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IRAK-M Is a Novel Member of the Pelle/Interleukin-1 Receptor-associated Kinase (IRAK) Family

1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês

10.1074/jbc.274.27.19403

1083-351X

Holger Wesche, Xiong Gao, Xiaoxia Li, Carsten J. Kirschning, George R. Stark, Zhaodan Cao,

Immune Cell Function and Interaction

The interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for interleukin-1 (IL-1) and has later been implicated in signal transduction of other members of the Toll/IL-1 receptor family. We now report the identification and characterization of a novel IRAK-like molecule. In contrast to the ubiquitously expressed IRAK and IRAK-2, this new IRAK-like molecule is found mainly in cells of monomyeloic origin and is, therefore, designated IRAK-M. Although IRAK-M and IRAK-2 exhibit only a negligible autophosphorylation activity, they can reconstitute the IL-1 response in a 293 mutant cell line lacking IRAK. In addition, we show for the first time that members of the IRAK family are indispensable elements of lipopolysaccharide signal transduction. The discovery of IRAK-M adds another level of complexity to our understanding of signaling by members of the Toll/IL-1 receptor family. The interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for interleukin-1 (IL-1) and has later been implicated in signal transduction of other members of the Toll/IL-1 receptor family. We now report the identification and characterization of a novel IRAK-like molecule. In contrast to the ubiquitously expressed IRAK and IRAK-2, this new IRAK-like molecule is found mainly in cells of monomyeloic origin and is, therefore, designated IRAK-M. Although IRAK-M and IRAK-2 exhibit only a negligible autophosphorylation activity, they can reconstitute the IL-1 response in a 293 mutant cell line lacking IRAK. In addition, we show for the first time that members of the IRAK family are indispensable elements of lipopolysaccharide signal transduction. The discovery of IRAK-M adds another level of complexity to our understanding of signaling by members of the Toll/IL-1 receptor family. interleukin-1 receptor Toll like receptor lipopolysaccharide IL-1R-associated kinase nuclear factor-κB tumor necrosis factor luciferase Rous sarcoma virus β-galactosidase 12-O-tetradecanoylphorbol-13-acetate TNF receptor-associated factor The Toll/IL-1R1receptor family consists of a large number of transmembrane proteins with conserved intracellular domains. Structural distinctions in the extracellular domains of these proteins divide this superfamily into two subgroups: the Toll-like receptors (TLRs) with leucine-rich repeats and IL-1R-related proteins with immunoglobulin-like motifs (1; for review, see Refs. 2Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar and 3Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (273) Google Scholar). All members of the Toll/IL-1R family with known functions are involved in host defense. For example, the proinflammatory and immune regulatory cytokines IL-1 and IL-18 signal through members of the IL-1R-related protein subfamily (4, 5; for review, see Refs. 6Dinarello C.A. Novick D. Puren A.J. Fantuzzi G. Shapiro L. Muhl H. Yoon D.Y. Reznikov L.L. Kim S.H. Rubinstein M. J. Leukocyte Biol. 1998; 63: 658-664Crossref PubMed Scopus (331) Google Scholar and 7O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (495) Google Scholar), whereas TLR-2 and TLR-4 have been shown recently to mediate cellular response to bacterial lipopolysaccharide (LPS) (8Du X. Thompson P. Chan E.K.L. Ledesma J. Roe B. Clifton S. Vogel S.N. Beutler B. Blood Cells Mol. Dis. 1998; 24: 340-355Crossref PubMed Scopus (297) Google Scholar, 9Kirschning K.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar, 10Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4379) Google Scholar, 11Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6382) Google Scholar, 12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1098) Google Scholar).The signal transduction pathways initiated by Toll/IL-1R family members ultimately lead to the activation of members of the rel and AP-1 family of transcription factors (for review, see Refs. 2Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar, 3Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (273) Google Scholar, 7O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (495) Google Scholar, and 13Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). The receptor proximal signaling events of the proinflammatory cytokine IL-1 have been studied in detail. The first signaling event for IL-1 is the ligand-induced complex formation of IL-1RI and IL-1RAcP (14Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 15Huang J. Gao X. Li S. Cao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12829-12832Crossref PubMed Scopus (194) Google Scholar, 16Korherr C. Hofmeister R. Wesche H. Falk W. Eur. J. Immunol. 1997; 27: 262-267Crossref PubMed Scopus (153) Google Scholar, 17Wesche H. Korherr C. Kracht M. Falk W. Resch K. Martin M.U. J. Biol. Chem. 1997; 272: 7727-7731Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The adaptor protein MyD88 is next recruited to this complex (18Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1990; 5: 1095-1097PubMed Google Scholar, 19Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar, 20Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), which in turn enables the association of the IL-1R-associated kinase (IRAK) (21Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (766) Google Scholar, 22Croston G.E. Cao Z. Goeddel D.V. J. Biol. Chem. 1995; 270: 16514-16517Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 23Martin M. Bol G.F. Eriksson A. Resch K. Brigelius-Flohe R. Eur. J. Immunol. 1994; 24: 1566-1571Crossref PubMed Scopus (80) Google Scholar). IRAK gets highly phosphorylated (21Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (766) Google Scholar, 24Yamin T.-T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), leaves the receptor complex, and interacts with TRAF6 (25Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1111) Google Scholar). The IRAK-TRAF6 interaction triggers kinase cascades that lead to the activation of IκB kinases and c-Jun NH2-terminal kinase, which phosphorylate IκB and c-Jun, respectively (for review, see Refs. 7O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (495) Google Scholar, 26Baeuerle P.A. Curr. Biol. 1998; 8: R19-R22Abstract Full Text Full Text PDF PubMed Google Scholar, and 27Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4247) Google Scholar).Accumulating evidence indicates that several components of the IL-1 pathway are also utilized by other members of the Toll/IL-1R family. TLR-4 has been shown to interact with MyD88, IRAK, and TRAF6 upon overexpression (28Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar, 29Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar). Dominant negative mutants of MyD88 and TRAF6 can block LPS-induced NF-κB activation mediated by TLR-2 (9Kirschning K.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar) as well as NF-κB activation induced by TLR-4 overexpression (28Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar, 29Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar). In addition, IL-18 induces IRAK phosphorylation and interaction with TRAF6 (30Kojima H. Takeuchi M. Ohta T. Nishida Y. Arai N. Ikeda M. Ikegami H. Kurimoto M. Biochem. Biophys. Res. Commun. 1998; 244: 183-186Crossref PubMed Scopus (123) Google Scholar, 31Robinson D. Shibuya K. Mui A. Zonin F. Murphy E. Sana T. Hartley S.B. Menon S. Kastelein R. Bazan F. O'Garra A. Immunity. 1997; 7: 571-581Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). Further evidence implicating IL-1 signaling components in the IL-18 pathway was provided by the observation that targeted disruption of the myd88 gene in mice results in a loss of response to both IL-1 and IL-18 (32Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1699) Google Scholar).The role of IRAK in cytokine signaling is also supported by genetic studies. Embryonic fibroblasts derived from IRAK-deficient mice show reduced NF-κB and c-Jun NH2-terminal kinase activities after IL-1 treatment (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar). IRAK knock-out mice are also severely compromised in IL-1-induced neutrophilia and IL-1-induced increase in serum levels of TNF and IL-6 (34Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; (in press)Google Scholar). Furthermore, splenocytes derived from the IRAK knock-out mice produced significantly reduced amounts of interferon-γ in response to IL-18 (34Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; (in press)Google Scholar). Nonetheless, in contrast to the phenotype of the MyD88 knock-out mice, IRAK-deficient mice retain partial IL-1 and IL-18 responses in all of the above mentioned assays, indicating the presence of a compensatory mechanism in animals.An IRAK-related molecule designated IRAK-2 has been identified in a computer-assisted EST data base search (19Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar). IRAK-2 is able to interact with other IL-1 signal transducers and activate NF-κB upon overexpression in 293 cells. Here we report the identification and characterization of a novel IRAK-like molecule that has both similarities and differences compared with other members of the IRAK family. Utilizing an IRAK-deficient cell line, we present evidence that both IRAK-2 and the newly identified IRAK-like molecule can provide functional redundancy in signal transduction by the Toll/IL-1R receptor family.DISCUSSIONMembers of the Toll/IL-1R family of transmembrane receptors play an important role in various immune responses (3Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (273) Google Scholar, 5Torigoe K. Ushio S. Okura T. Kobayashi S. Taniai M. Kunikata T. Murakami T. Sanou O. Kojima H. Fujii M. Ohta T. Ikeda M. Ikegami H. Kurimoto M. J. Biol. Chem. 1997; 272: 25737-25742Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 9Kirschning K.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar, 11Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6382) Google Scholar, 12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1098) Google Scholar, 13Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). Supported by both biochemical and genetic studies, many members of this receptor family engage similar intracellular signal transducers to generate cellular response (see the Introduction). In this study, we present for the first time genetic evidence for the involvement of IRAK in LPS signaling. The IRAK-deficient cell line 293I1A failed to respond to IL-1 as well as to LPS, and this defect can be corrected by introducing exogenous IRAK or IRAK-related proteins back into the cells. These results support an emerging scheme that the IL-1 intracellular signaling mechanism may be common to a class of receptors that share sequence homology with the IL-1 receptors.The observation that IRAK-deficient mice and cells retain significant residual IL-1 and IL-18 response (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar, 34Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; (in press)Google Scholar) implicates the presence of molecule(s) with overlapping functions in animals. The likely candidates for such molecules are IRAK-2 and IRAK-M, which share similarities in protein sequence as well as functional properties with IRAK. Compelling support for this notion came from the results that either IRAK-2 or IRAK-M could reconstitute IL-1 and LPS responses in the IRAK-deficient 293 cells. Also in agreement with the role of IRAK-2 and IRAK-M as signaling molecules with functions similar to those of IRAK, IRAK-deficient fibroblasts retained partial response to IL-1 (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar), whereas the IL-1 and LPS response is essentially abolished in IRAK-deficient 293 cells, which lack IRAK-M and express low levels of IRAK-2.The three IRAK-like proteins are unlikely to play completely redundant roles in animals. In contrast to IRAK and IRAK-2, whose mRNA is detected in multiple tissues and cell types, the expression of IRAK-M is limited mostly to cells of the monocytic lineage and is up-regulated during differentiation, suggesting a cell type-specific function of IRAK-M.The most striking feature that distinguishes IRAK from the other IRAK-like proteins is its potent autophosphorylation, which is not detected in IRAK-M and IRAK-2. Nevertheless, both IRAK-M and IRAK-2 are able to transduce IL-1- and LPS-mediated signals in the absence of IRAK. Assuming that autophosphorylation reflects the kinase activity, it is possible that the role of IRAK in signal transduction is not phosphorylating downstream targets. In nontransfected cells, IRAK autophosphorylation is detected only after IL-1 stimulation. Based on the observation that the phosphorylated form of IRAK is unable to bind to MyD88, which links IRAK to the receptor complex (20Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), we speculated that the phosphorylation of IRAK may trigger its release from the receptor complex and its subsequent interaction with TRAF6. If this assumption were correct, the requirement of the IRAK kinase activity in signaling would not be apparent in transfection experiments because of protein overexpression, which results in a high concentration of free IRAK. Consistent with this scenario, forms of IRAK proteins harboring mutations in their ATP binding pockets could still activate NF-κB upon overexpression in 293 cells, albeit with a lower potency (data not shown). Alternatively, the role of IRAK kinase activity may be to regulate IL-1 signaling negatively after cytokine induction. Supporting this hypothesis, IRAK phosphorylation is followed by its proteolytic degradation (24Yamin T.-T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Careful comparison of the duration of the IL-1 response in cells expressing physiological amounts of the wild type IRAK or the kinase-deficient form of IRAK will help to distinguish between these possibilities.Although IRAK-2 and IRAK-M have very low autophosphorylation activity on their own, the wild type forms can be phosphorylated better than their corresponding kinase-inactive mutants in the presence of IRAK (Fig. 5). Under the assumption that the mutation of a single amino acid residue in the ATP binding pocket does not transform IRAK-M or IRAK-2 into a poorer substrate for IRAK, it is reasonable to speculate that IRAK phosphorylates IRAK-2 and IRAK-M, thereby activating the intrinsic kinase activity of these molecules. Mapping the sites on IRAK-2 and IRAK-M which are phosphorylated by IRAK in combination with site-directed mutagenesis should help to address this issue.Our study shows that IRAK is more potent and efficacious than the other two IRAK-like proteins at restoring IL-1 and LPS responses in 293I1A cells. Although we cannot exclude that IRAK-M and IRAK-2 mainly participate in the signaling of other related receptors, our observations raise the possibility that IRAK may be the primary signal transducer. Because IRAK-2 and IRAK-M can form heterocomplexes with IRAK, they may help to amplify the cytokine signals by providing a critical mass. Under this scenario, the expression levels of IRAK-like molecules would influence the degree of the response of a given cell type to a set of cytokines such as IL-1, IL-18, or LPS. The Toll/IL-1R1receptor family consists of a large number of transmembrane proteins with conserved intracellular domains. Structural distinctions in the extracellular domains of these proteins divide this superfamily into two subgroups: the Toll-like receptors (TLRs) with leucine-rich repeats and IL-1R-related proteins with immunoglobulin-like motifs (1; for review, see Refs. 2Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar and 3Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (273) Google Scholar). All members of the Toll/IL-1R family with known functions are involved in host defense. For example, the proinflammatory and immune regulatory cytokines IL-1 and IL-18 signal through members of the IL-1R-related protein subfamily (4, 5; for review, see Refs. 6Dinarello C.A. Novick D. Puren A.J. Fantuzzi G. Shapiro L. Muhl H. Yoon D.Y. Reznikov L.L. Kim S.H. Rubinstein M. J. Leukocyte Biol. 1998; 63: 658-664Crossref PubMed Scopus (331) Google Scholar and 7O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (495) Google Scholar), whereas TLR-2 and TLR-4 have been shown recently to mediate cellular response to bacterial lipopolysaccharide (LPS) (8Du X. Thompson P. Chan E.K.L. Ledesma J. Roe B. Clifton S. Vogel S.N. Beutler B. Blood Cells Mol. Dis. 1998; 24: 340-355Crossref PubMed Scopus (297) Google Scholar, 9Kirschning K.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar, 10Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4379) Google Scholar, 11Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6382) Google Scholar, 12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1098) Google Scholar). The signal transduction pathways initiated by Toll/IL-1R family members ultimately lead to the activation of members of the rel and AP-1 family of transcription factors (for review, see Refs. 2Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar, 3Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (273) Google Scholar, 7O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (495) Google Scholar, and 13Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). The receptor proximal signaling events of the proinflammatory cytokine IL-1 have been studied in detail. The first signaling event for IL-1 is the ligand-induced complex formation of IL-1RI and IL-1RAcP (14Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 15Huang J. Gao X. Li S. Cao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12829-12832Crossref PubMed Scopus (194) Google Scholar, 16Korherr C. Hofmeister R. Wesche H. Falk W. Eur. J. Immunol. 1997; 27: 262-267Crossref PubMed Scopus (153) Google Scholar, 17Wesche H. Korherr C. Kracht M. Falk W. Resch K. Martin M.U. J. Biol. Chem. 1997; 272: 7727-7731Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The adaptor protein MyD88 is next recruited to this complex (18Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1990; 5: 1095-1097PubMed Google Scholar, 19Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar, 20Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), which in turn enables the association of the IL-1R-associated kinase (IRAK) (21Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (766) Google Scholar, 22Croston G.E. Cao Z. Goeddel D.V. J. Biol. Chem. 1995; 270: 16514-16517Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 23Martin M. Bol G.F. Eriksson A. Resch K. Brigelius-Flohe R. Eur. J. Immunol. 1994; 24: 1566-1571Crossref PubMed Scopus (80) Google Scholar). IRAK gets highly phosphorylated (21Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (766) Google Scholar, 24Yamin T.-T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), leaves the receptor complex, and interacts with TRAF6 (25Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1111) Google Scholar). The IRAK-TRAF6 interaction triggers kinase cascades that lead to the activation of IκB kinases and c-Jun NH2-terminal kinase, which phosphorylate IκB and c-Jun, respectively (for review, see Refs. 7O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (495) Google Scholar, 26Baeuerle P.A. Curr. Biol. 1998; 8: R19-R22Abstract Full Text Full Text PDF PubMed Google Scholar, and 27Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4247) Google Scholar). Accumulating evidence indicates that several components of the IL-1 pathway are also utilized by other members of the Toll/IL-1R family. TLR-4 has been shown to interact with MyD88, IRAK, and TRAF6 upon overexpression (28Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar, 29Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar). Dominant negative mutants of MyD88 and TRAF6 can block LPS-induced NF-κB activation mediated by TLR-2 (9Kirschning K.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar) as well as NF-κB activation induced by TLR-4 overexpression (28Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar, 29Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar). In addition, IL-18 induces IRAK phosphorylation and interaction with TRAF6 (30Kojima H. Takeuchi M. Ohta T. Nishida Y. Arai N. Ikeda M. Ikegami H. Kurimoto M. Biochem. Biophys. Res. Commun. 1998; 244: 183-186Crossref PubMed Scopus (123) Google Scholar, 31Robinson D. Shibuya K. Mui A. Zonin F. Murphy E. Sana T. Hartley S.B. Menon S. Kastelein R. Bazan F. O'Garra A. Immunity. 1997; 7: 571-581Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). Further evidence implicating IL-1 signaling components in the IL-18 pathway was provided by the observation that targeted disruption of the myd88 gene in mice results in a loss of response to both IL-1 and IL-18 (32Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1699) Google Scholar). The role of IRAK in cytokine signaling is also supported by genetic studies. Embryonic fibroblasts derived from IRAK-deficient mice show reduced NF-κB and c-Jun NH2-terminal kinase activities after IL-1 treatment (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar). IRAK knock-out mice are also severely compromised in IL-1-induced neutrophilia and IL-1-induced increase in serum levels of TNF and IL-6 (34Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; (in press)Google Scholar). Furthermore, splenocytes derived from the IRAK knock-out mice produced significantly reduced amounts of interferon-γ in response to IL-18 (34Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; (in press)Google Scholar). Nonetheless, in contrast to the phenotype of the MyD88 knock-out mice, IRAK-deficient mice retain partial IL-1 and IL-18 responses in all of the above mentioned assays, indicating the presence of a compensatory mechanism in animals. An IRAK-related molecule designated IRAK-2 has been identified in a computer-assisted EST data base search (19Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar). IRAK-2 is able to interact with other IL-1 signal transducers and activate NF-κB upon overexpression in 293 cells. Here we report the identification and characterization of a novel IRAK-like molecule that has both similarities and differences compared with other members of the IRAK family. Utilizing an IRAK-deficient cell line, we present evidence that both IRAK-2 and the newly identified IRAK-like molecule can provide functional redundancy in signal transduction by the Toll/IL-1R receptor family. DISCUSSIONMembers of the Toll/IL-1R family of transmembrane receptors play an important role in various immune responses (3Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (273) Google Scholar, 5Torigoe K. Ushio S. Okura T. Kobayashi S. Taniai M. Kunikata T. Murakami T. Sanou O. Kojima H. Fujii M. Ohta T. Ikeda M. Ikegami H. Kurimoto M. J. Biol. Chem. 1997; 272: 25737-25742Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 9Kirschning K.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar, 11Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6382) Google Scholar, 12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1098) Google Scholar, 13Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). Supported by both biochemical and genetic studies, many members of this receptor family engage similar intracellular signal transducers to generate cellular response (see the Introduction). In this study, we present for the first time genetic evidence for the involvement of IRAK in LPS signaling. The IRAK-deficient cell line 293I1A failed to respond to IL-1 as well as to LPS, and this defect can be corrected by introducing exogenous IRAK or IRAK-related proteins back into the cells. These results support an emerging scheme that the IL-1 intracellular signaling mechanism may be common to a class of receptors that share sequence homology with the IL-1 receptors.The observation that IRAK-deficient mice and cells retain significant residual IL-1 and IL-18 response (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar, 34Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; (in press)Google Scholar) implicates the presence of molecule(s) with overlapping functions in animals. The likely candidates for such molecules are IRAK-2 and IRAK-M, which share similarities in protein sequence as well as functional properties with IRAK. Compelling support for this notion came from the results that either IRAK-2 or IRAK-M could reconstitute IL-1 and LPS responses in the IRAK-deficient 293 cells. Also in agreement with the role of IRAK-2 and IRAK-M as signaling molecules with functions similar to those of IRAK, IRAK-deficient fibroblasts retained partial response to IL-1 (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar), whereas the IL-1 and LPS response is essentially abolished in IRAK-deficient 293 cells, which lack IRAK-M and express low levels of IRAK-2.The three IRAK-like proteins are unlikely to play completely redundant roles in animals. In contrast to IRAK and IRAK-2, whose mRNA is detected in multiple tissues and cell types, the expression of IRAK-M is limited mostly to cells of the monocytic lineage and is up-regulated during differentiation, suggesting a cell type-specific function of IRAK-M.The most striking feature that distinguishes IRAK from the other IRAK-like proteins is its potent autophosphorylation, which is not detected in IRAK-M and IRAK-2. Nevertheless, both IRAK-M and IRAK-2 are able to transduce IL-1- and LPS-mediated signals in the absence of IRAK. Assuming that autophosphorylation reflects the kinase activity, it is possible that the role of IRAK in signal transduction is not phosphorylating downstream targets. In nontransfected cells, IRAK autophosphorylation is detected only after IL-1 stimulation. Based on the observation that the phosphorylated form of IRAK is unable to bind to MyD88, which links IRAK to the receptor complex (20Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), we speculated that the phosphorylation of IRAK may trigger its release from the receptor complex and its subsequent interaction with TRAF6. If this assumption were correct, the requirement of the IRAK kinase activity in signaling would not be apparent in transfection experiments because of protein overexpression, which results in a high concentration of free IRAK. Consistent with this scenario, forms of IRAK proteins harboring mutations in their ATP binding pockets could still activate NF-κB upon overexpression in 293 cells, albeit with a lower potency (data not shown). Alternatively, the role of IRAK kinase activity may be to regulate IL-1 signaling negatively after cytokine induction. Supporting this hypothesis, IRAK phosphorylation is followed by its proteolytic degradation (24Yamin T.-T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Careful comparison of the duration of the IL-1 response in cells expressing physiological amounts of the wild type IRAK or the kinase-deficient form of IRAK will help to distinguish between these possibilities.Although IRAK-2 and IRAK-M have very low autophosphorylation activity on their own, the wild type forms can be phosphorylated better than their corresponding kinase-inactive mutants in the presence of IRAK (Fig. 5). Under the assumption that the mutation of a single amino acid residue in the ATP binding pocket does not transform IRAK-M or IRAK-2 into a poorer substrate for IRAK, it is reasonable to speculate that IRAK phosphorylates IRAK-2 and IRAK-M, thereby activating the intrinsic kinase activity of these molecules. Mapping the sites on IRAK-2 and IRAK-M which are phosphorylated by IRAK in combination with site-directed mutagenesis should help to address this issue.Our study shows that IRAK is more potent and efficacious than the other two IRAK-like proteins at restoring IL-1 and LPS responses in 293I1A cells. Although we cannot exclude that IRAK-M and IRAK-2 mainly participate in the signaling of other related receptors, our observations raise the possibility that IRAK may be the primary signal transducer. Because IRAK-2 and IRAK-M can form heterocomplexes with IRAK, they may help to amplify the cytokine signals by providing a critical mass. Under this scenario, the expression levels of IRAK-like molecules would influence the degree of the response of a given cell type to a set of cytokines such as IL-1, IL-18, or LPS. Members of the Toll/IL-1R family of transmembrane receptors play an important role in various immune responses (3Medzhitov R. Janeway Jr., C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (273) Google Scholar, 5Torigoe K. Ushio S. Okura T. Kobayashi S. Taniai M. Kunikata T. Murakami T. Sanou O. Kojima H. Fujii M. Ohta T. Ikeda M. Ikegami H. Kurimoto M. J. Biol. Chem. 1997; 272: 25737-25742Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 9Kirschning K.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar, 11Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6382) Google Scholar, 12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1098) Google Scholar, 13Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). Supported by both biochemical and genetic studies, many members of this receptor family engage similar intracellular signal transducers to generate cellular response (see the Introduction). In this study, we present for the first time genetic evidence for the involvement of IRAK in LPS signaling. The IRAK-deficient cell line 293I1A failed to respond to IL-1 as well as to LPS, and this defect can be corrected by introducing exogenous IRAK or IRAK-related proteins back into the cells. These results support an emerging scheme that the IL-1 intracellular signaling mechanism may be common to a class of receptors that share sequence homology with the IL-1 receptors. The observation that IRAK-deficient mice and cells retain significant residual IL-1 and IL-18 response (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar, 34Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; (in press)Google Scholar) implicates the presence of molecule(s) with overlapping functions in animals. The likely candidates for such molecules are IRAK-2 and IRAK-M, which share similarities in protein sequence as well as functional properties with IRAK. Compelling support for this notion came from the results that either IRAK-2 or IRAK-M could reconstitute IL-1 and LPS responses in the IRAK-deficient 293 cells. Also in agreement with the role of IRAK-2 and IRAK-M as signaling molecules with functions similar to those of IRAK, IRAK-deficient fibroblasts retained partial response to IL-1 (33Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar), whereas the IL-1 and LPS response is essentially abolished in IRAK-deficient 293 cells, which lack IRAK-M and express low levels of IRAK-2. The three IRAK-like proteins are unlikely to play completely redundant roles in animals. In contrast to IRAK and IRAK-2, whose mRNA is detected in multiple tissues and cell types, the expression of IRAK-M is limited mostly to cells of the monocytic lineage and is up-regulated during differentiation, suggesting a cell type-specific function of IRAK-M. The most striking feature that distinguishes IRAK from the other IRAK-like proteins is its potent autophosphorylation, which is not detected in IRAK-M and IRAK-2. Nevertheless, both IRAK-M and IRAK-2 are able to transduce IL-1- and LPS-mediated signals in the absence of IRAK. Assuming that autophosphorylation reflects the kinase activity, it is possible that the role of IRAK in signal transduction is not phosphorylating downstream targets. In nontransfected cells, IRAK autophosphorylation is detected only after IL-1 stimulation. Based on the observation that the phosphorylated form of IRAK is unable to bind to MyD88, which links IRAK to the receptor complex (20Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), we speculated that the phosphorylation of IRAK may trigger its release from the receptor complex and its subsequent interaction with TRAF6. If this assumption were correct, the requirement of the IRAK kinase activity in signaling would not be apparent in transfection experiments because of protein overexpression, which results in a high concentration of free IRAK. Consistent with this scenario, forms of IRAK proteins harboring mutations in their ATP binding pockets could still activate NF-κB upon overexpression in 293 cells, albeit with a lower potency (data not shown). Alternatively, the role of IRAK kinase activity may be to regulate IL-1 signaling negatively after cytokine induction. Supporting this hypothesis, IRAK phosphorylation is followed by its proteolytic degradation (24Yamin T.-T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Careful comparison of the duration of the IL-1 response in cells expressing physiological amounts of the wild type IRAK or the kinase-deficient form of IRAK will help to distinguish between these possibilities. Although IRAK-2 and IRAK-M have very low autophosphorylation activity on their own, the wild type forms can be phosphorylated better than their corresponding kinase-inactive mutants in the presence of IRAK (Fig. 5). Under the assumption that the mutation of a single amino acid residue in the ATP binding pocket does not transform IRAK-M or IRAK-2 into a poorer substrate for IRAK, it is reasonable to speculate that IRAK phosphorylates IRAK-2 and IRAK-M, thereby activating the intrinsic kinase activity of these molecules. Mapping the sites on IRAK-2 and IRAK-M which are phosphorylated by IRAK in combination with site-directed mutagenesis should help to address this issue. Our study shows that IRAK is more potent and efficacious than the other two IRAK-like proteins at restoring IL-1 and LPS responses in 293I1A cells. Although we cannot exclude that IRAK-M and IRAK-2 mainly participate in the signaling of other related receptors, our observations raise the possibility that IRAK may be the primary signal transducer. Because IRAK-2 and IRAK-M can form heterocomplexes with IRAK, they may help to amplify the cytokine signals by providing a critical mass. Under this scenario, the expression levels of IRAK-like molecules would influence the degree of the response of a given cell type to a set of cytokines such as IL-1, IL-18, or LPS. We thank Timothy Hoey for the PBL cDNA library, Keith Williamson and Joanne Waszuck for DNA Sequencing, David V. Goeddel and Mike Rothe for helpful comments on the manuscript, and Vishva Dixit for IRAK-2 cDNA.