IRAK-2 Participates in Multiple Toll-like Receptor Signaling Pathways to NFκB via Activation of TRAF6 Ubiquitination

Abstract

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFκB signaling axis, recent data have shown that it is dispensable for NFκB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFκB activation pathway. The poxviral TLR antagonist A52 inhibited NFκB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFκB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFκB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFκB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFκB. Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFκB signaling axis, recent data have shown that it is dispensable for NFκB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFκB activation pathway. The poxviral TLR antagonist A52 inhibited NFκB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFκB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFκB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFκB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFκB. Toll-like receptors (TLRs) 3The abbreviations used are: TLR, Toll-like receptor; TIR, Toll-IL-1 receptor-resistance; NFκB, nuclear factor κB; IRF, interferon regulatory factor; MyD88, myeloid differentiation factor 88; Mal, MyD88 adaptor-like; TRIF, TIR domain-containing adaptor inducing interferon-β; TRAM, TRIF-related adaptor molecule; IRAK, interleukin-1 receptor-associated kinase; TRAF6, tumor necrosis factor receptor-associated factor 6; LPS, lipopolysaccharide; Ab, antibody; mAb, monoclonal antibody; MAP, mitogen-activated protein; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin. recognize specific pathogen-associated molecular patterns found on infectious agents (1Akira S. Curr. Top. Microbiol. Immunol. 2006; 311: 1-16PubMed Google Scholar). TLRs are part of the larger IL-1R/TLR superfamily, defined by the presence of a cytoplasmic Toll-IL-1R-resistance (TIR) signaling domain, which also includes the IL-1, IL-18, and IL-33 receptors. Upon engagement of distinct TLRs by specific pathogen-associated molecular patterns, such as bacterial lipoprotein (for TLR2), viral dsRNA (for TLR3), or LPS (for TLR4), intracellular signaling cascades mediate activation of transcription factors such as NFκB and interferon regulatory factors (IRFs) leading to gene induction and the production of pro-inflammatory cytokines, chemokines and interferons. Upon activation, IL-1R and all of the TLRs, excluding only TLR3, recruit the TIR domain-containing adaptor molecule myeloid differentiation factor 88 (MyD88) through a TIR-TIR homotypic protein interaction, leading to NFκB activation (2Medzhitov 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 (1311) Google Scholar). For TLR2 and TLR4, the TIR adaptor Mal (3Fitzgerald 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: 78-83Crossref PubMed Scopus (1005) Google Scholar) recruits MyD88 to the receptor complex (4Kagan J.C. Medzhitov R. Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). TLR3 utilizes another TIR adaptor known as TIR domain-containing adaptor inducing interferon β (TRIF) (5Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat. Immunol. 2003; 4: 161-167Crossref PubMed Scopus (1014) Google Scholar, 6Hoebe K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar). TLR4 can also signal through TRIF, via a bridging adaptor called TRIF-related adaptor molecule (TRAM), resulting in a delayed MyD88-independent NFκB response (7Oshiumi H. Sasai M. Shida K. Fujita T. Matsumoto M. Seya T. J. Biol. Chem. 2003; 278: 49751-49762Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Formation of the TLR·adaptor complex leads to the recruitment of interleukin-1 receptor associated kinases (IRAKs) (8Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Crossref PubMed Scopus (344) Google Scholar). Four IRAKs have been identified namely IRAK-1, IRAK-2, IRAK-M, and IRAK-4. Both IRAK-1 and IRAK-4 are active Ser/Thr kinases, and phosphorylation of IRAK-1 by IRAK-4 is crucial for IRAK-1 activation during TLR signaling (9Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar, 10Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (549) Google Scholar). Mice lacking IRAK-4 are completely resistant to LPS treatment and, furthermore, display attenuated IL-1R-, TLR2-, TLR3-, and TLR9-induced cytokine responses (11Suzuki 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: 750-756Crossref PubMed Scopus (671) Google Scholar). Conversely, although IRAK-1 was originally thought to be critical for TLR-induced NFκB activation, IRAK-1-deficient mice show only partial defects in IL-1-, IL-18-, and LPS-induced signaling (12Kanakaraj 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 (177) Google Scholar, 13Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; 163: 978-984PubMed Google Scholar, 14Swantek J.L. Tsen M.F. Cobb M.H. Thomas J.A. J. Immunol. 2000; 164: 4301-4306Crossref PubMed Scopus (234) Google Scholar). IRAK-M on the other hand has been identified as an inhibitor of TLR signaling (15Kobayashi K. Hernandez L.D. Galan J.E. Janeway Jr., C.A. Medzhitov R. Flavell R.A. Cell. 2002; 110: 191-202Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar). Previous studies have implicated IRAK-2 in IL-1R and TLR4 signaling by showing that IRAK-2 can associate with MyD88 and Mal (3Fitzgerald 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: 78-83Crossref PubMed Scopus (1005) Google Scholar, 16Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google Scholar). Furthermore, the poxvirus protein A52, which interacts with both IRAK-2 and TRAF6, can inhibit TLR-induced NFκB activation (17Bowie A. Kiss-Toth E. Symons J.A. Smith G.L. Dower S.K. O'Neill L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10162-10167Crossref PubMed Scopus (391) Google Scholar, 18Harte M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). However, the extent of the role of IRAK-2 in TLR signaling and its mechanism of action remain to be established. Here we present evidence that A52 inhibits NFκB solely via its interaction with IRAK-2 and not TRAF6, and that IRAK-2 is required for all TLR-mediated pathways to NFκB. We show that IRAK-2, but not IRAK-1 causes TRAF6 ubiquitination, a critical step in the activation of NFκB by TLRs. Thus we propose that IRAK-2 has a more fundamental role than IRAK-1, in the TLR-NFκB axis. Expression Vectors—Generation of the A52 and ΔA52 expression vectors has been described previously (17Bowie A. Kiss-Toth E. Symons J.A. Smith G.L. Dower S.K. O'Neill L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10162-10167Crossref PubMed Scopus (391) Google Scholar, 19Maloney G. Schroder M. Bowie A.G. J. Biol. Chem. 2005; 280: 30838-30844Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The source of other plasmids was: IRAK-2-Myc, kIRAK-2-Myc, and AU1-MyD88 (M. Muzio, Mario Negri Institute, Milan, Italy), TLR3-FLAG (D. Golenbock, University of Massachusetts Medical School, Worchester, MA), CD4-TLR4 (R. Medzhitov, Yale University, New Haven, CT), IRAK-1 and FLAG-TRAF6 (Tularik Inc., San Francisco, CA), Mal, TRAM, and retinoic acid-inducible gene-I (K. Fitzgerald, University of Massachusetts Medical School, Worchester, MA), TRIF (C. Basler, Mount Sinai School of Medicine, New York, NY), and HA-ubiquitin (A. Mansell, Monash University, Melbourne, Australia). Antibodies and Reagents—Antibodies used were: anti-Myc mAb, anti-β-actin mAb, and anti-FLAG mAb (Sigma); anti-HA mAb (Covance); anti-IRAK-1 mAb and anti-TRAF6 polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-IRAK-2 polyclonal Ab (Upstate Biotechnologies). Human recombinant IL-1α was from NCI, National Institutes of TLR used were LPS from A. University, and and and activation as as induction were by gene or were and and with gene plasmids and expression plasmids the as described previously J. Haga I.R. Schroder M. Bartlett N.W. Maloney G. Reading P.C. K.A. Smith G.L. Bowie A.G. J. Exp. Med. 2005; PubMed Scopus Google Scholar, M. R. Schroder M. J. Bowie A.G. Nat. Immunol. 2006; PubMed Scopus Google Scholar). For all of was to data for and were (for or (for by the of For of NFκB of a gene was The was used to with of a protein expression with of the was by of of or protein expression vectors as required in with the were with TLR for to and of were in and data are as induction to for a of a of was by J. Haga I.R. Schroder M. Bartlett N.W. Maloney G. Reading P.C. K.A. Smith G.L. Bowie A.G. J. Exp. Med. 2005; PubMed Scopus Google Scholar). was in and data are as for a of the human gene the of were with by with and a a cell were and by and For gene in were with and with and a of to with or LPS for For in primary human were from in for and with as was and were for with LPS were and cytokine production by and were from as previously described J. Haga I.R. Schroder M. Bartlett N.W. Maloney G. Reading P.C. K.A. Smith G.L. Bowie A.G. J. Exp. Med. 2005; PubMed Scopus Google Scholar). For of was with were by of with the to protein or for were by and of were the IRAK-2 gene the to A52 IRAK-2 to from TLRs to we that poxvirus protein A52 could inhibit NFκB activation by TLR2, -3, -4, or in human and associate with both TRAF6 and IRAK-2 M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). we to that A52 expression NFκB activation through all TLRs and in A52 and NFκB induction in TLR2 and not it that A52 was of all TLR pathways to NFκB, both in human and cells. We the of A52 to inhibit NFκB on its interaction with TRAF6 the interaction has been shown to activation, a A52 protein of the could associate with TRAF6 activation G. Schroder M. Bowie A.G. J. Biol. Chem. 2005; 280: 30838-30844Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). ΔA52 could with IRAK-2, it a to IRAK-2 in NFκB activation. that A52 the induction of an gene by treatment of with of TLR3 and treatment or of a active CD4-TLR4 and ΔA52 was as a of NFκB activation via TLR3, and TLR4 as Thus the interaction of A52 with TRAF6 is dispensable for of and TLR-induced NFκB activation, that it is via its with IRAK-2 that A52 can TLR signaling to NFκB, to IRAK-2 as a critical downstream of signaling IRAK-2 NFκB and previously shown M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google Scholar) IRAK-2 IRAK-1, led to NFκB activation the of IRAK-2 on activation, another downstream event in TLR a gene on the phosphorylation of the was J. Haga I.R. Schroder M. Bartlett N.W. Maloney G. Reading P.C. K.A. Smith G.L. Bowie A.G. J. Exp. Med. 2005; PubMed Scopus Google Scholar). IRAK-2 of the to to for MyD88 expression IRAK-1 to activation in IRAK-1 has previously been implicated in the induction of by (12Kanakaraj 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 (177) Google and of IRAK-1 has been shown to phosphorylation of A. E. A. Mol. Biol. 2001; PubMed Scopus Google Scholar). gene induction is and and with expression of IRAK-2 in induction and in the production of in both to to for by TLRs by the IRAK-2 have previously shown although the poxvirus protein which both and could the induction of via TLR3, A52 could not J. Haga I.R. Schroder M. Bartlett N.W. Maloney G. Reading P.C. K.A. Smith G.L. Bowie A.G. J. Exp. Med. 2005; PubMed Scopus Google Scholar). that IRAK-2 is not required for by the role of IRAK-2 in TLR-mediated activation in a the of A52 to inhibit other TLR pathways to was the of was as a and its and was a by A52 on the activation of by TLR4 or by the and downstream TRIF from the and can also and activation via MyD88 A. E. N. K.A. J. Biol. 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Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google and we also found that inhibited TLR3-, and NFκB activation, NFκB activation through retinoic acid-inducible gene-I was not TLR3, retinoic acid-inducible gene-I is a viral RNA receptor that NFκB activation M. M. T. N. T. M. K. S. Fujita T. Nat. Immunol. PubMed Scopus Google Scholar). evidence for a role for IRAK-2 in signal from TLR3, TLR4, and TLR8 to NFκB, gene was used to IRAK-2 The of IRAK-2 was by showing that IRAK-2 protein expression from a expression was completely in the presence of IRAK-2 with with a and that NFκB activation by expression of IRAK-2 was also while treatment of with the on or NFκB the IRAK-2 of the NFκB gene by TLR by the for IRAK-2 in TLR4 signaling was in primary human by the of IRAK-2 on LPS-induced cytokine production in human cells. treatment not LPS-induced IRAK-2 by IRAK-2 of MyD88 but of IRAK-2 to in TLR signaling cascades to NFκB IRAK-2 to NFκB for TLR4 the adaptors MyD88, Mal, TRIF, and TRAM, TLR3 only TRIF and TLR8 solely through MyD88 S. S. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the of IRAK-2 on the induction of NFκB by MyD88, Mal, TRIF, and was IRAK-2 inhibited MyD88 signaling to NFκB and in particular NFκB by NFκB by Mal was in with both data are with of IRAK-2 downstream of MyD88 M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google Scholar) and Mal (3Fitzgerald 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: 78-83Crossref PubMed Scopus (1005) Google Scholar). although NFκB activation by was to IRAK-2 of NFκB activation was TRAM, which is upstream of TRIF in the TLR4 was data alternative usage of IRAK-2 in the and pathways to NFκB activation. The that TLR3 of MyD88 and Mal and solely utilizes TRIF, has a for IRAK-2, that IRAK-2 be the TLR3 signaling complex upstream of TRIF. IRAK-2 with IRAK-2 can associate with Mal (3Fitzgerald 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: 78-83Crossref PubMed Scopus (1005) Google Scholar) and a complex with the IL-1R M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google we that IRAK-2 could with TIR domain-containing in to IRAK-1, which via a interaction with We IRAK-2 could associate with the TLR3 receptor by an TLR3 and complex was in the of and the of the interaction was of TLR3 by with TLR3 and IRAK-2 the of IRAK-2 to associate with TLR3 the receptor complex was in the of which the of the IRAK-2 NFκB by TRAF6 can also with the domain-containing protein TRAF6 M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google which is an downstream molecule for transcription factor and activation by and TLRs. a of IRAK-2 the of TRAF6 IRAK-2, as previously by H. B. M. T. D. M. M. Du K. S. Y. Wu H. Nature. 2002; PubMed Scopus Google Scholar). were to the of TRAF6 as an of IRAK-2 The central of both were to because of the TRAF6 of its H. B. M. T. D. M. M. Du K. S. Y. Wu H. Nature. 2002; PubMed Scopus Google Scholar). that was to activate NFκB activation or to the of the TRAF6 IRAK-2 completely all IRAK-2 that TRAF6 is a critical for NFκB and activation. The of TRAF6 to IRAK-2 was by central to distinct mutants, and signal pathways as as the protein to was to NFκB or activation, or to the Both were a in the TRAF6 is for IRAK-2 the TRAF6 not to be for IRAK-2 signaling to NFκB or activation. of the central the TRAF6 of its to with TRAF6 in an in for the of H. B. M. T. D. M. M. Du K. S. Y. Wu H. Nature. 2002; PubMed Scopus Google we the of the to with was in it was of with TRAF6 the active also associated with TRAF6 the to TRAF6 in that the of with TRAF6 was not through the TRAF6 other are to TRAF6 is an ubiquitin and TRAF6 is a critical step in the activation of NFκB by TLRs C. M. J. Chen Nature. 2001; PubMed Scopus Google Scholar). Here we that expression of IRAK-2 is to of Further, although the IRAK-2 loss-of-function could with TRAF6 could TRAF6 an of HA-ubiquitin to TRAF6 was in the presence of and by but not by Thus the of the IRAK-2 to activate downstream signaling with to TRAF6 The of TRAF6 was of TRAF6 to the presence of ubiquitin of TRAF6 of TRAF6 was also in the presence of IRAK-2 the of ubiquitin to TRAF6 IRAK-2 is cells. it has been that causes TRAF6 of IRAK-2 to IRAK-1 in the that IRAK-1 the of IRAK-2 to TRAF6 to 6; the for step in the induction of NFκB in response to TLR signal IRAK-1 and IRAK-2 evidence for a more central role for IRAK-2 in TLR signaling to NFκB with identified M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google the role of IRAK-2 in TLR signaling has remained as has the extent of its in different TLR we have the of the poxvirus protein A52 to inhibit TLR signaling to IRAK-2 as a central role in NFκB activation. other known A52 to have on the and the of an A52 which only with IRAK-2 and not its other TRAF6, to the of A52 on NFκB to an with A52 IRAK-2 to be but that it IRAK-2 of signaling and it with other signaling M.T. Haga I.R. Maloney G. Gray P. Reading P.C. Bartlett N.W. Smith G.L. Bowie A. O'Neill L.A. J. Exp. Med. 2003; 197: 343-351Crossref PubMed Scopus (310) Google Scholar). shown that A52 inhibited NFκB activation by TLR2-, -3, -4, -5, -7, and we on the TLR3 because IRAK-1 been shown to have role in NFκB activation. NFκB activation by was in IRAK-1-deficient and IRAK-1 IRAK-4 was to the TLR3 receptor complex engagement Z. M. H. Li X. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). we that IRAK-2 is part of the TLR3 receptor complex and of IRAK-2 expression by suppressed NFκB activation in to and pathways to NFκB, IRAK-2 to upstream of the with the interaction of IRAK-2 with for in TLR signaling pathways to NFκB is shown in is TRIF is in NFκB activation by in the of TRIF, signaling to NFκB from TLR3 is K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar, M. S. H. K. T. H. M. M. K. S. Science. 2003; PubMed Scopus Google it is that IRAK-2 a TLR3 to NFκB of TRIF. However, because studies it to be TRIF in TLR3 signaling in studies have the originally central role of IRAK-1 in NFκB activation. For in IRAK-1 was dispensable for NFκB activation and pro-inflammatory cytokine production S. S. M. T. H. M. C. T. S. J. Exp. Med. 2005; PubMed Scopus Google Scholar). is thought to signal in a to and we show that inhibited NFκB activation. Further, alternative for IRAK-1 have such as TLR4-mediated activation and of the Y. Li T. Li J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that of IRAK-1 on LPS induction of the pro-inflammatory IRAK-2 inhibited LPS-induced NFκB activation, and, in primary human LPS production of the chemokine has been identified as a of IRAK-1 S. S. M. T. H. M. C. T. S. J. Exp. Med. 2005; PubMed Scopus Google Scholar). The of an complex by phosphorylation of to be a for induction by and the of of A52 or expression on -5, and activation that IRAK-2 has role in activation. IRAK-1, although in some pathways to NFκB, be more for TLR signaling to the that IRAK-2 but not IRAK-1 could TRAF6 evidence for the of IRAK-2 IRAK-1 in TLR-mediated NFκB activation, because the of TRAF6 is for IL-1R/TLR signaling to NFκB C. E. A. J. C. C. Chen Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Wu Y. Y. B. C. X. Z. 2006; PubMed Scopus Google although of TRAF6 is on by IRAK-2 is to be established. it been that IRAK-1 could TRAF6 ubiquitination, a recent demonstrated that the protein could TRAF6 in the of IRAK-1 E. E. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). Thus we have defined a in IRAK-1 and IRAK-2 which that are not but in TLR previously identified in IRAK-2 H. B. M. T. D. M. M. Du K. S. Y. Wu H. Nature. 2002; PubMed Scopus Google not been The is IRAK-2 the TRAF6 is with the of the which the of IRAK-2 expression to NFκB and activation and The that to TRAF6 and activate NFκB that is in critical for signal and with TRAF6 we have demonstrated that IRAK-2 has a central role in TLR signaling pathways to NFκB activation, and in to IRAK-1, is by all TLRs for IRAK-2 can with TLR, namely TLR3, and TRAF6 is a critical downstream of IRAK-2 that IRAK-2 is to be in activation, IRAK-2 be a to pro-inflammatory TLR via NFκB, while