Regulation of the TAK1 Signaling Pathway by Protein Phosphatase 2C

Protein phosphatase 2C (PP2C) is implicated in the negative regulation of stress-activated protein kinase cascades in yeast and mammalian cells. In this study, we determined the role of PP2Cβ-1, a major isoform of mammalian PP2C, in the TAK1 signaling pathway, a stress-activated protein kinase cascade that is activated by interleukin-1, transforming growth factor-β, or stress. Ectopic expression of PP2Cβ-1 inhibited the TAK1-mediated mitogen-activated protein kinase kinase 4-c-Jun amino-terminal kinase and mitogen-activated protein kinase kinase 6-p38 signaling pathways. In vitro, PP2Cβ-1 dephosphorylated and inactivated TAK1. Coimmunoprecipitation experiments indicated that PP2Cβ-1 associates with the central region of TAK1. A phosphatase-negative mutant of PP2Cβ-1, PP2Cβ-1 (R/G), acted as a dominant negative mutant, inhibiting dephosphorylation of TAK1 by wild-type PP2Cβ-1 in vitro. In addition, ectopic expression of PP2Cβ-1(R/G) enhanced interleukin-1-induced activation of an AP-1 reporter gene. Collectively, these results indicate that PP2Cβ negatively regulates the TAK1 signaling pathway by direct dephosphorylation of TAK1. Protein phosphatase 2C (PP2C) is implicated in the negative regulation of stress-activated protein kinase cascades in yeast and mammalian cells. In this study, we determined the role of PP2Cβ-1, a major isoform of mammalian PP2C, in the TAK1 signaling pathway, a stress-activated protein kinase cascade that is activated by interleukin-1, transforming growth factor-β, or stress. Ectopic expression of PP2Cβ-1 inhibited the TAK1-mediated mitogen-activated protein kinase kinase 4-c-Jun amino-terminal kinase and mitogen-activated protein kinase kinase 6-p38 signaling pathways. In vitro, PP2Cβ-1 dephosphorylated and inactivated TAK1. Coimmunoprecipitation experiments indicated that PP2Cβ-1 associates with the central region of TAK1. A phosphatase-negative mutant of PP2Cβ-1, PP2Cβ-1 (R/G), acted as a dominant negative mutant, inhibiting dephosphorylation of TAK1 by wild-type PP2Cβ-1 in vitro. In addition, ectopic expression of PP2Cβ-1(R/G) enhanced interleukin-1-induced activation of an AP-1 reporter gene. Collectively, these results indicate that PP2Cβ negatively regulates the TAK1 signaling pathway by direct dephosphorylation of TAK1. stress-activated protein kinase mitogen-activated protein kinase c-Jun amino-terminal kinase MAPK kinase MKK kinase protein serine/threonine phosphatase antibody hemagglutinin interleukin glutathioneS-transferase SDS-polyacrylamide gel electrophoresis mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase Stress-activated protein kinases (SAPKs)1 are a subfamily of the mitogen-activated protein kinase (MAPK) superfamily and are highly conserved from yeast to mammalian cells. SAPKs relay signals in response to various extracellular stimuli, including environmental stress and inflammatory cytokines. In mammalian cells, two distinct classes of SAPKs have been identified: the c-Jun amino-terminal kinases (JNKs) (JNK1, JNK2, and JNK3) and the p38 MAPKs (p38α, p38β, p38γ, and p38δ) (1Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1140) Google Scholar, 2Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1392) Google Scholar). Activation of SAPKs requires phosphorylation at conserved tyrosine and threonine residues in the catalytic domain. This phosphorylation is mediated by dual specificity protein kinases, which are the members of the MAPK kinase (MKK) family. Of these, MKK3 and MKK6 phosphorylate p38, MKK7 phosphorylates JNK, and MKK4 can phosphorylate either. These MKKs, in turn, are activated by phosphorylation of conserved serine and threonine residues (1Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1140) Google Scholar, 2Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1392) Google Scholar). Recently, several MKK-activating MKK kinases (MKKKs) have been identified. Some of these MKKKs are also known to be activated by phosphorylation, but the details are unclear at present. In the absence of signaling, SAPK cascades return to their inactive, dephosphorylated state, suggesting a possible role for phosphatases in SAPK regulation. In yeast cells, molecular genetic analysis has indicated that two distinct protein phosphatase groups, protein tyrosine phosphatase and protein serine/threonine phosphatase type 2C (PP2C), act as negative regulators of SAPK pathways (3Wurgler-Murphy S.M. Saito H. Trends. Biochem. Sci. 1997; 22: 172-176Abstract Full Text PDF PubMed Scopus (246) Google Scholar). InSchizosaccharomyces pombe, tyrosine phosphatase Pyp2 and the yeast homolog of PP2C (Ptc1 and Ptc3) have been shown to dephosphorylate and inactivate Spc1, the yeast homolog of SAPK (4Shiozaki K. Russell P. EMBO J. 1995; 14: 492-502Crossref PubMed Scopus (156) Google Scholar,5Nguyen A.N. Shiozaki K. Genes Dev. 1999; 13: 1653-1663Crossref PubMed Scopus (108) Google Scholar). PP2C is one of four major protein serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) in eukaryotes and is implicated in the regulation of various cellular functions. To date, at least six distinct PP2C gene products (2Cα, 2Cβ, 2Cγ, 2Cδ, Wip1, and Ca2+/calmodulin-dependent protein kinase phosphatase) have been found in mammalian cells (6Tamura S. Lynch K.R. Larner J. Fox J. Yasui A. Kikuchi K. Suzuki Y. Tsuiki S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1796-1800Crossref PubMed Scopus (109) Google Scholar, 7Wenk J. Trompeter H.I. Pettrich K.G. Cohen P.T.W. Campbell D.G. Mieskes G. FEBS Lett. 1992; 297: 135-138Crossref PubMed Scopus (72) Google Scholar, 8Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (48) Google Scholar, 9Guthridge M.A. Bellosta P. Tavoloni N. Basilico C. Mol. Cell. Biol. 1997; 17: 5485-5498Crossref PubMed Scopus (53) Google Scholar, 10Tong Y. Quirion R. Shen S.-H. J. Biol. Chem. 1998; 273: 35282-35290Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 11Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Appella E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6048-6053Crossref PubMed Scopus (470) Google Scholar, 12Kitani T. Ishida A. Okuno S. Takeuchi M. Kameshita I. Fujisawa H. J. Biochem. (Tokyo). 1999; 125: 1022-1028Crossref PubMed Scopus (51) Google Scholar). In addition, two distinct isoforms of the human PP2Cα (α-1 and -2) and five isoforms of the mouse PP2Cβ (β-1, -2, -3, -4, and -5) have been identified (13Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (62) Google Scholar, 14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar, 15Terasawa T. Kobayashi T. Murakami T. Ohnishi M. Kato S. Tanaka O. Kondo H. Yamamoto H. Takeuchi T. Tamura S. Arch. Biochem. Biophys. 1993; 307: 342-349Crossref PubMed Scopus (42) Google Scholar, 16Kato S. Terasawa T. Kobayashi T. Ohnishi M. Sasahara Y. Kusuda K. Yanagawa Y. Hiraga A. Matsui Y. Tamura S. Arch. Biochem. Biophys. 1995; 318: 387-393Crossref PubMed Scopus (30) Google Scholar). These isoforms are generated in each species as splicing variants of a single pre-mRNA. We have recently reported that ectopic expression of mouse PP2Cα or PP2Cβ-1 inhibited the stress-activated MKK3/6-p38 and MKK4/7-JNK pathways but not the mitogen-activated MKK1-ERK1 pathway. Thus, negative regulation by PP2Cα and PP2Cβ-1 is selective for different SAPK pathways (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). Essentially the same results were obtained in studies of human PP2Cα-1 and -2 in mammalian cells (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). Currently, the in vivo target molecule(s) of PP2C is unknown, although MKK4, MKK6, and p38 have been proposed as substrates of PP2Cα-2 (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). TAK1 was originally identified as an MKKK that functions in the transforming growth factor-β signaling pathway (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar). TAK1 can activate both the MKK4-JNK and MKK6-p38 pathways (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar). Recent studies have indicated that TAK1 is also activated by various stimuli, including environmental stress and inflammatory cytokines, and that it plays critical roles in various cellular responses (19Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 20Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (529) Google Scholar, 21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar, 22Ishitani T. Ninomiya-Tsuji J. Nagai S.-I. Nishita M. Meneghini M. Barker N. Waterman M. Bowerman B. Clevers H. Shibuya H. Matsumoto K. Nature. 1999; 399: 798-802Crossref PubMed Scopus (521) Google Scholar). Studies on the regulation of TAK1 activity have revealed that a TAK1-binding protein, TAB1, functions as an activator promoting TAK1 autophosphorylation (21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar, 23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, the protein phosphatase(s) responsible for inactivation of TAK1 has not been identified. In this study, we provide evidence indicating that PP2Cβ-1 selectively associates with TAK1 and inhibits the TAK1 signaling pathway by direct dephosphorylation. The restriction enzymes and other modifying enzymes used for DNA manipulation were obtained from Takara (Kyoto, Japan). Anti-6xHis, anti-Myc, and anti-TAK1 antibodies (Abs) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-MKK4 and anti-phospho-MKK3/6 Abs were supplied by New England Biolabs (Beverly, MA). Anti-hemagglutinin (HA; 12CA5) and anti-Flag (M2) Abs were purchased from Roche Molecular Biochemicals and Kodak Scientific Imaging Systems, respectively. Anti-PP2Cβ Ab was raised in rabbit against an oligopeptide of mouse PP2Cβ (RILSAENIPNLPPGGGLAGK). Human interleukin-1β (IL-1β) was from Roche Molecular Biochemicals. All the other reagents used were from Wako Pure Chemical (Osaka, Japan). Expression plasmids were constructed by standard procedures. Plasmids that express PP2C, TAK1, TAB1, MAPKs, MKKs, and MKKKs in mammalian cells were constructed using cDNAs encoding these proteins (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar, 21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar) under the control of the CMV promoter. Epitope tags were added to the constructs using synthesized oligonucleotides. Mutated cDNAs were generated by polymerase chain reaction. For bacterial expression of proteins, cDNAs encoding the proteins were subcloned into pGEX (Amersham Pharmacia Biotech) to generate glutathioneS-transferase (GST) fusion proteins or into pQE31 (Qiagen, Hilden, Germany) to generate hexahistidine-tagged protein and affinity-purified by standard procedures. Other expression plasmids were as described elsewhere (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 24Kusuda K. Kobayashi T. Ikeda S. Ohnishi M. Chida N. Yanagawa Y. Shineha R. T. S. Hiraga A. Tamura S. Biochem. J. 1998; PubMed Scopus (42) Google Scholar) and Z. Science. 1996; PubMed Scopus Google Scholar) cells were in with the cells were by the or using The of DNA was by with The cells were for and kinase were as The cells were in a and and the were with Abs for at The were with protein (Amersham Pharmacia with with and with or substrates in of kinase and of at for The were by and for Protein phosphatase were as cells were with and expression The was from with anti-Flag and phosphorylation was in kinase at for with the was with or in kinase at for the indicated proteins were by SDS-polyacrylamide gel electrophoresis and the into the proteins were with a Japan). in the and were by and The were with Abs at for and with Ab at for The of each was with an enhanced (Amersham Pharmacia were with a and The were with the indicated Abs for at The proteins were with and to were with the reporter J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the cells were with for activity was determined with the reporter was for We have reported that two mouse PP2C PP2Cα and PP2Cβ-1, selectively stress-activated MKK4, MKK6, and (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). However, the target molecule(s) of PP2C has not been identified. both the MKK4-JNK and MKK6-p38 signaling pathways are activated by TAK1 (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google we expression of PP2Cβ-1 phosphorylation of MKK4 and MKK6 at their serine or threonine of TAK1 and enhanced phosphorylation of MKK4 or MKK6 in cells A and However, expression of PP2Cβ-1 inhibited phosphorylation of MKK4 and We PP2Cβ-1 expression activation of and the and p38 kinases in cells were activated by the TAK1. However, these kinase were inhibited PP2Cβ-1 was and In expression of a mutant an to on activation of or These results that PP2Cβ-1 inhibits the TAK1 signaling pathway at TAK1 or of TAK1, and We have shown that TAK1, with TAB1, is activated by autophosphorylation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). TAK1 autophosphorylation can be by on and this was of TAK1, which is the of was to K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google also shown in To PP2Cβ-1 the phosphorylation of TAK1, we TAK1, TAB1, and PP2Cβ-1 in cells. shown in expression of wild-type PP2Cβ-1, but not a in the of TAK1 This that PP2Cβ-1 TAK1 To TAK1 is a of PP2C, we the phosphorylation and kinase activity of TAK1 with vitro. and were in cells, and was from with anti-Flag the TAK1 was with TAK1 This was with or TAK1 was found to be dephosphorylated by but not by in a The dephosphorylation was on the of We determined dephosphorylation of TAK1 by PP2Cβ-1 TAK1 were with and for TAK1 activity in vitro. The of PP2Cβ-1 the of TAK1 to phosphorylate and MKK6 Thus, PP2Cβ-1 and TAK1 in vitro. This the that PP2Cβ-1 negatively regulates the TAK1 signaling pathway by TAK1. Recent studies have indicated that one of the human PP2C and MKK4, MKK6, and p38 in (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). we PP2Cβ-1 also dephosphorylate and inactivate MKK6 in vitro. MKK6 is activated by autophosphorylation and is to phosphorylate p38 in T. N. Yamaguchi K. Gotoh Y. Irie K. T. Shirakabe K. Y. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We used this to the of PP2Cβ-1 on MKK6 We found that PP2Cβ-1 not MKK6 kinase activity under it TAK1 A we determined the of PP2Cβ-1 on phosphorylation of cells were with and to and was from the with anti-Flag The were with of on the phosphorylation of MKK6 these results indicate that PP2Cβ-1 not act To PP2Cβ-1 associates with TAK1, we and or in cells. Cell were with and was by with shown in both and with although the of the wild-type PP2Cβ-1 with TAK1 was that of This is for PP2Cβ-1, major mouse PP2C isoform S. Kobayashi T. Terasawa T. Ohnishi M. Sasahara Y. Kanamaru R. Tamura S. PubMed Scopus Google not with under the same the of PP2Cβ-1 with TAK1 is not by a protein The that the PP2Cβ has a for TAK1 that of wild-type PP2Cβ that PP2Cβ TAK1. The mutant, in which is by is in both phosphorylation and activation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). We PP2Cβ-1 and TAK1 or in cells and We found that an for PP2Cβ-1 to that of wild-type TAK1 indicating that phosphorylation at is not for with We PP2Cβ-1 and TAK1, at can also with one shown in the TAK1 was by anti-TAK1 Ab in the PP2Cβ from cells, but not in the rabbit To which region of TAK1 is for with PP2Cβ-1, we generated proteins, and the amino-terminal and of TAK1, We each mutant with in cells and from with anti-Flag analysis using Ab revealed that was with and but not with This that the central region of TAK1 is responsible for with To the specificity of the of PP2Cβ-1 with TAK1, we PP2Cβ-1 with other SAPK signaling pathway was with or in cells. was from with anti-Flag and the were to with of these proteins, for with PP2Cβ-1 Thus, PP2Cβ-1 with TAK1. PP2Cβ-1(R/G) to have a for TAK1 wild-type PP2Cβ-1 we PP2Cβ-1(R/G) act as a dominant negative To this we the of PP2Cβ-1(R/G) on TAK1 dephosphorylation in vitro. We found that PP2Cβ-1(R/G) inhibited the dephosphorylation of TAK1 by PP2Cβ-1 in a has recently been reported that of cells the signaling pathway activation of TAK1 (21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar). we the of PP2Cβ-1 to activation of TAK1 and AP-1 We cells with PP2Cβ-1 and TAK1 and determined the of PP2Cβ-1 expression on on and activation of TAK1. a of TAK1 on (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) However, the of PP2Cβ-1 the of TAK1. The expression of PP2Cβ-1 also inhibited the activation of TAK1 we cells with PP2Cβ-1 PP2Cβ-1(R/G) and AP-1 activity using an reporter gene. PP2Cβ-1 AP-1 activity in a However, of AP-1 activation by PP2Cβ-1 was by with PP2Cβ-1(R/G) ectopic expression of PP2Cβ-1(R/G) enhanced AP-1 activity in a MAPK cascades are signaling of of protein and MAPK (1Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1140) Google Scholar, 2Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1392) Google Scholar). phosphorylation of these is for the activation of the MAPK protein phosphatases be to roles in the regulation of these we recently that two major protein serine/threonine PP2Cα and inactivate the stress-activated and p38 MAPK pathways (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar) that PP2Cα inhibits the and p38 cascades by MKK4, MKK6, and TAK1 is a of the MKKK and the and p38 pathways. In this we the role of PP2Cβ in TAK1-mediated signaling pathways. We several of evidence suggesting that PP2Cβ negatively regulates the TAK1 pathways by and TAK1. ectopic expression of PP2Cβ inhibits the MKK4-JNK and MKK6-p38 pathways activated by TAK1 it is known that the TAK1-binding protein TAK1 by promoting autophosphorylation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). We found that PP2Cβ TAK1 autophosphorylation in PP2Cβ and TAK1 in but to dephosphorylate MKK6 PP2Cβ with TAK1 but not with MKK4, MKK6, JNK, or p38 and Collectively, these are with the that PP2Cβ TAK1-mediated signaling by with and TAK1. TAK1 functions in various including as a of transforming growth and (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar, 21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar) and as a negative in T. Ninomiya-Tsuji J. Nagai S.-I. Nishita M. Meneghini M. Barker N. Waterman M. Bowerman B. Clevers H. Shibuya H. Matsumoto K. Nature. 1999; 399: 798-802Crossref PubMed Scopus (521) Google it be to PP2Cβ to the control of these of with PP2Cβ-1 not in dephosphorylation of TAK1, as by the that the of TAK1 is that of TAK1 by cells a of we that the for the dephosphorylation be that the dephosphorylated TAK1 can be both and are in the cells. this that are other phosphorylation in TAK1 that are not substrates for TAK1 associates with PP2Cβ but not with PP2Cα Thus, the of TAK1 with PP2Cβ is TAK1 is activated autophosphorylation of in the activation kinase and of TAK1 to to both phosphorylation and activation of TAK1 (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). has an for PP2Cβ to that of wild-type TAK1 indicating that phosphorylation of TAK1 is not for with This that the of TAK1 with PP2Cβ not of the for but that PP2Cβ and TAK1 are This of PP2Cβ and the and of TAK1. The central region of TAK1 is for with PP2Cβ A region of TAK1 is in with H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (529) Google which that PP2Cβ the of TAK1 with However, this is we not and PP2Cβ in their with TAK1. J. M. K. R. K. and S. with TAK1 associates with in the absence of (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). the of TAK1 for with PP2Cβ and be is not PP2Cβ associates with TAK1 or However, the that PP2Cβ to with against the that the PP2Cβ and TAK1. To role PP2C in SAPK signaling it is to cellular PP2C activity is by extracellular In yeast cells, the expression of is enhanced by stress Shiozaki K. Russell P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In expression of PP2Cα and PP2Cβ-1 are not stress of cells (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). PP2Cα has been shown to to the of p38 and in the of the to p38 to the by stress (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). PP2Cβ an role in TAK1 TAK1 signaling (21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google and ectopic expression of PP2Cβ AP-1 a mutant, has a for TAK1 wild-type PP2Cβ and as a dominant negative the of wild-type PP2Cβ on AP-1 ectopic expression of AP-1 activation but not activation of These results the that PP2Cβ TAK1 activity PP2Cβ associates with TAK1 and not this it is to that regulation of PP2Cβ activity is in regulation of TAK1 PP2Cβ activity be and to TAK1 to the it is to the phosphatase activity of PP2Cβ is enhanced cells are to stress or with cytokines. (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar) recently reported that PP2Cα MKK4, MKK6, and p38 in vitro. In this study, we that PP2Cβ and TAK1. Thus, in mammalian cells, SAPK pathways are negatively by PP2C isoforms at different PP2Cβ inhibits the pathways at the TAK1 MKKK and PP2Cα at the MKK and MAPK In addition, two distinct of protein phosphatases other PP2C also in the regulation of the SAPK pathways. The of the dual specificity phosphatases known as MAPK that inactivate MAPKs by both tyrosine and threonine residues in the catalytic Of the MAPK and MAPK have been shown to selectively dephosphorylate and inactivate p38 and M. A. N. U. M. C. K. A. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The PP2A, which p38 kinase in J. Cohen P. S. M. A. T. Cell. Full Text PDF PubMed Scopus Google Scholar). with the enhanced MKK6 activity in cells T. N. Yamaguchi K. Gotoh Y. Irie K. T. Shirakabe K. Y. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). These results that also negatively SAPK pathways and the that several different of protein phosphatases each negatively distinct in SAPK pathways. We are to of and J. for with the expression plasmids of and respectively. We are also to for