Regulation of the Interleukin-1-induced Signaling Pathways by a Novel Member of the Protein Phosphatase 2C Family (PP2Cepsilon )*

Ming Guang LiDagger , Koji Katsura§, Hisayuki Nomiyama, Ken-ichiro KomakiDagger , Jun Ninomiya-Tsuji||, Kunihiro Matsumoto**, Takayasu KobayashiDagger DaggerDagger, and Shinri TamuraDagger §§

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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, PP2Cepsilon , in the regulation of the IL-1-TAK1 signaling pathway. PP2Cepsilon was composed of 303 amino acids, and the overall similarity of amino acid sequence between PP2Cepsilon and PP2Calpha was found to be 26%. Ectopic expression of PP2Cepsilon 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. PP2Cepsilon dephosphorylated TAK1 in vitro. Co-immunoprecipitation experiments indicated that PP2Cepsilon associates stably with TAK1 and attenuates the binding of TAK1 to MKK4 or MKK6. Ectopic expression of a phosphatase-negative mutant of PP2Cepsilon , PP2Cepsilon (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 PP2Cepsilon 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, PP2Cepsilon contributes to keeping the TAK1 signaling pathway in an inactive state by associating with and dephosphorylating TAK1.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 (p38alpha , p38beta , p38gamma , and p38delta ) (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.

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 (2Calpha , 2Cbeta , 2Cgamma , 2Cdelta , Wip1, and Ca2+/calmodulin-dependent protein kinase phosphatase) have been found in mammalian cells (5-12). In addition, two distinct isoforms of the human PP2Calpha (alpha -1 and -2) and five isoforms of the mouse PP2Cbeta (beta -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 (PP2Calpha , PP2Cbeta , 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 PP2Calpha or PP2Cbeta -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 PP2Calpha and PP2Cbeta -1 is selective for SAPK pathways. We have provided further evidence indicating that PP2Cbeta -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 PP2Calpha -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).

TAK1 was originally identified as a MKK kinase that functions in the transforming growth factor-beta signaling pathway (21). TAK1 can activate both the MKK4-JNK and MKK6-p38 pathways (18). Studies of the mechanism of transforming growth factor-beta -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 alpha ), 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.

In this study, we present evidence that a novel member of the PP2C family (PP2Cepsilon ) participates in the negative regulation of the TAK1 signaling pathway and suggest that PP2Cepsilon is involved in the IL-1-induced regulation of TAK1.

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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, [gamma -32P]ATP, and RedivueTML [35S]methionine were purchased from Amersham Biosciences. CDP-Star substrate was obtained from Applied Biosystems (Bedford, MA). The luciferase assay, beta -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-1beta 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).

Cloning of PP2Cepsilon 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).

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 PP2Cepsilon cDNA.

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-- alpha -Casein was phosphorylated by protein kinase A and [gamma -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 [gamma -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.

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-PP2Cepsilon or MBP-PP2Cbeta -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-PP2Cepsilon , together with recombinant GST-c-Jun and [gamma -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-PP2Cepsilon 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.

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 PP2Cepsilon cDNA was used for the hybridization.

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 beta -galactosidase reporter plasmid, under the control of a beta -actin promoter, was cotransfected to normalize the transfection efficiency.

    RESULTS
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INTRODUCTION
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DISCUSSION
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Primary Structure of PP2Cepsilon -- 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 PP2Cepsilon . Overall, the homogeneity between PP2Calpha and PP2Cepsilon was 26%.


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Fig. 1.   Nucleotide and deduced amino acid sequence, in vitro translation, and tissue-specific expression of PP2Cepsilon . 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 PP2Cepsilon 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 PP2Cepsilon cDNA (upper panel). The positions of 28 and 18 S rRNA are shown on the right, and the positions of PP2Cepsilon mRNA are shown on the left.

Expression of PP2Cepsilon in Escherichia coli Cells-- To determine whether the recombinant PP2Cepsilon possessed protein phosphatase activity, MBP-PP2Calpha or MBP-PP2Cepsilon fusion proteins were expressed in E. coli and then purified with amylose resin. The purified MBP-PP2Cepsilon fusion protein exhibited substantial Mg2+- or Mn2+-dependent and okadaic acid-insensitive protein phosphatase activity (data not shown). The specific activity of MBP-PP2Cepsilon was similar to that of MBP-PP2Calpha when phosphorylated alpha -casein was used as the substrate (data not shown).

Northern Blot Analysis-- Northern hybridization was performed on mouse tissues to clarify the tissue distribution of PP2Cepsilon 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 PP2Cepsilon mRNA signal was not observed in liver, lung, or skeletal muscle by Northern blot analysis, we were able to detect the PP2Cepsilon mRNA signal in these tissues by polymerase chain reaction, suggesting that PP2Cepsilon mRNA is ubiquitously expressed in a variety of tissues (data not shown).

PP2Cepsilon Inhibits the IL-1-induced Activation of AP-1 Reporter Gene in Mammalian Cells-- Three distinct PP2C family members (PP2Calpha , PP2Cbeta , and Wip1) have previously been implicated in the regulation of the SAPK systems (14, 17-19). Therefore, we examined the possibility that PP2Cepsilon 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 PP2Cepsilon 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 PP2Cbeta -1 (18), PP2Cepsilon inhibited the IL-1-induced activation of AP-1 in a concentration-dependent manner (Fig. 2A).


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Fig. 2.   PP2Cepsilon 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-PP2Cepsilon . Forty-eight hours after the transfection, the cells were treated with or without IL-1beta 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-PP2Cepsilon . 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 beta -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-PP2Cepsilon 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).

We next asked whether the TAK1-induced activation of AP-1 is also affected by the co-expression of PP2Cepsilon (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 PP2Cepsilon , suggesting that TAK1 itself or a component(s) that lies downstream of TAK1 is a substrate of PP2Cepsilon .

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 PP2Cepsilon . We expressed Myc-JNK (Fig. 2C) or FLAG-p38 (Fig. 2D) with HA-TAK1 in 293 cells and tested the effect of PP2Cepsilon co-expression on the TAK1-enhanced activation of JNK and p38. PP2Cepsilon 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 PP2Cepsilon .

MKK3b-enhanced Phosphorylation of p38 Was Not Suppressed by PP2Cepsilon -- We next determined whether MKK3b-induced activation of p38 is affected by PP2Cepsilon in 293 cells. The expression of PP2Calpha suppressed the MKK3b-enhanced phosphorylation of p38 in 293 cells (Fig. 3A), confirming our previous observation performed using COS7 cells (17). In contrast, PP2Cepsilon 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 PP2Cepsilon .


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Fig. 3.   PP2Cepsilon 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-PP2Calpha (A) or pCMV-HA-PP2Cepsilon (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 PP2Calpha or PP2Cepsilon was also immunoblotted with anti-HA antibody (bottom panels).

PP2Cepsilon Dephosphorylates TAK1 but Not MKK4 or JNK in Vitro-- To determine whether TAK1 is a substrate of PP2Cepsilon , we examined the dephosphorylation of TAK1 after incubation with PP2Cepsilon 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 [gamma -32P]ATP, TAK1 became autophosphorylated. The immunoprecipitate containing the phosphorylated TAK1 was washed and next incubated with bacterially produced MBP-PP2Cepsilon or GST-PP2Cbeta -1. TAK1 was dephosphorylated by PP2Cbeta -1 (Fig. 4A), confirming our previous observation (18). Similarly, TAK-1 was dephosphorylated by MBP-PP2Cepsilon in a dose-dependent manner (Fig. 4B).


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Fig. 4.   PP2Cepsilon 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 [gamma -32P]ATP, the phosphorylated proteins were washed, and aliquots were incubated with the indicated amounts of recombinant GST-PP2Cbeta (A) or MBP-PP2Cepsilon (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-PP2Cepsilon . 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-PP2Cepsilon 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, [gamma -32P]ATP, and the indicated amounts of MBP-PP2Cepsilon 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-PP2Cepsilon in the reaction mixture (lower panel).

We next tested whether PP2Cepsilon could dephosphorylate MKK4 or JNK in vitro (Fig. 4, C and D). Phosphorylated MKK4 or JNK was incubated with increasing concentrations of PP2Cepsilon in vitro. PP2Cepsilon 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 PP2Cepsilon suppresses TAK1 signaling pathways by directly dephosphorylating TAK1 itself.

A Point Mutant of PP2Cepsilon , PP2Cepsilon (D/A), Exhibits a Dominant Negative Effect-- To investigate the mechanism of PP2Cepsilon action on TAK1 in more detail, we searched for a dominant negative form of PP2Cepsilon in various point mutants of PP2Cepsilon , defective in protein phosphatase activity. We prepared expression constructs for five different point mutants of PP2Cepsilon , 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 alpha -casein (Fig. 5A). In addition, one of these mutants, PP2Cepsilon (D/A), in which Asp-245 had been replaced by Ala, was able to inhibit the dephosphorylation of TAK1 by PP2Cepsilon in vitro (Fig. 5B). PP2Cepsilon (D/A), expressed in 293 cells, reversed the inhibition of the TAK1-activated AP-1 reporter gene by the wild-type PP2Cepsilon (Fig. 5C). Furthermore, expression of PP2Cepsilon (D/A) in 293 cells enhanced further the TAK1-activated AP-1 reporter gene (Fig. 5D). These results support the ideas that PP2Cepsilon (D/A) acts as a dominant negative form and that the endogenous PP2Cepsilon may in fact participate in the negative regulation of the SAPK signaling pathways.


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Fig. 5.   PP2Cepsilon (D/A), a point mutant of PP2Cepsilon , acts as a dominant negative form. A, protein phosphatase activities of the recombinant MBP-PP2Cepsilon 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-PP2Cepsilon (D/A) or MBP alone and/or MBP-PP2Cepsilon 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 PP2Cepsilon (D/A) and PP2Cepsilon (C) or for PP2Cepsilon (D/A) alone (D). Luciferase activities were determined and normalized on the basis of beta -galactosidase expression 48 h after the transfection. The data shown are the mean ± S.E. (n = 3).

PP2Cepsilon Associates with TAK1-- Since TAK1 was found to be a substrate of PP2Cepsilon , we next asked whether the phosphatase was able to associate with TAK1. To answer this question, we co-expressed HA-TAK1 and HA-PP2Cepsilon or HA-PP2Cepsilon (D/A) in 293 cells (Fig. 6A). The cell extracts were immunoprecipitated with anti-TAK1 antibody. Co-precipitated HA-PP2Cepsilon was detected by immunoblotting using anti-HA antibody. The results demonstrated that both PP2Cepsilon and PP2Cepsilon (D/A) were co-immunoprecipitated with TAK1 (Fig. 6A).


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Fig. 6.   PP2Cepsilon 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-PP2Cepsilon (WT) (lane 2), or pCMV-HA-PP2Cepsilon (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 PP2Cepsilon 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-PP2Cepsilon (bottom panel). B, pCMV-HA-PP2Cepsilon 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-PP2Cepsilon , or MBP-PP2Cepsilon (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-PP2Cepsilon , and MBP-PP2Cepsilon (D/A) in the reaction mixture were detected by immunoblotting with anti-MBP antibody (right panel).

We considered the possibility that PP2Cepsilon might associate preferentially with the phosphorylated TAK1, the substrate of PP2Cepsilon . 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-PP2Cepsilon 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 PP2Cepsilon , indicating that phosphorylation at Ser-192 of TAK1 is not required for the association of TAK1 with PP2Cepsilon (Fig. 6B).

To test whether PP2Cepsilon 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-PP2Cepsilon , or MBP-PP2Cepsilon (D/A). The bound proteins were immunoblotted with anti-MBP antibody (Fig. 6C). Both PP2Cepsilon and PP2Cepsilon (D/A) were able to associate with TAK1 in vitro.

PP2Cepsilon Attenuates, but PP2Cepsilon (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 PP2Cepsilon . To this end, we investigated the effect of PP2Cepsilon or PP2Cepsilon (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-PP2Cepsilon (D/A), whereas it was attenuated by HA-PP2Cepsilon . These results raised the possibility that the association of TAK1 with PP2Cepsilon (D/A) enhances its association with MKK4 or MKK6 by forming a ternary complex composed of TAK1, PP2Cepsilon (D/A), and MKK4 or MKK6, a situation not found with PP2Cepsilon (Fig. 9).


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Fig. 7.   The association between TAK1 and MKK4 or MKK6 is suppressed and enhanced by PP2Cepsilon and PP2Cepsilon (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-PP2Cepsilon (lane 3) or pCMV-HA-PP2Cepsilon (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-PP2Cepsilon (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-PP2Cepsilon (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).

To test this possibility, we examined whether PP2Cepsilon (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 PP2Cepsilon (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. PP2Cepsilon (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, PP2Cepsilon (D/A), and MKK4 or MKK6 was formed in these cells (Fig. 9). These results also suggest that PP2Cepsilon (D/A) enhanced the activity of the TAK1-activated AP-1 reporter gene by inducing the association between MKK4 and TAK1 (Fig. 5D).

IL-1 Induces Transient Dissociation of PP2Cepsilon from TAK1-- The evidence that the PP2Cepsilon expressed in cells attenuates the association between TAK1 and MKK4 or MKK6 prompted us to speculate that, in the absence of an activating signal, PP2Cepsilon may associate with TAK1 and contribute to keeping it in an inactive state by dephosphorylation. Upon activation of TAK1 by an upstream signal, PP2Cepsilon 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 PP2Cepsilon with TAK1. We expressed PP2Cepsilon in 293IL-1RI cells and tested the effect of IL-1 treatment on the association between the endogenous TAK1 and the expressed PP2Cepsilon . In the absence of IL-1, PP2Cepsilon 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 PP2Cepsilon 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 PP2Cepsilon from TAK1, and PP2Cepsilon 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 PP2Cepsilon (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 PP2Cepsilon (Fig. 2A). The evidence that a substantial amount of the endogenous TAK1 still associated with the exogenous PP2Cepsilon upon IL-1 treatment of the cells for 15 min (Fig. 8A, top panel, lanes 7 and 8) may explain why the PP2Cepsilon -induced partial suppression of the AP-1 reporter gene activity was observed 6 h after the onset of the IL-1 treatment (Fig. 2A).


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Fig. 8.   IL-1 induces transient dissociation of PP2Cepsilon from TAK1. A, 293IL-1RI cells were transfected with pCMV-HA-PP2Cepsilon (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 PP2Cepsilon using alpha -casein as the substrate.

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 PP2Cepsilon from TAK1, and this mobility shift was canceled when PP2Cepsilon reassociated with TAK1 (Fig. 8A, middle panel, lanes 11 and 12). These results support the conclusion that IL-1-induced dissociation of PP2Cepsilon contributes to the activation of TAK1.

We were interested in determining whether the activity of PP2Cepsilon was affected by IL-1 treatment. HA-PP2Cepsilon was expressed in 293IL-1RI cells, and the expressed HA-PP2Cepsilon 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 alpha -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.

We finally asked whether, similar to IL-1 treatment, activation of TAK1 by TAB1 also induces the dissociation of PP2Cepsilon from TAK1. We found that co-immunoprecipitation of exogenous TAK1 and exogenous PP2Cepsilon were not suppressed by the co-expression of TAB1 in 293 cells (data not shown). These results suggest that the dissociation of PP2Cepsilon from TAK1 is rather specifically induced by IL-1 treatment of the cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PP2Cbeta -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 (PP2Cepsilon ) and elucidated its role in TAK1-mediated signaling pathways. PP2Cepsilon 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 PP2Cepsilon exhibited Mg2+/Mn2+-dependent and okadaic acid-insensitive protein phosphatase activity. We present several lines of evidence suggesting that PP2Cepsilon negatively regulates the TAK1 pathways by dephosphorylating TAK1 itself. First, PP2Cepsilon expressed in 293 cells inhibited the TAK-1-induced activation of JNK and p38 (Fig. 2, C and D). Second, PP2Cepsilon dephosphorylated TAK1 but had no effect on the phosphorylation of MKK4 or JNK in vitro (Fig. 4, B-D). Additionally, PP2Cepsilon 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 PP2Cepsilon , PP2Cepsilon (D/A), inhibited the dephosphorylation of TAK1 by PP2Cepsilon 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 PP2Cepsilon suppresses TAK1-mediated signaling by associating with and dephosphorylating TAK1.

Studies of the modes of action of PP2Cepsilon and PP2Cepsilon (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 PP2Cepsilon (D/A) enhanced the association between TAK1 and MKK4 or MKK6, whereas PP2Cepsilon expression attenuated the association. PP2Cepsilon (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 PP2Cepsilon (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 PP2Cepsilon (D/A) does not dephosphorylate TAK1 and prevents the dephosphorylation of TAK1 by endogenous PP2Cepsilon , 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 PP2Cepsilon is co-expressed in the cells, the PP2Cepsilon would be able to associate with TAK1 and dephosphorylate it, and the dephosphorylated TAK1 may lose its affinity for MKK4 or MKK6 (Fig. 9).


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Fig. 9.   A model of PP2Cepsilon 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. PP2Cepsilon binding to TAK1 interferes with this association and thereby interrupts the activation of MKK4 and MKK6 and the subsequent downstream signaling. The binding of PP2Cepsilon 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 PP2Cepsilon (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 PP2Cepsilon with TAK1 attenuates the binding of MKK4 or MKK6 to TAK1 raises the possibility that the association between PP2Cepsilon 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 PP2Cepsilon (Fig. 8). However, IL-1 treatment for 6 h rather enhanced the association. These results suggest that PP2Cepsilon contributes to maintaining the SAPK system in an inactive state in the absence of IL-1-induced signals, and the dissociation of PP2Cepsilon from TAK1 upon IL-1 treatment may contribute to the activation of TAK1 (Figs. 8A and 9). These results also suggest that PP2Cepsilon 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 PP2Cepsilon was not inhibited by the IL-1 treatment for 15 min, suggesting that dissociation from TAK1 does not induce the inactivation of PP2Cepsilon . 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 PP2Cepsilon 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 PP2Cepsilon 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 PP2Cbeta -1 also suppresses the TAK1 signaling pathways by dephosphorylating TAK1 (18). Similar to PP2Cepsilon , PP2Cbeta -1 associated stably with TAK1, and a dominant negative mutant of PP2Cbeta -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-beta , IL-1, tumor necrosis factor alpha , 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 PP2Cbeta -1 may regulate TAK1 activated by a stimulus distinct from IL-1. Alternatively, both PP2Cepsilon and PP2Cbeta -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 PP2Cbeta -1 and PP2Cepsilon .

    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.

Dagger Dagger 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.

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RESULTS
DISCUSSION
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