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Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

2009; Elsevier BV; Volume: 284; Issue: 33 Linguagem: Inglês

10.1074/jbc.m109.007146

1083-351X

Sung Il Kim, Joon Hyeok Kwak, Hee-Jun Na, Jin Kuk Kim, Yan Ding, Mary E. Choi,

Bone Metabolism and Diseases

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI. Members of the transforming growth factor-β (TGF-β) 3The abbreviations used are: TGF-βtransforming growth factor βTAK1TGF-β-activated kinase 1TABTAK1-binding proteinMAPKmitogen-activated protein kinaseMKK3MAPK kinase kinase 3TβRItype I TGF-β receptorTβRIItype II TGF-β receptorTNF-αtumor necrosis factor αTRAFTNF receptor-associated factorsiRNAsmall Interfering RNAJNKc-Jun N-terminal kinaseILinterleukinIRAKIL-1 receptor-associated kinaseXIAPX-linked inhibitor of apoptosisHAhemagglutininMMCmouse mesangial cellFBSfetal bovine serumPBSphosphate-buffered salineWTwild typeIPimmunoprecipitateIBimmunoblotBSAbovine serum albuminKDkinase deficient. superfamily are key regulators of various biological processes such as cellular differentiation, proliferation, apoptosis, and wound healing (1Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar, 2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). 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In embryonic fibroblasts from TAB1 null mice, IL-1 and TNF-α could induce TAK1-mediated NF-κB and JNK activation (45Shim J.H. Xiao C. Paschal A.E. Bailey S.T. Rao P. Hayden M.S. Lee K.Y. Bussey C. Steckel M. Tanaka N. Yamada G. Akira S. Matsumoto K. Ghosh S. Genes Dev. 2005; 19: 2668-2681Crossref PubMed Scopus (597) Google Scholar). TAK1 activation induced by TNF-α, IL-1, and T-cell receptor requires TAB2 or its homologous protein TAB3 (46Takaesu 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 (495) Google Scholar, 47Jiang Z. Ninomiya-Tsuji J. Qian Y. Matsumoto K. Li X. Mol. Cell. Biol. 2002; 22: 7158-7167Crossref PubMed Scopus (238) Google Scholar, 48Ishitani T. Takaesu G. Ninomiya-Tsuji J. Shibuya H. Gaynor R.B. Matsumoto K. EMBO J. 2003; 22: 6277-6288Crossref PubMed Scopus (219) Google Scholar, 49Jin G. Klika A. Callahan M. Faga B. Danzig J. Jiang Z. Li X. Stark G.R. 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On the other hand, it has been demonstrated that hematopoietic progenitor kinase 1 plays a role as an upstream mediator of TGF-β-induced TAK1 activation, which in turn activates the MKK4-JNK signaling cascade in 293T cells (56Wang W. Zhou G. Hu M.C. Yao Z. Tan T.H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 57Zhou G. Lee S.C. Yao Z. Tan T.H. J. Biol. Chem. 1999; 274: 13133-13138Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Besides hematopoietic progenitor kinase 1, it has been also suggested that X-linked inhibitor of apoptosis (XIAP) might link TAK1 to TGF-β/BMP receptors through the capability of XIAP to interact with TGF-β/BMP receptors and TAB1 (58Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (326) Google Scholar). Thus, although various molecules participate in the activation of TAK1, the precise mechanism by which TGF-β1 induces TAK1 activation is incompletely understood. Here, we provide evidence that the association of TAK1 with TGF-β receptors is important for TGF-β1-induced activation of TAK1 in mouse mesangial cells. TGF-β1 stimulation induces interaction of TβRI and TβRII, triggering dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation, independent of receptor kinase activity of TβRI. Recombinant human TGF-β1 was obtained from R&D Systems (Minneapolis, MN). Antibodies against HA (Y-11), His6, TAK1, TβRI, TβRII, and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA). Anti-p-Smad2 and anti-Smad2/3 antibodies were from Upstate Biotechnology (Lake Placid, NY). Anti-FLAG (M2) and anti-Myc (9E10) antibodies were from Sigma. Cy3-conjugated goat anti-rabbit IgG antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody and normal goat serum were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Anti-V5 antibodies and pcDNA3.1, pcDNA3.1/ V5-His TOPO TA expression kit, and Lipofectamine PlusTM reagent were purchased from Invitrogen. Glomerular mesangial cells from male C57BL/6 mice were isolated and characterized as previously described (22Wang L. Ma R. Flavell R.A. Choi M.E. J. Biol. Chem. 2002; 277: 47257-47262Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Primary mouse mesangial cells (MMC) established in culture were maintained in RPMI 1640 medium supplemented with 15% FBS, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. Transfection of expression vectors was performed using Lipofectamine PlusTM reagent (Invitrogen) according to the manufacturer舗s instructions. In brief, cells grown to ∼60% confluence on either 100- or 60-mm dishes were washed with phosphate-buffered saline (PBS) and transfected with 1 μg (for 100-mm dish) or 0.3 μg (for 60-mm dish) of the respective plasmids for 4 h under serum-free conditions. The total amount of DNA was adjusted with empty vector, pcDNA3.1. After transfection, cells were washed with PBS and incubated in medium supplemented with 15% FBS for 16 h before each experiment. Mammalian expression constructs for HA-tagged wild type TAK1 (HA-TAK1) and kinase-deficient mutant TAK1 (HA-TAK1-KD (K63W), FLAG-TAB1, and Myc-TAB1 were kindly provided by K. Matsumoto (Nagoya University) (60Hanada M. Ninomiya-Tsuji J. Komaki K. Ohnishi M. Katsura K. Kanamaru R. Matsumoto K. Tamura S. J. Biol. Chem. 2001; 276: 5753-5759Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). FLAG-tagged wild type TAK1 (FLAG-TAK1), kinase-deficient mutant TAK1 (FLAG-TAK1- KD (K63W)), and C-terminal-truncated mutant TAK1 (FLAG-TAK1ΔC) were kindly provided by G. Gross (42Hoffmann A. Preobrazhenska O. Wodarczyk C. Medler Y. Winkel A. Shahab S. Huylebroeck D. Gross G. Verschueren K. J. Biol. Chem. 2005; 280: 27271-27283Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To amplify respective cDNAs, various PCR primer sets were synthesized according to corresponding DNA sequences (mouse TAK1, BC006665; rat TβRI; NM_012775). TAK1 cDNAs for wild type and kinase-deficient mutant were amplified by using PCR with TAK1 primer set (forward primer, 5′-GGATCCGGGATCATGTCGACAGCCTCCGC-3′; reverse primer, 5′-CGCGGTACCTGAAGTGCCTTGTCGTTTCTG-3′) and re-cloned using pcDNA3.1/V5-His TOPO TA expression kit, and V5/His-TAK1 and V5/His-TAK1-KD were obtained. The phosphorylation site mutant of TAK1 (V5/His-TAK1-TA (T187A)) and polyubiquitination site mutant of TAK1 (V5/His-TAK1-K34R) were produced by PCR-based mutagenesis with mutagenic primer sets (TAK1-T187A forward primer, 5′-CAAACACACATGGCCAATAATAAAG-3′; TAK1-T187A reverse primer, 5′-CTTTATTATTGGTCATGTGTGTTTG-3′; TAK1-K34R forward primer, 5′-CCTGAACTTCGAGGAGATCGACTACAGGGAGATCGAGGTGGAA-3′; TAK1-K34R-reverse primer, 5′-CCACCTCGATCTCCCTGTAGTCGATCTCTCGAAGTTCAGGACC-3′) and the TAK1 primer set. The resulting construct was confirmed by sequencing. HA-tagged wild type TβRI (pCMV5B-HA-TβRI-WT), kinase-deficient mutant TβRI (pCMV5B-HA-TβRI-KD (K232R)), His-tagged TβRII (pCMV5B-His-TβRII-WT), and kinase-deficient TβRII (pCMV5B-His-TβRII-KD (K227R)) were obtained from J. Wrana (62Choi M.E. Am. J. Physiol. 1999; 276: F88-F95Crossref PubMed Google Scholar) through Addgene (Cambridge, MA). V5/His-tagged wild type TβRI (V5/His-ΤβRΙ) was re-cloned in pcDNA3.1/V5-His TOPO TA expression vector after PCR using rat TβRI cDNA as a template (62Choi M.E. Am. J. Physiol. 1999; 276: F88-F95Crossref PubMed Google Scholar, 63Chin B.Y. Mohsenin A Li S.X. Choi A.M. Choi M.E. Am. J. Physiol. Renal Physiol. 2001; 280: F495-F504Crossref PubMed Google Scholar) and RI primer set (RI forward primer, 5′-GCCTCGAGGGGACCATGGAGGCGTCG-3′; RI reverse primer, 5′-CCCAAGCTTCATTTTGATGCCTTCCTGTTG-3′). Various point mutations were introduced into V5/His-tagged TβRI by PCR-based mutagenesis with the RI primer set and respective mutagenic primer set. The constitutively active mutant of rat TβRI (V5/His-TβRI-CA (T198D)) was obtained with the mutagenic primer set (RI-T198D forward primer, 5′-GATATCGTGCTACAAGAAAGC-3′; RI-T198D reverse primer, 5′-GCTTTCTTGTAGCACGATATC-3′). The kinase-deficient mutant of rat TβRI (V5/His-TβRI-KD (K226R)) was produced with the primer set RI-K226R forward primer (5′-GGAATATTCTCTTCTAGAGAAGAA-3′) and RI-K226R reverse primer (5′-TTCTTCTCTAGAAGAGAATATTCC-3′). The GS domain mutant of rat TβRI (V5/His-TβRI-GS) bearing mutations of five phosphorylation sites in the GS domain (T185V, T186V, S187A, S189A, S191A) was produced with the RI-GS mutagenic primer set (RI-GS forward primer, 5′-GTAGTTGCAGGGGCAGGAGCCGGCTTACCACTGCTTGTTCAA-3′; RI-GS reverse primer, 5′-TTGAACAAGCAGTGGTAAGCCGGCTCCTGCCCCTGCAACTAC-3′). Each of the PCR products was re-cloned in pcDNA3.1/V5-His TOPO TA expression vector, and correct clones were confirmed by sequencing. Deletion mutant versions of rat TβRI (V5/His-ΤβRI-LC and V5/His-ΤβRI-SC) were generated by PCR with the RI forward primer and respective reverse primers (RI-LC reverse primer, 5′-CCCAAGCTTTTCATGGCGTAACATTACAGTCTG-3′ or RI-SC reverse primer, 5′-CCCAAGCTTCACGCGGTGGTGAATGACAGTG-3′). V5/His-TβRI-SC (amino acids 1–151) contains only nine amino acids with the partial TRAF6 binding motif after the transmembrane domain, whereas V5/His-TβRI-LC (amino acids 1–255) harbors the juxtamembrane region, GS domain, and a portion of the kinase domain excluding the L45 loop. The construction of rat HA-tagged TβRII and C-terminal-truncated mutant TβRII (HA-TβRII and HA-TβRIIΔC) was previously described (63Chin B.Y. Mohsenin A Li S.X. Choi A.M. Choi M.E. Am. J. Physiol. Renal Physiol. 2001; 280: F495-F504Crossref PubMed Google Scholar). siGENOME SMARTpool targeted against mouse TAB1 (L-042328-00) and siCONTROL Non-Targeting Pool (D-001206-13) were purchased from Dharmacon Inc. Transfection of siRNA using DharmaFECTTM4 reagent was carried out according to the manufacturer舗s instructions. Briefly, cells were grown to 50% confluence on 60-mm dishes in 3 ml of media supplemented with 15% FBS. Twenty microliters of respective siRNA (total 400 μmol) were added to 380 μl of serum-free media and mixed with 400 μl of serum-free media containing 8 μl of DharmaFECTTM4. After 20 min, 3.2 ml of media containing 15% serum were added to prepare siRNA transfection media. For transfection, culture media were replaced with the siRNA transfection media. The final concentration of respective siRNA was 100 nm for each transfection. After transfection, cells were incubated in medium containing 15% FBS for 48 h, then rendered quiescent in medium supplemented with 0.5% FBS for 16 h before each experiment. Cells were washed once with ice-cold PBS and lysed in buffer containing 1% Nonidet P-40, 20 mm Tris (pH 8.0), 150 mm NaCl, 12.5 mm β-glycerophosphate, 1.5 mm MgCl2, 2 mm EGTA, 1 mm NaF, 2 mm dithiothreitol, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, and 20 μm aprotinin. Cells were disrupted by using sonication and then centrifuged for 15 min at 14,000 × g at 4 °C to remove cellular debris. The protein concentration of cell lysates was determined by BCA protein assay reagent kit (Pierce). For Western blotting, protein samples (50–100 μg) were subjected to 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with either 5% nonfat milk or 5% bovine serum albumin (BSA) for 1 h and then incubated with primary antibodies overnight on a rocker at 4 °C. The membranes were washed 3 times (15 min each) with TTBS buffer (10 mm Tris (pH 7.5), 50 mm NaCl, and 0.05% Tween 20) and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature. The target proteins were detected with LumiGLO (Cell Signaling Technologies). In the case of immunoprecipitation experiments, 200–500 μg of cell lysates were pre-cleaned with either normal mouse or rabbit IgG with protein A/G-agarose for 2 h at 4 °C. Cell lysates were transferred to fresh tube and reacted with 2 μg of primary antibodies for 30 min at 4 °C followed by precipitation with 20 μl of protein A/G-agarose for 2 h for proteins overexpressed or overnight for endogenous proteins at 4 °C. The immunoprecipitates were then washed three times with the lysis buffer and subjected to Western blotting. MMC grown to 70% confluence were rendered quiescent in medium supplemented with 0.5% FBS for 16 h, then stimulated with TGF-β1 (2 ng/ml) for the indicated times. Cells were washed 3 times with PBS and then treated with freshly prepared solution containing 2% paraformaldehyde, 0.1% Triton X-100 in PBS for 15 min to fix and permeabiliz