Phosphorylation of Thr-178 and Thr-184 in the TAK1 T-loop Is Required for Interleukin (IL)-1-mediated Optimal NFκB and AP-1 Activation as Well as IL-6 Gene Expression
2008; Elsevier BV; Volume: 283; Issue: 36 Linguagem: Inglês
10.1074/jbc.m802825200
ISSN1083-351X
AutoresYang Yu, Ningling Ge, Min Xie, Wenjing Sun, Susan M. Burlingame, Amy Kun Pass, Jed G. Nuchtern, Dekai Zhang, Songbin Fu, Michael Schneider, Jia Fan, Jianhua Yang,
Tópico(s)interferon and immune responses
ResumoTAK1 (transforming growth factor-β-activated kinase 1), a mitogen-activated protein kinase kinase kinase, is activated by various cytokines, including interleukin-1 (IL-1). However, the precise regulation for TAK1 activation at the molecular level is still not fully understood. Here we report that dual phosphorylation of Thr-178 and Thr-184 residues within the kinase activation loop of TAK1 is essential for TAK1-mediated NFκB and AP-1 activation. Once co-overexpressed with TAB1, TAK1 mutant with alanine substitution of these two residues fails to activate IKKβ-mediated NFκB and JNK-mediated AP-1, whereas TAK1 mutant with replacement of these two sites with acidic residues acts like the TAK1 wild type. Consistently, TAK1 mutant with alanine substitution of these two residues severely inhibits IL-1-induced NFκB and AP-1 activities, whereas TAK1 mutant with replacement of these two sites with acidic residues slightly enhances IL-1-induced NFκB and AP-1 activities compared with the TAK1 wild-type. IL-1 induces the phosphorylation of endogenous TAK1 at Thr-178 and Thr-184. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with wild-type TAK1 or a TAK1 mutant containing threonine 178 and 184 to alanine mutations revealed the importance of these two sites in IL-1-mediated IKK-NFκB and JNK-AP-1 activation as well as IL-1-induced IL-6 gene expression. Our finding is the first report that substitution of key serine/threonine residues with acidic residues mimics the phosphorylated state of TAK1 and renders TAK1 active during its induced activation. TAK1 (transforming growth factor-β-activated kinase 1), a mitogen-activated protein kinase kinase kinase, is activated by various cytokines, including interleukin-1 (IL-1). However, the precise regulation for TAK1 activation at the molecular level is still not fully understood. Here we report that dual phosphorylation of Thr-178 and Thr-184 residues within the kinase activation loop of TAK1 is essential for TAK1-mediated NFκB and AP-1 activation. Once co-overexpressed with TAB1, TAK1 mutant with alanine substitution of these two residues fails to activate IKKβ-mediated NFκB and JNK-mediated AP-1, whereas TAK1 mutant with replacement of these two sites with acidic residues acts like the TAK1 wild type. Consistently, TAK1 mutant with alanine substitution of these two residues severely inhibits IL-1-induced NFκB and AP-1 activities, whereas TAK1 mutant with replacement of these two sites with acidic residues slightly enhances IL-1-induced NFκB and AP-1 activities compared with the TAK1 wild-type. IL-1 induces the phosphorylation of endogenous TAK1 at Thr-178 and Thr-184. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with wild-type TAK1 or a TAK1 mutant containing threonine 178 and 184 to alanine mutations revealed the importance of these two sites in IL-1-mediated IKK-NFκB and JNK-AP-1 activation as well as IL-1-induced IL-6 gene expression. Our finding is the first report that substitution of key serine/threonine residues with acidic residues mimics the phosphorylated state of TAK1 and renders TAK1 active during its induced activation. Interleukin-1 (IL-1) 4The abbreviations used are:ILinterleukinIKKIκB kinaseJNKc-Jun-NH2-terminal kinaseNFκBnuclear transcription factor κBIL-1RIL-1 receptorMEFmouse embryo fibroblastHAhemagglutinin. is a proinflammatory cytokine and plays a crucial role in inflammation, stress, and disease in many cell types(1Dinarello C.A. Cytokine Growth Factor Rev. 1997; 8: 253-265Crossref PubMed Scopus (612) Google Scholar, 2O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (499) Google Scholar, 3Arend W.P. Cytokine Growth Factor Rev. 2002; 13: 323-340Crossref PubMed Scopus (559) Google Scholar, 4Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1261) Google Scholar). Cellular responses to IL-1 are mediated by intracellular signaling pathways that activate nuclear transcription factor κB (NFκB) and AP-1 (activator protein 1) (1Dinarello C.A. Cytokine Growth Factor Rev. 1997; 8: 253-265Crossref PubMed Scopus (612) Google Scholar, 5Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1376) Google Scholar). interleukin IκB kinase c-Jun-NH2-terminal kinase nuclear transcription factor κB IL-1 receptor mouse embryo fibroblast hemagglutinin. TAK1 (transforming growth factor-β-activated kinase 1), a member of the evolutionarily conserved mitogen-activated protein kinase kinase kinase family, was originally found to function in signaling of the transforming growth factor-β (6Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1175) Google Scholar). TAK1 is activated by various cellular stresses, including IL-1, tumor necrosis factor-α, lipopolysaccharide, osmotic stress, and latent membrane protein 1 from Epstein-Barr virus (7Irie T. Muta T. Takeshige K. FEBS Lett. 2000; 467: 160-164Crossref PubMed Scopus (164) Google Scholar, 8Takaesu G. Surabhi R.M. Park K.J. Ninomiya-Tsuji J. 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Once activated, TAK1 translocates from the membrane to the cytosol along with TRAF6 and its association partners, TAB1 (TAK1-binding protein 1), TAB2, and TAB3 (16Cheung P.C. Nebreda A.R. Cohen P. Biochem. J. 2004; 378: 27-34Crossref PubMed Scopus (135) Google Scholar, 17Ishitani T. Takaesu G. Ninomiya-Tsuji J. Shibuya H. Gaynor R.B. Matsumoto K. EMBO J. 2003; 22: 6277-6288Crossref PubMed Scopus (219) Google Scholar, 18Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (520) Google Scholar, 19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). TAK1 activation subsequently leads to the activations of IκB kinase (IKK) and c-Jun NH2-terminal kinase (JNK) as well as p38. Activated IKK phosphorylates IκB proteins, and phosphorylated IκB proteins are degraded by the ubiquitin-mediated proteosome pathway (20Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4086) Google Scholar). Degradation of IκB proteins leads to NFκB translocation into the nucleus and activation of NFκB-dependent gene transcription in the nucleus (20Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4086) Google Scholar, 21Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4599) Google Scholar). Activated JNKs phosphorylate specific sites on the amino-terminal trans-activation domain of transcription factor c-Jun, an important component of transcriptional activator AP-1. Phosphorylation of these sites stimulates the ability of c-Jun to activate AP-1-dependent gene expression (22Davis R.J. Biochem. Soc. Symp. 1999; 64: 1-12PubMed Google Scholar, 23Davis R.J. 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Schuttelkopf A.W. Cohen P. Van Aalten D.M. Biochem. J. 2006; 399: 427-434Crossref PubMed Scopus (52) Google Scholar). The 67 amino acids at the COOH-terminal end of TAB1 were demonstrated to be sufficient for full TAK1 activation (27Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). This claim was supported by a crystal structure study of an TAK1-TAB1 chimeric protein (28Brown K. Vial S.C. Dedi N. Long J.M. Dunster N.J. Cheetham G.M. J. Mol. Biol. 2005; 354: 1013-1020Crossref PubMed Scopus (70) Google Scholar). Phosphorylation and dephosphorylation of critical serine and threonine residues in the activation loop of serine/threonine protein kinases are essential for kinase activation and inactivation (29Alessi D.R. Saito Y. Campbell D.G. Cohen P. Sithanandam G. Rapp U. Ashworth A. Marshall C.J. Cowley S. EMBO J. 1994; 13: 1610-1619Crossref PubMed Scopus (468) Google Scholar, 30Johnson L.N. Noble M.E. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1178) Google Scholar, 31Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (841) Google Scholar, 32Resing K.A. Mansour S.J. Hermann A.S. Johnson R.S. Candia J.M. Fukasawa K. Vande Woude G.F. Ahn N.G. Biochemistry. 1995; 34: 2610-2620Crossref PubMed Scopus (68) Google Scholar, 33Zheng C.F. Guan K.L. EMBO J. 1994; 13: 1123-1131Crossref PubMed Scopus (300) Google Scholar). Many studies have shown that substitution of these critical residues with acidic residues to mimic the phosphorylation state renders the kinase constitutively active (34Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (752) Google Scholar, 35Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar). In the case of TAK1, several studies have been carried out to identify the phosphorylation sites and examine the effect of phosphorylation on TAK1 activity. For example, phosphorylation of Thr-187 and Ser-192 within the activation loop of TAK1 has been shown to be promoted by cytokine tumor necrosis factor-α. However, instead of rendering the kinase constitutively active, replacement of these residues with acidic residues leads to inactivation of TAK1 kinase (36Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 37Sakurai H. Miyoshi H. Mizukami J. Sugita T. FEBS Lett. 2000; 474: 141-145Crossref PubMed Scopus (142) Google Scholar, 38Sakurai H. Nishi A. Sato N. Mizukami J. Miyoshi H. Sugita T. Biochem. Biophys. Res. Commun. 2002; 297: 1277-1281Crossref PubMed Scopus (69) Google Scholar, 39Singhirunnusorn P. Suzuki S. Kawasaki N. Saiki I. Sakurai H. J. Biol. Chem. 2005; 280: 7359-7368Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In this report, we further examine the potential phosphorylation sites in the activation loop of TAK1 responsible for TAK1-mediated NFκB and AP-1 activations. Using mutational analysis and reporter assays, we identified two key amino acids at positions Thr-178 and Thr-184 that are additional regulatory phosphorylation sites required for TAK1-mediated optimal NFκB and AP-1 activation. We confirmed the phosphorylation of both Thr-178 and Thr-184 residues on TAK1 by using a specific antibody that recognizes the dual phosphorylation of these two sites. These two residues are located within the TAK1 kinase subdomains VII and VIII. Interestingly, phosphorylation of these two residues can be induced by IL-1 stimulation and are required for IL-1-induced optimal IKK-NFκB and JNK-AP-1 activation as well as IL-6 gene expression. Cell Culture and Transfection—TAK1-deficient MEF cells have been described before (40Liu H.H. Xie M. Schneider M.D. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11677-11682Crossref PubMed Scopus (132) Google Scholar, 41Wan Y.Y. Chi H. Xie M. Schneider M.D. Flavell R.A. Nat. Immunol. 2006; 7: 851-858Crossref PubMed Scopus (221) Google Scholar, 42Xie M. Zhang D. Dyck J.R. Li Y. Zhang H. Morishima M. Mann D.L. Taffet G.E. Baldini A. Khoury D.S. Schneider M.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 17378-17383Crossref PubMed Scopus (287) Google Scholar). HeLa, HEK293T, and MEF-TAK1 knock-out cells were maintained in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. HeLa, HEK293T, and MEF-TAK1 knock-out cells were transfected with expression plasmids using Fugene6 (Roche Applied Science) and Lipofectamine 2000 reagents (Invitrogen), respectively. Expression Vectors—A cDNA construct containing the full-length open reading frame of the wild-type human TAK1 was subcloned into the 3FLAG-tagged mammalian expression vector pcDNA3.1. Point mutations were made by site-directed PCR mutagenesis and verified by DNA sequencing analysis. The NFκB-dependent firefly luciferase reporter plasmid and pCMV promoter-dependent Renilla luciferase reporter were purchased from Clontech. The retroviral expression vectors were constructed by subcloning the TAK1-wild type or TAK1-T178A/T184A cDNA fragment into the pBabe-puro vector. Mammalian expression vectors for TAB1, TRAF6, and IKKβ were constructed by subcloning cDNAs encoding the full-length wild-type human proteins into the pcDNA3.1 vector. Antibody and Reagents—A specific anti-phospho-TAK1 (pT178/pT184) antibody was generated by immunizing rabbits with the synthetic phosphopeptide corresponding to amino acids VLKICDFGpTACDIQpTHM (where pT represents phosphothreonine) of human TAK1 by Genemed Synthesis, Inc. (South San Francisco, CA). Antibodies against TAK1, phospho-TAK1 (Thr-184), p38, phospho-p38, JNK, phospho-JNK, IκBα, phospho-IκBα, phospho-IKKα/β, phospho-p65, and secondary antibodies conjugated to horseradish peroxidase were purchased from Cell Signaling Technology (Beverly, MA). Antibodies for HA epitope, proliferating cell nuclear antigen (PC-10), and NFκB-p65 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-FLAG antibody (M5) and anti-actin antibodies were obtained from Sigma. Recombinant human IL-1β and mouse IL-1β were obtained from R & D Systems (Minneapolis, MN). Establishment of Stable TAK1-deficient MEF Cell Lines Expressing TAK1 Wild Type and TAK1-T178A/T184A—The pBabe-TAK1 wild type or pBabe-TAK1-T178A/T184A or pBabe empty vector were co-transfected with retrovirus packing vector Pegpam 3e and PLC-ECO in HEK293T cells to obtain retroviral supernatants. Viral supernatants were collected after 48 and 72 h. MEF-TAK1 knock-out cells were incubated with virus-containing medium in the presence of 4 μg/ml Polybrene (hexadimethrine bromide; Sigma). Stable cell lines were established after 10 days of puromycin (2 μg/ml) selection. Immunoprecipitation and Immunoblotting—To prepare total cell lysates, cells were placed on ice and washed three times with ice-cold phosphate-buffered saline. Cells were lysed by adding lysis buffer (25 mm HEPES (pH 7.7), 135 mm NaCl, 3 mm EDTA, 1% Triton X-100, 25 mm β-glycerophosphate, 0.1 mm sodium orthovanadate, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm benzamidine, 20 mm disodium p-nitrophenyl phosphate, 1 mm phenylmethylsulfonyl fluoride, phosphatase inhibitor mixture 1 and 2 (Sigma)). After centrifuging the lysate at 13,000 × g for 15 min at 4 °C, antibody was added to the supernatant and incubated with rotation for 3 h at 4 °C. After adding a protein A-agarose bead suspension (protein A; Santa Cruz Biotechnology), the mixture was further incubated with rotation for 3 h at 4 °C. After three washes with the washing buffer (20 mm HEPES (pH 7.7), 50 mm NaCl, 2.5 mm MgCl2, 0.1 mm EDTA, and 0.05% Triton X-100), the beads were resuspended in Laemmli sample buffer and boiled for 5 min. The immunoprecipitates or the whole cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were probed with appropriate antibodies. The IgG horseradish peroxidase-conjugated antibodies were used as the secondary antibodies. The proteins were detected using the ECL-Plus Western blotting detection system (Amersham Biosciences) and visualized by autography. Preparation of Cytoplasmic and Nuclear Extracts—Cytoplasmic and nuclear extracts were obtained as described by Singhirunnusorn et al. (39Singhirunnusorn P. Suzuki S. Kawasaki N. Saiki I. Sakurai H. J. Biol. Chem. 2005; 280: 7359-7368Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Cytoplasmic extracts were prepared by adding Buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 mm glycerophosphate, 0.1 mm sodium orthovanadate, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) to cell pellets. The cells were suspended and chilled on ice for 15 min. Next, 25 μl of 10% Nonidet P-40 was added, and the suspension was vortexed vigorously for 10 s. Cytoplasmic extracts were collected after centrifugation at 15,000 rpm for 5 min. To prepare nuclear extracts, the nuclear pellets were washed twice using Buffer A. Then Buffer B (20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 mm glycerophosphate, 1 mm sodium orthovanadate, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) was added to the nuclear pellets. The resuspension was put on ice for 15 min, followed by centrifugation at 15,000 rpm for 5 min. The supernatants were collected as nuclear extracts. Reverse Transcription-Polymerase Chain Reaction—Mouse IL-6 expression in MEF cells was analyzed by reverse transcription-PCR. Total RNA was isolated with TRIzol reagent (Invitrogen), and cDNA was prepared using SuperScript III gene expression tools (Invitrogen) according to the manufacturer's protocol. The primers used were as follows: mouse IL-6/F442, 5′-caaagccagagtccttcagag-3′; mouse IL-6/R593, 5′-tggtccttagccactccttc-3′; mouse Actb/F305, 5′-tgttaccaactgggacgac-3′; mouse mActb/R711, 5′-cacgcacgatttccctctc-3′. The PCR products were subjected to electrophoresis on a 2% agarose gel. Enzyme-linked Immunosorbent Assay—Supernatants of stable MEF-TAK1 knock-out cell lines expressing TAK1 wild type and TAK1-T178A/T184A, which were treated with or without mouse IL-1β (10 ng/ml), were collected at different time points. Mouse IL-6 concentrations in medium were determined by enzyme-linked immunosorbent assay using a kit (BD Biosciences) according to the manufacturer's instructions. In Vitro Phosphatase Treatment—HEK293T cells were transfected with FLAG-TAK1 and TAB1 expression vectors, and cell extracts were prepared in protein lysis buffer. Epitope-tagged TAK1 was immunoprecipitated with anti-FLAG antibody and washed three times with washing buffer and then two times with 1× phosphatase reaction buffer (50 mm HEPES, 100 mm NaCl, 2 mm dithiothreitol, 0.1 mm EGTA, 0.025% Tween 20). Then 40 μl of 1× phosphatase reaction buffer with 1 μl of λ-protein phosphatase (New England Biolabs, Ipswich, MA) was added to the immunoprecipitates at 30 °C for 30 min. Luciferase Reporter Assay—MEF-TAK1 cells were seeded at a concentration of 3 × 105 cells/well and cultured overnight in 6-well plates. NFκB-dependent firefly luciferase reporter and effector plasmids were co-transfected along with the Renilla luciferase plasmid. Thirty-six hours after transfection, cells were harvested in lysis buffer (Promega, Madison, WI), and luciferase assays were performed using the Dual-Luciferase reporter assay system (Promega). The relative luciferase activity was calculated by dividing the firefly luciferase activity by the Renilla luciferase activity. Data represent three independent experiments performed in duplicate. Thr-178 and Thr-184 Residues in the Activation Loop of Human TAK1 Are Two Potential Phosphorylation Sites Essential for TAK1/TAB1-mediated NFκB and AP-1 Activation—Phosphorylation of serine and/or threonine residue(s) between kinase subdomains VII and VIII is essential for the activation of many kinases (29Alessi D.R. Saito Y. Campbell D.G. Cohen P. Sithanandam G. Rapp U. Ashworth A. Marshall C.J. Cowley S. EMBO J. 1994; 13: 1610-1619Crossref PubMed Scopus (468) Google Scholar, 30Johnson L.N. Noble M.E. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1178) Google Scholar, 31Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (841) Google Scholar, 32Resing K.A. Mansour S.J. Hermann A.S. Johnson R.S. Candia J.M. Fukasawa K. Vande Woude G.F. Ahn N.G. Biochemistry. 1995; 34: 2610-2620Crossref PubMed Scopus (68) Google Scholar, 33Zheng C.F. Guan K.L. EMBO J. 1994; 13: 1123-1131Crossref PubMed Scopus (300) Google Scholar). Furthermore, substitution of these critical serine or threonine residues with acidic residues mimics the phosphorylation and renders these kinases constitutively active (34Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (752) Google Scholar, 35Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar). TAK1 has previously been identified to play a critical role in the cytokine-mediated NFκB and JNK signal transduction pathways (9Vidal S. Khush R.S. Leulier F. Tzou P. Nakamura M. Lemaitre B. Genes Dev. 2001; 15: 1900-1912Crossref PubMed Scopus (250) Google Scholar, 43Boutros M. Agaisse H. Perrimon N. Dev. Cell. 2002; 3: 711-722Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 44Park J.M. Brady H. Ruocco M.G. Sun H. Williams D. Lee S.J. Kato Jr., T. Richards N. Chan K. Mercurio F. Karin M. Wasserman S.A. Genes Dev. 2004; 18: 584-594Crossref PubMed Scopus (151) Google Scholar, 45Silverman N. Zhou R. Erlich R.L. Hunter M. Bernstein E. Schneider D. Maniatis T. J. Biol. Chem. 2003; 278: 48928-48934Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). To investigate which serine or threonine residue within the activation loop is essential for TAK1-mediated NFκB and AP-1 activation, we aligned amino acid sequences in a region of the kinase activation loop between domains DFG and M(A/S)PE of TAK1 from various species (Fig. 1A). Four conserved Ser/Thr sites (Thr-178, Thr-184, Thr-187, and Ser-192) were found in this region. Phosphorylation of Thr-187 and Ser-192 residues has been shown to be involved in the regulation of TAK1 kinase activity (36Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 39Singhirunnusorn P. Suzuki S. Kawasaki N. Saiki I. Sakurai H. J. Biol. Chem. 2005; 280: 7359-7368Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). However, replacement of these two sites with acidic residues inactivates TAK1 kinase activity. These studies suggest that Thr-178 and Thr-184 residues might also be the potential phosphorylation sites that are involved in the regulation of TAK1 activity. To characterize the physiological role of Thr-178 and Thr-184 on human TAK1, NFκB and AP-1-dependent luciferase reporter assays were used to assess the effect of point mutations (threonine to alanine or glutamic acid) at Thr-178 and/or Thr-184 on TAK1-induced NFκB and AP-1 activities. In these assays, the expression plasmid encoding the wild-type, active site lysine to arginine mutant (K63R) or each point mutant of TAK1 was transfected into TAK1-deficient cells, along with plasmids containing TAB1-, NFκB-, and AP-1-dependent firefly and control pCMV Renilla luciferase reporter genes. As shown in Fig. 1, B–D, TAK1 T178A and T184A single mutation partially impaired TAK1-induced NFκB and AP-1-dependent luciferase activities compared with the wild-type TAK1, whereas the TAK1 T178A/T184A double mutation as well as the K63R kinase-inactive mutation completely abrogated TAK1/TAB1-induced NFκB and AP-1 activities. In contrast, T178E and T184E single or double mutant TAK1 induced NFκB and AP-1 activation in the reporter assays at a similar or slightly higher levels compared with the wild type. These results strongly suggest that the TAK1 T178E/T184E mutant is active in the assays, and the phosphorylation of both Thr-178 and Thr-184 residues is required for the TAK1/TAB1-induced NFκB and AP-1 activities. Overexpression of TAK1 and TAB1 Induces Phosphorylation of TAK1 at Thr-178 and Thr-184—To examine directly whether Thr-178 and Thr-184 are phosphorylated during TAK1 activation, we generated a new phospho-specific antibody recognizing human TAK1 phosphorylated at Thr-178 and Thr-184 (pT178/pT184) by immunizing rabbits with a synthetic phosphopeptide (KLH-coupled) corresponding to residues surrounding Thr-178 and Thr-184 of human TAK1. Antibodies were purified by peptide affinity chromatography (Genemed Synthesis, Inc., South San Francisco, CA). As shown in Fig. 2A, our antibody specific for phospho-TAK1 (pT178/pT184) recognized the wild type, T178A, and T184A in a TAB1-dependent manner but not T178A/T184A double mutant TAK1 proteins. To further test the specificity of our antibody, another phosphoantibody recognizing the phospho-Thr-184 of TAK1 was used to detect the phosphorylation of the two sites. As shown in Fig. 2B, anti-phospho-TAK1 (pT184) only recognized TAK1 T178A and not the T184A mutant. These results demonstrate that our antibody can recognize both TAK1 T178A and T184A single mutant and suggest that Ala substitution at Thr-178 had no effect on the phosphorylation of Thr-184 on the activated TAK1. However, Ala substitution at Thr-187 and Ser-192 abolished TAK1/TAB1 overexpression-induced TAK1 phosphorylation at Thr-178 and Thr-184 (Fig. 2C). To further validate the specificity of our antibody to the phosphorylation at these two sites, we coexpressed FLAG-tagged wild-type TAK1 and TAB1 in HEK293T cells. TAK1 immunoprecipitates were treated in vitro with λ-protein phosphatase. This treatment, whose efficacy was demonstrated by extinction of the anti-phospho-TAK1 (pT178/pT184) signal, resulted in complete abrogation of the phosphorylation of the two sites (Fig. 2D). Taken together, these results demonstrate that our antibody specifically detects TAK1 only when phosphorylated at threonine 178 and/or 184 residues. Phosphorylation at these two sites could be induced by TAK1 and TAB1 co-overexpression in the cells. Phosphorylation at Thr-178 and Thr-184 Is Required for TAK1/TAB1-induced IKK-NFκB and JNK-AP-1 Activation—IKKβ plays a critical role in the activation of NFκB, and the phosphorylation of Ser-176 and Ser-180 residues at the activation loop is essential for IKKβ activation by tumor necrosis factor-α and IL-1 (34Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (752) Google Scholar). Overexpression of TAK1 and TAB1 results in IKKβ activation (18Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (520) Google Scholar, 36Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). To test whether dual phosphorylation of both Thr-178 and Thr-184 is required for TAK1/TAB1-mediated phosphorylation of IKKβ at Ser-176 and Ser-180 residues, we transfected the full-length wild-type and mutant TAK1 expression plasmids with HA-tagged IKKβ and TAB1 into TAK1-deficient mouse embryonic fibroblasts, immunoprecipitated HA-tagged IKKβ from the cell lysates with an anti-HA antibody, and immunoblotted with an antibody specific for phospho-IK
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