TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8–mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)–dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.
CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.
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. Nishihira T. Satomi S. Hiraga A. Tamura S. Biochem. J. 1998; 332: 243-250Crossref PubMed Scopus (42) Google Scholar) COS7, 293, and 293IL-1RI (25Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (781) Google Scholar) cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum. At 50–80% confluency the cells were transfected by the DEAE-dextran method or using LipofectAMINE (Life Technologies, Inc.). The total amount of DNA (0.5–2 μg per 35-mm dish) was kept constant by supplementing with empty vector. The cells were cultured for 24–48 h after transfection and then harvested. Immune complex kinase assays were performed as follows. The cells were lysed in a buffer containing 20 mm Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 150 mm NaCl, 1 mm EGTA, 1 mm sodium orthovanadate, 50 mm NaF, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride, and the lysates were incubated with appropriate Abs for 1 h at 4 °C. The resulting immune complexes were recovered with protein G-Sepharose (Amersham Pharmacia Biotech), washed twice with Tris-buffered saline (20 mm Tris-HCl, pH 7.5, 150 mm NaCl), twice with 20 mm Tris-HCl, pH 7.5, and then incubated with or without appropriate substrates in 25 μl of kinase buffer (20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, and 1 mm dithiothreitol) containing 0.5–3 μCi of [γ-32P]ATP (NEG-002A, PerkinElmer Life Sciences) at 30 °C for 10–30 min. The reactions were stopped by adding SDS-sample buffer and boiled for 2 min. Protein phosphatase assays were carried out as follows. COS7 cells seeded onto 10-cm dishes were cotransfected with Flag-TAK1 and Myc-TAB1 expression plasmids. The Flag-TAK1-Myc-TAB1 complex was immunoprecipitated from cell extracts with anti-Flag Ab, and phosphorylation was carried out in kinase buffer containing [γ-32P]ATP at 30 °C for 30 min. After washing three times with 20 mm Tris-HCl, pH 7.5, the immune complex was then incubated with or without recombinant GST-PP2Cβ in kinase buffer at 30 °C for the indicated times. Phosphorylated proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the radioactivities incorporated into the proteins were detected with a BAS 2000 image analyzer (Fuji, Tokyo, Japan). Proteins in the cell lysates and immunoprecipitates were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The membranes were incubated with primary Abs at 4 °C for 16 h and then incubated with horseradish peroxidase-conjugated secondary Ab at 25 °C for 1 h. The chemiluminescence of each blot was detected with an enhanced chemiluminescence system (Amersham Pharmacia Biotech). Cells were lysed with a buffer containing 20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% (v/v) Triton X-100, and 1 mmphenylmethylsulfonyl fluoride. The cell lysates were incubated with the indicated Abs for 1 h at 4 °C. The immunoprecipitated proteins were washed three times with Tris-buffered saline and submitted to Western blot analysis. Cells were transfected with the AP-1-luciferase reporter plasmid (26Beltman J. Erickson J.R. Martin G.A. Lyons J.F. Cook S.J. J. Biol. Chem. 1999; 274: 3772-3780Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). After the transfection, cells were treated with IL-1β for 6 h. Luciferase activity was determined with the Luciferase Assay System (Promega). β-actin-β-galactosidase reporter plasmid was cotransfected for normalizing transfection efficiencies. We have previously reported that two mouse PP2C isoforms, PP2Cα and PP2Cβ-1, selectively inhibit stress-activated MKKs (MKK3, MKK4, MKK6, and MKK7) (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. Because 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 Scholar), we examined whether expression of PP2Cβ-1 affects TAK1-induced phosphorylation of MKK4 and MKK6 at their serine or threonine residues. Coexpression of TAK1 and TAB1 enhanced phosphorylation of MKK4 or MKK6 when expressed together in COS7 cells (Fig. 1, A and B). However, concomitant expression of PP2Cβ-1 markedly inhibited TAK1-induced phosphorylation of MKK4 and MKK6. We then tested whether PP2Cβ-1 expression affects TAK1-induced activation of JNK1 and p38α. Both the JNK1 and p38 kinases expressed in COS7 cells were activated by the exogenous TAK1. However, these kinase activities were inhibited when PP2Cβ-1 was coexpressed (Fig.1, C and D). In contrast, expression of PP2Cβ-1(R/G), a phosphatase-defective mutant containing an Arg-179 to Gly mutation, had no inhibitory effect on TAK1-induced activation of JNK1 or p38. These results suggest that PP2Cβ-1 inhibits the TAK1 signaling pathway at TAK1 or downstream of TAK1, e.g. MKKs and MAPKs. We have previously shown that TAK1, when coexpressed 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 monitored by decreased mobility on SDS-PAGE, and this mobility shift was cancelled when Ser-192 of TAK1, which is the site of autophosphorylation, was mutated to alanine (Ref.23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar; also shown in Fig. 2). To determine whether PP2Cβ-1 affects the phosphorylation state of TAK1, we coexpressed TAK1, TAB1, and PP2Cβ-1 in COS7 cells. As shown in Fig. 2, expression of wild-type PP2Cβ-1, but not PP2Cβ-1(R/G), caused a substantial decrease in the levels of TAK1 phosphorylation. This result suggests that PP2Cβ-1 acts upon TAK1 directly. To investigate whether TAK1 is a substrate of PP2C, we examined the phosphorylation and kinase activity of TAK1 incubated with PP2Cin vitro. Flag-TAK1 and TAB1 were coexpressed in COS7 cells, and Flag-TAK1 was immunoprecipitated from cell extracts with anti-Flag Ab. When the immunopurified TAK1 complex was incubated with [γ-32P]ATP, TAK1 became autophosphorylated. This reaction mixture was next incubated with bacterially produced GST-PP2Cβ-1 or GST-PP2Cβ-1(R/G). TAK1 was found to be dephosphorylated by GST-PP2Cβ-1, but not by GST-PP2Cβ-1(R/G), in a dose-dependent manner (Fig.3 A). The PP2Cβ-1-mediated dephosphorylation reaction was dependent on the presence of Mg2+ (Fig. 3 B). We then determined whether dephosphorylation of TAK1 by PP2Cβ-1 reduces TAK1 activity. Flag-TAK1 immunoprecipitates were treated with GST-PP2Cβ-1 and measured for TAK1 activity in vitro. The presence of PP2Cβ-1 decreased the ability of TAK1 to phosphorylate itself and MKK6 (Fig. 3 C). Thus, PP2Cβ-1 dephosphorylates and inactivates TAK1 in vitro. This supports the possibility that PP2Cβ-1 negatively regulates the TAK1 signaling pathway by dephosphorylating TAK1. Recent studies have indicated that one of the human PP2C isoforms, PP2Cα-2, dephosphorylates and inactivates MKK4, MKK6, and p38 in vitro (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). Therefore, we tested whether PP2Cβ-1 could also dephosphorylate and inactivate MKK6 in vitro. Bacterially produced MKK6 is activated by autophosphorylation and is able to phosphorylate p38 in vitro (27Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). We used this system to determine the effect of recombinant PP2Cβ-1 on MKK6 activity. We found that PP2Cβ-1 treatment did not affect MKK6 kinase activity under conditions where it inactivates TAK1 (Fig. 3, A andC versus Fig.4 A). Next, we determined the effect of PP2Cβ-1 on stress-induced phosphorylation of MKK6. COS7 cells were transfected with Flag-MKK6 and subjected to hyperosmotic stress, and Flag-MKK6 was immunoprecipitated from the cell lysates with anti-Flag Ab. The immunoprecipitates were then incubated with GST-PP2Cβ-1. Increasing concentrations of GST-PP2Cβ-1 had no effect on the phosphorylation level of MKK6 (Fig.4 B). Taken together, these results indicate that PP2Cβ-1 does not act upon MKK6. To determine whether PP2Cβ-1 associates with TAK1, we coexpressed Myc-TAK1 and HA-PP2Cβ-1 or HA-PP2Cβ-1(R/G) in COS7 cells. Cell extracts were immunoprecipitated with anti-Myc Ab, and coprecipitated HA-PP2Cβ-1 was detected by immunoblotting with anti-HA Ab. As shown in Fig.5 A, both HA-PP2Cβ-1 and HA-PP2Cβ-1(R/G) coimmunoprecipitated with Myc-TAK1, although the interaction of the wild-type PP2Cβ-1 with TAK1 was substantially weaker than that of PP2Cβ-1(R/G). This interaction is specific for PP2Cβ-1, because HA-PP2Cα, another major mouse PP2C isoform (28Kato S. Kobayashi T. Terasawa T. Ohnishi M. Sasahara Y. Kanamaru R. Tamura S. Gene. 1994; 145: 311-312Crossref PubMed Scopus (11) Google Scholar), did not coimmunoprecipitate with Myc-TAK1 under the same conditions (Fig. 5 B). Therefore, the association of PP2Cβ-1 with TAK1 is not caused by a nonspecific protein interaction. The observation that the catalytically inactive PP2Cβ has a higher affinity for TAK1 than that of wild-type PP2Cβ suggested that PP2Cβ might preferentially bind phosphorylated TAK1. The TAK1(S/A) mutant, in which Ser-192 is replaced by Ala, is defective in both phosphorylation and activation (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 coexpressed PP2Cβ-1 and TAK1 or TAK1(S/A) in 293 cells and performed coimmunoprecipitation experiments. We found that TAK1(S/A) had an affinity for PP2Cβ-1 similar to that of wild-type TAK1 (Fig. 5 C), indicating that phosphorylation at Ser-192 is not required for association with PP2Cβ-1. We next examined whether endogenous PP2Cβ-1 and TAK1, expressed at lower physiological levels, can also interact with one another. As shown in Fig. 5 D, the 70-kDa endogenous TAK1 was specifically detected by anti-TAK1 Ab in the endogenous PP2Cβ immunoprecipitates from 293 cells, but not in the rabbit IgG immunoprecipitates. To determine which region of TAK1 is required for its interaction with PP2Cβ-1, we generated three Myc-tagged, truncated proteins, Myc-TAK1(N400), Myc-TAK1(C366), and Myc-TAK1(C176), containing the amino-terminal 400, carboxyl-terminal 366, and carboxyl-terminal 176 amino acids of TAK1, respectively (Fig.6 A). We coexpressed each deletion mutant along with Flag-PP2Cβ-1(R/G) in 293 cells and immunoprecipitated Flag-PP2Cβ-1(R/G) from cell extracts with anti-Flag Ab. Subsequent immunoblot analysis using anti-Myc Ab revealed that Flag-PP2Cβ-1 was associated with Myc-TAK1(N400) and Myc-TAK1(C366) but not with Myc-TAK1(C176) (Fig. 6 B). This result indicates that the central region of TAK1 is responsible for its association with PP2Cβ-1. To evaluate the specificity of the association of PP2Cβ-1 with TAK1, we examined whether PP2Cβ-1 could associate with other SAPK signaling pathway components. Flag-PP2Cβ-1 was coexpressed with Myc-TAK1, Myc-MEKK3, Myc-MKK4, Myc-MKK6, Myc-JNK1, or Myc-p38α in 293 cells. Flag-PP2Cβ-1 was immunoprecipitated from cell extracts with anti-Flag Ab, and the immune complexes were subjected to immunoblotting with anti-Myc Ab. None of these proteins, except for Myc-TAK1, coimmunoprecipitated with PP2Cβ-1 (Fig.7). Thus, PP2Cβ-1 specifically interacts with TAK1. Because PP2Cβ-1(R/G) appeared to have a higher affinity for TAK1 than did wild-type PP2Cβ-1 (Fig. 5 A), we asked whether PP2Cβ-1(R/G) could act as a dominant negative mutant. To test this possibility, we examined the effect of PP2Cβ-1(R/G) on PP2Cβ-1-mediated TAK1 dephosphorylation in vitro. We found that PP2Cβ-1(R/G) inhibited the dephosphorylation of TAK1 by PP2Cβ-1 in a dose-dependent manner (Fig.8 A). It has recently been reported that IL-1 treatment of cells activates the JNK signaling pathway through 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). Therefore, we examined the ability of PP2Cβ-1 to affect activation of TAK1 and AP-1 following IL-1 stimulation. We transfected 293IL-1RI cells with PP2Cβ-1 and TAK1 and determined the effect of PP2Cβ-1 expression on IL-1-induced mobility shift on SDS-PAGE and activation of TAK1. IL-1 treatment caused a slight mobility shift of TAK1 on SDS-PAGE, confirming our previous observation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) (Fig. 8 B,lower panel). However, the coexpression of PP2Cβ-1 totally reversed the mobility shift of TAK1. The expression of PP2Cβ-1 also inhibited the IL-1-induced activation of TAK1 (Fig. 8 B,upper panel). Next, we transfected 293IL-1RI cells with PP2Cβ-1 or/and PP2Cβ-1(R/G) and assayed AP-1 activity using an AP-1-dependent luciferase reporter gene. PP2Cβ-1 blocked IL-1-induced AP-1 activity in a dose-dependent manner (Fig.8 C). However, inhibition of IL-1-induced AP-1 activation by PP2Cβ-1 was reversed by cotransfection with PP2Cβ-1(R/G) (Fig.8 D). Furthermore, ectopic expression of PP2Cβ-1(R/G) enhanced IL-1-induced AP-1 activity in a dose-dependent manner (Fig. 8 E). MAPK cascades are intracellular signaling modules composed of three tiers of sequentially activating protein kinases: MKKK, MKK, 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). Because phosphorylation of these components is essential for the activation of the MAPK cascades, protein phosphatases may be expected to play important roles in the regulation of these cascades. Indeed, we recently demonstrated that two major protein serine/threonine phosphatases, PP2Cα and PP2Cβ, inactivate the stress-activated JNK 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). Furthermore, Takekawaet al. (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar) showed that PP2Cα inhibits the JNK and p38 cascades by dephosphorylating MKK4, MKK6, and p38. TAK1 is a member of the MKKK family and activates the JNK and p38 pathways. In this study we elucidated the role of PP2Cβ in TAK1-mediated signaling pathways. We present several lines of evidence suggesting that PP2Cβ negatively regulates the TAK1 pathways by dephosphorylating and inactivating TAK1. First, ectopic expression of PP2Cβ inhibits the MKK4-JNK and MKK6-p38 pathways activated by TAK1 (Fig. 1). Second, it is known that the TAK1-binding protein TAB1 activates TAK1 by promoting its autophosphorylation (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 overexpression decreased TAB1-induced TAK1 autophosphorylation in vivo(Fig. 2). Third, PP2Cβ dephosphorylates and inactivates TAK1 in vitro (Fig. 3) but fails to dephosphorylate MKK6 (Fig. 4). Finally, PP2Cβ interacts with TAK1 but not with MEKK3, MKK4, MKK6, JNK, or p38 (Figs. 5 and 7). Collectively, these data are consistent with the idea that PP2Cβ suppresses TAK1-mediated signaling by associating with and dephosphorylating TAK1. Because TAK1 functions in various biological responses, including acting as a positive regulator of transforming growth factor-β- and IL-1-induced signal transduction (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 regulator in Wnt-induced signal transduction (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), it would be interesting to examine whether PP2Cβ contributes to the control of these physiological responses. Coexpression of TAK1/TAB1 with PP2Cβ-1 did not result in complete dephosphorylation of TAK1, as judged by the fact that the mobility of TAK1 is still slower than that of TAK1 expressed by itself (Fig. 2). COS7 cells contain a substantial amount of free, endogenous TAB1. Therefore, we speculate that the reason for the incomplete dephosphorylation may be that the dephosphorylated TAK1 can be rephosphorylated, because both TAB1 and ATP are present in the cells. Alternatively, this may suggest that there are other phosphorylation sites in TAK1 that are not substrates for PP2C. TAK1 associates with PP2Cβ but not with PP2Cα (Fig. 5). Thus, the interaction of TAK1 with PP2Cβ is rather specific. TAK1 is activated via autophosphorylation of Ser-192 in the activation loop between kinase domains VII and VIII. Mutation of TAK1 Ser-192 to Ala to create TAK1(S/A) abolishes both phosphorylation and activation of TAK1 (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(S/A) has an affinity for PP2Cβ similar to that of wild-type TAK1 (Fig. 5 C), indicating that phosphorylation of TAK1 is not required for its association with PP2Cβ. This suggests that the association of TAK1 with PP2Cβ does not occur simply through affinity of the enzyme (PP2Cβ) for its substrate (phosphorylated TAK1), but rather that PP2Cβ and TAK1 are stably associated. This may ensure appropriate localization of PP2Cβ and facilitate the specific and rapid deactivation of TAK1. The central region of TAK1 is required for its association with PP2Cβ (Fig. 6). A similar region of TAK1 is involved in its association with TAB1 (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), which suggests that PP2Cβ might prevent the association of TAK1 with TAB1. However, this possibility is unlikely, because we did not observe any competition between TAB1 and PP2Cβ in their association with TAK1. 2M. Hanada, J. Ninomiya-Tsuji, K.-i. Komaki, M. Ohnishi, K. Katsura, R. Kanamaru, K. Matsumoto, and S. Tamura, unpublished observation. Consistent with this, endogenous TAK1 constitutively associates with TAB1 in the absence of ligand stimulation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Therefore, the minimum regions of TAK1 required for association with PP2Cβ and TAB1 must be different. It is still not clear whether PP2Cβ associates with TAK1 directly or indirectly. However, the observation that PP2Cβ fails to interact with TAB12 argues against the possibility that TAB1 mediates the association between PP2Cβ and TAK1. To understand what role PP2C may play in regulating SAPK signaling pathways, it is important to determine how cellular PP2C activity is affected by extracellular stimuli. In fission yeast cells, the expression of Ptc1 is enhanced by hyperosmotic stress (29Gaits F. Shiozaki K. Russell P. J. Biol. Chem. 1997; 272: 17873-17879Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In contrast, expression levels of PP2Cα and PP2Cβ-1 are not altered following stress treatment 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 preferentially bind to the phosphorylated form of p38 and may function in the adaptive phase of the stimulation cycle to restore p38 to the inactive state following stimulation by stress (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). PP2Cβ may play an analogous role in maintaining TAK1 signaling. TAK1 mediates IL-1-induced JNK signaling (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 ectopic expression of PP2Cβ blocks IL-1-induced AP-1 activation. PP2Cβ(R/G), a catalytically inactive mutant, has a higher affinity for TAK1 than does wild-type PP2Cβ and acts as a dominant negative factor, antagonizing the inhibitory effect of wild-type PP2Cβ on IL-1-induced AP-1 activation. Furthermore, ectopic expression of PP2Cβ(R/G) enhances IL-1-stimulated AP-1 activation but does not cause constitutive activation of AP-1.2 These results raise the possibility that PP2Cβ may down-regulate TAK1 activity after ligand stimulation. Because endogenous PP2Cβ constitutively associates with TAK1 (Fig. 5 D), and ligand stimulation does not affect this association,2 it is tempting to speculate that regulation of PP2Cβ enzymatic activity is involved in regulation of TAK1 signaling. Alternatively, PP2Cβ activity may be constitutive and serve to restore TAK1 to the inactive state following stimulation. Therefore it is important to determine whether the phosphatase activity of PP2Cβ is enhanced when cells are subjected to stress or treated with pro-inflammatory cytokines. Takekawa et al. (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar) recently reported that PP2Cα dephosphorylates MKK4, MKK6, and p38 in vitro. In this study, we show that PP2Cβ dephosphorylates and inactivates TAK1. Thus, in mammalian cells, SAPK pathways are negatively regulated by multiple PP2C isoforms at different levels; PP2Cβ inhibits the pathways at the TAK1 MKKK level, and PP2Cα acts at the MKK and MAPK levels. In addition, two distinct groups of protein phosphatases other than PP2C also participate in the regulation of the SAPK pathways. The first group consists of the dual specificity phosphatases (also known as MAPK phosphatases) that inactivate MAPKs by dephosphorylating both tyrosine and threonine residues in the catalytic domains. Of the nine isolated MAPK phosphatases, M3/6 and MAPK phosphatase-5 have been shown to selectively dephosphorylate and inactivate p38 and JNK (30Muda M. Theodosius A. Rodrigues N. Boschert U. Camps M. Gillieron C. Davies K. Ashworth A. Arkinstall S. J. Biol. Chem. 1996; 271: 27205-27208Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The second group includes PP2A, which inactivates partially purified p38 kinase in vitro (32Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1523) Google Scholar). Cells treated with the PP2A inhibitor okadaic acid show enhanced MKK6 activity in epithelial cells (27Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). These results suggest that PP2A may also negatively regulate SAPK pathways and raise the possibility that several different groups of protein phosphatases each negatively regulate distinct targets in SAPK pathways. We are grateful to Drs. Ulrich Siebenlist (National Institutes of Health) and Simon J. Cook (the Babraham Institute) for providing us with the expression plasmids of MEKK3 and AP-1-luciferase, respectively. We are also grateful to Kimio Konno for technical assistance.
Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is suggested to be involved in TGF-β-induced gene expression, but the signaling mechanism from TAK1 to the nucleus remains largely undefined. We have found that p38 mitogen-activated protein kinase, and its direct activator MKK6 are rapidly activated in response to TGF-β. Expression of dominant negative MKK6 or dominant negative TAK1 inhibited the TGF-β-induced transcriptional activation as well as the p38 activation. Constitutive activation of the p38 pathway in the absence of TGF-β induced the transcriptional activation, which was enhanced synergistically by coexpression of Smad2 and Smad4 and was inhibited by expression of the C-terminal truncated, dominant negative Smad4. Furthermore, we have found that activating transcription factor-2 (ATF-2), which is known as a nuclear target of p38, becomes phosphorylated in the N-terminal activation domain in response to TGF-β, that ATF-2 forms a complex with Smad4, and that the complex formation is enhanced by TGF-β. In addition, expression of a nonphosphorylatable form of ATF-2 inhibited the TGF-β-induced transcriptional activation. These results show that the p38 pathway is activated by TGF-β and is involved in the TGF-β-induced transcriptional activation by regulating the Smad-mediated pathway.
Transforming growth factor beta-activated kinase 1 (TAK1) functions downstream of inflammatory cytokines to activate c-Jun N-terminal kinase (JNK) as well as NF-kappaB in several cell types. However, the functional role of TAK1 in an in vivo setting has not been determined. Here we have demonstrated that TAK1 is the major regulator of skin inflammation as well as keratinocyte death in vivo. Epidermal-specific deletion of TAK1 causes a severe inflammatory skin condition by postnatal day 6-8. The mutant skin also exhibits massive keratinocyte death. Analysis of keratinocytes isolated from the mutant skin revealed that TAK1 deficiency results in a striking increase in apoptosis in response to tumor necrosis factor (TNF). TAK1-deficient keratinocytes cannot activate NF-kappaB or JNK upon TNF treatment. These results suggest that TNF induces TAK1-deficient keratinocyte death because of the lack of NF-kappaB (and possibly JNK)-mediated cell survival signaling. Finally, we have shown that deletion of the TNF receptor can largely rescue keratinocyte death as well as inflammatory skin condition in epidermal-specific TAK1-deficient mice. Our results demonstrate that TAK1 is a master regulator of TNF signaling in skin and regulates skin inflammation and keratinocyte death.
Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766-778.
Osmotic stress activates MAPKs, including JNK and p38, which play important roles in cellular stress responses. Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the MAPK kinase kinase (MAPKKK) family and can activate JNK and p38. TAK1 can also activate IkappaB kinase (IKK) that leads to degradation of IkappaB and subsequent NF-kappaB activation. We found that TAK1 is essential for osmotic stress-induced activation of JNK but is not an exclusive mediator of p38 activation. Furthermore, we found that although TAK1 was highly activated upon osmotic stress, it could not induce degradation of IkappaB or activation of NF-kappaB. These results suggest that TAK1 activity is somehow modulated to function specifically in osmotic stress signaling, leading to the activation of JNK but not of IKK. To elucidate the mechanism underlying this modulation, we screened for potential TAK1-binding proteins. We found that TAO2 (thousand-and-one amino acid kinase 2) associates with TAK1 and can inhibit TAK1-mediated activation of NF-kappaB but not of JNK. We observed that TAO2 can interfere with the interaction between TAK1 and IKK and thus may regulate TAK1 function. TAK1 is activated by many distinct stimuli, including cytokines and stresses, and regulation by TAO2 may be important to activate specific intracellular signaling pathways that are unique to osmotic stress.
