From the Department of Biochemistry, Institute of
Development, Aging, and Cancer, Tohoku University, 4-1 Seiryomachi,
Aoba-ku, Sendai 980-8575, Japan, the § Biological Resources
Division, Japan International Research Center for Agricultural
Sciences, 1-1 Ohwashi, Tsukuba 305-0851, Japan, the ¶ Department
of Biochemistry, Kumamoto University Medical School, Honjo, Kumamoto
860-0811, Japan, the
Department of Environmental and Molecular
Toxicology, North Carolina State University, Raleigh, North
Carolina 27695-7633, and the ** Department of Molecular
Biology, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan
Received for publication, November 11, 2002, and in revised form, January 22, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although TAK1 signaling plays essential roles in
eliciting cellular responses to interleukin-1 (IL-1), a proinflammatory
cytokine, how the IL-1-TAK1 signaling pathway is positively and
negatively regulated remains poorly understood. In this study, we
investigated the possible role of a novel protein phosphatase 2C (PP2C)
family member, PP2C Stress-activated protein kinases
(SAPKs)1 are a subfamily of
the mitogen-activated protein kinase 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 N-terminal kinases (JNK1, JNK2, and JNK3) and the p38 mitogen-activated protein kinases (p38 In the absence of a signal, the constituents of the SAPK cascade return
to their dephosphorylated, inactive state, suggesting an essential role
for phosphatases in SAPK regulation. Protein phosphatases are
classified into three groups, Ser/Thr phosphatases, Ser/Thr/Tyr
phosphatases, and Tyr phosphatases, depending on their phosphoamino
acid specificity. Dephosphorylation of SAPK signal pathway components
requires the participation of a variety of phosphatases. In fact,
participation by members of all three groups in the negative regulation
of SAPK signaling pathways has been reported (4).
PP2C is one of four major protein serine/threonine phosphatases (PP1,
PP2A, PP2B, and PP2C) found in eukaryotes. At least six distinct PP2C
gene products (2C TAK1 was originally identified as a MKK kinase that functions in the
transforming growth factor- In this study, we present evidence that a novel member of the PP2C
family (PP2C Materials--
Restriction enzymes and other modifying enzymes
used for DNA manipulation were obtained from Takara (Kyoto, Japan).
Glutathione-agarose beads, protein A-agarose beads, Hybond-P membranes,
Hybond-N+ membranes, [ Cloning of PP2C Construction of Expression Plasmids--
Plasmids expressing
PP2C, TAK1, TAB1, mitogen-activated protein kinases, and MKKs in
mammalian cells were constructed using cDNAs encoding these
proteins, under the control of the CMV promoter. Epitope tags were
added to the constructs using synthesized oligonucleotides. Mutated
cDNAs were generated by polymerase chain reactions. For bacterial
expression of proteins, cDNAs encoding the proteins were subcloned
into pGEX (Amersham Biosciences) or into pMAL-C2X (New England Biolabs)
to generate glutathione S-transferase (GST) fusion proteins
or maltose-binding protein (MBP) fusion proteins. Other expression
plasmids were prepared as described elsewhere (23, 26).
In Vitro Transcription and Translation--
TNT Quick Coupled
Transcription/Translation Systems (Promega) were used to determine the
start codon of PP2C Cell Culture and Transfection--
293 cells and 293IL-1RI (27)
cells were grown in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% (v/v) fetal bovine serum and
penicillin/streptomycin. At ~50-70% confluence the cells were
transfected by the calcium phosphate transfection method or using
LipofectAMINE (Invitrogen). The total amount of DNA used for
transfection was 1 µg per 12-well plate, 2 µg/6-cm plate, and 10 µg/10-cm plate. After transfection, the cells were cultured for
48 h and harvested.
Preparation of Protein Phosphatase Substrates--
Protein Phosphatase Assay--
Casein phosphatase activity was
assayed by measuring the release of [32P]phosphate
from 32P-labeled casein, essentially as described
previously (28). TAK1 phosphatase activity was determined by incubating
the 32P-labeled TAK1 with the indicated amounts of
recombinant MBP-PP2C Immunoprecipitation and Immunoblot Analysis--
Cells
transfected with the indicated expression plasmids were washed twice
with phosphate-buffered saline and lysed with ice-cold lysis buffer.
Immunoprecipitation was performed with the indicated antibodies and
protein A-Sepharose beads. The immunoprecipitates were subjected to
10% (w/v) SDS-PAGE and then transferred onto polyvinylidene difluoride
membranes. The membranes were incubated overnight with the primary
antibodies at 4 °C, incubated with alkaline phosphatase-conjugated
secondary antibody for 1 h at 25 °C, and developed by
chemiluminescence using CDP-Star as the substrate.
Northern Blot Analysis--
Total RNA was extracted from
the mouse tissues with RNAzol B (Biotecx Laboratories, Inc.). The
denatured RNA (15 µg) was electrophoresed in a 1.0% (w/v) agarose
gel and transferred onto a Hybond-N+ membrane, and Northern
hybridization was carried out as described previously (15). A
32P-labeled probe representing the entire coding sequence
of the PP2C Reporter Gene Assay--
A reporter gene activity assay
was performed as described previously (18, 32). The
pGL3-AP-1/luciferase reporter gene was used to measure
AP-1-dependent transcriptional activity. Luciferase activity was determined with a luciferase assay system (Promega). A
Primary Structure of PP2C Expression of PP2C Northern Blot Analysis--
Northern hybridization was performed
on mouse tissues to clarify the tissue distribution of PP2C PP2C
We next asked whether the TAK1-induced activation of AP-1 is also
affected by the co-expression of PP2C
Since TAK1 activates both JNK and p38 signaling pathways via MKK4/7 and
MKK3/6, respectively, we also tested whether TAK1-induced activation of
JNK or p38 was suppressed by PP2C MKK3b-enhanced Phosphorylation of p38 Was Not Suppressed by
PP2C PP2C
We next tested whether PP2C A Point Mutant of PP2C PP2C
We considered the possibility that PP2C
To test whether PP2C PP2C
To test this possibility, we examined whether PP2C IL-1 Induces Transient Dissociation of PP2C
It has been established that the activation of TAK1 by IL-1 induces the
upward mobility shift of TAK1 (due to autophosphorylation) on SDS-PAGE
(23). As shown in lanes 7 and 8 of the
middle panel of Fig. 8A, mobility
shift of TAK1 was observed in accordance with the IL-1-induced
dissociation of PP2C
We were interested in determining whether the activity of PP2C
We finally asked whether, similar to IL-1 treatment, activation of TAK1
by TAB1 also induces the dissociation of PP2C SAPK cascades are intracellular signaling modules composed of
three tiers of sequentially activated protein kinases: MKK kinase, MKK,
and SAPK. Because phosphorylation of these components is essential for
the activation of the SAPK cascades, protein phosphatases may be
expected to play important roles in their regulation. Indeed, accumulating evidence indicates that a number of protein phosphatases belonging to three different protein phosphatase groups (Ser/Thr phosphatase, Ser/Thr/Tyr phosphatase, and Tyr phosphatase) participate in the regulation of SAPK systems (4).
We recently reported that PP2C Studies of the modes of action of PP2C, in the regulation of the IL-1-TAK1 signaling
pathway. PP2C
was composed of 303 amino acids, and the overall
similarity of amino acid sequence between PP2C
and PP2C
was found
to be 26%. Ectopic expression of PP2C
inhibited the IL-1- and
TAK1-induced activation of mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase or MKK3-p38 signaling pathway. PP2C
dephosphorylated TAK1 in vitro. Co-immunoprecipitation
experiments indicated that PP2C
associates stably with TAK1 and
attenuates the binding of TAK1 to MKK4 or MKK6. Ectopic expression of a
phosphatase-negative mutant of PP2C
, PP2C
(D/A), which acted as a
dominant negative form, enhanced both the association between TAK1 and
MKK4 or MKK6 and the TAK1-induced activation of an AP-1 reporter gene.
The association between PP2C
and TAK1 was transiently suppressed by
IL-1 treatment of the cells. Taken together, these results suggest
that, in the absence of IL-1-induced signal, PP2C
contributes to
keeping the TAK1 signaling pathway in an inactive state by associating
with and dephosphorylating TAK1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p38
, p38
, and p38
) (1, 2). Activation of SAPKs requires phosphorylation of conserved tyrosine and threonine residues present in the catalytic domain. This phosphorylation is mediated by
dual specificity protein kinases, which are members of the mitogen-activated protein kinase kinase (MKK) family. Of these, MKK3
and MKK6 phosphorylate p38, MKK7 phosphorylates JNK, and MKK4 can
phosphorylate either. These MKKs, in turn, are similarly activated by
the phosphorylation of conserved serine and threonine residues (1, 2).
Recently, several MKK-activating MKK kinases have been
identified (3). Some of these MKK kinases are also known to be
activated by phosphorylation.
, 2C
, 2C
, 2C
, Wip1, and Ca2+/calmodulin-dependent protein kinase
phosphatase) have been found in mammalian cells (5-12). 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 as splicing variants
of a single pre-mRNA. Of the six different members of the PP2C
family, three (PP2C
, PP2C
, and Wip1) have recently been
implicated in the negative regulation of SAPK signaling pathways (14,
17-19). We and others have 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
(14, 17). Thus, negative regulation by PP2C
and PP2C
-1 is
selective for SAPK pathways. We have provided further evidence
indicating that PP2C
-1 associates with another upstream kinase,
TAK1, and inhibits the SAPK signaling pathways by direct
dephosphorylation of TAK1 (18). Takekawa et al. (14) have
found that PP2C
-2 dephosphorylates and inactivates MKK4, MKK6 and
p38, both in vivo and in vitro. In addition, they
reported that Wip1, whose expression is induced by ionizing radiation
in a p53-dependent manner, inactivates p38 by specific
dephosphorylation of a conserved threonine residue and suppresses
subsequent p53 activation (10, 19).
signaling pathway (21). TAK1 can
activate both the MKK4-JNK and MKK6-p38 pathways (18). Studies of the
mechanism of transforming growth factor-
-induced activation of TAK1
have revealed that a TAK1-binding protein, TAB1, functions as an
activator promoting TAK1 autophosphorylation (22, 23). Recent studies
have indicated that TAK1 is also activated by various stimuli,
including environmental stress and inflammatory cytokines (IL-1 and
tumor necrosis factor
), and TAB2, another TAK1 binding protein, has
been found to link TAK1 and TRAF6 and acts as a mediator of TAK1 action
in the IL-1-induced signaling pathway (24, 25). However, the detailed
mechanism of positive and negative regulation of TAK1 is not yet fully understood.
) participates in the negative regulation of the TAK1
signaling pathway and suggest that PP2C
is involved in the
IL-1-induced regulation of TAK1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, and RedivueTML
[35S]methionine were purchased from Amersham Biosciences.
CDP-Star substrate was obtained from Applied Biosystems (Bedford, MA). The luciferase assay,
-galactosidase enzyme assay, and TNT Quick Coupled Transcription/Translation Systems were supplied by Promega (Madison, WI). Amylose resin and anti-MBP antibody were purchased from
New England Biolabs (Beverly, MA). Anti-hemagglutinin antibody (HA;
12CA5) and human IL-1
were purchased from Roche Molecular Biochemicals. Anti-phospho-JNK, anti-phospho-p38, anti-phospho-MKK4, and anti-phospho-MKK3/6 antibodies were obtained from Cell Signaling (Beverly, MA). Anti-TAK1, anti-Myc, and anti-His antibodies were from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-FLAG (M2)
antibody was from Kodak Scientific Imaging Systems (New Haven, CT). All
other reagents used were from Wako Pure Chemical (Osaka, Japan).
cDNA--
We searched the expressed
sequence tag data base for DNA clones encoding the amino acid sequences
of the unique motifs conserved in mouse PP2C family members. Three
different clones, each potentially encoding a novel member of the PP2C
family, were found. These three cDNAs were designated clone-1, -2 (to be published elsewhere), and -3 (to be published elsewhere). A
putative full-length cDNA of clone-1 was obtained by 5'- and
3'-rapid amplification of cDNA ends methods using the total RNA
fraction isolated from the hearts of adult mice as the template.
Nucleotide sequence data for the full-length cDNA are available in
the GenBankTM data base under the accession number
AY184801. This sequence has been scanned against the data base, and all
sequences with significant relatedness to the new sequence were
identified (BF466920, BB611990, BB636411, BU052859, BB611559, AI613837, BM876958, AF117832, BF471931, AA509367, BE980311, BF464085, AA498729,
AI481407, BG296608, BE948499, BB628580, BF465398, BB643596, BE983821,
BB866395, BB662653, BB622909, BI220774, BE859571).
cDNA.
-Casein
was phosphorylated by protein kinase A and [
-32P]ATP
as described previously (28, 29). The catalytic subunit of protein
kinase A was purified as described by Reimann and Beham (30). The
reaction mixture was gel-filtered by Sephadex G-25 and the isolated
32P-labeled casein was stored at
20 °C before use. To
obtain 32P-labeled TAK1, 293 cells (2 × 106) were cotransfected with 5 µg of pcDNA3-HA-TAK1
and 5 µg of pcDNA3-HA-TAB1, using the calcium phosphate
transfection method. The cell lysates were prepared 48 h after the
transfection, and the TAK1-TAB1 complex was immunoprecipitated
with anti-TAK1 antibody and protein A-Sepharose beads (Amersham
Biosciences). After immunoprecipitation, the beads were washed with
Tris-buffered saline (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl) containing 0.05% (v/v) Tween 20 and incubated with [
-32P]ATP in kinase buffer (20 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM dithiothreitol) for 30 min at 30 °C. The immune
complex containing the autophosphorylated TAK1 was washed with 50 mM Tris-HCl, pH 7.5, and subsequently with phosphatase
buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% (v/v) Brij 35, and 2 mM MnCl2). Aliquots were stored at
20 °C
before use. In order to prepare the phosphorylated JNK, 2 µg of
pcDNA3-Myc-JNK was transfected into 293 cells (2 × 105) using LipofectAMINE. IL-1 (500 units/ml) was added to
the medium 48 h after the transfection, and the cells were
incubated for 15 min. 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 cell
lysates were immunoprecipitated with anti-Myc antibody and protein
A-Sepharose beads. The immune complex was washed with Tris-buffered
saline containing 0.05% (v/v) Tween 20 and then with the kinase buffer
and stored in aliquots at
20 °C before use. In order to prepare
the GST-phospho-MKK4, pEBG2T-MKK4, pcDNA3-HA-TAK1, and
pcDNA3-HA-TAB1 were cotransfected into 293 cells. The cells were
harvested 48 h after the transfection, and the GST-phospho-MKK4
was isolated from the cell lysates with glutathione-Sepharose beads.
The phosphorylated GST-MKK4 was eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 5 mM glutathione, 0.25 M sucrose, 0.5 mM dithiothreitol, and 0.1 mM EGTA) and stored at
20 °C before use.
or MBP-PP2C
-1 in phosphatase buffer for 30 min at 30 °C. The reaction was stopped by the addition of SDS-sample
buffer, and the reaction mixture was subjected to SDS-PAGE followed by
autoradiography. To determine the JNK phosphatase activity, the
phosphorylated JNK was incubated with the indicated amounts of
recombinant MBP-PP2C
, together with recombinant GST-c-Jun and
[
-32P]ATP (0.5-3 µCi) in the kinase buffer. The
reaction mixtures were incubated for 30 min at 30 °C. The reactions
were stopped by adding SDS-sample buffer. The proteins were separated
by SDS-PAGE and analyzed by autoradiography. In order to determine the
MKK4 phosphatase activity, the phospho-MKK4 was incubated with the recombinant MBP-PP2C
for 30 min at 30 °C in phosphatase buffer. The proteins in the reaction mixture were separated by SDS-PAGE and
immunoblotted with anti-phospho-MKK4 and anti-MBP antibodies.
cDNA was used for the hybridization.
-galactosidase reporter plasmid, under the control of a
-actin promoter, was cotransfected to normalize the transfection efficiency.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The obtained cDNA
clone (clone-1) contained a single oligonucleotide of 1566 bp (Fig.
1A). The first Met codon and
the first stop codon downstream were at nucleotide 238 and 1318, respectively. However, the fourth Met codon at nucleotide 409 was in
the context of the translational start consensus sequence (A at
3 and
G at +4, where the A in ATG is +1) (33). Therefore, we performed an in vitro translation of the cDNA clone to determine
which Met was the actual translational initiation codon. The largest
size of the proteins synthesized in vitro was 34 kDa (Fig.
1B). This size matched that of the protein from the fourth
Met codon, which is composed of 303 amino acids. The bands of the
smaller sizes may presumably be the proteolytic products of the 34-kDa
protein. Therefore, we tentatively concluded that the open reading
frame resided between 409 and 1318 nt. The primary sequence contained six motifs commonly conserved in PP2C family members in addition to a
basic amino acid cluster unique to this 34-kDa protein (34). This
suggested that the encoded protein was a novel PP2C family member, and
it was designated PP2C
. Overall, the homogeneity between PP2C
and
PP2C
was 26%.
View larger version (53K):
[in a new window]
Fig. 1.
Nucleotide and deduced amino acid sequence,
in vitro translation, and tissue-specific expression
of PP2C . A, the nucleotides
are numbered on the left, and the amino acids on the
right. The open reading frame, starting from nucleotide 409 and ending at 1318, marked by asterisks, encompasses 303 amino acid residues. Five methionine residues located at the N-terminal
region are numbered with Roman numerals (I-V). The six
motifs uniquely conserved in the PP2C family members are
boxed. The cluster of basic amino acids is
underlined. B, the PP2C
cDNA subcloned
into pT7Blue was translated in vitro using RNA
polymerase-coupled reticulocyte lysates in the presence of
[35S]methionine. The synthesized proteins were separated
in a 10% (w/v) SDS-PAGE. The gel was dried and autoradiographed.
C, total RNA fractions (15 µg) obtained from the indicated
mouse tissues were separated in a 1% (w/v) agarose gel. After staining
with ethidium bromide (lower panel), the RNAs in
the gel were transferred to a nylon membrane and hybridized with a
random primed 32P-labeled full-length PP2C
cDNA
(upper panel). The positions of 28 and 18 S rRNA
are shown on the right, and the positions of PP2C
mRNA are shown on the left.
in Escherichia coli Cells--
To determine
whether the recombinant PP2C
possessed protein phosphatase activity,
MBP-PP2C
or MBP-PP2C
fusion proteins were expressed in
E. coli and then purified with amylose resin. The purified
MBP-PP2C
fusion protein exhibited substantial Mg2+- or
Mn2+-dependent and okadaic acid-insensitive
protein phosphatase activity (data not shown). The specific activity of
MBP-PP2C
was similar to that of MBP-PP2C
when phosphorylated
-casein was used as the substrate (data not shown).
mRNA. A strong signal of 5.9-kb mRNA was observed in the brain,
and a weak signal corresponding to the same size was also found in the
heart (Fig. 1C). Interestingly, a mRNA signal of 2.2 kb
was observed only in the testis (Fig. 1C). Although the
PP2C
mRNA signal was not observed in liver, lung, or skeletal
muscle by Northern blot analysis, we were able to detect the PP2C
mRNA signal in these tissues by polymerase chain reaction,
suggesting that PP2C
mRNA is ubiquitously expressed in a variety
of tissues (data not shown).
Inhibits the IL-1-induced Activation of AP-1 Reporter Gene
in Mammalian Cells--
Three distinct PP2C family members (PP2C
,
PP2C
, and Wip1) have previously been implicated in the regulation of
the SAPK systems (14, 17-19). Therefore, we examined the possibility
that PP2C
was also involved in the regulation of SAPK signaling
pathways. In 293 cells, IL-1 stimulates the activity of the
transcription factor complex AP-1 through the activation of TAK1 and
JNK (35). We transfected 293 IL-1RI cells, which express the IL-1
receptor, with the expression plasmid of PP2C
and then assayed the
AP-1 activity using an AP-1-dependent luciferase reporter
gene (Fig. 2A). Similar to our
previously reported data with transfected PP2C
-1 (18), PP2C
inhibited the IL-1-induced activation of AP-1 in a
concentration-dependent manner (Fig. 2A).
View larger version (20K):
[in a new window]
Fig. 2.
PP2C inhibits the
IL-1-induced SAPK signaling pathways. A, 293IL-1RI
cells were transfected with an AP-1-luciferase reporter plasmid by the
lipofection method with or without the cotransfection of
pCMV-HA-PP2C
. Forty-eight hours after the transfection, the cells
were treated with or without IL-1
for 6 h. Then the luciferase
activities of the cell lysates were determined. B, 293 cells
were transfected with an AP1-luciferase reporter plasmid with or
without pCDNA3-HA-TAK1 and the indicated amounts of the
pCMV-HA-PP2C
. The luciferase activities of the cell lysates were
determined 48 h after the transfection. Differences in the
transfection efficiency were normalized by cotransfection of a
-galactosidase expression plasmid for A and B.
The data shown are the mean ± S.E. (n = 3).
C and D, pcDNA3-Myc-JNK (0.2 µg;
C) or pcDNA3-FLAG-p38 (0.2 µg; D) was
cotransfected with or without pcDNA3-HA-TAK1 (0.2 µg;
C and D) and the indicated amounts of
pCMV-HA-PP2C
to 293 cells. The total amount of DNA was adjusted to 1 µg with the empty vector. Western blot analysis was performed with
the cell lysates 48 h after the transfection. Anti-phospho-JNK
antibody (C) and anti-phospho-p38 (D) antibody
were used to determine the phosphorylation levels of JNK and p38,
respectively (top panels). Aliquots of the
lysates were immunoblotted with anti-Myc (C) or anti-FLAG
(D) antibody (middle panels). Anti-HA
antibody was used to detect the co-expressed PP2C (bottom
panels).
(Fig. 2B). The
expression of TAK1 alone in 293 IL-1RI cells activated the AP-1
reporter gene. This enhanced activity was substantially suppressed by
PP2C
, suggesting that TAK1 itself or a component(s) that lies
downstream of TAK1 is a substrate of PP2C
.
. We expressed Myc-JNK (Fig.
2C) or FLAG-p38 (Fig. 2D) with HA-TAK1 in 293 cells and tested the effect of PP2C
co-expression on the
TAK1-enhanced activation of JNK and p38. PP2C
was found to inhibit
the TAK1-enhanced activation of either JNK or p38. These results
indicated that a signaling component(s) situated between TAK1 and
JNK/p38 could be a substrate of PP2C
.
--
We next determined whether MKK3b-induced activation of
p38 is affected by PP2C
in 293 cells. The expression of PP2C
suppressed the MKK3b-enhanced phosphorylation of p38 in 293 cells (Fig.
3A), confirming our previous
observation performed using COS7 cells (17). In contrast, PP2C
expressed in the cells exhibited no effect on the phosphorylation level
of p38 (Fig. 3B), indirectly suggesting no direct effect on
MKK3 and thereby raising the possibility that TAK1, which lies upstream
of MKK3, might be the substrate of PP2C
.
View larger version (14K):
[in a new window]
Fig. 3.
PP2C does not
inhibit the MKK3b enhanced phosphorylation of p38. A
and B, 293 cells were transfected with pcDNA3-FLAG-p38
(0.25 µg) with or without the cotransfection of the
pcDNA3-His-MKK3b (0.25 µg) and the indicated amounts of
pcDNA3-HA-PP2C
(A) or pCMV-HA-PP2C
(B).
The total amount of DNA was adjusted to 1 µg with the empty vector.
Western blot analysis was performed with the cell lysates 48 h
after the transfection using anti-phospho-p38 antibody (top
panels) or anti-FLAG antibody (middle
panels). The co-expressed PP2C
or PP2C
was also
immunoblotted with anti-HA antibody (bottom
panels).
Dephosphorylates TAK1 but Not MKK4 or JNK in Vitro--
To
determine whether TAK1 is a substrate of PP2C
, we examined the
dephosphorylation of TAK1 after incubation with PP2C
in vitro (Fig. 4B). HA-TAK1
and its promoter HA-TAB1 were co-expressed in 293 cells, and HA-TAK1
was immunoprecipitated from cell extracts with anti-TAK1 antibody. When
the immunoprecipitated TAK1 complex was incubated with
[
-32P]ATP, TAK1 became autophosphorylated. The
immunoprecipitate containing the phosphorylated TAK1 was washed and
next incubated with bacterially produced MBP-PP2C
or GST-PP2C
-1.
TAK1 was dephosphorylated by PP2C
-1 (Fig. 4A), confirming
our previous observation (18). Similarly, TAK-1 was dephosphorylated by
MBP-PP2C
in a dose-dependent manner (Fig.
4B).
View larger version (36K):
[in a new window]
Fig. 4.
PP2C
dephosphorylates TAK1 but not MKK4 or JNK in
vitro. A and B, 293 cells were
cotransfected with pcDNA3-HA-TAK1 and pcDNA3-HA-TAB1. The cell
lysates were immunoprecipitated with anti-TAK1 antibody. The
immunoprecipitates were incubated with [
-32P]ATP, the
phosphorylated proteins were washed, and aliquots were incubated with
the indicated amounts of recombinant GST-PP2C
(A) or
MBP-PP2C
(B). The proteins were separated by SDS-PAGE and
analyzed by autoradiography (upper panels) or
stained with Coomassie Brilliant Blue (lower
panels). C, 293 cells were cotransfected with
pEBG2T-MKK4, pcDNA3-HA-TAK1, and pcDNA3-HA-TAB1. The cell
lysates were harvested 48 h after the transfection, and the
phosphorylated GST-MKK4 was extracted by glutathione-Sepharose beads.
The phosphorylated GST-MKK4 was eluted and incubated with the indicated
amounts of recombinant MBP-PP2C
. The phosphorylation levels of
GST-MKK4 was determined by Western blot analysis using
anti-phospho-MKK4 antibody (upper panel).
Anti-MBP antibody was used to detect the MBP-PP2C
in the reaction
mixture (lower panel). D,
pcDNA3-Myc-JNK was transfected into 293 cells. Forty-eight hours
after the transfection, the cells were cultured in the presence of IL-1
for 15 min. The Myc-JNK was immunoprecipitated from the cell lysates
with anti-Myc antibody. Aliquots were incubated with c-Jun,
[
-32P]ATP, and the indicated amounts of MBP-PP2C
for 30 min at 30 °C. The phosphorylation levels of the c-Jun were
determined by autoradiography following SDS-PAGE of the reaction
mixture (upper panel). The gel was stained with
Coomassie Brilliant Blue to detect the MBP-PP2C
in the reaction
mixture (lower panel).
could dephosphorylate MKK4 or JNK
in vitro (Fig. 4, C and D).
Phosphorylated MKK4 or JNK was incubated with increasing concentrations
of PP2C
in vitro. PP2C
did not dephosphorylate either
MKK4 (Fig. 4C) or JNK (Fig. 4D) at the
concentrations high enough to dephosphorylate TAK1. Based on these
results, we propose that PP2C
suppresses TAK1 signaling pathways by
directly dephosphorylating TAK1 itself.
, PP2C
(D/A), Exhibits a Dominant
Negative Effect--
To investigate the mechanism of PP2C
action on
TAK1 in more detail, we searched for a dominant negative form of
PP2C
in various point mutants of PP2C
, defective in protein
phosphatase activity. We prepared expression constructs for five
different point mutants of PP2C
, in which each of five different
amino acids, known to be conserved in PP2C family members and to play essential roles in the enzyme activity, was replaced by another amino
acid. We found that three of the resulting recombinant proteins exhibited essentially no activity against phosphorylated
-casein (Fig. 5A). In addition, one of
these mutants, PP2C
(D/A), in which Asp-245 had been replaced by Ala,
was able to inhibit the dephosphorylation of TAK1 by PP2C
in
vitro (Fig. 5B). PP2C
(D/A), expressed in 293 cells,
reversed the inhibition of the TAK1-activated AP-1 reporter gene by the
wild-type PP2C
(Fig. 5C). Furthermore, expression of
PP2C
(D/A) in 293 cells enhanced further the TAK1-activated AP-1
reporter gene (Fig. 5D). These results support the ideas that PP2C
(D/A) acts as a dominant negative form and that the endogenous PP2C
may in fact participate in the negative regulation of the SAPK signaling pathways.
View larger version (21K):
[in a new window]
Fig. 5.
PP2C (D/A), a point
mutant of PP2C
, acts as a dominant negative
form. A, protein phosphatase activities of the
recombinant MBP-PP2C
and its five point mutants were determined in
the presence of 10 mM MnCl2 and 1 µM okadaic acid using 32P-labeled casein as
the substrate. B, aliquots of the phosphorylated TAK1,
prepared as described in the legend to Fig. 4, A and
B, were incubated with the indicated amounts of
MBP-PP2C
(D/A) or MBP alone and/or MBP-PP2C
for 30 min at
30 °C. The proteins were separated by SDS-PAGE and analyzed by
autoradiography. C and D, AP-1-luciferase
reporter plasmid was cotransfected with or without the TAK1 expression
plasmid and the indicated amounts of the expression plasmids for
PP2C
(D/A) and PP2C
(C) or for PP2C
(D/A) alone
(D). Luciferase activities were determined and normalized on
the basis of
-galactosidase expression 48 h after the
transfection. The data shown are the mean ± S.E.
(n = 3).
Associates with TAK1--
Since TAK1 was found to be a
substrate of PP2C
, we next asked whether the phosphatase was able to
associate with TAK1. To answer this question, we co-expressed HA-TAK1
and HA-PP2C
or HA-PP2C
(D/A) in 293 cells (Fig.
6A). The cell extracts were
immunoprecipitated with anti-TAK1 antibody. Co-precipitated HA-PP2C
was detected by immunoblotting using anti-HA antibody. The results
demonstrated that both PP2C
and PP2C
(D/A) were
co-immunoprecipitated with TAK1 (Fig. 6A).
View larger version (33K):
[in a new window]
Fig. 6.
PP2C associates with
TAK1 both in vivo and in vitro.
A, 293 cells were cotransfected with pcDNA3-HA-TAK1 and
empty vector (lane 1), pCMV-HA-PP2C
(WT)
(lane 2), or pCMV-HA-PP2C
(D/A)
(lane 3). Aliquots of the lysates were
immunoprecipitated with anti-TAK1 antibody 48 h after the
transfection, and the immunoprecipitates were immunoblotted with
anti-HA antibody to determine whether the PP2C
was
co-immunoprecipitated with TAK1 (top panel). The
immunoprecipitated TAK1 was also detected by anti-HA antibody
(middle panel). Aliquots of the lysates were also
immunoblotted with anti-HA antibody to detect the expressed HA-PP2C
(bottom panel). B, pCMV-HA-PP2C
was
cotransfected with empty vector (lanes 1 and
3), pcDNA3-Myc-TAK1 (lane 2), or
pcDNA3-Myc-TAK1(S/A) (lane 4) into 293 cells.
Aliquots of the lysates were immunoprecipitated with anti-Myc antibody,
and the immunoprecipitates were immunoblotted with anti-HA antibody
(top panel) or anti-Myc antibody
(middle panel). Aliquots of the lysates were also
immunoblotted with anti-HA antibody (bottom
panel). C, 293 cells were transfected with
pcDNA3-HA-TAK1. Immunoprecipitation was performed with anti-TAK1
antibody 48 h after the transfection. The immune complexes were
isolated with protein A-Sepharose beads and incubated with the
recombinant MBP, MBP-PP2C
, or MBP-PP2C
(D/A) in the lysis buffer.
The protein A-Sepharose beads were isolated by centrifugation, and the
proteins bound to TAK1 were immunoblotted with anti-MBP antibody
(left upper panel). The HA-TAK1
recovered in the immunoprecipitates was immunoblotted with anti-HA
antibody (left lower panel). The
recombinant MBP, MBP-PP2C
, and MBP-PP2C
(D/A) in the reaction
mixture were detected by immunoblotting with anti-MBP antibody
(right panel).
might associate
preferentially with the phosphorylated TAK1, the substrate of PP2C
. The TAK1(S/A) mutant, in which Ser-192 is replaced by Ala, is defective
in both phosphorylation and activation (23). We co-expressed HA-PP2C
and Myc-TAK1 or Myc-TAK1(S/A) in 293 cells and immunoprecipitated the
Myc-TAK1 or Myc-TAK1(S/A) by anti-Myc antibody from the cell extracts.
Immunoblot analysis using anti-HA antibody revealed that, similar to
TAK1, TAK1(S/A) was co-immunoprecipitated with PP2C
, indicating that
phosphorylation at Ser-192 of TAK1 is not required for the association
of TAK1 with PP2C
(Fig. 6B).
associates with TAK1 also in vitro,
we expressed HA-TAK1 in 293 cells and immunoprecipitated it with anti-TAK1 antibody. The immune complex, attached to protein A beads,
was incubated with recombinant MBP, MBP-PP2C
, or MBP-PP2C
(D/A). The bound proteins were immunoblotted with anti-MBP antibody (Fig. 6C). Both PP2C
and PP2C
(D/A) were able to associate
with TAK1 in vitro.
Attenuates, but PP2C
(D/A) Enhances, the Association of
TAK1 with MKK4 or MKK6 in 293 Cells--
We were interested in
elucidating the mechanism of the inhibition of signal transmission
between TAK1 and MKK4 or MKK6 by PP2C
. To this end, we investigated
the effect of PP2C
or PP2C
(D/A) on the interaction between TAK1
and MKK4 or MKK6. HA-TAK1, together with Myc-MKK4 or His-MKK6, was
first co-expressed in 293 cells, and the Myc-MKK4 or His-MKK6 was
immunoprecipitated. Immunoblot analysis of the immunoprecipitates with
anti-TAK1 antibody revealed that TAK1 was co-immunoprecipitated with
both MKK4 (Fig. 7A) and MKK6
(Fig. 7B). Moreover, this association was substantially
enhanced by the co-expression of HA-PP2C
(D/A), whereas it was
attenuated by HA-PP2C
. These results raised the possibility that the
association of TAK1 with PP2C
(D/A) enhances its association with
MKK4 or MKK6 by forming a ternary complex composed of TAK1,
PP2C
(D/A), and MKK4 or MKK6, a situation not found with PP2C
(Fig. 9).
View larger version (39K):
[in a new window]
Fig. 7.
The association between TAK1 and MKK4 or MKK6
is suppressed and enhanced by PP2C and
PP2C
(D/A), respectively. A and
B, pcDNA3-Myc-MKK4 (A) or
pcDNA3-His-MKK6 (B) was cotransfected with
(lanes 2-4) or without (lane
1) pcDNA3-HA-TAK1, together with pCMV-HA-PP2C
(lane 3) or pCMV-HA-PP2C
(D/A) (lane
4). Immunoprecipitation using anti-Myc antibody
(A) or anti-His antibody (B) was performed on the
cell lysates 48 h after the transfection. The immunoprecipitates
were immunoblotted with anti-TAK1 (A and B),
anti-Myc (A), or anti-His (B) antibodies, as
indicated. Aliquots of the lysates were also immunoblotted with anti-HA
antibody to detect the co-expressed HA-TAK1 (A and
B) and HA-PP2C
(A and B), as
indicated. C and D, pcDNA3-Myc-MKK4
(C) or pcDNA3-His-MKK6 (D) was cotransfected
with (lanes 3 and 4) or without
(lanes 1 and 2) pcDNA3-HA-TAK1
together with empty vector (lanes 1 and
3) or pcDNA3-HA-PP2C
(D/A) (lanes
2 and 4). Immunoprecipitation using anti-Myc
(C) or anti-His (D) antibody was performed with
the cell lysates. The immunoprecipitates were immunoblotted with
anti-HA (C and D), anti-Myc (C), or
anti-His (D) antibodies, as indicated. Aliquots of the
lysates were also immunoblotted with anti-HA antibody
(bottom panel).
(D/A)
co-immunoprecipitates with MKK4 or MKK6 only in the presence of
co-expressed TAK1 in the cells. Myc-MKK4 or His-MKK6 was co-expressed
with PP2C
(D/A) in the presence or absence of co-expression of
HA-TAK1 in 293 cells. Myc-MKK4 and His-MKK6 was immunoprecipitated with anti-Myc and anti-His antibodies, respectively. Then the
immunoprecipitates were immunoblotted with anti-HA antibody.
PP2C
(D/A) was indeed co-immunoprecipitated with either MKK4 (Fig.
7C) or MKK6 (Fig. 7D) when TAK1 was co-expressed
in the cells, whereas no such co-immunoprecipitation was observed in
the absence of the co-expressed TAK1. These results indicated that a
ternary complex composed of TAK1, PP2C
(D/A), and MKK4 or MKK6 was
formed in these cells (Fig. 9). These results also suggest that
PP2C
(D/A) enhanced the activity of the TAK1-activated AP-1 reporter
gene by inducing the association between MKK4 and TAK1 (Fig.
5D).
from TAK1--
The
evidence that the PP2C
expressed in cells attenuates the association
between TAK1 and MKK4 or MKK6 prompted us to speculate that, in the
absence of an activating signal, PP2C
may associate with TAK1 and
contribute to keeping it in an inactive state by dephosphorylation.
Upon activation of TAK1 by an upstream signal, PP2C
may dissociate
from TAK1, thereby inducing its activation as well as its association
with MKK4 or MKK6. To test this possibility, we determined whether IL-1
treatment of the cells influenced the association of PP2C
with TAK1.
We expressed PP2C
in 293IL-1RI cells and tested the effect of IL-1
treatment on the association between the endogenous TAK1 and the
expressed PP2C
. In the absence of IL-1, PP2C
expressed in the
293IL-1RI cells was co-immunoprecipitated with the endogenous TAK1
(Fig. 8A, top
panel, lanes 3 and 4). The
co-immunoprecipitation between PP2C
and TAK1 was substantially attenuated when the cells were treated with IL-1 for 15 min (Fig. 8A, top panel, lanes
3 and 4 versus lanes
7 and 8), whereas the co-immunoprecipitation was
rather enhanced by treatment of the cells with IL-1 for 6 h (Fig.
8A, top panel, lanes
3 and 4 versus lanes
11 and 12). These results suggest that IL-1
treatment induces transient dissociation of PP2C
from TAK1, and
PP2C
reassociates with TAK1 later on in a feedback mechanism. IL-1
treatment of the cells for 15 min did not induce the activation of the
AP-1 reporter gene irrespective of the co-expression of PP2C
(data not shown). In contrast, the AP-1 reporter gene activity was
substantially enhanced 6 h after the initiation of IL-1 treatment,
and this IL-1-induced activation of the AP-1 reporter gene was
partially suppressed by co-expression of PP2C
(Fig. 2A).
The evidence that a substantial amount of the endogenous TAK1 still
associated with the exogenous PP2C
upon IL-1 treatment of the cells
for 15 min (Fig. 8A, top panel,
lanes 7 and 8) may explain why the
PP2C
-induced partial suppression of the AP-1 reporter gene activity
was observed 6 h after the onset of the IL-1 treatment (Fig.
2A).
View larger version (36K):
[in a new window]
Fig. 8.
IL-1 induces transient dissociation of
PP2C from TAK1. A, 293IL-1RI
cells were transfected with pCMV-HA-PP2C
(lanes
3, 4, 7, 8, 11,
and 12) or empty vector (lanes 1,
2, 5, 6, 9, and
10). Forty-eight hours after the transfection, the cells
were treated with IL-1 for 15 min (lanes 5,
6, 7, and 8) or 6 h (lanes
9, 10, 11, and 12), and the
cell lysates were prepared. Aliquots of the lysates were
immunoprecipitated with anti-TAK1 antibody. The immunoprecipitates were
immunoblotted with anti-HA antibody (top panel)
or anti-TAK1 antibody (middle panel). Aliquots of
the lysates were also immunoblotted with anti-HA antibody
(bottom panel). B, aliquots of the
lysates, described in A, were also immunoprecipitated with
anti-HA antibody to determine the phosphatase activity of PP2C
using
-casein as the substrate.
from TAK1, and this mobility shift was canceled
when PP2C
reassociated with TAK1 (Fig. 8A,
middle panel, lanes 11 and
12). These results support the conclusion that IL-1-induced
dissociation of PP2C
contributes to the activation of TAK1.
was
affected by IL-1 treatment. HA-PP2C
was expressed in 293IL-1RI
cells, and the expressed HA-PP2C
was immunoprecipitated with anti-HA
antibody 15 min and 6 h after the IL-1 treatment. The protein
phosphatase activities of the immunoprecipitates were determined using
-casein as the substrate. No difference in the activity was observed
between the immunioprecipitates obtained 0 and 15 min after the onset
of the IL-1 treatment (Fig. 8B). However, the activity level
of the immunoprecipitates obtained 6 h after the onset of IL-1
treatment was 26% higher than that of the 0-min control.
from TAK1. We found
that co-immunoprecipitation of exogenous TAK1 and exogenous PP2C
were not suppressed by the co-expression of TAB1 in 293 cells (data not
shown). These results suggest that the dissociation of PP2C
from
TAK1 is rather specifically induced by IL-1 treatment of the cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1 suppresses the SAPK signaling
pathways by associating with and dephosphorylating TAK1 (18). In this
study, we revealed a novel member of the PP2C family (PP2C
) and
elucidated its role in TAK1-mediated signaling pathways. PP2C
is
composed of 303 amino acids and contains the six unique motifs conserved in all members of mammalian PP2C family (Fig. 1A)
(34). The recombinant PP2C
exhibited
Mg2+/Mn2+-dependent and okadaic
acid-insensitive protein phosphatase activity. We present several lines
of evidence suggesting that PP2C
negatively regulates the TAK1
pathways by dephosphorylating TAK1 itself. First, PP2C
expressed in
293 cells inhibited the TAK-1-induced activation of JNK and p38 (Fig.
2, C and D). Second, PP2C
dephosphorylated TAK1 but had no effect on the phosphorylation of MKK4 or JNK in vitro (Fig. 4, B-D). Additionally, PP2C
expressed
in 293 cells did not inactivate either MKK3 or p38 co-expressed (Fig.
3, A and B) but associated stably with TAK1 (Fig.
6B). Finally, a dominant negative mutant of PP2C
,
PP2C
(D/A), inhibited the dephosphorylation of TAK1 by PP2C
in vitro and further enhanced the TAK1-induced activation of
the AP-1 reporter gene (Fig. 5D). Taken together, these data
are consistent with the idea that PP2C
suppresses TAK1-mediated
signaling by associating with and dephosphorylating TAK1.
and PP2C
(D/A) revealed
that they exhibit opposite effects on the association between TAK1 and
MKK4 or MKK6 (Fig. 7, A and B). Thus, the
expression of PP2C
(D/A) enhanced the association between TAK1 and
MKK4 or MKK6, whereas PP2C
expression attenuated the association.
PP2C
(D/A) was co-immunoprecipitated with MKK4 or MKK6 only when TAK1
was co-expressed (Fig. 7, C and D). These
observations indicate that the PP2C
(D/A) expressed in cells
associates with TAK1, and this association enhances the binding of MKK4
or MKK6 to TAK1, thereby forming a ternary complex (Fig.
9). Since PP2C
(D/A) does not dephosphorylate TAK1 and prevents the dephosphorylation of TAK1 by
endogenous PP2C
, TAK1 might activate MKK4 or MKK6 more profoundly than in the absence of the mutant (Fig. 9). We have demonstrated that
the expression of TAK1 alone enhances the phosphorylation of both JNK
and p38 (Fig. 2, C and D). Therefore, the
expressed TAK1 should be activated and able to activate MKK4 and MKK6.
When PP2C
is co-expressed in the cells, the PP2C
would be able to associate with TAK1 and dephosphorylate it, and the
dephosphorylated TAK1 may lose its affinity for MKK4 or MKK6 (Fig.
9).
View larger version (32K):
[in a new window]
Fig. 9.
A model of PP2C
function in the regulation of TAK1 signaling pathway. The
activation of MKK4 or MKK6 by TAK1 requires a physical interaction
between TAK1 and MKK4 or MKK6. PP2C
binding to TAK1 interferes with
this association and thereby interrupts the activation of MKK4 and MKK6
and the subsequent downstream signaling. The binding of PP2C
to TAK1
is transiently inhibited by IL-1. This dissociation may induce the
activation of TAK1 and its association with MKK4/6. In contrast,
binding of PP2C
(D/A) to TAK1 enhances the association of TAK1 with
MKK4 or MKK6 and stimulates the TAK1 signaling pathway.
The evidence that the association of PP2C with TAK1 attenuates the
binding of MKK4 or MKK6 to TAK1 raises the possibility that the
association between PP2C
and TAK1 itself may be regulated by an
upstream signal(s) in vivo. Indeed, our study indicated that
IL-1 treatment of 293IL-1RI cells for 15 min attenuated the association
between TAK1 and PP2C
(Fig. 8). However, IL-1 treatment for 6 h
rather enhanced the association. These results suggest that PP2C
contributes to maintaining the SAPK system in an inactive state in the
absence of IL-1-induced signals, and the dissociation of PP2C
from
TAK1 upon IL-1 treatment may contribute to the activation of TAK1
(Figs. 8A and 9). These results also suggest that PP2C
reassociates with TAK1 following the initial dissociation, thereby inhibiting the TAK1 signaling pathways by dephosphorylation of TAK1 in
a feedback mechanism. Phosphatase activity of the expressed PP2C
was
not inhibited by the IL-1 treatment for 15 min, suggesting that
dissociation from TAK1 does not induce the inactivation of PP2C
. In
contrast, the phosphatase activity level of the immunoprecipitates obtained 6 h after the onset of IL-1 treatment was 26% higher than that of the 0-min control. This enhancement of the PP2C
activity may also be involved in the mechanism of feedback regulation of TAK1.
A number of protein phosphatases have been known to participate in the
negative regulation of SAPK signaling pathways (4). To date, studies of
these phosphatases have suggested that they inhibit the SAPK system by
a feedback mechanism. Thus, their activation or expression is induced
by SAPK signaling pathway-dependent mechanisms, and they,
in turn, negatively regulate the SAPK systems by dephosphorylation (4,
19, 20, 36-39). Alternatively, the phosphatases may have high affinity
only for the phosphorylated form of the SAPK systems' components and
again suppress the systems by a feedback mechanism (14). In this study,
we suggested that PP2C participates both in switching on and off of
TAK1 signaling pathways by dissociating from and associating with TAK1,
respectively. Our study, in fact, may propose a novel mechanism for
regulation of SAPK signaling pathways by protein phosphatases.
We previously reported that PP2C-1 also suppresses the TAK1
signaling pathways by dephosphorylating TAK1 (18). Similar to PP2C
,
PP2C
-1 associated stably with TAK1, and a dominant negative mutant
of PP2C
-1 further enhanced the TAK1-activated AP-1 reporter gene
activity. TAK1 has been found to be activated by a variety of
extracellular stimuli such as transforming growth factor-
, IL-1,
tumor necrosis factor
, RANKL, and stress (21, 24, 31), yet similar
kinase cascades downstream of TAK1 commonly participate in the
signaling pathways. This raises the possibility that the signaling
system activated by each stimulus behaves independently, and in each
system TAK1 may be negatively regulated by a different protein
phosphatase. Therefore, it is tempting to speculate that PP2C
-1 may
regulate TAK1 activated by a stimulus distinct from IL-1.
Alternatively, both PP2C
and PP2C
-1 may act on TAK1 in the same
signaling pathway but with different detailed mechanisms. Further
studies are required to elucidate the significance of the dual
regulation of TAK1 by PP2C
-1 and PP2C
.
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Kimio Konno for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY184801.
To whom correspondence may be addressed. Tel.: 81-22-717-8471;
Fax: 81-22-717-8476; E-mail: takayasu@idac.tohoku.ac.jp.
§§ To whom correspondence may be addressed. Tel.: 81-22-717-8471; Fax: 81-22-717-8476; E-mail: tamura@idac.tohoku.ac.jp.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M211474200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SAPK, stress-activated protein kinase; MKK, mitogen-activated protein kinase kinase; PP1, PP2A, PP2B, and PP2C, protein phosphatase 1, 2A, 2B, and 2C, respectively; IL, interleukin; HA, hemagglutinin; JNK, Jun N-terminal kinase; CMV, cytomegalovirus; GST, glutathione S-transferase; MBP, maltose-binding protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Garrington, T. P., and Johnson, G. L. (1999) Curr. Opin. Cell Biol. 11, 211-218[CrossRef][Medline] [Order article via Infotrieve] |
2. | Ip, Y. T., and Davis, R. J. (1998) Curr. Opin. Cell Biol. 10, 205-219[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Widmann, C.,
Gibson, S.,
Jarpe, M. B.,
and Johnson, G. L.
(1999)
Physiol. Rev.
79,
143-180 |
4. |
Tamura, S.,
Hanada, M.,
Ohnishi, M.,
Katsura, K.,
Sasaki, M.,
and Kobayashi, T.
(2002)
Eur. J. Biochem.
269,
1060-1066 |
5. | Tamura, S., Lynch, K. R., Larner, J., Fox, J., Yasui, A., Kikuchi, K., Suzuki, Y., and Tsuiki, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1796-1800[Abstract] |
6. | Wenk, J., Trompeter, H. I., Pettrich, K. G., Cohen, P. T. W., Campbell, D. G., and Mieskes, G. (1992) FEBS Lett. 297, 135-138[CrossRef][Medline] [Order article via Infotrieve] |
7. | Travis, S. M., and Welsh, M. J. (1997) FEBS Lett. 412, 415-419[CrossRef][Medline] [Order article via Infotrieve] |
8. | Guthridge, M. A., Bellosta, P., Tavoloni, N., and Basilico, C. (1997) Mol. Cell. Biol. 17, 5485-5498[Abstract] |
9. |
Tong, Y.,
Quirion, R.,
and Shen, S-H.
(1998)
J. Biol. Chem.
273,
35282-35290 |
10. |
Fiscella, M.,
Zhang, H.,
Fan, S.,
Sakaguchi, K.,
Shen, S.,
Mercer, W. E.,
Vande Woude, G. F.,
O'Connor, P. M.,
and Appella, E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6048-6053 |
11. | Kitani, T., Ishida, A., Okuno, S., Takeuchi, M., Kameshita, I., and Fujisawa, H. (1999) J. Biochem. (Tokyo) 125, 1022-1028[Abstract] |
12. |
Leung-Hagesteijn, C.,
Mahendra, A.,
Naruszewicz, I.,
and Hannigan, G. E.
(2001)
EMBO J.
20,
2160-2170 |
13. | Mann, D. J., Campbell, D. G., McGowan, C. H., and Cohen, P. T. (1992) Biochim. Biophys. Acta 1130, 100-104[Medline] [Order article via Infotrieve] |
14. |
Takekawa, M.,
Maeda, T.,
and Saito, H.
(1998)
EMBO J.
17,
4744-4752 |
15. | Terasawa, T., Kobayashi, T., Murakami, T., Ohnishi, M., Kato, S., Tanaka, O., Kondo, H., Yamamoto, H., Takeuchi, T., and Tamura, S. (1993) Arch. Biochem. Biophys. 307, 342-349[CrossRef][Medline] [Order article via Infotrieve] |
16. | Kato, S., Terasawa, T., Kobayashi, T., Ohnishi, M., Sasahara, Y., Kusuda, K., Yanagawa, Y., Hiraga, A., Matsui, Y., and Tamura, S. (1995) Arch. Biochem. Biophys. 318, 387-393[CrossRef][Medline] [Order article via Infotrieve] |
17. | Hanada, M., Kobayashi, T., Ohnishi, M., Ikeda, S., Wang, H., Katsura, K., Yanagawa, Y., Hiraga, A., Kanamaru, R., and Tamura, S. (1998) FEBS Lett. 437, 172-176[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Hanada, M.,
Ninomiya-Tsuji, J.,
Komaki, K.,
Ohnishi, M.,
Katsura, K.,
Kanamaru, R.,
Matsumoto, K.,
and Tamura, S.
(2001)
J. Biol. Chem.
276,
5753-5759 |
19. | Takekawa, M., Adachi, M., Nakahata, A., Nakayama, I., Itoh, F., Tsukuda, H., Taya, Y., and Imai, K. (2000) EMB0 J. 19, 6517-6526[CrossRef] |
20. | Merlot, S., Gosti, F., Guerrier, D., Vavasseur, A., and Giraudat, J. (2001) Plant J. 25, 295-303[CrossRef][Medline] [Order article via Infotrieve] |
21. | Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 2008-2011[Abstract] |
22. | Shibuya, H., Yamaguchi, K., Shirakabe, K., Tonegawa, A., Gotoh, Y., Ueno, N., Irie, K., Nishida, E., and Matsumoto, K. (1996) Science 272, 1179-1182[Abstract] |
23. |
Kishimoto, K.,
Matsumoto, K.,
and Ninomiya-Tsuji, J.
(2000)
J. Biol. Chem.
275,
7359-7364 |
24. |
Shirakabe, K.,
Yamaguchi, K.,
Shibuya, H.,
Irie, K.,
Matsuda, S.,
Moriguchi, T.,
Gotoh, Y.,
Matsumoto, K.,
and Nishida, E.
(1997)
J. Biol. Chem.
272,
8141-8144 |
25. | Takaesu, G., Kishida, S., Hiyama, A., Yamaguchi, K., Shibuya, H., Irie, K., Ninomiya-Tsuji, J., and Matsumoto, K. (2000) Mol. Cell 5, 649-658[Medline] [Order article via Infotrieve] |
26. | Kusuda, K., Kobayashi, T., Ikeda, S., Ohnishi, M., Chida, N., Yanagawa, Y., Shineha, R., Nishihira, T., Satomi, S., Hiraga, A., and Tamura, S. (1998) Biochem. J. 332, 243-250[Medline] [Order article via Infotrieve] |
27. | Cao, Z., Henzel, W. J., and Gao, X. (1996) Science 271, 1128-1131[Abstract] |
28. | Tamura, S., Kikuchi, K., Hiraga, A., Hosokawa, M., and Tsuiki, S. (1978) Biochim. Biophys. Acta 524, 349-356[Medline] [Order article via Infotrieve] |
29. | McGowan, C. H., and Cohen, P. (1988) Methods Enzymol. 159, 416-425[Medline] [Order article via Infotrieve] |
30. | Reimann, E. M., and Beham, R. A. (1983) Methods Enzymol. 99, 51-55[Medline] [Order article via Infotrieve] |
31. |
Mizukami, J.,
Takaesu, G.,
Akatsuka, H.,
Sakurai, H.,
Ninomiya-Tsuji, J.,
Matsumoto, K.,
and Sakurai, N.
(2002)
Mol. Cell. Biol.
22,
992-1000 |
32. |
Beltman, J.,
Erickson, J. R.,
Martin, G. A.,
Lyons, J. F.,
and Cook, S. J.
(1999)
J. Biol. Chem.
274,
3772-3780 |
33. | Kozak, M. (1986) Cell. 44, 283-292[Medline] [Order article via Infotrieve] |
34. | Pilgrim, D., McGregor, A., Jackle, P., Johnson, T., and Hansen, D. (1995) Mol. Biol. Cell 6, 1159-1171[Abstract] |
35. | Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J-I., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252-256[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Gaits, F.,
Shiozaki, K.,
and Russell, P.
(1997)
J. Biol. Chem.
272,
17873-17879 |
37. |
Franklin, C. C.,
and Kraft, A. S.
(1997)
J. Biol. Chem.
272,
16917-16923 |
38. |
Camps, M.,
Nichols, A.,
Gillieron, C.,
Antonsson, B.,
Muda, M.,
Chabert, C.,
Boschert, U.,
and Arkinstall, S.
(1998)
Science
280,
1262-1265 |
39. | Tanoue, T., Moriguchi, T., and Nishida, E. (1999) J. Biol. Chem. 274, 19490-19956 |