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TIRP, a Novel Toll/Interleukin-1 receptor (TIR) Domain-containing Adapter Protein Involved in TIR Signaling

2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês

10.1074/jbc.m303451200

ISSN

1083-351X

Autores

Lianghua Bin, Liang‐Guo Xu, Hong‐Bing Shu,

Tópico(s)

Neutrophil, Myeloperoxidase and Oxidative Mechanisms

Resumo

The Toll/interleukin-1 receptor (TIR) family members play important roles in host defense. These receptors signal through TIR domain-containing adapter proteins. In this report, we identified a novel TIR domain-containing adapter protein designated as TIRP. Co-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors. TIRP also interacts with kinase-inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-M, and TRAF6. Overexpression of TIRP activates NF-κB and potentiates IL-1 receptor-mediated NF-κB activation. A dominant negative mutant of TIRP inhibits IL-1- but not tumor necrosis factor-triggered NF-κB activation. Moreover, TIRP-mediated NF-κB activation is inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKβ. Our findings suggest that TIRP is involved in IL-1-triggered NF-κB activation and functions upstream of IRAK, IRAK-2, TRAF6, and IKKβ The Toll/interleukin-1 receptor (TIR) family members play important roles in host defense. These receptors signal through TIR domain-containing adapter proteins. In this report, we identified a novel TIR domain-containing adapter protein designated as TIRP. Co-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors. TIRP also interacts with kinase-inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-M, and TRAF6. Overexpression of TIRP activates NF-κB and potentiates IL-1 receptor-mediated NF-κB activation. A dominant negative mutant of TIRP inhibits IL-1- but not tumor necrosis factor-triggered NF-κB activation. Moreover, TIRP-mediated NF-κB activation is inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKβ. Our findings suggest that TIRP is involved in IL-1-triggered NF-κB activation and functions upstream of IRAK, IRAK-2, TRAF6, and IKKβ The Toll/interleukin-1 receptor (TIR) 1The abbreviations used are: TIR, Toll/interleukin-1 receptor; TIRP, TIR domain-containing protein; TLR, Toll-like receptor; IRAK, interleukin-1 receptor-associated kinase; TRIF, TIR domain-containing adapter-inducing IFN-β; JNK, c-Jun NH2-terminal kinase; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; IKK, IκB kinase; IFN, interferon; HA, hemagglutinin; CMV, cytomegalovirus; RSV, Rous sarcoma virus; AcP, accessary protein; TIRAP, TIR domain-containing adapter protein; MAL, MyD88-adapter-like. 1The abbreviations used are: TIR, Toll/interleukin-1 receptor; TIRP, TIR domain-containing protein; TLR, Toll-like receptor; IRAK, interleukin-1 receptor-associated kinase; TRIF, TIR domain-containing adapter-inducing IFN-β; JNK, c-Jun NH2-terminal kinase; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; IKK, IκB kinase; IFN, interferon; HA, hemagglutinin; CMV, cytomegalovirus; RSV, Rous sarcoma virus; AcP, accessary protein; TIRAP, TIR domain-containing adapter protein; MAL, MyD88-adapter-like. family members are evolutionary conserved proteins that are critically involved in host defense from plants to humans (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). The TIR family is divided into two subfamilies, the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) subfamilies. In mammals, the IL-1R subfamily members are important mediators of inflammation and adaptive immune responses, whereas the TLR subfamily members recognize microbial products termed PAMPs (pathogen-associated molecular patterns) and are critical components of the innate immune system (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). The IL-1R and TLR subfamilies are distinguished through structural divergence in the extracellular domains. The IL-1R subfamily members contain immunoglobulin-like motifs, whereas the TLR subfamily members contain leucine-rich repeats. However, both subfamily members share a conserved cytoplasmic domain, the TIR domain (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). Various studies have shown that the TIR domain is required for TIR family member-mediated signaling that leads to activation of transcription factors NF-κB, activating protein-1, and ATF-2 and subsequent induction of various chemokines and cytokines that are involved in host defense against the pathogens (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). The signaling pathways initiated by IL-1 receptors have become a paradigm on how other TIR family receptors signal. Upon IL-1 stimulation, IL-1R1 forms a complex with IL-1RAcP, a co-receptor (6Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Google Scholar, 7Wesche H. Korherr C. Kracht M. Falk W. Resch K. Martin M.U. J. Biol. Chem. 1997; 272: 7727-7731Google Scholar, 8Huang J. Gao X. Li S. Cao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12829-12832Google Scholar, 9Cullinan E.B. Kwee L. Nunes P. Shuster D.J. Ju G. McIntyre K.W. Chizzonite R.A. Labow M.A. J. Immunol. 1998; 161: 5614-5620Google Scholar). This leads to recruitment of the adapter protein MyD88 to the receptor signaling complex (10Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 11Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Google Scholar, 12Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. DiMarco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Google Scholar). MyD88, in turn, recruits the serine/threonine kinase IRAK (11Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Google Scholar). Once IRAK is recruited to the receptor complex, it is activated and then dissociated from the receptor complex (13Cao Z. Yiong J. Takeuchi M. Kurama T. Goeddel D. Nature. 1996; 383: 443-446Google Scholar). Dissociated IRAK interacts with TRAF6, which then activates NF-κB through a kinase cascade containing TAK1 and IKK (13Cao Z. Yiong J. Takeuchi M. Kurama T. Goeddel D. Nature. 1996; 383: 443-446Google Scholar, 14Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Google Scholar, 15Takesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 7: 2475-2484Google Scholar, 16Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Google Scholar). In addition to IL-1 receptor signaling, MyD88 is also involved in signaling by TLRs (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). Gene knock-out studies have suggested that MyD88 is required for cytokine induction triggered by a variety of ligands that signal through the TIR family members, such as IL-1β, IL-18, lipopolysaccharide, MALP-2, CpG-DNA, and poly(I·C) (17Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Google Scholar). However, it has been shown that poly(I·C) and lipopolysaccharide-induced NF-κB and JNK activation was delayed but not abolished in MyD88–/– cells. In addition, poly(I·C) and lipopolysaccharide still triggered dendritic cell maturation (17Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Google Scholar). These studies suggest that MyD88-independent pathways exist. Recently, two additional TIR domain-containing adapter molecules, TIRAP/Mal and TRIF/TICAM, were identified (18Horng T. Barton G. Medzhitov R. Nature Immunol. 2001; 2: 835-841Google Scholar, 19Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Google Scholar, 20Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Google Scholar, 21Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nature Immunol. 2003; 4: 161-167Google Scholar). TIRAP is associated with TLR4. A dominant negative mutant of TIRAP inhibits NF-κB activation triggered by TLR4 but not by TLR9 or IL-1R (18Horng T. Barton G. Medzhitov R. Nature Immunol. 2001; 2: 835-841Google Scholar, 19Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Google Scholar). Furthermore, gene knock-out studies have indicated that TIRAP is essential for lipopolysaccharide-induced cytokine production, suggesting that TIRAP is required for TLR4 signaling (22Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Google Scholar, 23Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Google Scholar). It has also been shown that TIRAP is involved in signaling triggered by ligands for TLR1, TRL2, and TLR6, but not by IL-1 and IL-18, and ligands for TLR3, TLR7, and TLR9 (22Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Google Scholar, 23Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Google Scholar). TRIF is the third identified TIR domain-containing adapter molecule. In addition to NF-κB, TRIF also activates IFN-β promoter (20Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Google Scholar, 21Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nature Immunol. 2003; 4: 161-167Google Scholar). A dominant negative mutant of TRIF inhibits TLR2, TLR3, TLR4, and TLR7-mediated NF-κB activation and TLR3-mediated activation of IFN-β promoter (20Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Google Scholar, 21Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nature Immunol. 2003; 4: 161-167Google Scholar). So far, TRIF is the only TIR-containing adapter protein that is implied in TLR3-mediated production of IFN-β, suggesting that TRIF is involved in TLR3-activating NF-κB as well as IFN-β (20Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Google Scholar, 21Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nature Immunol. 2003; 4: 161-167Google Scholar). Similar with the TIR domain-containing adapter proteins, four IRAK-like molecules have been identified (24Suzuki N. Suzuki S. Yeh W.C. Trends Immunol. 2002; 23: 503-506Google Scholar). Although IRAK was firstly identified as a component of the IL-1R signaling complex and later shown to be recruited to the TLR signaling complexes upon ligand stimulation, gene knock-out studies have indicated that IRAK deficiency has a relatively mild effect on IL-1R and TLR signaling (25Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; 163: 978-984Google Scholar, 26Kanakaraj P. Ngo K. Wu Y. Angulo A. Ghazal P. Harris C.A. Siekierka J.J. Peterson P.A. Fung-Leung W.P. J. Exp. Med. 1999; 189: 1129-1138Google Scholar). It has been shown that the kinase activity of IRAK is dispensable for IRAK function as a mediator of TIR-triggered NF-κB activation (27Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Google Scholar). IRAK-2 and IRAK-M, two other members of the IRAK family, have no kinase activity but activate NF-κB when overexpressed in 293 cells and partially restore IL-1 signaling in IRAK–/– cells (10Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 28Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Google Scholar, 29Kobayashi K. Hernandez L.D. Galan J.E. Janeway Jr., C.A. Medzhitov R. Flavell R.A. Cell. 2002; 110: 191-202Google Scholar). IRAK-4, the fourth member of the IRAK family, has unique features that distinguish it from all of the other IRAK proteins. First, overexpression of IRAK-4 does not result in robust NF-κB activation. Second, expression of a kinase-inactive mutant of IRAK-4 is sufficient to inhibit IL-1-mediated NF-κB activation in cells (30Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Google Scholar). By contrast, corresponding point mutations of IRAK, IRAK-2, or IRAK-M do not have the same dominant negative effects (10Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 28Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Google Scholar, 31Cao Z. Henzel S.J. Gao X. Science. 1996; 271: 1128-1131Google Scholar). Third, IRAK is a direct substrate of IRAK-4, but IRAK-4 can not be phosphorylated by IRAK (30Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Google Scholar). Finally, gene knock-out studies indicate that IRAK-4 is required for signaling triggered by IL-1R and TLR engagement (32Suzuki N. Suzuki S. Duncan G.S. Millar D.G. Wada T. Mirtsos C. Takada H. Wakeham A. Itie A. Li S. Penninger J.M. Wesche H. Ohashi P.S. Mak T.W. Yeh W.C. Nature. 2002; 416: 750-756Google Scholar). These results suggest that IRAK-4, as well as its kinase activity, is required for TIR signaling and that IRAK-4 functions upstream of IRAK. Recently, it has been shown that IRAK-M prevents dissociation of IRAK and IRAK-4 from MyD88 and formation of IRAK·TRAF6 complexes (28Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Google Scholar). IRAK-M–/– cells exhibited increased cytokine production upon TLR/IL-1 stimulation and bacterial challenge. IRAK-M–/– mice showed increased inflammatory responses to bacterial infection and decreased tolerance to endotoxin shock (28Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Google Scholar). These data suggest that IRAK-M is a negative regulator of signaling by the TIR family members. In this study, we identified a novel TIR domain-containing adapter protein, designated as TIRP (for TIR-containing protein). Our findings suggest that TIRP interacts with various proteins involved in the TIR signaling pathways and plays a role in NF-κB activation triggered by the IL-1 receptors. Reagents—Recombinant TNF-α and IL-1 (R&D Systems, Minneapolis, MN), monoclonal antibodies against FLAG, and the HA (Sigma) epitopes were purchased from the indicated resources. Northern Blot Hybridization—Human multiple tissue mRNA blots were purchased from Clontech (Palo Alto, CA). The blots were hybridized with [32P]dCTP and [32P]dATP-labeled cDNA probe corresponding to TIRP coding sequence. The hybridization was performed in the rapid hybridization buffer (Clontech) under high stringency condition. Constructs—NF-κB luciferase reporter construct (Dr. Gary Johnson, University of Colorado Health Sciences Center) and mammalian expression plasmids for FLAG-TRAF6, FLAG-TRAF6-(289–522), IKKβ, IκBα(SS/AA) (Dr. David Goeddel, Tularik Inc.), FLAG-IRAK, FLAG-IRAK-(K239S), FLAG-IRAK-4, FLAG-IRAK-4-KA, FLAG-IRAK-4-(1–191), FLAG-IRAK-M, FLAG-IL-1R1, FLAG-IL-1RAcP, Myc-MyD88 (Dr. Zhaodan Cao, Tularik Inc.), pCMV1-FLAG-TLR2, and pCMV1-FLAG-TLR4 (Dr. Bruce Beutler, Scripps Research Institute) were provided by the indicated investigators. Mammalian expression plasmids for HA-TIRP and its deletion mutants, FLAG-tagged IRAK-2, IRAK-(1–215), IRAK-2-(97–590), TRIF, and TIRAP, were constructed by PCR amplification of the corresponding cDNA fragments and subsequent cloning into a CMV promoter-based vector containing a 5′-HA or FLAG tag. IFN-β luciferase reporter was constructed by PCR amplification of the human IFN-β promoter fragment (–300 to + 25) and cloning into pGL3-Basic vector (Promega, Madison, WI). Cell Transfection and Reporter Gene Assays—293 cells (2 × 105) were seeded in 6-well dishes and transfected the following day by the standard calcium phosphate precipitation (33Sambrook J. Fritch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual,2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Within the same experiment, each transfection was performed in triplicate, and where necessary, empty control plasmid was added to ensure that each transfection receives the same amount of total DNA. To normalize for transfection efficiency, 0.2 μg of RSV-β-galactosidase luciferase reporter plasmid was added to each transfection. Approximately sixteen hours after transfection, luciferase reporter assays were performed using a luciferase assay kit (BD Biosciences) by following the manufacturer's protocol. β-Galactosidase activity was measured using the Galacto-Light chemiluminescent kit (TROPIX, Bedford, MA). Luciferase activities were normalized on the basis of β-galactosidase luciferase expression levels. All of the reporter gene assays were repeated for at least three times. Data shown were from one representative experiment Co-immunoprecipitation and Western Blot Analysis—For transient transfection and co-immunoprecipitation experiments, 293 cells (2 × 106) were transfected for 24 h. Transfected cells were lysed in 1 ml of lysis buffer (15 mm Tris, 120 mm NaCl, 1% Triton X-100, 25 mm KCl, 2 mm EGTA, 2 mm EDTA, 0.1 mm dithiothreitol, 0.5% Triton X-100, 10 μg/ml leupeptin, 0.5 mm phenylmethylsulfonyl fluoride, pH 7.5). For each immunoprecipitation, a 0.4-ml aliquot of lysate was incubated with 0.5 μg of the indicated monoclonal antibody or control mouse IgG and 25 μl of a 1:1 slurry of GammaBind G Plus-Sepharose (Amersham Biosciences) for 2 h. The Sepharose beads were washed three times with 1 ml of lysis buffer with 500 mm NaCl. The precipitates were fractionated on SDS-PAGE, and subsequent Western blot analysis was performed as described. All of the immunoprecipitation experiments were repeated for at least three times, and similar data were obtained. Identification and Cloning of TIRP—It has been suggested that several TIR domain-containing adapters are involved in the TIR signaling pathways and that differential use of these adapters provides the specificity in the TIR signaling pathways (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). We reasoned that additional TIR domain-containing adapters might exist. Therefore, we searched the GenBank™ EST databases for sequences that encode TIR domain-containing proteins. This effort identified multiple human EST clones that encode a protein designated as TIRP. GenBank™ accession number for the longest EST clone is BQ438847. Further BLAST searches also identified mouse ortholog of human TIRP. Sequence comparisons suggest that human and mouse TIRPs share ∼70% identity at amino acid level (Fig. 1A). TIRP contains a TIR domain at the middle, which is mostly conserved with that of TRIF/TICAM (Fig. 1B) (18Horng T. Barton G. Medzhitov R. Nature Immunol. 2001; 2: 835-841Google Scholar, 19Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Google Scholar, 20Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Google Scholar, 21Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nature Immunol. 2003; 4: 161-167Google Scholar). Northern blot analysis suggests that human TIRP mRNA is expressed in most examined tissues including the spleen, prostate, testis, uterus, small intestine, colon, peripheral blood leukocytes, heart, placenta, lung, liver, skeletal muscle, and pancreas (Fig. 1C). Three transcripts of different sizes, ∼3.8, 3.6, and 2.0 kb, respectively, were detected, and these transcripts were differentially expressed in the tissues (Fig. 1C). TIRP Interacts with IL-1R1 and IL-1RAcP—Previously, it has been shown that the TIR domain-containing adapter proteins interact with TIR receptors (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). We determined whether TIRP interacts with IL-1R1, IL-1RAcP, and TLR2. To do this, we transfected expression plasmids for C-terminal FLAG-tagged receptors and N-terminal HA-tagged TIRP into 293 cells. Co-immunoprecipitation experiments indicated that TIRP interacted with IL-1R1 and IL-1RAcP but not with TLR2 and TLR4 (Fig. 2A). These data suggest that TIRP is a component of the IL-1 receptor signaling complex. TIRP Interacts with Other TIR-containing Adapter Proteins—It has been reported that TIR-containing adapter proteins can form heterodimers (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Google Scholar, 2Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Google Scholar, 3O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Google Scholar, 4Akira S. Takeda K. Kaisho T. Nature Immunol. 2001; 2: 675-680Google Scholar, 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Google Scholar). To test whether TIRP can interact with known TIR-containing adapter proteins, we transfected expression plasmids for TIRP and MyD88, TIRAP, and TRIF into 293 cells, respectively. Co-immunoprecipitation experiments suggest that TIRP interacts with TIRAP and TRIF (Fig. 2B) but not with MyD88 (data not shown). These data suggest that TIRP selectively interacts with other TIR-containing adapter proteins. TIRP Interacts with IRAKs—MyD88, TIRAP, and TRIF function as adapters to recruit IRAKs into the signaling complexes of TIR family members (10Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 11Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Google Scholar, 12Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. DiMarco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Google Scholar, 13Cao Z. Yiong J. Takeuchi M. Kurama T. Goeddel D. Nature. 1996; 383: 443-446Google Scholar, 20Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Google Scholar, 21Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nature Immunol. 2003; 4: 161-167Google Scholar, 22Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Google Scholar, 23Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Google Scholar). We examined whether TIRP also interacts with IRAKs. In transient transfection and co-immunoprecipitation experiments, TIRP did not interact with wild-type IRAK and IRAK-4 but interacted with kinase-inactive mutants of IRAK and IRAK-4 (Fig. 3A). These results are similar to MyD88, which has been shown to interact with kinase-inactive mutant but not with wild type IRAK. In transient transfection and co-immunoprecipitation experiments, TIRP also interacted with wild-type IRAK-2 and IRAK-M (Fig. 3A), two IRAK family members that have no kinase activity. Taken together, these data suggest that TIRP only interacts with kinase-inactive IRAKs. TIRP Interacts with TRAF6 —It has been shown that TRAF6 is associated with activated IRAK (13Cao Z. Yiong J. Takeuchi M. Kurama T. Goeddel D. Nature. 1996; 383: 443-446Google Scholar). We examined whether TIRP is associated with TRAF6. To do this, we transfected FLAG-tagged TRAF6 and HA-tagged TIRP into 293 cells and performed co-immunoprecipitation experiments. These experiments indicated that TIRP interacted with TRAF6 but not with TRAF5 (Fig. 3B). Also, the TRAF domain of TRAF6 was sufficient for its interaction with TIRP (Fig. 3B). These results suggest that TIRP specifically interacts with TRAF6. We next determined which domain of TIRP is responsible for its interaction with TRAF6. TIRP contains a conserved TIR domain at the middle flanked by unconserved N- and C-terminal domains. We made HA-tagged TIRP deletion mutants containing one individual domain or combinations of different domains. Transient transfection and co-immunoprecipitation experiments suggest that the TIR domain is required and sufficient for the interaction of TIRP with TRAF6 (Fig. 3C). Interestingly, we found that the TIR domain of TIRP contains a PRERT motif at amino acid 181, which is conserved with the defined TRAF6 binding motif PXEXX (34Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde 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). Overexpression of TIRP Activates NF-κB but Not IFN-β Promoter—Since TIRP interacts with proteins involved in signaling by TIR family members, we determined whether TIRP activates NF-κB. We transfected a mammalian expression plasmid for TIRP into 293 cells and performed NF-κB luciferase reporter gene assays. These experiments indicated that TIRP could activate NF-κB (Fig. 4A). However, TIRP activated NF-κB to a lesser degree than MyD88, TRIF, and TIRAP in the same experiments (Fig. 4A). Previously, it has been shown that TRIF can potently activate IFN-β promoter. To examine whether TIRP can activate IFN-β promoter, we transfected 293 cells (2 × 105) with various amounts of TIRP plasmid ranging from 0.01 to 3.2 μg and then performed IFN-β luciferase reporter gene assays. The result indicated that TIRP could not activate IFN-β promoter regardless of the transfected TIRP plasmid concentration (Fig. 4B) (data not shown). In these experiments, MyD88 and TIRAP also did not activate IFN-β promoter, whereas TRIF potently activated IFN-β promoter (Fig. 4B). Because TIRP can weakly activate NF-κB, we determined whether it potentiates IL-1R1- and IL-1AcP-mediated NF-κB activation. Previously, it has been shown that the overexpression of IL-1R1 or IL-1RAcP alone weakly activates NF-κB. In reporter gene assays, we found that TIRP could synergy with IL-1R1 or IL-1-RAcP to activate NF-κB (Fig. 4C). A TIRP Dominant Negative Mutant Inhibits NF-κB Activation Mediated by IL-1 Receptors but Not by TRAF6 and IKKβ— Previously, it has been shown that the TIR domains of MyD88, TRIF, and TIRAP function as dominant negative mutants to inhibit signaling by the TIR family members (10Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 11Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Google Scholar, 12Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. DiMarco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Google Scholar, 13Cao Z. Yiong J. Takeuchi M. Kurama T. Goeddel D. Nature. 1996; 383: 443-446Google Scholar, 20Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Google Scholar, 21Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nature Immunol. 2003; 4: 161-167Google Scholar, 22Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Google Scholar, 23Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Google Scholar). We examined whether the TIR domain of TIRP can inhibit NF-κB activation triggered by IL-1 receptors. In reporter gene assays, we found that the TIR domain of TIRP, TIRP-(78–171), significantly inhibited NF-κB activation mediated by overexpression of IL-1R1 and IL-1RacP but not by overexpression of TRAF6 and IKKβ (Fig. 5A). In these experiments, TIRP-(78–171) did not inhibit TLR4 and only slightly inhibited TLR2-mediated NF-κB activation (Fig. 5A). In reporter gene assays, TIRP-(78–171) also inhibited NF-κB activation triggered by IL-1 but not by TNF (Fig. 5B). These data suggest that TIRP is specifically involved in IL-1-triggered NF-κB activation. NF-κB Activation by TIRP Is Inhibited by Dominant Negative Mutants of IRAK, IRAK-2, TRAF6, and IKKβ—To determine the molecular order of TIRP in the IL-1 signaling pathways, we determined whether TIRP-mediated NF-κB activation is inhibited by dominant negative mutants of proteins involved in IL-1 signaling pathways. In reporter gene assays, we found that dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKβ as well as IκBα-undegradable mutant potently inhibited TIRP-induced NF-κB activation (Fig. 5C). In these experiments, dominant negative mutants of MyD88 and IRAK-4 did not inhibit TIRP-mediated NF-κB activation (Fig. 5C). These data suggest that TIRP functions upstream of IRAK, IRAK-2, TRAF6, and IKKβ in IL-1 receptor-mediated NF-κB activation pathways. Previously, three TIR-containing adapter proteins have been reported, including MyD88, TIRAP/Mal, and TRIF/TICAM. In this paper, we describe the identification of TIRP, the fourth member of this family. TIRP shares many similar properties with the other three TIR-containing adapter proteins. It contains a conserved TIR domain and is associated with IL-1RI and IL-1RAcP, suggesting that TIRP plays a role in IL-1 signaling. Previous studies have shown that MyD interacts with kinase-inactive mutant of IRAK but not wild-type IRAK. It has been proposed that MyD88 recruits inactive IRAK to IL-1R complex. Once IRAK is activated in the complex, it dissociates with MyD88 and then interacts with TRAF6 (10Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Google Scholar, 11Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Google Scholar, 12Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. DiMarco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Google Scholar). Similar to MyD88, TIRP interacts with kinase-inactive mutants of IRAK and IRAK-4 but not with their wild-type counterparts. These data suggest that TIRP may also interact with inactive IRAK and IRAK-4 in cells and dissociate when they are activated. In addition to kinase-inactive IRAK and IRAK-4 mutants, TIRP also interacts with IRAK-2 and IRAK-M, two IRAK family members that are kinase-inactive. These data further support our hypothesis that TIRP interacts with kinase-inactive IRAKs. In addition to IRAKs, TIRP also interacts with TRAF6. This interaction is mediated by the TRAF domain of TRAF6. TIRP does not interact with TRAF5, a TRAF family member that is involved in signaling by TNF receptor family members. The TIR domain of TIRP is required and sufficient for its interaction with TRAF6. These data suggest that TIRP specifically interacts with TRAF6 and other signaling proteins involved in IL-1 signaling pathways. Overexpression of TIRP activates NF-κB but not IFN-β promoter. A dominant negative mutant of TIRP (TIRP-(78–171)) inhibits IL-1-induced but not TNF-induced NF-κB activation. Conversely, dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKβ inhibited TIRP-mediated NF-κB activation. These data suggest that TIRP functions upstream of IRAK, IRAK-2, TRAF6, and IKKβ in the IL-1-induced NF-κB activation pathway. Because IL-1 is unable to activate NF-κB in certain MyD88-deficient cells, it is possible that these cells do not express TIRP. Alternatively, TIRP is not able to complement MyD88 deficiency and both proteins are required for IL-1-induced NF-κB activation. Although TIRP interacts with kinase-inactive mutant of IRAK-4, a dominant negative mutant of IRAK-4 did not inhibit TIRP-induced NF-κB activation. It is possible that IRAK-4 functions upstream of TIRP in IL-1 signaling. In conclusion, we identified TIRP, a novel TIR domain-containing adapter protein that is involved in IL-1-triggered NF-κB activation pathways. It should be pointed out that our characterization of TIRP was based on the mammalian overexpression systems. Further experiments, such as analysis of TIRP knock-out mice, will be needed to confirm a role of TIRP in IL-1 or other TIR signaling pathways. We thank Drs. David Goeddel, Zhaodan Cao, Gary Johnson, and Bruce Beutler for reagents.

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