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IRAK4 Kinase Activity Is Redundant for Interleukin-1 (IL-1) Receptor-associated Kinase Phosphorylation and IL-1 Responsiveness

IRAK4 Kinase Activity Is Redundant for Interleukin-1 (IL-1) Receptor-associated Kinase... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 25, Issue of June 18, pp. 26748 –26753, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. IRAK4 Kinase Activity Is Redundant for Interleukin-1 (IL-1) Receptor-associated Kinase Phosphorylation and IL-1 Responsiveness* Received for publication, January 23, 2004, and in revised form, March 15, 2004 Published, JBC Papers in Press, April 14, 2004, DOI 10.1074/jbc.M400785200 Jinzhong Qin‡§, Zhengfan Jiang‡, Youcun Qian‡, Jean-Laurent Casanova , and Xiaoxia Li‡§ From the ‡Department of Immunology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the Unite ´ d’Immunologie et d’He ´matologie Pe ´diatriques, Laboratoire de Ge ´ne ´tique Humaine des Maladies Infectieuses Unite ´ Mixte de Recherche Universite ´ Rene ´ Descartes-INSERM U550, 75270 Paris Cedex 06, France, and the §Department of Biology, Cleveland State University, Cleveland, Ohio 44115 taining receptors (1) and the leucine-rich repeat motif-contain- Interleukin-1 (IL-1) stimulation leads to the recruit- ment of interleukin-1 receptor-associated kinase (IRAK) ing receptors (2). The Ig domain subgroup includes IL-1R1, to the IL-1 receptor, where IRAK is phosphorylated, IL-18R, and T1/ST2. IL-1 has been demonstrated to be one of ubiquitinated, and eventually degraded. Kinase-inac- the key orchestrators of the immune response, eliciting a wide tive mutant IRAK is still phosphorylated in response to range of biological responses, including fever, lymphocyte acti- IL-1 stimulation when it is transfected into IRAK-defi- vation, and leukocyte infusion to the site of injury and infection cient cells, suggesting that there must be an IRAK ki- (3). The leucine-rich repeat subgroup consists of at least 10 nase in the pathway. The fact that IRAK4, another IRAK Toll-like receptors (TLRs) (2, 4 –7), which detect invasion of family member necessary for the IL-1 pathway, is able to pathogens by recognizing the pathogen-associated molecular phosphorylate IRAK in vitro suggests that IRAK4 might patterns, leading to the activation of innate and adaptive im- be the IRAK kinase. However, we now found that the mune responses. IRAK4 kinase-inactive mutant had the same ability as The IL-1R/TLR superfamily delivers biological activities the wild-type IRAK4 in restoring IL-1-mediated signal- mainly by activating the transcription of various genes in dif- ing in human IRAK4-deficient cells, including NFB-de- ferent target cells. Tremendous effort has been devoted to pendent reporter gene expression, the activation of understanding the signaling pathways mediated by this recep- NFB and JNK, and endogenous IL-8 gene expression. tor superfamily. Due to the similarities in their cytoplasmic These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. domains, these receptors employ related yet distinct signaling Furthermore, we showed that the kinase activity of components and downstream pathways. Because IL-1R was IRAK4 was not necessary for IL-1-induced IRAK phos- the first discovered receptor in this superfamily, the IL-1-me- phorylation, suggesting that IRAK phosphorylation can diated signaling pathway serves as a “prototype” for other probably be achieved either by autophosphorylation or family members. Genetic and biochemical studies revealed that by trans-phosphorylation through IRAK4. In support of IL-1R mediates a very complex pathway, involving a cascade of this, only the impairment of the kinase activity of both kinases organized by multiple adapter molecules into sequen- IRAK and IRAK4 efficiently abolished the IL-1 pathway, tial signaling complexes, leading to the activation of the tran- demonstrating that the kinase activity of IRAK and scription factors NFB, activating transcription factor, and IRAK4 is redundant for IL-1-mediated signaling. More- AP-1 (8 –10). Based on studies by our group and others (11–13), over, consistent with the fact that IRAK4 is a necessary we postulated a model of the IL-1 pathway (Fig. 1). Upon IL-1 component of the IL-1 pathway, we found that IRAK4 stimulation, adapter molecules (including MyD88 (14) and was required for the efficient recruitment of IRAK to the Tollip (15)) are first recruited to the IL-1 receptor followed by IL-1 receptor complex. the recruitment of two serine-threonine kinases, IRAK4 (16, 17) and IRAK (18, 19), and the adapter TRAF6 (20), resulting in the formation of the receptor complex (Complex I). During The Toll-IL-1 receptor superfamily, a large family of pro- the formation of Complex I, IRAK is phosphorylated, which teins defined by the presence of an intracellular Toll-IL-1 re- creates an interface for its interaction with adapter Pellino 1 ceptor (TIR) domain, plays crucial roles in the immune re- (11). The formation of Pellino 1-IRAK4-IRAK-TRAF6 causes sponses. This superfamily can be divided into two main conformational changes in the receptor complex (Complex I), subgroups based on their extracellular domains, the Ig-con- releasing these signaling molecules from the receptor. The released components then interact with the membrane-bound preassociated TAK1-TAB1-TAB2 (12), resulting in the forma- * This work was supported by National Institutes of Health Grant tion of Complex II (IRAK-TRAF6-TAK1-TAB1-TAB2). TAK1 GM 600020 (to X. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must and TAB2 are phosphorylated in the membrane-bound Com- therefore be hereby marked “advertisement” in accordance with 18 plex II, triggering the dissociation and translocation of TRAF6- U.S.C. Section 1734 solely to indicate this fact. TAK1-TAB1-TAB2 (Complex III) from the membrane to the To whom correspondence should be addressed: Dept. of Immunol- ogy, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH cytosol. The translocated Complex III interacts with additional 44195. Tel.: 216-445-8706; Fax: 216-444-9329; E-mail: [email protected]. factors in the cytosol, leading to TAK1 activation. The activa- The abbreviations used are: IL-1, interleukin-1; IL-1R, IL-1 recep- tion of TAK1 eventually leads to the activation of IB kinase, in tor; IRAK, IL-1R-associated kinase; TLR, Toll-like receptor; JNK, c-Jun turn leading to the phosphorylation and degradation of IB NH -terminal kinase; TIR, Toll-IL-1R; TIRAP, TIR-containing adapter protein; wt, wild-type; KD, kinase-inactive; luc, luciferase. proteins and liberation of NFB to activate transcription in the 26748 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. The Kinase Activity of Human IRAK4 26749 FIG.1. Model of IL-1-mediated signaling pathway. IKK,IB kinase. FIG.3. IL-1-induced activation of NFB and JNK. A,NFB gel shift assay. Cell extracts were made from untreated or IL-1-treated (100 units/ml for 15 or 30 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, human wild-type, or kinase-inactive IRAK4. The NFB binding site from the IP-10 gene was used as a probe. The two bands in the gel shift assay are due mainly to p50-p65 het- erodimers (bottom) and p65-p65 homodimers (top). B, JNK activation induced by IL-1. Whole cell lysates were prepared from untreated or IL-1-treated (100 units/ml for 15 or 20 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, wild-type, or kinase- inactive IRAK4 and subjected to Western blot analysis using anti-phos- pho-c-Jun (P-JNK) and anti-JNK (JNK) antibodies (loading control). FIG.2. Complementation of human IRAK4-deficient cells. Hu- man IRAK4-deficient fibroblasts were co-transfected transiently with E-selectin-luc (A) or AP-1-dependent luc (B) with increasing amounts of ing transcription factor and AP1, thereby also activating gene human wild-type, kinase-inactive, or truncated IRAK4 retroviral ex- transcription. pression constructs. Thirty-six hours later, the cells were either un- Although the above model of the IL-1 pathway is well sup- treated or stimulated for 6 h with IL-1 (100 units/ml). Luciferase activ- ported by our published studies and work from other groups, ities were normalized to -galactosidase. Data are presented as fold induction of luciferase activity in the treated cells. The experiments the detailed signaling mechanism for the pathway is still un- were repeated four times. Shown are the data from a typical experi- clear. We recently further investigated the formation and acti- ment. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT, vation of the IL-1 receptor complex (Complex I), especially IRAK4KD, and IRAK4-(1–191)) are shown by Western analysis of regarding the mechanism of IRAK and IRAK4 in the receptor whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control (Actin). complex. IRAK is hyperphosphorylated at the receptor com- plex. The phosphorylation of IRAK is likely to play an impor- tant role in IL-1-mediated signaling, although the kinase ac- nucleus (21–24). Activated TAK1 has also been implicated in tivity of IRAK is dispensable for its function (19). We have the IL-1-induced activation of MKK6 and JNK (25), leading to previously shown that kinase-inactive mutant IRAK is still the activation of other transcription factors, including activat- phosphorylated in response to IL-1 stimulation when it is 26750 The Kinase Activity of Human IRAK4 FIG.4. Northern analysis of IL-8 gene expression. Total RNAs were made from human IRAK4-deficient fibroblasts stably transfected with empty vector, wild- type, or kinase-inactive IRAK4 untreated or treated with IL-1 (100 units/ml for 1– 4 h). Human IL-8 cDNA was used as a probe, and the signals were normalized after rep- robing with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT and IRAK4KD) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti- FLAG)), and actin was used as a loading control (Actin). FIG.5. Western analysis of IL-1-induced IRAK phosphorylation and ubiquitination. Human IRAK4-deficient fibroblasts stably trans- fected with control vector DNA, wild-type, or kinase-inactive IRAK4 were untreated or treated with IL-1 (100 units/ml for 30 or 60 min). Cell extracts were analyzed by the Western procedure with anti-IRAK. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT and IRAK4KD) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control (Actin). U-IRAK, ubiquitinated IRAK. P-IRAK, phosphorylated IRAK. 4-deficient fibroblasts (26) were maintained in Dulbecco’s modified Ea- transfected into IRAK-null cells, suggesting that there must be gle’s medium, supplemented with 10% fetal calf serum, penicillin G an IRAK kinase in the pathway (19). Recent studies indicate (100 g/ml), and streptomycin (100 g/ml). that another member of the IRAK family, IRAK4, is likely to Recombinant Plasmids and Transfection—pE-selectin-luc, an NFB- function as the IRAK kinase. Severe impairment of IL-1R/TLR- dependent E-selectin-luciferase reporter plasmid, and AP-1-dependent- mediated signaling is observed in mice lacking IRAK4 and in luciferase reporter construct were described by Woronicz et al. (23). human patients deficient in IRAK4 (16, 17, 26, 27). Further- N-terminal FLAG-tagged wild-type, kinase-inactive (KK213AA, two lysine residues in the ATP binding pocket were mutated to alanine, more, IRAK4 is able to phosphorylate IRAK in vitro (16). abbreviated as IRAK4KD) and truncated (1–191aa) IRAK-4 cDNA were Therefore, it has been postulated that IRAK4 is recruited to the cloned into the retrovirus vector, pBabe-puro. Transfection of the IRAK- IL-1 receptor upon IL-1 stimulation, where it is activated, 4-deficient fibroblasts was performed using the FuGENE 6 transfection leading to the phosphorylation of IRAK. However, we now reagent, as recommended by the manufacturer (Roche Diagnostics). found that the kinase activity of human IRAK4 is dispensable Transfection solution was prepared by mixing 1 g of plasmid DNA and for IL-1-mediated signaling. The presence of kinase activity 3 l of FuGENE 6 transfection reagent in 100 l of serum-free medium. After incubation at room temperature for 15 min, the mixture was from either IRAK or IRAK4 is sufficient to phosphorylate added to tissue culture wells containing 1  10 cells in 2 ml of complete IRAK. Although IRAK4 is not necessary for IRAK phosphoryl- culture medium. ation, IRAK4 facilitates the recruitment of IRAK to the IL-1 Coimmunoprecipitation and Immunoblotting—Cells that were not receptor complex. treated or treated with IL-1 (100 units/ml) were lysed in a Triton- containing lysis buffer (0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 MATERIALS AND METHODS mM NaCl, 12.5 mM -glycerophosphate, 1.5 mM MgCl ,10mM NaF, 2 Biological Reagents and Cell Culture—Recombinant human IL-1 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM EGTA, 20 M was provided by the National Cancer Institute. Anti-JNK, anti-phos- aprotinin, 1 mM phenylmethylsulfonyl fluoride). Cell extracts were in- pho-JNK, anti-IL-1R, and anti-IRAK polyclonal antibodies were from cubated with 1 g of antibody or preimmune serum (negative control) Santa Cruz (Santa Cruz Biotechnology). Anti-IRAK4 polyclonal anti- for 2 h followed by a 2-h incubation with 20 l of protein A-Sepharose body was kindly provided by Dr. Holger Wesche (Tularik, South San beads (prewashed and resuspended in phosphate-buffered saline at a Francisco, CA). 293-TK/Zeo cells (19), I1A cells (19), and human IRAK- 1:1 ratio). After incubation, the beads were washed four times with lysis The Kinase Activity of Human IRAK4 26751 FIG.6. Kinase-dependent E-selectin luciferase activation by IL-1 in the IRAK-deficient I1A cells. Wild-type or mutant IRAK (100 ng) and increasing amounts of kinase-inactive IRAK4 expression constructs were co-transfected with E-selectin-luc (100 ng) into the human IRAK-deficient I1A cells. The transfected cells were untreated or treated with IL-1 (100 units/ml for 6 h) followed by luciferase reporter assay. Data are presented as -fold induction of luciferase activity in the treated cells. Shown are the averages from three independent experiments. Cells transfected with vector DNA (100 ng) and E-selectin-luc DNA (100 ng) only were used as controls. Levels of the transfected FLAG-tagged IRAK4KD and IRAK (IRAKwt and IRAKmt) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)) and anti-IRAK (IRAK), and actin was used as a loading control (Actin). buffer, separated by SDS-PAGE, transferred to Immobilon-P mem- We then compared the ability of IRAK4KD (kinase-inactive branes (Millipore), and analyzed by immunoblotting. mutant IRAK4) with IRAK4wt (wild-type IRAK4) in restoring Reporter Assays—2  10 cells were transfected by the same proce- IL-1-induced NFB and JNK activation. Equal amounts of dure as described above with 1 g of pE-selectin-luc, 1 gofpSV -- human IRAK4wt and IRAK4KD were stably co-transfected galactosidase, and 100 ng of each expression construct. After 48 h, the with pBaBePURO into human IRAK4-deficient cells. The pu- cells were split onto two 35-mm plates and stimulated with IL-1 the next day for 4 h before harvest. Luciferase and -galactosidase activi- romycin-resistant clones were pooled and then either untreated ties were determined by using the luciferase assay system and chemi- or treated with IL-1 for the indicated times followed by NFB luminescent reagents from Promega (Madison, WI). gel shift assay and Western analysis with anti-phospho-JNK. Gel Shift Assays and Northern blotting—An NFB binding site (5- As shown in Fig. 3, IRAK4wt (wild-type IRAK4) and IRAK4KD GAGCAGAGGGAAATTCCGTAACTT-3) from the IP-10 gene was used (kinase-inactive mutant IRAK4) restored similar levels of as a probe. Complementary oligonucleotides, end-labeled with polynucle- otide kinase (Roche Applied Science) and [- P]ATP, were annealed by NFB and JNK activation in human IRAK4-deficient cells in slow cooling. Approximately 20,000 cpm of probe were used per assay. response to IL-1 stimulation, confirming that the kinase activ- Whole cell extracts were used for the assay. The binding reaction was ity of human IRAK4 is dispensable for IL-1-mediated signaling. carried out at 4 °C for 20 min in a total volume of 20 l containing 20 mM We also examined IL-1-induced gene expression in human Hepes buffer, pH 7.0, 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM dithiothre- IRAK4-deficient cells stably transfected with either IRAK4wt itol, 0.25 mM phenylmethanesulfonyl fluoride, and 10% glycerol. For Northern analysis, total RNA was isolated by using the TRIzol (human wild-type IRAK4) or IRAK4KD (human kinase-inactive reagent (Invitrogen). Appropriate gene-specific probes were made with mutant IRAK4). As shown in Fig. 4, the kinase inactive mutant a random priming kit (Amersham Biosciences). Transfers to the posi- IRAK4 (IRAK4KD) was able to restore similar levels of IL-8 gene tively charged nylon membrane Hybond-N were performed according to expression in response to IL-1 stimulation as compared with the procedure provided by Amersham Biosciences. wild-type IRAK4 (IRAK4wt). Taken together, the above results RESULTS clearly demonstrate that the kinase activity of human IRAK4 is not necessary for IL-1-mediated signaling pathway. The Kinase Activity of Human IRAK4 Is Dispensable for IL-1- The Kinase Activity of Human IRAK and IRAK4 Is Redundant mediated Signaling—Severe impairment of IL-1R/TLR-mediated for IL-1-mediated Signaling—We have previously taken a ge- signaling is observed in mice lacking IRAK4 and in IRAK4- netic approach to study IL-1-dependent signaling pathways; deficient cells derived from human patients, indicating that through random mutagenesis, we generated IL-1-unresponsive IRAK4 is required for IL-1-mediated signaling (16, 17, 26). How- cell lines lacking specific components of the pathways from hu- ever, it is unclear whether the kinase activity of IRAK4 is nec- man embryonic kidney 293 cells (19). Mutant cell line I1A, which essary for the pathway. To address this question, increasing lacks both IRAK protein and mRNA, has been used effectively to amounts of DNA of the human wild-type (IRAK4wt) and kinase- study structure-function relationships of IRAK in IL-1-depend- inactive mutant IRAK4 (KK213AA, abbreviated as IRAK4KD) ent signaling. Neither NFB nor JNK is activated in IL-1-treated were co-transfected into human IRAK4-deficient cells with NFB- I1A cells, but these responses are restored in I1A-IRAK cells, dependent-E-selectin-luc and AP-1-dependent-luc reporter con- indicating that IRAK is required for both. However, the kinase structs. As shown in Fig. 2, wild-type (IRAK4wt) and the kinase- activity of IRAK is not required for IL-1-dependent signaling inactive mutant IRAK4 (IRAK4KD) displayed similar ability in restoring the IL-1-induced NFB- and AP-1-dependent luciferase since kinase-dead IRAK mutants were still able to restore IL-1 responsiveness. The fact that the kinase-inactive mutant IRAK is activity, suggesting that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. still phosphorylated in response to IL-1 stimulation when it is 26752 The Kinase Activity of Human IRAK4 IRAK modification in IRAK4-deficeint cells stably transfected with the kinase-inactive mutant IRAK4 could very well be due to IRAK auto-phosphorylation in response to IL-1 stimulation. Taken together, the above results showed that although both human IRAK and IRAK4 are required for IL-1 signaling, their kinase activity is not necessary for the pathway, suggesting that the kinase activity of human IRAK4 and IRAK might be redundant for IL-1 signaling. To test this hypothesis, we co- transfected increasing amounts of DNA of IRAK4KD (kinase- inactive mutant IRAK4) into I1A cells either with wild-type IRAK (IRAKwt) or kinase-inactive mutant IRAK (K239A, ab- breviated as IRAKmt). Interestingly, although the kinase-in- active mutant IRAK4 (IRAK4KD) had minimum effect on E-selectin-promoter-driven luciferase activity when it is co- transfected with wild-type IRAK (IRAKwt) into the I1A cells, the IRAK4 mutant (IRAK4KD) efficiently inhibited the lucif- erase activity upon its co-transfection with the kinase-inactive mutant IRAK (IRAKmt) into the I1A cells (Fig. 6). These re- sults showed that only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, suggesting that the kinase activity of human IRAK and IRAK4 is probably indeed redundant for IL-1-mediated signaling. IRAK4 Facilitates the Recruitment of IRAK to the IL-1 Re- ceptor Complex—Previous studies show that upon IL-1 stimu- lation, the adapter molecules MyD88 and Tollip are recruited to the IL-1 receptor followed by the recruitment of two serine- threonine kinases, IRAK4 and IRAK, and the adapter TRAF6, resulting in the formation of the receptor complex (Complex I) (11–13). As shown in Fig. 7A, IRAK4 was still efficiently re- cruited to the receptor in both IRAK-null I1A and MyD88-null I3A cells, indicating that the recruitment of IRAK4 does not require MyD88 or IRAK. On the other hand, IRAK was not recruited to the IL-1 receptor in MyD88-null I3A cells, confirm- ing that MyD88 is responsible for recruiting IRAK to the re- ceptor upon IL-1 stimulation (Fig. 7A). Taken together, these results indicate that the IL-1-induced recruitment of IRAK4 to the IL-1 receptor is upstream of MyD88. Although the above results clearly indicate that the recruit- ment of IRAK4 to the IL-1 receptor is upstream and independ- FIG.7. IL-1-induced IL-1R immune complex. A, extracts of 293-WT, ent of IRAK, we examined the role of IRAK4 in the recruitment I1A, I2A, or I3A, untreated or stimulated with IL-1 (100 units/ml for 10 or of IRAK to the IL-1 receptor. As shown in Fig. 7B, whereas a 20 min), were immunoprecipitated with anti-IRAK-4 followed by Western small amount of IRAK was recruited to the IL-1 receptor in analyses with antibodies against IL-1R, MyD88, and IRAK. The expres- IRAK4-deficient cells upon IL-1 stimulation, transfection of sion of IRAK and MyD88 in wild-type 293 cells (WT) and mutant cell lines (I1A, I2A, and I3A) is shown by Western analysis of whole cell extracts IRAK4 facilitated the recruitment of IRAK to the receptor in (WCE) with anti-IRAK (IRAK) and anti-MyD88 (MyD88). B, extracts of these cells. IRAK-4-deficient fibroblasts transfected with control vector DNA or wild- type IRAK4 were untreated or stimulated with IL-1 (100 units/ml for 30 DISCUSSION or 60 min) and then immunoprecipitated (IP) with anti-IRAK followed by Western analyses with antibodies against IL-1R. The expression of In this report, we showed that IRAK4 kinase-inactive mu- IRAK4 in the transfected cells is shown by Western analysis of whole cell tant had the same ability as the wild-type IRAK4 in restoring extracts with anti-IRAK4 (IRAK4). IL-1-mediated signaling in IRAK4-deficient cells, indicating that the kinase activity of IRAK4 is not necessary for the IL-1 transfected into I1A cells suggests that there must be an IRAK pathway. The fact that only the impairment of the kinase kinase in the pathway (19). As IRAK4 was able to phosphorylate activity of both IRAK and IRAK4 efficiently abolished the IL-1 IRAK in vitro, it was suggested that IRAK4 might be the IRAK pathway suggests that the kinase activity of IRAK and IRAK4 kinase. To examine the role of IRAK4 in IL-1-induced IRAK is probably redundant for IL-1-mediated signaling. Further- phosphorylation, we examined IL-1-induced IRAK modification more, we showed that although the kinase activity of IRAK4 in human IRAK4-deficient cells stably transfected with wild-type was not necessary for IL-1-induced IRAK phosphorylation, IRAK4 and kinase-inactive IRAK4 mutant. As shown in Fig. 5, IRAK4 facilitated the recruitment of IRAK to the IL-1 receptor IRAK was not phosphorylated or ubiquitinated upon IL-1 stim- complex. Based on these results, we hypothesize that upon IL-1 ulation in IRAK4-deficient cells transfected with vector DNA stimulation, through receptor adapter molecules, IRAK4 is re- (Vector). Transfection of wild-type IRAK4 (IRAK4wt) into the cruited to the IL-1 receptor, which then facilitates the recruit- IRAK4-deficient cells restored IL-1-induced IRAK phosphoryla- ment of IRAK to the receptor, where IRAK is hyperphosphory- tion and ubiquitination in these cells. Interestingly, IL-1-induced lated. The IL-1-induced IRAK phosphorylation can be achieved IRAK modification was also restored in IRAK4-deficient cells either by autophosphorylation or by trans-phosphorylation transfected with the kinase-inactive mutant IRAK4 (IRAK4KD), through IRAK4. The phospho-IRAK then interacts with recep- indicating that the kinase activity of human IRAK4 is not nec- tor proximal signaling components TRAF6 and Pellino1 and is essary for IL-1-induced IRAK phosphorylation. 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IRAK4 Kinase Activity Is Redundant for Interleukin-1 (IL-1) Receptor-associated Kinase Phosphorylation and IL-1 Responsiveness

Qin, Jinzhong; Jiang, Zhengfan; Qian, Youcun; Casanova, Jean-Laurent; Li, Xiaoxia
Journal of Biological Chemistry – Jun 1, 2004
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0021-9258
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10.1074/jbc.m400785200
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 25, Issue of June 18, pp. 26748 –26753, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. IRAK4 Kinase Activity Is Redundant for Interleukin-1 (IL-1) Receptor-associated Kinase Phosphorylation and IL-1 Responsiveness* Received for publication, January 23, 2004, and in revised form, March 15, 2004 Published, JBC Papers in Press, April 14, 2004, DOI 10.1074/jbc.M400785200 Jinzhong Qin‡§, Zhengfan Jiang‡, Youcun Qian‡, Jean-Laurent Casanova , and Xiaoxia Li‡§ From the ‡Department of Immunology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the Unite ´ d’Immunologie et d’He ´matologie Pe ´diatriques, Laboratoire de Ge ´ne ´tique Humaine des Maladies Infectieuses Unite ´ Mixte de Recherche Universite ´ Rene ´ Descartes-INSERM U550, 75270 Paris Cedex 06, France, and the §Department of Biology, Cleveland State University, Cleveland, Ohio 44115 taining receptors (1) and the leucine-rich repeat motif-contain- Interleukin-1 (IL-1) stimulation leads to the recruit- ment of interleukin-1 receptor-associated kinase (IRAK) ing receptors (2). The Ig domain subgroup includes IL-1R1, to the IL-1 receptor, where IRAK is phosphorylated, IL-18R, and T1/ST2. IL-1 has been demonstrated to be one of ubiquitinated, and eventually degraded. Kinase-inac- the key orchestrators of the immune response, eliciting a wide tive mutant IRAK is still phosphorylated in response to range of biological responses, including fever, lymphocyte acti- IL-1 stimulation when it is transfected into IRAK-defi- vation, and leukocyte infusion to the site of injury and infection cient cells, suggesting that there must be an IRAK ki- (3). The leucine-rich repeat subgroup consists of at least 10 nase in the pathway. The fact that IRAK4, another IRAK Toll-like receptors (TLRs) (2, 4 –7), which detect invasion of family member necessary for the IL-1 pathway, is able to pathogens by recognizing the pathogen-associated molecular phosphorylate IRAK in vitro suggests that IRAK4 might patterns, leading to the activation of innate and adaptive im- be the IRAK kinase. However, we now found that the mune responses. IRAK4 kinase-inactive mutant had the same ability as The IL-1R/TLR superfamily delivers biological activities the wild-type IRAK4 in restoring IL-1-mediated signal- mainly by activating the transcription of various genes in dif- ing in human IRAK4-deficient cells, including NFB-de- ferent target cells. Tremendous effort has been devoted to pendent reporter gene expression, the activation of understanding the signaling pathways mediated by this recep- NFB and JNK, and endogenous IL-8 gene expression. tor superfamily. Due to the similarities in their cytoplasmic These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. domains, these receptors employ related yet distinct signaling Furthermore, we showed that the kinase activity of components and downstream pathways. Because IL-1R was IRAK4 was not necessary for IL-1-induced IRAK phos- the first discovered receptor in this superfamily, the IL-1-me- phorylation, suggesting that IRAK phosphorylation can diated signaling pathway serves as a “prototype” for other probably be achieved either by autophosphorylation or family members. Genetic and biochemical studies revealed that by trans-phosphorylation through IRAK4. In support of IL-1R mediates a very complex pathway, involving a cascade of this, only the impairment of the kinase activity of both kinases organized by multiple adapter molecules into sequen- IRAK and IRAK4 efficiently abolished the IL-1 pathway, tial signaling complexes, leading to the activation of the tran- demonstrating that the kinase activity of IRAK and scription factors NFB, activating transcription factor, and IRAK4 is redundant for IL-1-mediated signaling. More- AP-1 (8 –10). Based on studies by our group and others (11–13), over, consistent with the fact that IRAK4 is a necessary we postulated a model of the IL-1 pathway (Fig. 1). Upon IL-1 component of the IL-1 pathway, we found that IRAK4 stimulation, adapter molecules (including MyD88 (14) and was required for the efficient recruitment of IRAK to the Tollip (15)) are first recruited to the IL-1 receptor followed by IL-1 receptor complex. the recruitment of two serine-threonine kinases, IRAK4 (16, 17) and IRAK (18, 19), and the adapter TRAF6 (20), resulting in the formation of the receptor complex (Complex I). During The Toll-IL-1 receptor superfamily, a large family of pro- the formation of Complex I, IRAK is phosphorylated, which teins defined by the presence of an intracellular Toll-IL-1 re- creates an interface for its interaction with adapter Pellino 1 ceptor (TIR) domain, plays crucial roles in the immune re- (11). The formation of Pellino 1-IRAK4-IRAK-TRAF6 causes sponses. This superfamily can be divided into two main conformational changes in the receptor complex (Complex I), subgroups based on their extracellular domains, the Ig-con- releasing these signaling molecules from the receptor. The released components then interact with the membrane-bound preassociated TAK1-TAB1-TAB2 (12), resulting in the forma- * This work was supported by National Institutes of Health Grant tion of Complex II (IRAK-TRAF6-TAK1-TAB1-TAB2). TAK1 GM 600020 (to X. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must and TAB2 are phosphorylated in the membrane-bound Com- therefore be hereby marked “advertisement” in accordance with 18 plex II, triggering the dissociation and translocation of TRAF6- U.S.C. Section 1734 solely to indicate this fact. TAK1-TAB1-TAB2 (Complex III) from the membrane to the To whom correspondence should be addressed: Dept. of Immunol- ogy, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH cytosol. The translocated Complex III interacts with additional 44195. Tel.: 216-445-8706; Fax: 216-444-9329; E-mail: [email protected]. factors in the cytosol, leading to TAK1 activation. The activa- The abbreviations used are: IL-1, interleukin-1; IL-1R, IL-1 recep- tion of TAK1 eventually leads to the activation of IB kinase, in tor; IRAK, IL-1R-associated kinase; TLR, Toll-like receptor; JNK, c-Jun turn leading to the phosphorylation and degradation of IB NH -terminal kinase; TIR, Toll-IL-1R; TIRAP, TIR-containing adapter protein; wt, wild-type; KD, kinase-inactive; luc, luciferase. proteins and liberation of NFB to activate transcription in the 26748 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. The Kinase Activity of Human IRAK4 26749 FIG.1. Model of IL-1-mediated signaling pathway. IKK,IB kinase. FIG.3. IL-1-induced activation of NFB and JNK. A,NFB gel shift assay. Cell extracts were made from untreated or IL-1-treated (100 units/ml for 15 or 30 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, human wild-type, or kinase-inactive IRAK4. The NFB binding site from the IP-10 gene was used as a probe. The two bands in the gel shift assay are due mainly to p50-p65 het- erodimers (bottom) and p65-p65 homodimers (top). B, JNK activation induced by IL-1. Whole cell lysates were prepared from untreated or IL-1-treated (100 units/ml for 15 or 20 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, wild-type, or kinase- inactive IRAK4 and subjected to Western blot analysis using anti-phos- pho-c-Jun (P-JNK) and anti-JNK (JNK) antibodies (loading control). FIG.2. Complementation of human IRAK4-deficient cells. Hu- man IRAK4-deficient fibroblasts were co-transfected transiently with E-selectin-luc (A) or AP-1-dependent luc (B) with increasing amounts of ing transcription factor and AP1, thereby also activating gene human wild-type, kinase-inactive, or truncated IRAK4 retroviral ex- transcription. pression constructs. Thirty-six hours later, the cells were either un- Although the above model of the IL-1 pathway is well sup- treated or stimulated for 6 h with IL-1 (100 units/ml). Luciferase activ- ported by our published studies and work from other groups, ities were normalized to -galactosidase. Data are presented as fold induction of luciferase activity in the treated cells. The experiments the detailed signaling mechanism for the pathway is still un- were repeated four times. Shown are the data from a typical experi- clear. We recently further investigated the formation and acti- ment. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT, vation of the IL-1 receptor complex (Complex I), especially IRAK4KD, and IRAK4-(1–191)) are shown by Western analysis of regarding the mechanism of IRAK and IRAK4 in the receptor whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control (Actin). complex. IRAK is hyperphosphorylated at the receptor com- plex. The phosphorylation of IRAK is likely to play an impor- tant role in IL-1-mediated signaling, although the kinase ac- nucleus (21–24). Activated TAK1 has also been implicated in tivity of IRAK is dispensable for its function (19). We have the IL-1-induced activation of MKK6 and JNK (25), leading to previously shown that kinase-inactive mutant IRAK is still the activation of other transcription factors, including activat- phosphorylated in response to IL-1 stimulation when it is 26750 The Kinase Activity of Human IRAK4 FIG.4. Northern analysis of IL-8 gene expression. Total RNAs were made from human IRAK4-deficient fibroblasts stably transfected with empty vector, wild- type, or kinase-inactive IRAK4 untreated or treated with IL-1 (100 units/ml for 1– 4 h). Human IL-8 cDNA was used as a probe, and the signals were normalized after rep- robing with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT and IRAK4KD) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti- FLAG)), and actin was used as a loading control (Actin). FIG.5. Western analysis of IL-1-induced IRAK phosphorylation and ubiquitination. Human IRAK4-deficient fibroblasts stably trans- fected with control vector DNA, wild-type, or kinase-inactive IRAK4 were untreated or treated with IL-1 (100 units/ml for 30 or 60 min). Cell extracts were analyzed by the Western procedure with anti-IRAK. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT and IRAK4KD) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control (Actin). U-IRAK, ubiquitinated IRAK. P-IRAK, phosphorylated IRAK. 4-deficient fibroblasts (26) were maintained in Dulbecco’s modified Ea- transfected into IRAK-null cells, suggesting that there must be gle’s medium, supplemented with 10% fetal calf serum, penicillin G an IRAK kinase in the pathway (19). Recent studies indicate (100 g/ml), and streptomycin (100 g/ml). that another member of the IRAK family, IRAK4, is likely to Recombinant Plasmids and Transfection—pE-selectin-luc, an NFB- function as the IRAK kinase. Severe impairment of IL-1R/TLR- dependent E-selectin-luciferase reporter plasmid, and AP-1-dependent- mediated signaling is observed in mice lacking IRAK4 and in luciferase reporter construct were described by Woronicz et al. (23). human patients deficient in IRAK4 (16, 17, 26, 27). Further- N-terminal FLAG-tagged wild-type, kinase-inactive (KK213AA, two lysine residues in the ATP binding pocket were mutated to alanine, more, IRAK4 is able to phosphorylate IRAK in vitro (16). abbreviated as IRAK4KD) and truncated (1–191aa) IRAK-4 cDNA were Therefore, it has been postulated that IRAK4 is recruited to the cloned into the retrovirus vector, pBabe-puro. Transfection of the IRAK- IL-1 receptor upon IL-1 stimulation, where it is activated, 4-deficient fibroblasts was performed using the FuGENE 6 transfection leading to the phosphorylation of IRAK. However, we now reagent, as recommended by the manufacturer (Roche Diagnostics). found that the kinase activity of human IRAK4 is dispensable Transfection solution was prepared by mixing 1 g of plasmid DNA and for IL-1-mediated signaling. The presence of kinase activity 3 l of FuGENE 6 transfection reagent in 100 l of serum-free medium. After incubation at room temperature for 15 min, the mixture was from either IRAK or IRAK4 is sufficient to phosphorylate added to tissue culture wells containing 1  10 cells in 2 ml of complete IRAK. Although IRAK4 is not necessary for IRAK phosphoryl- culture medium. ation, IRAK4 facilitates the recruitment of IRAK to the IL-1 Coimmunoprecipitation and Immunoblotting—Cells that were not receptor complex. treated or treated with IL-1 (100 units/ml) were lysed in a Triton- containing lysis buffer (0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 MATERIALS AND METHODS mM NaCl, 12.5 mM -glycerophosphate, 1.5 mM MgCl ,10mM NaF, 2 Biological Reagents and Cell Culture—Recombinant human IL-1 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM EGTA, 20 M was provided by the National Cancer Institute. Anti-JNK, anti-phos- aprotinin, 1 mM phenylmethylsulfonyl fluoride). Cell extracts were in- pho-JNK, anti-IL-1R, and anti-IRAK polyclonal antibodies were from cubated with 1 g of antibody or preimmune serum (negative control) Santa Cruz (Santa Cruz Biotechnology). Anti-IRAK4 polyclonal anti- for 2 h followed by a 2-h incubation with 20 l of protein A-Sepharose body was kindly provided by Dr. Holger Wesche (Tularik, South San beads (prewashed and resuspended in phosphate-buffered saline at a Francisco, CA). 293-TK/Zeo cells (19), I1A cells (19), and human IRAK- 1:1 ratio). After incubation, the beads were washed four times with lysis The Kinase Activity of Human IRAK4 26751 FIG.6. Kinase-dependent E-selectin luciferase activation by IL-1 in the IRAK-deficient I1A cells. Wild-type or mutant IRAK (100 ng) and increasing amounts of kinase-inactive IRAK4 expression constructs were co-transfected with E-selectin-luc (100 ng) into the human IRAK-deficient I1A cells. The transfected cells were untreated or treated with IL-1 (100 units/ml for 6 h) followed by luciferase reporter assay. Data are presented as -fold induction of luciferase activity in the treated cells. Shown are the averages from three independent experiments. Cells transfected with vector DNA (100 ng) and E-selectin-luc DNA (100 ng) only were used as controls. Levels of the transfected FLAG-tagged IRAK4KD and IRAK (IRAKwt and IRAKmt) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)) and anti-IRAK (IRAK), and actin was used as a loading control (Actin). buffer, separated by SDS-PAGE, transferred to Immobilon-P mem- We then compared the ability of IRAK4KD (kinase-inactive branes (Millipore), and analyzed by immunoblotting. mutant IRAK4) with IRAK4wt (wild-type IRAK4) in restoring Reporter Assays—2  10 cells were transfected by the same proce- IL-1-induced NFB and JNK activation. Equal amounts of dure as described above with 1 g of pE-selectin-luc, 1 gofpSV -- human IRAK4wt and IRAK4KD were stably co-transfected galactosidase, and 100 ng of each expression construct. After 48 h, the with pBaBePURO into human IRAK4-deficient cells. The pu- cells were split onto two 35-mm plates and stimulated with IL-1 the next day for 4 h before harvest. Luciferase and -galactosidase activi- romycin-resistant clones were pooled and then either untreated ties were determined by using the luciferase assay system and chemi- or treated with IL-1 for the indicated times followed by NFB luminescent reagents from Promega (Madison, WI). gel shift assay and Western analysis with anti-phospho-JNK. Gel Shift Assays and Northern blotting—An NFB binding site (5- As shown in Fig. 3, IRAK4wt (wild-type IRAK4) and IRAK4KD GAGCAGAGGGAAATTCCGTAACTT-3) from the IP-10 gene was used (kinase-inactive mutant IRAK4) restored similar levels of as a probe. Complementary oligonucleotides, end-labeled with polynucle- otide kinase (Roche Applied Science) and [- P]ATP, were annealed by NFB and JNK activation in human IRAK4-deficient cells in slow cooling. Approximately 20,000 cpm of probe were used per assay. response to IL-1 stimulation, confirming that the kinase activ- Whole cell extracts were used for the assay. The binding reaction was ity of human IRAK4 is dispensable for IL-1-mediated signaling. carried out at 4 °C for 20 min in a total volume of 20 l containing 20 mM We also examined IL-1-induced gene expression in human Hepes buffer, pH 7.0, 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM dithiothre- IRAK4-deficient cells stably transfected with either IRAK4wt itol, 0.25 mM phenylmethanesulfonyl fluoride, and 10% glycerol. For Northern analysis, total RNA was isolated by using the TRIzol (human wild-type IRAK4) or IRAK4KD (human kinase-inactive reagent (Invitrogen). Appropriate gene-specific probes were made with mutant IRAK4). As shown in Fig. 4, the kinase inactive mutant a random priming kit (Amersham Biosciences). Transfers to the posi- IRAK4 (IRAK4KD) was able to restore similar levels of IL-8 gene tively charged nylon membrane Hybond-N were performed according to expression in response to IL-1 stimulation as compared with the procedure provided by Amersham Biosciences. wild-type IRAK4 (IRAK4wt). Taken together, the above results RESULTS clearly demonstrate that the kinase activity of human IRAK4 is not necessary for IL-1-mediated signaling pathway. The Kinase Activity of Human IRAK4 Is Dispensable for IL-1- The Kinase Activity of Human IRAK and IRAK4 Is Redundant mediated Signaling—Severe impairment of IL-1R/TLR-mediated for IL-1-mediated Signaling—We have previously taken a ge- signaling is observed in mice lacking IRAK4 and in IRAK4- netic approach to study IL-1-dependent signaling pathways; deficient cells derived from human patients, indicating that through random mutagenesis, we generated IL-1-unresponsive IRAK4 is required for IL-1-mediated signaling (16, 17, 26). How- cell lines lacking specific components of the pathways from hu- ever, it is unclear whether the kinase activity of IRAK4 is nec- man embryonic kidney 293 cells (19). Mutant cell line I1A, which essary for the pathway. To address this question, increasing lacks both IRAK protein and mRNA, has been used effectively to amounts of DNA of the human wild-type (IRAK4wt) and kinase- study structure-function relationships of IRAK in IL-1-depend- inactive mutant IRAK4 (KK213AA, abbreviated as IRAK4KD) ent signaling. Neither NFB nor JNK is activated in IL-1-treated were co-transfected into human IRAK4-deficient cells with NFB- I1A cells, but these responses are restored in I1A-IRAK cells, dependent-E-selectin-luc and AP-1-dependent-luc reporter con- indicating that IRAK is required for both. However, the kinase structs. As shown in Fig. 2, wild-type (IRAK4wt) and the kinase- activity of IRAK is not required for IL-1-dependent signaling inactive mutant IRAK4 (IRAK4KD) displayed similar ability in restoring the IL-1-induced NFB- and AP-1-dependent luciferase since kinase-dead IRAK mutants were still able to restore IL-1 responsiveness. The fact that the kinase-inactive mutant IRAK is activity, suggesting that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. still phosphorylated in response to IL-1 stimulation when it is 26752 The Kinase Activity of Human IRAK4 IRAK modification in IRAK4-deficeint cells stably transfected with the kinase-inactive mutant IRAK4 could very well be due to IRAK auto-phosphorylation in response to IL-1 stimulation. Taken together, the above results showed that although both human IRAK and IRAK4 are required for IL-1 signaling, their kinase activity is not necessary for the pathway, suggesting that the kinase activity of human IRAK4 and IRAK might be redundant for IL-1 signaling. To test this hypothesis, we co- transfected increasing amounts of DNA of IRAK4KD (kinase- inactive mutant IRAK4) into I1A cells either with wild-type IRAK (IRAKwt) or kinase-inactive mutant IRAK (K239A, ab- breviated as IRAKmt). Interestingly, although the kinase-in- active mutant IRAK4 (IRAK4KD) had minimum effect on E-selectin-promoter-driven luciferase activity when it is co- transfected with wild-type IRAK (IRAKwt) into the I1A cells, the IRAK4 mutant (IRAK4KD) efficiently inhibited the lucif- erase activity upon its co-transfection with the kinase-inactive mutant IRAK (IRAKmt) into the I1A cells (Fig. 6). These re- sults showed that only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, suggesting that the kinase activity of human IRAK and IRAK4 is probably indeed redundant for IL-1-mediated signaling. IRAK4 Facilitates the Recruitment of IRAK to the IL-1 Re- ceptor Complex—Previous studies show that upon IL-1 stimu- lation, the adapter molecules MyD88 and Tollip are recruited to the IL-1 receptor followed by the recruitment of two serine- threonine kinases, IRAK4 and IRAK, and the adapter TRAF6, resulting in the formation of the receptor complex (Complex I) (11–13). As shown in Fig. 7A, IRAK4 was still efficiently re- cruited to the receptor in both IRAK-null I1A and MyD88-null I3A cells, indicating that the recruitment of IRAK4 does not require MyD88 or IRAK. On the other hand, IRAK was not recruited to the IL-1 receptor in MyD88-null I3A cells, confirm- ing that MyD88 is responsible for recruiting IRAK to the re- ceptor upon IL-1 stimulation (Fig. 7A). Taken together, these results indicate that the IL-1-induced recruitment of IRAK4 to the IL-1 receptor is upstream of MyD88. Although the above results clearly indicate that the recruit- ment of IRAK4 to the IL-1 receptor is upstream and independ- FIG.7. IL-1-induced IL-1R immune complex. A, extracts of 293-WT, ent of IRAK, we examined the role of IRAK4 in the recruitment I1A, I2A, or I3A, untreated or stimulated with IL-1 (100 units/ml for 10 or of IRAK to the IL-1 receptor. As shown in Fig. 7B, whereas a 20 min), were immunoprecipitated with anti-IRAK-4 followed by Western small amount of IRAK was recruited to the IL-1 receptor in analyses with antibodies against IL-1R, MyD88, and IRAK. The expres- IRAK4-deficient cells upon IL-1 stimulation, transfection of sion of IRAK and MyD88 in wild-type 293 cells (WT) and mutant cell lines (I1A, I2A, and I3A) is shown by Western analysis of whole cell extracts IRAK4 facilitated the recruitment of IRAK to the receptor in (WCE) with anti-IRAK (IRAK) and anti-MyD88 (MyD88). B, extracts of these cells. IRAK-4-deficient fibroblasts transfected with control vector DNA or wild- type IRAK4 were untreated or stimulated with IL-1 (100 units/ml for 30 DISCUSSION or 60 min) and then immunoprecipitated (IP) with anti-IRAK followed by Western analyses with antibodies against IL-1R. The expression of In this report, we showed that IRAK4 kinase-inactive mu- IRAK4 in the transfected cells is shown by Western analysis of whole cell tant had the same ability as the wild-type IRAK4 in restoring extracts with anti-IRAK4 (IRAK4). IL-1-mediated signaling in IRAK4-deficient cells, indicating that the kinase activity of IRAK4 is not necessary for the IL-1 transfected into I1A cells suggests that there must be an IRAK pathway. The fact that only the impairment of the kinase kinase in the pathway (19). As IRAK4 was able to phosphorylate activity of both IRAK and IRAK4 efficiently abolished the IL-1 IRAK in vitro, it was suggested that IRAK4 might be the IRAK pathway suggests that the kinase activity of IRAK and IRAK4 kinase. To examine the role of IRAK4 in IL-1-induced IRAK is probably redundant for IL-1-mediated signaling. Further- phosphorylation, we examined IL-1-induced IRAK modification more, we showed that although the kinase activity of IRAK4 in human IRAK4-deficient cells stably transfected with wild-type was not necessary for IL-1-induced IRAK phosphorylation, IRAK4 and kinase-inactive IRAK4 mutant. As shown in Fig. 5, IRAK4 facilitated the recruitment of IRAK to the IL-1 receptor IRAK was not phosphorylated or ubiquitinated upon IL-1 stim- complex. Based on these results, we hypothesize that upon IL-1 ulation in IRAK4-deficient cells transfected with vector DNA stimulation, through receptor adapter molecules, IRAK4 is re- (Vector). Transfection of wild-type IRAK4 (IRAK4wt) into the cruited to the IL-1 receptor, which then facilitates the recruit- IRAK4-deficient cells restored IL-1-induced IRAK phosphoryla- ment of IRAK to the receptor, where IRAK is hyperphosphory- tion and ubiquitination in these cells. Interestingly, IL-1-induced lated. The IL-1-induced IRAK phosphorylation can be achieved IRAK modification was also restored in IRAK4-deficient cells either by autophosphorylation or by trans-phosphorylation transfected with the kinase-inactive mutant IRAK4 (IRAK4KD), through IRAK4. The phospho-IRAK then interacts with recep- indicating that the kinase activity of human IRAK4 is not nec- tor proximal signaling components TRAF6 and Pellino1 and is essary for IL-1-induced IRAK phosphorylation. 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Published: Jun 1, 2004

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