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• W2171342661 abstract "Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFκΒ and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFκΒ and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.AB062111AB062110 Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFκΒ and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFκΒ and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.AB062111AB062110 interleukin-1 Toll-like receptor IL-1 receptor-associated kinase-1 tumor necrosis factor receptor-associated factor 6 transforming growth factor ॆ-activated kinase 1 c-Jun amino-terminal kinase glutathione S-transferase maltose-binding protein mouse embryonic fibroblast forkhead-associated domain dominant negative mitogen-activated kinase kinase II Interleukin-1 (IL-1)1 is a principal cytokine responsible for the induction of mediators that orchestrate immune and inflammatory responses by activating transcription factors NFκB and AP-1 (1Fitzgerald K.A. O'Neill L.A. Microbes Infect. 2000; 2: 933-943Google Scholar). The Toll-like receptors (TLRs) have also recently emerged as critical molecules in the establishment of innate immune and inflammatory responses due to their ability to recognize various pathogen-associated molecules (2Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Google Scholar). Thus, the Toll/IL-1 receptor family members play a critical role in the switch from innate to adaptive immunity. The signal transduction pathways of the Toll/IL-1 receptor family members consist of common intermediates including MyD88, members of IL-1 receptor-associated kinase (IRAK) family, and tumor necrosis factor receptor-associated factor 6 (TRAF6), whereas TLR3 and TLR4 have MyD88-independent pathways (3O'Neill L.A.J. Mol. Cell. 2002; 10: 969-971Google Scholar). Furthermore, the signaling cascade is evolutionarily conserved with that initiated by the Drosophila Toll receptor (2Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Google Scholar). Therefore, the MyD88/IRAK/TRAF6 pathway is an essential protein linkage required for the immune and inflammatory systems. IL-1 signaling is initiated by ligand-induced formation of a receptor complex that consists of the IL-1 receptor and IL-1 receptor accessory protein. Then, the cytosolic protein MyD88 (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 5Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Google Scholar) and Tollip (6Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Google Scholar) are recruited to this complex. MyD88 in turn recruits members of the IRAK family including IRAK-1, IRAK-2, IRAK-M (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Google Scholar, 8Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Google Scholar), and IRAK-4 (9Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Google Scholar) via interaction between their death domains. IRAK-1 is activated presumably via phosphorylation by IRAK-4, leaving the receptor complex to interact with TRAF6. Subsequently, TRAF6 activates transforming growth factor ॆ-activated kinase 1 (TAK1), which activates a number of downstream signaling cascades, including those of IκB kinase, p38, and c-Jun amino-terminal kinase (JNK), leading to the activation of transcription factors such as NFκB and AP-1 (10Ninomiya T.J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Google Scholar). All members of the IRAK family activate NFκΒ when overexpressed in cells. However, only IRAK-4 requires kinase activity to activate NFκΒ, and IRAK-4 phosphorylates IRAK-1 in vitro (9Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Google Scholar). Furthermore, introduction of IRAK-1, IRAK-2, or IRAK-M can restore IL-1-induced NFκΒ activation in IRAK-1-deficient cells, whereas IRAK-4 cannot compensate for the loss of IRAK-1. Thus, IRAK-4 may act upstream of IRAK-1 and phosphorylate IRAK-1 in a stimulation-dependent manner. TRAF6-deficient mice are defective in IL-1 signaling (11Naito A. Azuma S. Tanaka S. Miyazaki T. Takaki S. Takatsu K. Nakao K. Nakamura K. Katsuki M. Yamamoto T. Inoue J. Genes Cells. 1999; 4: 353-362Google Scholar, 12Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Google Scholar). Furthermore, TRAF6 activates TAK1 when TRAF6 was artificially oligomerized without IL-1 stimulation (13Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Google Scholar, 14Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Google Scholar). Although these recent data indicate that IRAK-1 and TRAF6 play pivotal roles in the Toll/IL-1 receptor signaling, the molecular mechanisms underlying the interaction of TRAF6 with IRAK-1 released from the receptor complex and how the IRAK-1·TRAF6 complex activates downstream signals remain to be elucidated. In this study, we identified an adapter protein that is likely to link IRAK-1 to TRAF6 and activates TRAF6 upon IL-1 stimulation. Full-length mouse TRAF6 was subcloned into pBD-GAL4-Cam (Stratagene, La Jolla, CA) and used as bait in a two-hybrid screening of an ICR (8-week-old female) bone marrow cDNA library fused to the activation domain of Gal4 in pAD-GAL4–2.1 (Stratagene). The cDNA encoding human TIFA was identified from a human B-cell cDNA library. Sequence data for mouse, and human TIFA have been submitted to DDBJ/EMBL/GenBankTM databases under accession numberAB062111 and AB062110, respectively. A mouse multiple tissue RNA blot (Clontech, Palo Alto, CA) was incubated with32P-labeled full-length mouse TIFA cDNA and ॆ-actin cDNA at 65 °C. The filter was washed with 0.5× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate), 0.2 (w/v)7 SDS at 65 °C for 30 min. pME-FLAG-TRAF6 and pME-FLAG-TRAF6-(275–530) were described previously (15Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Google Scholar). pME-FLAG-TIFA and pME-Myc-TIFA were constructed by insertion of a DNA fragment encoding FLAG- or Myc-tagged TIFA, respectively, into pME18S (16Shiio Y. Yamamoto T. Yamaguchi N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5206-5210Google Scholar). For expression of GST fusion proteins, a DNA fragment encoding GST-tagged TIFA or a mutant was inserted into pME18S. A cDNA encoding IRAK-1-(1–211) was cloned by PCR and inserted into pME18S. pEF-MyD88 and pEF-MyD88-(152–296) were kindly provided by S. Akira (Osaka University); pEF-TLR4 and pEF-MD-2 were provided by K. Miyake (University of Tokyo); pEF-TAK1, pEF-TAK1(K63W), and pEF-TAB1 were provided by K. Matsumoto (Nagoya University); pEF-IRAK-1 was provided by T. Naka (Osaka University); 3xκB-luc and 3xMκB-luc were provided by S. Miyamoto (University of Wisconsin); pEF-T7-JNK was provided by S. Ohno (Yokohama City University); pEF-Ubc13(C87A) was provided by Z. J. Chen (University of Texas). Anti-TRAF6, anti-IRAK-1, and anti-Myc antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG M2 antibody (Sigma) and anti-T7 antibody (Novagen, Madison, WI) were purchased. Anti-GST, anti-TIFA, and anti-maltose-binding protein (MBP) antibodies were generated by injection of recombinant GST protein or MBP-TIFA fusion protein into rabbits. For the GST pull-down assay, HEK293T cells were cotransfected with pME-GST-TIFA and either pME-FLAG-TRAF6, pME-FLAG-TIFA, or pEF-IRAK-1. Thirty-six hours after transfection, cells were lysed in 500 ॖl of TNE buffer (50 mm Tris-HCl, pH 8.0, 17 Nonidet P-40, 1 mm EDTA, 150 mmNaCl, 0.5 mm dithiothreitol) and centrifuged to remove cellular debris. The resulting supernatant was used as a cell lysate. Cell lysates were incubated with glutathione-Sepharose (Amersham Biosciences), and the GST fusion protein complexes were separated on 8.57 polyacrylamide/SDS gels. For analysis of the IRAK-1·TIFA·TRAF6 complex, HEK293T cells were cotransfected with the indicated combinations of pEF-IRAK-1, pME-Myc-TIFA, and pME-FLAG-TRAF6. Cell lysates were subjected to immunoprecipitation by the addition of anti-FLAG antibody and protein G-Sepharose (Amersham Biosciences). The resulting immunoprecipitates were separated on 107 or 12.57 polyacrylamide/SDS gels. Cell lysates were also used to analyze the expression level of each protein. Establishment of mouse embryonic fibroblasts (MEFs) and TRAF6-deficient MEFs and introduction of FLAG-TIFA into MEFs by retrovirus vector were performed as described (17Kobayashi N. Kadono Y. Naito A. Matsumoto K. Yamamoto T. Tanaka S. Inoue J. EMBO J. 2001; 20: 1271-1280Google Scholar). Mouse 70Z and MEF cells were either untreated or treated with IL-1 (20 ng/ml) for 5 min. Cell lysates were subjected to immunoprecipitation with 1 ॖg of the appropriate antibody and 20 ॖl of protein G-Sepharose (Amersham Biosciences). Immunoprecipitates or whole-cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were immunoblotted with specific antibodies and visualized with appropriate horseradish peroxidase-conjugated second antibody and the ECL Western blotting system (Amersham Biosciences). MBP-tagged TIFA protein was expressed inEscherichia coli BL21 and affinity purified with amylose resin (New England BioLabs, Beverly, MA). MBP-TIFA (24 ॖg) was cleaved with 2.4 units of PreScission Protease (Amersham Biosciences) at 4 °C for 4 h. Fifty microliters of the reaction mixture was applied to a Superdex 75 column (0.32 × 30 cm), and fractions (20 ॖl) were collected. The fractions were analyzed on 107 polyacrylamide-SDS gel, and proteins were visualized by Coomassie Brilliant Blue Staining. Molecular weight marker proteins were analyzed under the column conditions described above. HEK293T cells were transfected with 1 ng of 3xκB-luc or 3xMκB-luc (mutant κB sites), 10 ng of ॆ-actin-ॆ-galactosidase and the indicated amounts of various expression plasmids. Thirty-six hours after transfection, luciferase activity was measured with the PicaGene luciferase assay system (TOYO INK, Tokyo), and ॆ-galactosidase activity was used to standardize transfection efficiency. For thein vitro JNK kinase assay, HEK293T cells were transfected with 10 ng of pEF-T7-JNK and the indicated amounts of pME-FLAG-TIFA. Thirty-six hours after transfection, His-JNK was immunoprecipitated with anti-T7 antibody. Immunoprecipitates were incubated with 2 ॖg of GST-c-JUN (1–89) fusion protein (New England BioLabs) in 20 ॖl of kinase buffer (20 mm HEPES, pH 7.5, 20 mmMgCl2, 5 ॖCi of [γ-32P]ATP (3000 Ci/mmol)) at 30 °C for 30 min. To identify the protein(s) that regulate association of IRAK-1 with TRAF6, we performed yeast two-hybrid screening of a mouse bone marrow cDNA library using murine TRAF6 as bait. We identified a cDNA encoding a 184-amino acid protein, identical to T2BP previously identified as a TRAF2-binding protein, that activates NFκB and JNK in transient transfection assays (Ref. 18Kanamori M. Suzuki H. Saito R. Muramatsu M. Hayashizaki Y. Biochem. Biophys. Res. Commun. 2002; 290: 1108-1113Google Scholar and data not shown). Because the present study showed that T2BP associates with TRAF6 and a forkhead-associated (FHA) domain of the T2BP (Fig.1A) is likely to play a critical role in the signaling (see 舠Discussion舡), we designated T2BP as a TRAF-interactingprotein with an FHA domain, TIFA. TIFA mRNA was expressed highly in spleen and moderately in other tissues, whereas it was not detectable in skeletal muscle (Fig.1B). FHA domains are conserved sequences of 60–100 amino acids found mainly in eukaryotic nuclear proteins (19Hofmann K. Bucher P. Trends Biochem. Sci. 1995; 20: 347-349Google Scholar) that participate in establishing or maintaining cell-cycle checkpoints, DNA repair, or transcriptional regulation (20Li J. Lee G.I. Van Doren S.R. Walker J.C. J. Cell Sci. 2000; 113: 4143-4149Google Scholar). More importantly, some of them were shown to bind directly to phosphoserine/phosphothreonine (Ser(P)/Thr(P)) residues (21Sun Z. Hsiao J. Fay D.S. Stern D.F. Science. 1998; 281: 272-274Google Scholar, 22Li J. Smith G.P. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7821-7826Google Scholar, 23Durocher D. Henckel J. Fersht A.R. Jackson S.P. Mol. Cell. 1999; 4: 387-394Google Scholar, 24Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. Mol. Cell. 2000; 6: 1169-1182Google Scholar) in the same way that SH2 domains interact with phosphotyrosine residues (20Li J. Lee G.I. Van Doren S.R. Walker J.C. J. Cell Sci. 2000; 113: 4143-4149Google Scholar, 25Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Google Scholar). To determine whether interaction of TIFA with TRAF6 is required for TIFA-induced NFκΒ and JNK activation, we identified the TRAF6 binding site in TIFA. Wild-type TIFA and various mutants were expressed as GST fusion proteins in HEK293T cells (Fig. 1C) in conjunction with FLAG-TRAF6. Subsequent GST pull-down assays revealed that the carboxyl-terminal portion of TIFA (C domain) is sufficient for TRAF6 binding and that the amino-terminal portion (N domain) and the FHA domain are not necessary (Fig. 1D). Recent analysis of the crystal structure of the TRAF-C domain of TRAF6 in complex with TRAF6-binding peptides from CD40 and RANK revealed that eight amino acid residues of the TRAF6-binding peptide contact the TRAF-C domain of TRAF6 and identifiedXXPXEXX-(aromatic/acidic) as a consensus sequence for TRAF6 binding (26Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevede N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Google Scholar) (Fig. 1C). Substitution of Ala for Glu at the fifth amino acid position of the TRAF6 binding site in CD40 abolished binding to TRAF6 (27Pullen S.S. Miller H.G. Everdeen D.S. Dang T.T.A. Crute J.J. Kehry M.R. Biochemistry. 1998; 37: 11836-11845Google Scholar, 28Tsukamoto N. Kobayashi N. Azuma S. Yamamoto T. Inoue J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1234-1239Google Scholar). A consensus TRAF6 binding motif is present in the C domain of both human and mouse TIFA (Fig. 1C). Substitution of Ala for Glu-178 (E178A) abolished binding of TIFA to TRAF6 (Fig. 1D), which is consistent with the notion that the TRAF domain of TRAF6 is responsible for binding to both CD40 (15Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Google Scholar) and TIFA (data not shown). We then analyzed the ability of various TIFA mutants to activate NFκB and JNK. Both the FHA and the C domain were required, whereas the N domain was not necessary for NFκB activation (Fig.2A). GST-E178A did not activate NFκB or JNK (Fig. 2, A and B), indicating that interaction of TIFA with TRAF6 is essential for TIFA-mediated NFκΒ and JNK activation. To clarify the role of the FHA domain of TIFA in NFκΒ and JNK activation, Gly-50 and Ser-66 were replaced with Glu and Ala, respectively (G50ES66A, Fig. 1,A and C), since identical mutations in the FHA domain of the Arabidopsis kinase-associated protein phosphatase abolished its binding to the phosphorylated receptor-like protein kinase (22Li J. Smith G.P. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7821-7826Google Scholar). Interestingly, GST-G50ES66A, which binds to TRAF6 (Fig. 1D), did not activate NFκB or JNK (Fig. 2,A and B), indicating that interaction of TIFA with TRAF6 is necessary but not sufficient for activation. Thus, the FHA domain of TIFA plays a critical role in the NFκB and JNK activation. TRAF6 transduces signals when oligomerized (13Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Google Scholar,14Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Google Scholar). Because a part of the expressed TIFA is phosphorylated (data not shown), homo-oligomerization of TIFA via the FHA domain/phosphopeptide interaction may facilitate or induce oligomerization of TRAF6. To address whether TIFA forms a homo-oligomer, the binding of FLAG-TIFA to various GST-TIFA mutants was analyzed (Fig.3A). GST-TIFA, but not GST, associated with FLAG-TIFA, indicating that TIFA forms a homo-oligomer. Both the FHA domain and the C domain were required, whereas the N domain was not necessary for the TIFA self-association. The structural requirement for the self-association is similar to that for NFκB and JNK activation, suggesting that the self-association of TIFA may be a prerequisite for activation of NFκB and JNK. However, GST-G50ES66A associated with FLAG-G50ES66A, suggesting that the FHA domain/phosphopeptide interaction is not likely to be involved in the self-association. More importantly, because the G50ES66A mutant does not activate downstream signals, homo-oligomer of wild-type TIFA and that of the G50ES66A mutant could have different physical properties. To address this question, recombinant TIFA was generated as an MBP-tagged protein with a cleavage site for human rhinovirus type-14 3C protease (29Walker P.A. Leong L.E. Ng P.W. Tan S.H. Waller S. Murphy D. Porter A.G. Biotechnology. 1994; 12: 601-605Google Scholar) at the junction (MBP-TIFA). Purified MBP-TIFA was digested almost completely by a fusion protein of GST with the 3C protease (GST-3C), and the reaction mixture was analyzed by Superdex 75 gel filtration chromatography (Fig. 3B, upper). According to the elution profile of the marker proteins, MBP (43 kDa) was present as a monomer, and GST-3C (46 kDa) formed a dimer. TIFA (24 kDa) was eluted at around fraction 14, which corresponds to TIFA trimer. However, when recombinant TIFA-G50ES66A protein was subjected to a similar experiment, the mutant protein was eluted at around fraction 7, which corresponds to TIFA pentamer or hexamer (Fig3B, lower). These results indicate that introduction of the G50ES66A mutation result in loss of the ability to form a trimer, presumably by altering the conformation of the FHA and the C domains, which are responsible for the self-association. Taken together, the self-association of TIFA may be required for activation of downstream signals, but the size or the conformation of homo-oligomer could be important. We next addressed whether TIFA interacts with IRAK-1. Coexpression of GST-TIFA with IRAK-1 followed by GST pull-down assay revealed that TIFA associates with IRAK-1 (Fig.4A). Both GST-G50ES66A and GST-E178A bind to IRAK-1 as efficiently as wild-type TIFA, indicating that the IRAK-1/TIFA interaction is not mediated by recognition of the phosphopeptide by the FHA domain. Because TIFA binds both TRAF6 and IRAK-1, we hypothesized that TIFA may affect the IRAK-1/TRAF6 interaction. To test this, the interaction between TRAF6 and IRAK-1 was analyzed in both the absence and presence of increasing expression of TIFA. Transfection experiments were carried out using 0.2 ॖg of TRAF6 and IRAK-1 expression vectors to minimize the IRAK-1/TRAF6 interaction in the absence of TIFA expression. The amount of IRAK-1 coprecipitated with TRAF6 was increased dramatically at the optimal expression of TIFA (Fig. 4B), indicating that TIFA, when overexpressed, can link TRAF6 to IRAK-1. For investigation of the physiological significance of the IRAK-1·TIFA·TRAF6 complex, the interactions of endogenous proteins were analyzed. For identification of endogenous TIFA protein, rabbit anti-TIFA polyclonal antibody was generated. When lysates prepared from 70Z mouse pre-B cells were subjected to immunoprecipitation followed by Western blotting with anti-TIFA antibody, two bands at ∼24 kDa that comigrated with protein expressed from TIFA cDNA in HEK293T cells were identified (Fig. 5A,left four lanes). These two bands were not observed when control IgG was used for immunoprecipitation, indicating that they represent endogenous TIFA. We next addressed whether TIFA interacts with TRAF6. Endogenous TRAF6 was immunoprecipitated from either unstimulated 70Z cells or from cells stimulated with IL-1 for 5 min, and coprecipitated TIFA was analyzed by Western blotting with anti-TIFA antibody. Similar amounts of TIFA were detected irrespective of IL-1 stimulation (Fig. 5A, right two lanes), indicating that TIFA interacts with TRAF6 constitutively in the 70Z pre-B cell line. To determine whether TIFA interacts with TRAF6 in a different cell type, wild-type MEF and TRAF6-deficient MEF (17Kobayashi N. Kadono Y. Naito A. Matsumoto K. Yamamoto T. Tanaka S. Inoue J. EMBO J. 2001; 20: 1271-1280Google Scholar) were analyzed. Because the reactivity of anti-TIFA antibody was relatively weak, wild-type MEF and TRAF6−/− MEF expressing FLAG-TIFA were generated by retrovirus vector-mediated gene transfer. To avoid overexpression of FLAG-TIFA, infection with retrovirus carrying FLAG-TIFA and a puromycin resistance gene was performed with a low titer of virus followed by puromycin selection. The amount of FLAG-TIFA expressed in wild-type MEF and TRAF6−/− MEF was ∼3 times that of endogenous TIFA protein in MEF and, because of this low expression of FLAG-TIFA, the basal activity of NFκΒ and JNK in MEF was not affected (data not shown). Thus, we may characterize the FLAG-TIFA as endogenous TIFA. When lysates from wild-type or TRAF6−/− MEF were subjected to immunoprecipitation with anti-TRAF6 antibody followed by Western blotting with anti-FLAG antibody, TIFA was precipitated only in the presence of TRAF6 (Fig.5B). Therefore, anti-TRAF6 antibody did not immunoprecipitate TIFA directly due to its cross-reactivity. From these data, we conclude that TIFA binds TRAF6 constitutively both in 70Z pre-B cells and in MEF in vivo. We next investigated the interaction of TIFA and IRAK-1 in vivo. When FLAG-TIFA was immunoprecipitated with anti-FLAG antibody from wild-type MEF in the absence or presence of IL-1 stimulation for 5 min followed by Western blotting with anti-IRAK-1 antibody, phosphorylated IRAK-1 was coprecipitated only in the presence of IL-1 stimulation (Fig. 5C). Because activated phospho-IRAK-1 is reported to be released from the receptor-MyD88 complex (5Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Google Scholar), it is possible that IRAK-1 associated with TIFA after release from the receptor complex, consistent with the previous finding that only activated phospho-IRAK-1 is coprecipitated with TRAF6 upon IL-1 stimulation (30Kobayashi K. Hernandez L.D. Galan J.E. Janeway Jr., C.A. Medzhitov R. Flavell R.A. Cell. 2002; 110: 191-202Google Scholar). IRAK-1 was not precipitated from MEFs infected with control virus. These results clearly indicate that TIFA interacts with IRAK-1 in an IL-1 signal-dependent manner in vivo and suggest that TIFA links TRAF6 to IRAK-1 upon IL-1 stimulation. To further investigate the notion that TIFA functions by interacting with IRAK-1 and TRAF6, the effect of a dominant-negative mutant (DN) of TIFA and DN of previously identified proteins involved in IL-1 signaling on NFκB-mediated transcription was analyzed. GST-G50ES66A suppressed IL-1- and TLR4-induced NFκB activation but not TNFα-induced activation (Fig. 6A), suggesting that TIFA functions in TRAF6-mediated signaling. MyD88- and IRAK-1-induced NFκB activation was also suppressed by GST-G50ES66A, whereas TAK1/TAB1-induced activation (10Ninomiya T.J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Google Scholar) was not affected (Fig.6B, left, luciferase activity without GST-G50ES66A was set to 100). Unexpectedly, TRAF6-induced NFκB activation was further augmented by GST-G50ES66A (Fig. 6B,right, luciferase activity with reporter alone was set to 1), which binds to TRAF6 but does not activate NFκΒ without expressing exogenous TRAF6. Transfection of a suboptimal dose of TRAF6 leads to the activation of NFκΒ to some extent, suggesting that exogenously expressed TRAF6 can be partially activated presumably by its conformational change or by its oligomerization under this transient transfection condition. GST-G50ES66A may be able to activate partially activated exogenous TRAF6. TIFA-induced NFκB activation was blocked by TRAF6-DN and TAK1-DN (Fig. 6C). An inactive form of Ubc13 (Ubc13(C87A)) also suppressed TIFA-induced activation (Fig. 6C), suggesting that the TIFA signal leads to Lys-63-linked polyubiquitination of TRAF6, which is required for TAK1-mediated activation of IκB kinase and MKK6 (13Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Google Scholar, 31Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Google Scholar). TIFA-induced activation was not affected by MyD88-DN, but it was blocked by IRAK-1-DN (Fig. 6C). Because TIFA binds IRAK-1 when overexpressed (Fig. 4A), it is possible that IRAK-1-DN inhibited TIFA-mediated NFκΒ activation by interfering with the activation process of TIFA even if IRAK-1 functions upstream of TIFA in vivo. Taken together, these findings support the notion that TIFA links TRAF6 to IRAK-1. We report the identification of TIFA as a TRAF6-binding protein that can mediate IL-1 signaling. In transient transfection assays, TIFA binds both TRAF6 and IRAK-1. Furthermore, IRAK-1 is efficiently coprecipitated with TRAF6 under conditions of optimal TIFA expression. Thus, TIFA, when overexpressed, can link TRAF6 to IRAK-1 in the absence of IL-1 stimulation. By analyzing the interaction of endogenous TIFA with TRAF6 in two different types of cells, we showed that TIFA can bind TRAF6 irrespective of IL-1 stimulation. In contrast, interaction of TIFA and IRAK-1 is dependent on IL-1 stimulation in vivo. These results strongly suggest that TIFA links TRAF6 to IRAK-1 in an IL-1 stimulation-dependent manner in vivo. TIFA may play a similar role in the signaling from members of the TLR family, because the TLR signal transduction pathways are also mediated by IRAK-1 and TRAF6 (3O'Neill L.A.J. Mol. Cell. 2002; 10: 969-971Google Scholar, 32Kawai T. Takeuchi O. Fujita T. Inoue J. Mühlradt P.F. Sato S. Hoshino K. Akira S. J. Immunol. 2001; 167: 5887-5894Google Scholar). In fact, TLR4-mediated NFκB activation was inhibited by expression of the DN of TIFA. Thus, TIFA may function in innate immunity. Although T2BP, identical to TIFA, was shown to associate with TRAF2, when they overexpressed, the T2BP/TRAF2 interaction in vivo has never been demonstrated (18Kanamori M. Suzuki H. Saito R. Muramatsu M. Hayashizaki Y. Biochem. Biophys. Res. Commun. 2002; 290: 1108-1113Google Scholar). Nevertheless, TIFA may regulate both TRAF2 and TRAF6 in vivo. The association of signal transducers including kinases and adapter proteins is largely regulated through protein phosphorylation, allowing dissociation as the balance shifts from kinase to phosphatase activity. Phosphorylation of proteins on serine and threonine residues has traditionally been recognized as a way to regulate enzyme conformation. Recent studies have revealed that serine/threonine phosphorylation can play critical roles in the formation of multimolecular signaling complexes through specific interaction of phosphorylated peptides and Ser(P)/Thr(P) binding modules including FHA domains, 14-3-3, WW domains, and WD40 and leucine-rich repeat domains (25Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Google Scholar). Among the identified FHA domain-containing proteins, a specific target protein has been determined for some. In each case, phosphorylation-dependent interaction of the FHA domain with the target protein is thought to be responsible for the physiologically significant regulation of cell function (20Li J. Lee G.I. Van Doren S.R. Walker J.C. J. Cell Sci. 2000; 113: 4143-4149Google Scholar). The FHA2 domain of Rad53p, a protein kinase involved in the DNA damage response and in cell cycle arrest in Saccharomyces cerevisiae, binds Rad9p when Rad9p is phosphorylated by DNA damage signals. Mutation of the FHA2 domain of Rad53p abolishes DNA damage-induced G2/M cell cycle arrest, indicating the biological relevance of the Rad53p-Rad9p interaction (21Sun Z. Hsiao J. Fay D.S. Stern D.F. Science. 1998; 281: 272-274Google Scholar). In addition, the FHA domain ofArabidopsis kinase-associated protein phosphatase interacts with a plasma membrane-integrated receptor, leucine-rich repeat receptor-like protein kinase, when receptor-like protein kinase is autophosphorylated upon ligand binding (22Li J. Smith G.P. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7821-7826Google Scholar). Receptor-like protein kinase is a product of the CLAVATA1 (CLV1) gene, mutations that result in plants with enlarged shoots and floral meristems (33Clark S.E. Williams R.W. Meyerowitz E.M. Cell. 1997; 89: 575-585Google Scholar). These observations and the fact that the G50ES66A mutant of TIFA is not able to activate NFκΒ or JNK suggest that a specific target protein for the FHA domain of TIFA may exist and play a role in the regulation of TIFA-mediated signaling. In this study, we identified three TIFA-binding proteins including TRAF6, IRAK-1, and TIFA itself. However, they were able to bind the G50ES66A TIFA mutant as well as wild-type TIFA, which suggests that none of them is a candidate protein that binds to the FHA domain in a phosphorylation-dependent manner. Because TRAF6 transduces signals when oligomerized (13Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Google Scholar, 14Baud V. Liu Z.G. Bennett B. Suzuki N. Xia Y. Karin M. Genes Dev. 1999; 13: 1297-1308Google Scholar) and recombinant TIFA can form trimers, one may postulate that TIFA is involved in the oligomerization of TRAF6 in concert with IRAK-1 to activate NFκΒ and JNK. Interestingly, recombinant TIFA G50ES66A mutant formed a pentamer or hexamer, not a trimer. Moreover, the G50ES66A mutant is able to activate NFκB in the presence of the suboptimal dose of TRAF6 but not in concert with endogenous TRAF6 in the absence of exogenous TRAF6. Given that transient transfection of the suboptimal dose of TRAF6 results in the partial activation of TRAF6, the intact structure of the FHA domain of TIFA may be required for the initial step of the TRAF6 activation that may require specific oligomer formation of TRAF6. Further studies are required to clarify exact roles of the FHA domain of TIFA; that is, binding to the putative phosphoprotein, holding the TIFA protein in an active conformation, or some other functions. Whether TIFA is essential for the IRAK-1/TRAF6 interaction or whether it stabilizes the interaction is not clear. One interesting possibility is that TIFA may augment the signal strength. IRAK-1 contains three consensus TRAF6 binding sites (26Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevede N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Google Scholar), and TIFA contains a single consensus TRAF6 binding site. Because TIFA and IRAK-1 interacts upon IL-1 stimulation, the number of TRAF6 molecules involved in signaling in cells abundantly expressing TIFA could be higher than that in cells with less TIFA expression. Furthermore, IRAK-1/TIFA interaction may induce their structural changes and increase their affinity for TRAF6. It is possible that the FHA domain of TIFA may bind to the substrate of IRAK-1 or IRAK-4, which leads to the stabilization of IRAK-1·TIFA·TRAF6 complex. It has been reported that kinase activity of IRAK-1 is not required for IL-1 signaling in a human embryonic kidney 293 cell line (34Takaesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Google Scholar). Perhaps it can affect signal strength in other specific cell types. In this sense, it is interesting that TIFA is highly expressed in spleen but scarcely expressed in skeletal muscle. During the preparation of our manuscript, Pellino 1 was reported to be associated with IRAK-1·IRAK-4·TRAF6 complex in an IL-1 signal-dependent manner (35Jiang Z. Johnson H.J. Nie H. Qin J. Bird T.A. Li X. J. Biol. Chem. 2003; 278: 10952-10956Google Scholar). Thus, Pellino 1 and TIFA may have similar roles. However, they have distinct tissue specificities; Pellino 1 is weakly expressed in spleen but significantly expressed in skeletal muscle. TRAF6 is also involved in mediating signals from members of the TNF receptor superfamily, including CD40, RANK, X-linked ectodysplatin-A2 receptor (XEDAR), and p75 neutrophin nerve growth factor receptor (11Naito A. Azuma S. Tanaka S. Miyazaki T. Takaki S. Takatsu K. Nakao K. Nakamura K. Katsuki M. Yamamoto T. Inoue J. Genes Cells. 1999; 4: 353-362Google Scholar,12Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Google Scholar, 36Naito A. Yoshida H. Nishioka E. Satoh M. Azuma S. Yamamoto T. Nishikawa S. Inoue J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8766-8771Google Scholar, 37Khursigara G. Orlinick J.R. Chao M.V. J. Biol. Chem. 1999; 274: 2597-2600Google Scholar). Although a recent study showed that the p75 nerve growth factor receptor recruits IRAK-1 upon ligand stimulation (38Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Google Scholar), other receptors are thought to form trimers and do not require IRAK-1 (39Inoue J. Ishida T. Tsukamoto N. Kobayashi N. Naito A. Azuma S. Yamamoto T. Exp. Cell Res. 2000; 254: 14-24Google Scholar). In our preliminary experiments, TIFA did not bind the cytoplasmic tail of CD40 or RANK, suggesting that TIFA may not be required for oligomerization of TRAF6 to transduce signals from members of the TNF receptor superfamily with the exception of the p75 nerve growth factor receptor. TIFA may regulate signals that are mediated by both TRAF6 and IRAK-1. To elucidate the molecular mechanism underlying TIFA signaling, especially to identify a role of the FHA domain, crystallographic studies of TIFA and identification of a target protein of the FHA domain are required. Further studies of TIFA may reveal a novel regulation of inflammation and innate immunity. We thank Z. J. Chen, S. Miyamoto, K. Miyake, S. Akira, K. Matsumoto, T. Naka, and S. Ohno for providing reagents. We thank H. Hayashi, R. Ajima, and T. Yamamoto for technical support and valuable discussions. We are grateful to Z. J. Chen and S. Miyamoto for critical reading of the manuscript." @default.
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• W2171342661 title "Identification of TIFA as an Adapter Protein That Links Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) to Interleukin-1 (IL-1) Receptor-associated Kinase-1 (IRAK-1) in IL-1 Receptor Signaling" @default.
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