February 1, 2004Open Access

Sequential Autophosphorylation Steps in the Interleukin-1 Receptor-associated Kinase-1 Regulate its Availability as an Adapter in Interleukin-1 Signaling

CKChristian KolleweFraunhofer Institute for Molecular Biology and Applied Ecology AMAnne-Christin MackensenMedizinische Hochschule Hannover DNDetlef NeumannMedizinische Hochschule Hannover

Key Points

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Abstract

The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr209. By fluorescence energy transfer experiments, we demonstrate that Thr209 phosphorylation results in a conformational change of the kinase domain, permitting further phosphorylations to take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1. Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity. Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain and kinase domain. Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. This identifies IRAK-1 as a novel type of adapter protein, which employs its own kinase activity to introduce negative charges adjacent to the protein interaction domain, which anchors IRAK-1 at the active receptor complex. Thus, IRAK-1 regulates its own availability as an adapter molecule by sequential autophosphorylation. The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr209. By fluorescence energy transfer experiments, we demonstrate that Thr209 phosphorylation results in a conformational change of the kinase domain, permitting further phosphorylations to take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1. Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity. Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain and kinase domain. Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. This identifies IRAK-1 as a novel type of adapter protein, which employs its own kinase activity to introduce negative charges adjacent to the protein interaction domain, which anchors IRAK-1 at the active receptor complex. Thus, IRAK-1 regulates its own availability as an adapter molecule by sequential autophosphorylation. The interleukin-1 receptor-associated kinase (IRAK-1) 1The abbreviations used are: IRAK-1interleukin-1 receptor-associated kinase 1ILinterleukinTIRToll/interleukin-1 receptorTLRToll-like receptorTNFtumor necrosis factorHEKhuman embryonic kidneyHPLChigh pressure liquid chromatographyaaamino acidsco-IPcoimmunoprecipitationEGFPenhanced green fluorescent proteinFRETfluorescence resonance energy transfer.1The abbreviations used are: IRAK-1interleukin-1 receptor-associated kinase 1ILinterleukinTIRToll/interleukin-1 receptorTLRToll-like receptorTNFtumor necrosis factorHEKhuman embryonic kidneyHPLChigh pressure liquid chromatographyaaamino acidsco-IPcoimmunoprecipitationEGFPenhanced green fluorescent proteinFRETfluorescence resonance energy transfer. is the prototype of a small family of serine/threonine protein kinases that are key molecules in the signaling cascade of the Toll/IL-1 receptor (TIR) family (reviewed in Ref. 1Martin M.U. Wesche H. Biochim. Biophys. Acta. 2002; 1592: 265-280Crossref PubMed Scopus (335) Google Scholar). The TIR family comprises the IL-1 receptor subfamily, recognizing the endogenous proinflammatory cytokines IL-1 and IL-18, and the members of the Toll-like receptor (TLR) subfamily, recognizing pathogen-associated molecular patterns (reviewed in Ref. 2O'Neill L.A. Dinarello C.A. Immunol. Today. 2000; 21: 206-209Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). After ligand binding, all members of the TIR family form multimeric receptor complexes. These receptors share the cytoplasmic TIR domain, which is indispensable for signal transduction. The TIR domain serves as a scaffold for the recruitment of adapter proteins. This results in the activation of a core signaling module consisting of MyD88, IRAK-4 and IRAK-1, and TRAF6. Subsequently, several central signaling pathways are activated in parallel, the activation of NF-κB being a hallmark of the inflammatory response. The additional use of individual adapter proteins like TIRAP/Mal (3Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (825) Google Scholar, 4Fitzgerald 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 (995) Google Scholar) by TLR2 or TLR4 and TICAMI/TRIF by TLR3 (5Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat. Immunol. 2003; 4: 161-167Crossref PubMed Scopus (1001) Google Scholar, 6Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1014) Google Scholar) accounts for the differential biological response of cells observed after stimulation with distinct pathogens. interleukin-1 receptor-associated kinase 1 interleukin Toll/interleukin-1 receptor Toll-like receptor tumor necrosis factor human embryonic kidney high pressure liquid chromatography amino acids coimmunoprecipitation enhanced green fluorescent protein fluorescence resonance energy transfer. interleukin-1 receptor-associated kinase 1 interleukin Toll/interleukin-1 receptor Toll-like receptor tumor necrosis factor human embryonic kidney high pressure liquid chromatography amino acids coimmunoprecipitation enhanced green fluorescent protein fluorescence resonance energy transfer. Presently, the family of human IRAK molecules consists of four members: IRAK-1 (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar), IRAK-2 (8Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (976) Google Scholar), IRAK-M (9Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), and IRAK-4 (10Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (534) Google Scholar). Although these molecules share significant sequence and structural homologies, they also show pronounced differences. Thus, deletion of IRAK-4 abrogates cell responsiveness to IL-1 and a series of other ligands of the TIR family (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 (657) Google Scholar), whereas deletion of IRAK-1 only drastically reduces IL-1, IL-18, and lipopolysaccharide responsiveness (12Thomas 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, 13Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson C.A. J. PubMed Scopus Google Scholar, P. Ngo K. Wu Y. A. P. C.A. Peterson J. 1999; PubMed Scopus Google Scholar), that IRAK-2 and IRAK-M for IRAK-1 X. M. C. K. Cao Z. Stark G.R. Biol. 1999; PubMed Scopus Google Scholar). IRAK-1 the family to its with the activated IL-1 receptor complex M. A. K. R. J. Immunol. PubMed Scopus Google Scholar) and its pronounced (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar). IL-1 IRAK-1 is to the IL-1 receptor is to the adapter proteins MyD88 (8Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (976) Google Scholar, K. C. H. P. J.L. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Henzel W.J. Li S. Cao Z. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and a molecule also as of IRAK-1 G. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. C. A. K. J. Nat. Biol. 2000; 2: PubMed Scopus Google Scholar). additional adapter molecules that with IRAK-1 and in the of a signaling complex at the with IRAK-1 and Z. H. J. Bird T.A. Li X. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) and E. J. Immunol. 2002; 169: PubMed Scopus Google Scholar). interaction of IRAK-1 with the downstream adapter TRAF6 as H. H. J. T. A. Y. Y. M. K. K. J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). IRAK-1 phosphorylated, to a pronounced shift in as a hallmark of IRAK IRAK-1 leaves the receptor complex and with TRAF6 Z. J. Takeuchi M. T. Nature. 1996; PubMed Scopus Google Scholar) and and Z. J. Y. Matsumoto K. Li X. Biol. 2002; PubMed Scopus Google Scholar). in vitro, IRAK-1 that is an active protein for IRAK-1 downstream of IRAK-1 the only are IRAK-1 Tollip G. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and an adapter molecule that is phosphorylated by IRAK-1 and IRAK-4 A. C. Wesche H. 2003; PubMed Scopus Google Scholar). that the consequences of phosphorylation of IRAK-1 dissociation from MyD88 (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar), of the interaction with Tollip K. J. C. A. K. J. Nat. Biol. 2000; 2: PubMed Scopus Google Scholar), and of the of IRAK-1 A.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of these the individual or the amino acids that phosphorylated in IRAK-1 or IRAK-1 a shift in The of downstream and the that IRAK-1 kinase activity is for IL-1 signaling (9Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, X. M. C. K. Cao Z. Stark G.R. Biol. 1999; PubMed Scopus Google Scholar, J. M.U. 1999; PubMed Scopus Google Scholar, K. K. J. 1999; PubMed Scopus Google Scholar) that the kinase activity of IRAK-1 other and IL-1 We to and the of IRAK-1 and to the consequences of for the function of IRAK-1 in IL-1 we show that phosphorylation of IRAK-1 is to sequential phosphorylation which in are We Thr209 as the amino for activation of IRAK-1 kinase activity and demonstrate that Thr387 in the activation loop is to full enzymatic activity. The of full kinase activation is hyperphosphorylation in the ProST We show that this hyperphosphorylation regulates the interaction of IRAK-1 with its upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. These that IRAK-1 its kinase activity to and its own availability as an adapter protein in IL-1 human by D. and human from from to human IRAK-1, and MyD88 by Z. Cao cells at in with 1 amino and kidney cells and in with and human IRAK-1 and human IRAK-1 with a of Z. Cao and (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar). The from by of the and of the of IRAK-1 by in the of the as by the and as (9Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, H. Henzel W.J. Li S. Cao Z. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). IRAK-1 deletion by the for as The to the to with in a at the of The the at M. Sci. 1996; 21: Full Text PDF PubMed Scopus Google Scholar) used with to cells and the by with a of of The of used for and By the of in all After an the and cells and for in 1 1 and and by with or by the and After with the to in kinase or in by to and with the with by an enhanced with the as (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar, H. Henzel W.J. Li S. Cao Z. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). in a kinase and 1 of and for at The kinase by in proteins by The and IRAK-1 and IRAK-1 from and with in by an of The for at mass an mass with a and a The of to the mass of of by of of to with of and 1 of the IRAK-1 with a by the sequence and The the and and with the enhanced green fluorescent protein and The by M. for cells with of for type or fluorescent after cells with and in 1 of 1 1 by for and by an additional of from with an of of enhanced fluorescent After of the of from the fluorescence the fluorescence at at the proteins with and at the of the with the of U. 1 cells the type IL-1 and the with 1 for the of with with and with as with and with protein 1 for 1 at in the 1 After the in at times with IRAK-1 is a protein four individual an N-terminal death domain E. A. D. M. E. Sci. Full Text PDF PubMed Scopus Google a in and residues the ProST region by and the region by Li X. M. Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google a protein kinase domain and a TRAF6 interaction H. M. T. D. M. M. K. S. Y. Wu H. Nature. 2002; PubMed Scopus Google Scholar). of IRAK-1 for a that IRAK-1 phosphorylated IL-1 stimulation of cells or the phosphorylated or the individual amino acids in vitro, IRAK-1 is of which results in a shift to the observed in cells with IRAK-1 in cells and to the IRAK-1 shift after in an in kinase in the of further proteins we the and amino acids phosphorylated in IRAK-1. these with endogenous IRAK-1, we in which IRAK-1 is to (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar). This to IRAK-1 individual or series of the individual and these for or they as a for IRAK-1 proteins an N-terminal in or in with IRAK-1. and the to an in kinase We this in the in IRAK-1 or that form by the interaction of IRAK-1 death at the receptor complex (9Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), by MyD88 K. C. H. P. J.L. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these of death domain and of the ProST region and active as a for IRAK-1 The kinase domain the death domain the phosphorylated by IRAK-1 These results the ProST region and the kinase domain as the of IRAK-1 that of IRAK-1 the of the protein the sequence of the kinase domain whereas a protein of the ProST region active We to which active This N-terminal amino acids and By these amino acids with alanine Thr209 as the amino for kinase activity. Thr209 alanine in the kinase domain of IRAK-1 or in IRAK-1 Substitution of Thr209 by the amino acids or in a active kinase but these IRAK-1 Thr209 proteins as as IRAK-1 in which the These results demonstrate that Thr209 is for enzymatic and they that Thr209 is a of to phosphorylated, we liquid mass from IRAK-1 IRAK-1 as The to and the mass of the the phosphorylated the of of with J. J. PubMed Scopus Google Scholar, P. 2000; PubMed Scopus Google Scholar), the to the of but a series of and also to a series of which a and which a that the phosphorylation at of further the phosphorylation of this a by and the the of the observed of the of the with the series from to of that and that these as the phosphorylation in the The of a in the of is at the of the kinase domain. Thus, we that Thr209 in a conformational change of the IRAK-1 kinase domain. the of kinase activity observed in we used this protein to this We proteins that of conformational by in the of or These proteins of an N-terminal an the IRAK-1 and a enhanced fluorescent protein These in and proteins with the The fluorescence as and all cells and green fluorescence after The protein the type kinase domain in an in kinase that the of the fluorescent at the the kinase domain the of cells with the fluorescent the of and of enhanced fluorescent protein at and of the fluorescence energy transfer at observed after by that the further in the form in the type molecule in which fluorescence energy transfer from the enhanced fluorescent protein to the results with proteins Full of IRAK-1 enzymatic activity of protein kinases is after phosphorylation of or amino acids in the activation the kinase domain of IRAK-1 is we that full enzymatic activity in the activation The activation loop of IRAK-1 and residues that as for By we Thr387 as the amino for full kinase activity. The in of the First, phosphorylation with and only a shift observed also other of or in the activation loop these proteins and like in kinases of negative charges at amino acids phosphorylation and is to kinases we Thr387 for or these proteins like the protein and active kinases phosphorylations in amino acids to we the of Although in the with that of and in the activation loop an IRAK-1 These show that Thr387 is to full of IRAK-1, which results in the shift in Thus, Thr387 regulates hyperphosphorylation of IRAK-1. The ProST the of IRAK-1 the ProST region of IRAK-1 as a for autophosphorylation. We observed that phosphorylation of proteins the ProST region in several phosphorylated phosphorylations The ProST region and residues in a of amino of this sequence high of and in a to M. Sci. 1996; 21: Full Text PDF PubMed Scopus Google Scholar, S. R. M. Science. PubMed Scopus Google Scholar) and of IRAK-1 the death domain and of of the adjacent ProST region with and the phosphorylation in deletion of and several amino acids in the ProST region and in phosphorylation patterns or phosphorylation in the phosphorylation after IRAK-1 to IRAK-1 with a change observed after to IRAK-1 that the is for after to IRAK-1 which is the IRAK-1 death domain. The death domain phosphorylated by IRAK-1 in The in IRAK-1 or by that the shift of IRAK-1 is to These results the ProST region as the of of endogenous IRAK-1 IL-1 cells with 1 for the After and the phosphorylation of IRAK-1 by the full shift of IRAK-1 observed after a gel to demonstrate the of IRAK-1 after of IRAK-1 is The shift by of the with protein 1 Hyperphosphorylation in the ProST of IRAK-1 with the MyD88 and the ProST region and the kinase domain as the structural that phosphorylated in IRAK-1. We also the sequence of phosphorylation and the consequences of the and phosphorylation We the of the hyperphosphorylation in the ProST which is the phosphorylation MyD88 and Tollip in the recruitment of IRAK-1 to the active receptor complex. to which of the individual phosphorylation in IRAK-1 the interaction with MyD88 or we these molecules in cells with of IRAK-1 that enzymatic activity IRAK-1 the ProST region or the kinase domain we We that interaction of IRAK-1 with MyD88 or Tollip to whereas interaction with the downstream adapter TRAF6 with MyD88 or whereas TRAF6 with IRAK-1. the kinase domain and the ProST region this deletion like and only with TRAF6 and with MyD88 or deletion of the ProST as the of the interaction with and downstream adapters to IRAK-1 or IRAK-1 This that the protein interaction of the death of MyD88 and IRAK-1 is to the of in the ProST whereas the interaction of IRAK-1 with which is by the is to the interaction of Tollip with IRAK-1, we that of the kinase domain interaction with whereas of the ProST region Thus, the ProST region is for the interaction of Tollip with IRAK-1, but hyperphosphorylation of the ProST region the interaction These results that of in the ProST region serves the function of activated and IRAK-1 from the adapter molecules at the receptor whereas the protein interaction with the downstream adapter TRAF6. of IRAK-1 IL-1 in or in to show the of in the we cells with in a endogenous IRAK-1, and its phosphorylation by only the form of IRAK-1 is After of IRAK-1 and the high molecular of IRAK-1 are after of stimulation The of that IRAK-1 is we the we IRAK-1 after with the of IRAK-1 the shift of IRAK-1 by with protein 1 that the shift is to phosphorylation or The IRAK family members IRAK-1 and IRAK-4 are protein kinases in the signaling cascade of the TIR family. are active and for the of TIR family members are in the and of inflammatory Presently, the of the kinase activity of IRAK-1. receptor-associated protein kinases use enzymatic activity to downstream to and the signal at the receptor complex. IRAK-4 this its kinase activity is for IL-1 signaling (10Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (534) Google Scholar). IRAK-1, this is that Although IRAK-1 phosphorylated after IL-1 at in to IRAK-1 kinase activity to for IL-1 signaling (9Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, X. M. C. K. Cao Z. Stark G.R. Biol. 1999; PubMed Scopus Google Scholar, J. M.U. 1999; PubMed Scopus Google Scholar, K. K. J. 1999; PubMed Scopus Google Scholar). downstream that is in signal This that the kinase activity of IRAK-1 other and to the function of IRAK-1 enzymatic we to the and amino acids in IRAK-1 and to the molecular of activation of the enzymatic activity of IRAK-1. we to the consequences of IRAK-1 phosphorylation for its function as a signaling a prototype for the TIR we the IL-1 receptor for We the ProST region and the kinase domain as phosphorylation in IRAK-1. that in the death domain phosphorylated K. J. C. A. K. J. Nat. Biol. 2000; 2: PubMed Scopus Google Scholar, K. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We phosphorylation in death domain or we only or of IRAK-1, we that IRAK-1 is a for other protein Thus, that is phosphorylated by a protein kinase with for NF-κB signaling Wu J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the kinase domain in whereas phosphorylation in the ProST region to several that several phosphorylations take in the ProST region that are for the IRAK-1 We observed that the core kinase domain, as by in protein and as a for N-terminal the Thr209 to autophosphorylation. in the Thr209 the enzymatic activity of the kinase domain or IRAK-1. Thus, Thr209 as a amino that the kinase activity of IRAK-1. By mass we that Thr209 is phosphorylated in By we that Thr209 a conformational change of the kinase domain. the of Thr209 the kinase domain as a for type IRAK-1, whereas in the of Thr209 this molecule these results that Thr209 phosphorylation regulates the of IRAK-1 kinase domain, enzymatic activity and to the activation Full enzymatic activity is after phosphorylation of Thr387 in the activation loop of IRAK-1. of Thr387 in a of and in a This is in with the that Thr209 phosphorylation a enzymatic which to a phosphorylated an This also that Thr387 regulates full enzymatic activity for the pronounced shift of IRAK-1. of Thr387 kinase activity an additional phosphorylation in the activation We a distinct phosphorylation in the activation is that IRAK-1 in its the activation loop and residues in IRAK-4 phosphorylation we Thr387 in the activation loop of IRAK-1 as a for IRAK-4 (10Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (534) Google Scholar), that IRAK-4 the activation loop of IRAK-1 in IRAK-1 from cells and to full kinase and the IRAK-1 shift we that at in IRAK-1 at the Thr209 and phosphorylation that IRAK-4 Thr209 of IRAK-1 IRAK-1 with IRAK-4 show a significant shift (10Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (534) Google Scholar), that IRAK-4 IRAK-1 at the sequence which results in the hyperphosphorylation of IRAK-1. We hyperphosphorylation of IRAK-1 to the ProST a sequence of amino acids in and which in the of protein by as for phosphorylation and also for M. Sci. 1996; 21: Full Text PDF PubMed Scopus Google Scholar, S. R. M. Science. PubMed Scopus Google Scholar). The the ProST region is phosphorylated by with ProST and we only which by with that observed with IRAK-1 This that hyperphosphorylation of the ProST region is by IRAK-1. Although phosphorylation of IRAK-1 is for its function as an adapter molecule in experiments, consequences for IRAK-1 in that interaction with the receptor complex or the upstream adapters MyD88 and Tollip after IRAK-1 phosphorylation (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar, M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (976) Google Scholar, K. C. H. P. J.L. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Henzel W.J. Li S. Cao Z. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). we demonstrate that hyperphosphorylation in the ProST region the interaction with adapter in the of IRAK-1 from the active receptor complex. Hyperphosphorylation interaction with the downstream adapter TRAF6 as by Cao (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar). phosphorylation with the of IRAK-1 protein by A.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. R. C. Li J. Immunol. 2002; PubMed Scopus Google Scholar). We that hyperphosphorylation in or adjacent to the in the ProST region regulates phosphorylation is the which proteins to the Although we to IRAK-1 several in cell IRAK-1 in X. M. C. K. Cao Z. Stark G.R. Biol. 1999; PubMed Scopus Google Scholar, X. M. Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). We the for this but the that the form of IRAK-1 is in a of IRAK-1 the IRAK-1 as a novel type of adapter molecule that its kinase activity to its own availability in IL-1 signaling by autophosphorylation. is in The results leading to the of IRAK-1 activation in The sequential phosphorylation steps of Thr209 and Thr387 and the hyperphosphorylation in the ProST region in of IRAK-1 in gel electrophoresis. We cells with IL-1 and by the of endogenous human IRAK-1. We observed the as in a form in cells the of to IRAK-1 phosphorylated at Thr209 and and the form of We to the shift in from IL-1 cells by that the the shift to phosphorylation a these results to the that the sequential in also with endogenous IRAK-1 in cells after IL-1 results for the of IRAK-1 to kinase activity is for signal the kinase activity of IRAK-1 to in an to the that dissociation of IRAK-1 from the receptor complex and at the the of IRAK-1. This to an and of the IL-1 signal an The availability of kinase to the from the results We and for The of in the proteins is We to for and in the proteins for and to for in We for

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