fig. 4.
PP2C does not
dephosphorylate MKK6 in vitro. A,
recombinant His-MKK6 (0.1 µg) and GST-p38
(1 µg) were incubated
in the presence of [
-32P]ATP with or without the
indicated amounts of recombinant GST-PP2C
-1 for 30 min at 30 °C.
The proteins in the samples were separated by SDS-PAGE and analyzed by
autoradiography (upper panel) or Coomassie Brilliant Blue
(CBB) staining (lower panel). B,
expression plasmid for Flag-MKK6 was transfected into COS7 cells. Cells
were subjected to osmotic shock (0.7 M NaCl for 20 min at
37 °C). The cell lysates were immunoprecipitated with anti-Flag Ab,
divided equally, and incubated with the indicated amounts of
recombinant GST-PP2C
-1 for 30 min at 30 °C. Samples were
immunoblotted with anti-phospho-MKK3/6 Ab (upper panel) or
stained with Amido Black (AB) (lower
panel).
-1 on stress-induced
phosphorylation of MKK6. COS7 cells were transfected with Flag-MKK6 and
subjected to hyperosmotic stress, and Flag-MKK6 was immunoprecipitated from the cell lysates with anti-Flag Ab. The immunoprecipitates were
then incubated with GST-PP2C
-1. Increasing concentrations of
GST-PP2C
-1 had no effect on the phosphorylation level of MKK6 (Fig.
4B). Taken together, these results indicate that PP2C
-1 does not act upon MKK6.
Associates with TAK1 in Mammalian Cells--
To determine
whether PP2C
-1 associates with TAK1, we coexpressed Myc-TAK1 and
HA-PP2C
-1 or HA-PP2C
-1(R/G) in COS7 cells. Cell extracts were
immunoprecipitated with anti-Myc Ab, and coprecipitated HA-PP2C
-1
was detected by immunoblotting with anti-HA Ab. As shown in Fig.
5A, both HA-PP2C
-1 and
HA-PP2C
-1(R/G) coimmunoprecipitated with Myc-TAK1, although the
interaction of the wild-type PP2C
-1 with TAK1 was substantially
weaker than that of PP2C
-1(R/G). This interaction is specific for
PP2C
-1, because HA-PP2C
, another major mouse PP2C isoform (28),
did not coimmunoprecipitate with Myc-TAK1 under the same conditions
(Fig. 5B). Therefore, the association of PP2C
-1 with TAK1
is not caused by a nonspecific protein interaction.
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Fig. 5.
PP2C associates with
TAK1 in mammalian cells. A, expression plasmid for
HA-PP2C
-1(R/G) (R/G, lanes 1, 2,
and 4) or wild-type HA-PP2C
-1 (WT, lane
3) was cotransfected with control plasmid (lane 1) or
expression plasmid for Myc-TAK1 (lanes 2-4) into COS7
cells. Aliquots of the lysates were immunoprecipitated with anti-Myc
Ab, and the immunoprecipitates were immunoblotted with anti-HA
(top panel) and anti-Myc (middle panel) Abs.
Aliquots of the lysates were also immunoblotted with anti-HA Ab
(bottom panel). B, expression plasmid for
HA-PP2C
or HA-PP2C
-1 was cotransfected with Myc-TAK1 into COS7
cells. Aliquots of the lysates were immunoprecipitated with anti-Myc
Ab, and the immunoprecipitates were immunoblotted with anti-HA
(top panel) and anti-Myc (middle panel) Abs.
Aliquots of the lysates were also immunoblotted with anti-HA Ab
(bottom panel). C, expression plasmid for
Flag-PP2C
-1 was cotransfected with expression plasmids for wild-type
Myc-TAK1 or Myc-TAK1(S/A) into 293 cells. Aliquots of the lysates were
immunoprecipitated with control Ab (C) or anti-Flag Ab
(F), and the immunoprecipitates were immunoblotted with
anti-Myc (top panel) and anti-Flag (middle panel)
Abs. Aliquots of the lysates were also immunoblotted with anti-Myc Ab
(bottom panel). D, 293 cells were lysed and
immunoprecipitated with control rabbit IgG (C) or
anti-PP2C
Ab (
). The immunoprecipitates were immunoblotted with
anti-TAK1 Ab (top panel). Aliquots of the lysates were also
immunoblotted with anti-PP2C
Ab (middle panel) and
anti-TAK1 Ab (bottom panel). IP,
immunoprecipitation; IB, immunoblot.
has a higher
affinity for TAK1 than that of wild-type PP2C
suggested that PP2C
might preferentially bind phosphorylated TAK1. The TAK1(S/A) mutant, in
which Ser-192 is replaced by Ala, is defective in both phosphorylation
and activation (23). We coexpressed PP2C
-1 and TAK1 or TAK1(S/A) in
293 cells and performed coimmunoprecipitation experiments. We found
that TAK1(S/A) had an affinity for PP2C
-1 similar to that of
wild-type TAK1 (Fig. 5C), indicating that phosphorylation at
Ser-192 is not required for association with PP2C
-1.
-1 and TAK1, expressed at
lower physiological levels, can also interact with one another. As
shown in Fig. 5D, the 70-kDa endogenous TAK1 was
specifically detected by anti-TAK1 Ab in the endogenous PP2C
immunoprecipitates from 293 cells, but not in the rabbit IgG immunoprecipitates.
--
To determine which region of TAK1 is required for its
interaction with PP2C
-1, we generated three Myc-tagged, truncated
proteins, Myc-TAK1(N400), Myc-TAK1(C366), and Myc-TAK1(C176),
containing the amino-terminal 400, carboxyl-terminal 366, and
carboxyl-terminal 176 amino acids of TAK1, respectively (Fig.
6A). We coexpressed each
deletion mutant along with Flag-PP2C
-1(R/G) in 293 cells and
immunoprecipitated Flag-PP2C
-1(R/G) from cell extracts with anti-Flag Ab. Subsequent immunoblot analysis using anti-Myc Ab revealed
that Flag-PP2C
-1 was associated with Myc-TAK1(N400) and
Myc-TAK1(C366) but not with Myc-TAK1(C176) (Fig. 6B). This result indicates that the central region of TAK1 is responsible for its
association with PP2C
-1.
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Fig. 6.
The central region of TAK1 is required for
association with PP2C . A,
full-length (FL) TAK1 and its deletion mutants are depicted.
TAK1(N400), TAK1(C366), and TAK1(C176) contain the amino-terminal 400 amino acids and the carboxyl-terminal 366 and 176 amino acids of TAK1,
respectively. B, expression plasmid for Flag-PP2C
-1(R/G)
was cotransfected with expression plasmid for 1xMyc-TAK1,
1xMyc-TAK1(N400), 6xMyc-TAK1(C366), or 6xMyc-TAK1(C176) into 293 cells.
Aliquots of the lysates were immunoprecipitated with anti-Flag Ab, and
the immunoprecipitates were immunoblotted with anti-Myc (upper
left panel) and anti-Flag (lower panel) Abs. Aliquots
of the lysates were also immunoblotted with anti-Myc Ab (right
panel). IP, immunoprecipitation; IB,
immunoblot.
Does Not Associate with MEKK3, MKK4, MKK6, JNK, or
p38--
To evaluate the specificity of the association of PP2C
-1
with TAK1, we examined whether PP2C
-1 could associate with other SAPK signaling pathway components. Flag-PP2C
-1 was coexpressed with
Myc-TAK1, Myc-MEKK3, Myc-MKK4, Myc-MKK6, Myc-JNK1, or Myc-p38
in 293 cells. Flag-PP2C
-1 was immunoprecipitated from cell extracts with
anti-Flag Ab, and the immune complexes were subjected to immunoblotting
with anti-Myc Ab. None of these proteins, except for Myc-TAK1,
coimmunoprecipitated with PP2C
-1 (Fig.
7). Thus, PP2C
-1 specifically
interacts with TAK1.
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Fig. 7.
PP2C selectively
associates with TAK1. Expression plasmid for Flag-PP2C
-1(R/G)
was cotransfected with expression plasmid for Myc-TAK1, Myc-MEKK3,
Myc-MKK4, Myc-MKK6, Myc-JNK1, or Myc-p38
into 293 cells. Aliquots of
the lysates were immunoprecipitated with anti-Flag Ab, and the
immunoprecipitates were immunoblotted with anti-Myc (upper left
panel) and anti-Flag (lower panel) Abs. Aliquots of the
lysates were also immunoblotted with anti-Myc Ab (right
panel). IP, immunoprecipitation; IB,
immunoblot.
on IL-1-stimulated AP-1 Activation--
Because
PP2C
-1(R/G) appeared to have a higher affinity for TAK1 than did
wild-type PP2C
-1 (Fig. 5A), we asked whether
PP2C
-1(R/G) could act as a dominant negative mutant. To test this
possibility, we examined the effect of PP2C
-1(R/G) on
PP2C
-1-mediated TAK1 dephosphorylation in vitro. We found
that PP2C
-1(R/G) inhibited the dephosphorylation of TAK1 by
PP2C
-1 in a dose-dependent manner (Fig.
8A).
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Fig. 8.
Effect of PP2C on
the IL-1-induced AP-1 activation. A, aliquots of the
phosphorylated TAK1 prepared as described in the legend to Fig. 3 were
incubated with the indicated amounts of GST-PP2C
-1(R/G) and/or
GST-PP2C
-1 for 30 min at 30 °C. The proteins were separated by
SDS-PAGE and analyzed by autoradiography. B, the expression
plasmids of Flag-PP2C
-1 and/or HA-TAK1 were transfected into 293 IL-1RI cells. After the treatment with IL-1 for 10 min, the cell
lysates were subjected to immune complex kinase assay using MKK6 as the
substrate (upper panel) or immunoblotted with anti-HA Ab
(lower panel). C-E, 293IL-1RI cells were
transfected with AP-1-luciferase reporter plasmid with or without the
indicated amounts of the expression plasmids for PP2C
-1
(C), PP2C
-1(R/G) and PP2C
-1 (D), or
PP2C
-1(R/G) (E). After the treatment with IL-1 for 6 h, cells were lysed, and luciferase activities were determined and
normalized on the basis of
-galactosidase expression. The data shown
are the mean ± S.D. (n = 3).
-1 to affect activation of
TAK1 and AP-1 following IL-1 stimulation. We transfected 293IL-1RI
cells with PP2C
-1 and TAK1 and determined the effect of PP2C
-1
expression on IL-1-induced mobility shift on SDS-PAGE and activation of
TAK1. IL-1 treatment caused a slight mobility shift of TAK1 on
SDS-PAGE, confirming our previous observation (23) (Fig. 8B,
lower panel). However, the coexpression of PP2C
-1 totally
reversed the mobility shift of TAK1. The expression of PP2C
-1 also
inhibited the IL-1-induced activation of TAK1 (Fig. 8B,
upper panel). Next, we transfected 293IL-1RI cells with
PP2C
-1 or/and PP2C
-1(R/G) and assayed AP-1 activity using an
AP-1-dependent luciferase reporter gene. PP2C
-1 blocked
IL-1-induced AP-1 activity in a dose-dependent manner (Fig.
8C). However, inhibition of IL-1-induced AP-1 activation by
PP2C
-1 was reversed by cotransfection with PP2C
-1(R/G) (Fig.
8D). Furthermore, ectopic expression of PP2C
-1(R/G) enhanced IL-1-induced AP-1 activity in a dose-dependent
manner (Fig. 8E).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PP2C
, inactivate the
stress-activated JNK and p38 MAPK pathways (17). Furthermore, Takekawa
et al. (14) showed that PP2C
inhibits the JNK and p38
cascades by dephosphorylating MKK4, MKK6, and p38. TAK1 is a member of
the MKKK family and activates the JNK and p38 pathways. In this study
we elucidated the role of PP2C
in TAK1-mediated signaling pathways.
negatively
regulates the TAK1 pathways by dephosphorylating and inactivating TAK1.
First, ectopic expression of PP2C
inhibits the MKK4-JNK and MKK6-p38
pathways activated by TAK1 (Fig. 1). Second, it is known that the
TAK1-binding protein TAB1 activates TAK1 by promoting its
autophosphorylation (23). We found that PP2C
overexpression
decreased TAB1-induced TAK1 autophosphorylation in vivo
(Fig. 2). Third, PP2C
dephosphorylates and inactivates TAK1 in
vitro (Fig. 3) but fails to dephosphorylate MKK6 (Fig. 4).
Finally, PP2C
interacts with TAK1 but not with MEKK3, MKK4, MKK6,
JNK, or p38 (Figs. 5 and 7). Collectively, these data are consistent
with the idea that PP2C
suppresses TAK1-mediated signaling by
associating with and dephosphorylating TAK1. Because TAK1 functions in
various biological responses, including acting as a positive regulator
of transforming growth factor-
- and IL-1-induced signal transduction (18, 21) and as a negative regulator in Wnt-induced signal
transduction (22), it would be interesting to examine whether PP2C
contributes to the control of these physiological responses.
-1 did not result in complete
dephosphorylation of TAK1, as judged by the fact that the mobility of
TAK1 is still slower than that of TAK1 expressed by itself (Fig. 2).
COS7 cells contain a substantial amount of free, endogenous TAB1.
Therefore, we speculate that the reason for the incomplete
dephosphorylation may be that the dephosphorylated TAK1 can be
rephosphorylated, because both TAB1 and ATP are present in the cells.
Alternatively, this may suggest that there are other phosphorylation
sites in TAK1 that are not substrates for PP2C.
but not with PP2C
(Fig. 5). Thus, the
interaction of TAK1 with PP2C
is rather specific. TAK1 is activated
via autophosphorylation of Ser-192 in the activation loop between
kinase domains VII and VIII. Mutation of TAK1 Ser-192 to Ala to create
TAK1(S/A) abolishes both phosphorylation and activation of TAK1 (23).
TAK1(S/A) has an affinity for PP2C
similar to that of wild-type TAK1
(Fig. 5C), indicating that phosphorylation of TAK1 is not
required for its association with PP2C
. This suggests that the
association of TAK1 with PP2C
does not occur simply through affinity
of the enzyme (PP2C
) for its substrate (phosphorylated TAK1), but
rather that PP2C
and TAK1 are stably associated. This may ensure
appropriate localization of PP2C
and facilitate the specific and
rapid deactivation of TAK1.
(Fig. 6). A similar region of TAK1 is involved in its association with
TAB1 (20), which suggests that PP2C
might prevent the association of
TAK1 with TAB1. However, this possibility is unlikely, because we did
not observe any competition between TAB1 and PP2C
in their
association with TAK1.2
Consistent with this, endogenous TAK1 constitutively associates with
TAB1 in the absence of ligand stimulation (23). Therefore, the minimum
regions of TAK1 required for association with PP2C
and TAB1 must be
different. It is still not clear whether PP2C
associates with TAK1
directly or indirectly. However, the observation that PP2C
fails to
interact with TAB12 argues against the possibility
that TAB1 mediates the association between PP2C
and TAK1.
and PP2C
-1 are not altered
following stress treatment of cells (17). PP2C
has been shown to
preferentially bind to the phosphorylated form of p38 and may function
in the adaptive phase of the stimulation cycle to restore p38 to the
inactive state following stimulation by stress (14). PP2C
may play
an analogous role in maintaining TAK1 signaling. TAK1 mediates
IL-1-induced JNK signaling (21), and ectopic expression of PP2C
blocks IL-1-induced AP-1 activation. PP2C
(R/G), a catalytically
inactive mutant, has a higher affinity for TAK1 than does wild-type
PP2C
and acts as a dominant negative factor, antagonizing the
inhibitory effect of wild-type PP2C
on IL-1-induced AP-1 activation.
Furthermore, ectopic expression of PP2C
(R/G) enhances
IL-1-stimulated AP-1 activation but does not cause constitutive
activation of AP-1.2 These results raise the
possibility that PP2C
may down-regulate TAK1 activity after ligand
stimulation. Because endogenous PP2C
constitutively associates with
TAK1 (Fig. 5D), and ligand stimulation does not affect this
association,2 it is tempting to speculate that
regulation of PP2C
enzymatic activity is involved in regulation of
TAK1 signaling. Alternatively, PP2C
activity may be constitutive and
serve to restore TAK1 to the inactive state following stimulation.
Therefore it is important to determine whether the phosphatase activity
of PP2C
is enhanced when cells are subjected to stress or treated
with pro-inflammatory cytokines.
dephosphorylates MKK4, MKK6, and p38 in vitro. In this
study, we show that PP2C
dephosphorylates and inactivates TAK1.
Thus, in mammalian cells, SAPK pathways are negatively regulated by
multiple PP2C isoforms at different levels; PP2C
inhibits the
pathways at the TAK1 MKKK level, and PP2C
acts at the MKK and MAPK
levels. In addition, two distinct groups of protein phosphatases other
than PP2C also participate in the regulation of the SAPK pathways. The
first group consists of the dual specificity phosphatases (also known
as MAPK phosphatases) that inactivate MAPKs by dephosphorylating both
tyrosine and threonine residues in the catalytic domains. Of the nine
isolated MAPK phosphatases, M3/6 and MAPK phosphatase-5 have been shown
to selectively dephosphorylate and inactivate p38 and JNK (30, 31). The
second group includes PP2A, which inactivates partially purified p38
kinase in vitro (32). Cells treated with the PP2A inhibitor
okadaic acid show enhanced MKK6 activity in epithelial cells (27).
These results suggest that PP2A may also negatively regulate SAPK
pathways and raise the possibility that several different groups of
protein phosphatases each negatively regulate distinct targets in SAPK pathways.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Ulrich Siebenlist (National Institutes of Health) and Simon J. Cook (the Babraham Institute) for providing us with the expression plasmids of MEKK3 and AP-1-luciferase, respectively. We are also grateful to Kimio Konno for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by the Smoking Research Foundation and the Takeda Science Foundation.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.
** To whom correspondence should be addressed. Tel.: 81-22-717-8471; Fax: 81-22-717-8476; E-mail: tamura@idac.tohoku.ac.jp.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M007773200
2 M. Hanada, J. Ninomiya-Tsuji, K.-i. Komaki, M. Ohnishi, K. Katsura, R. Kanamaru, K. Matsumoto, and S. Tamura, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun amino-terminal kinase; MKK, MAPK kinase; MKKK, MKK kinase; PP, protein serine/threonine phosphatase; Ab, antibody; HA, hemagglutinin; IL, interleukin; GST, glutathione S-transferase; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase.
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