Identification of Critical Residues of the MyD88 Death Domain Involved in the Recruitment of Downstream Kinases

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88E52A) and 58 (MyD88Y58A) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88K95A) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27–72)), comprising the Glu52 and Tyr58 residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27–72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-κB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling. MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88E52A) and 58 (MyD88Y58A) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88K95A) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27–72)), comprising the Glu52 and Tyr58 residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27–72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-κB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling. MyD88 was first discovered during studies addressing the differentiation of mouse myeloid cells in response to growth-inhibitory stimuli (1Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1990; 5: 387-396PubMed Google Scholar). Subsequent investigations revealed that MyD88 possesses a modular organization (2Hardiman G. Rock F.L. Balasubramanian S. Kastelein R.A. Bazan J.F. Oncogene. 1996; 13: 2467-2475PubMed Google Scholar), with an amino-terminal death domain (DD), 3The abbreviations used are: DDdeath domainTIRToll-interleukin-1 receptorTLRToll-like receptorILinterleukinIL-1Rinterleukin-1 receptorIDintermediate domainGFPgreen fluorescent proteinHEKhuman embryonic kidneyPBSphosphate-buffered salineIRAKinterleukin-1 receptor-associated kinase. 3The abbreviations used are: DDdeath domainTIRToll-interleukin-1 receptorTLRToll-like receptorILinterleukinIL-1Rinterleukin-1 receptorIDintermediate domainGFPgreen fluorescent proteinHEKhuman embryonic kidneyPBSphosphate-buffered salineIRAKinterleukin-1 receptor-associated kinase. found in proteins involved in cell death (3Hofmann K. Tschopp J. FEBS Lett. 1995; 371: 321-323Crossref PubMed Scopus (103) Google Scholar, 4Park H.H. Lo Y.C. Lin S.C. Wang L. Yang J.K. Wu H. Annu. Rev. Immunol. 2007; 25: 561-586Crossref PubMed Scopus (393) Google Scholar), and a carboxyl-terminal Toll-interleukin-1 receptor (TIR) domain, present in the intracytoplasmic tail of receptors belonging to the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily (5Hultmark D. Biochem. Biophys. Res. Commun. 1994; 199: 144-146Crossref PubMed Scopus (97) Google Scholar). MyD88 also has an intermediate domain (ID) that is crucial in TLR signaling due to its interaction with IRAK4 (6Burns K. Janssens S. Brissoni B. Olivos N. Beyaert R. Tschopp J. J. Exp. Med. 2003; 197: 263-268Crossref PubMed Scopus (412) Google Scholar). The role of MyD88 as a signal transducer was first shown in the pathways triggered by the activation of IL-1R (7Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (982) Google Scholar, 8Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar) and TLR4 (9Medzhitov 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 (1301) Google Scholar). Further studies showed that all TLRs, with the sole exception of TLR3, and the IL-1R family utilize the adaptor protein MyD88 to initiate their signaling pathway (10Janssens S. Beyaert R. Trends Biochem. Sci. 2002; 27: 474-482Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). death domain Toll-interleukin-1 receptor Toll-like receptor interleukin interleukin-1 receptor intermediate domain green fluorescent protein human embryonic kidney phosphate-buffered saline interleukin-1 receptor-associated kinase. death domain Toll-interleukin-1 receptor Toll-like receptor interleukin interleukin-1 receptor intermediate domain green fluorescent protein human embryonic kidney phosphate-buffered saline interleukin-1 receptor-associated kinase. By virtue of its modular organization, MyD88 critically bridges activated receptor complexes to downstream adaptors/effectors. Upon activation, MyD88 is recruited through its TIR domain by the homologous domain of the activated TLR/IL-1R (11Ulrichts P. Peelman F. Beyaert R. Tavernier J. FEBS Lett. 2007; 581: 629-636Crossref PubMed Scopus (25) Google Scholar, 12Brikos C. Wait R. Begum S. O'Neill L.A. Saklatvala J. Mol. Cell. Proteomics. 2007; 6: 1551-1559Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). MyD88, in turn, has been shown to interact with a family of downstream kinases, namely IRAK1 (13Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (772) Google Scholar), IRAK2 (7Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (982) Google Scholar), IRAK-M (15Wesche 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 (339) Google Scholar), and IRAK4 (16Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 5567-5572Crossref PubMed Scopus (540) Google Scholar), through the interaction of its DD with the respective DDs present in the amino-terminal region of IRAKs (17Neumann D. Kollewe C. Resch K. Martin M.U. Biochem. Biophys. Res. Commun. 2007; 354: 1089-1094Crossref PubMed Scopus (31) Google Scholar). At this stage, this multimeric complex is competent to elicit the propagation of the signal downstream of the receptor(s). Although MyD88 recruits IRAK-1 via DD-DD interactions, its recruitment of IRAK-4 appears to be rather unusual. Burns et al. (6Burns K. Janssens S. Brissoni B. Olivos N. Beyaert R. Tschopp J. J. Exp. Med. 2003; 197: 263-268Crossref PubMed Scopus (412) Google Scholar) first demonstrated that an alternatively spliced variant of MyD88 (MyD88s), lacking the ID domain, failed to interact with IRAK-4, suggesting that residues located in both the DD and ID of MyD88 are crucially involved in the recruitment of IRAK-4. Nevertheless, no information is available on the specific residues in the DD in MyD88 required for its interaction with either IRAK1 or IRAK4. The DD was initially defined as the region of homology between the cytoplasmic tails of the FAS/Apo1/CD95 and TNF receptors required for their induction of cytotoxic signaling (18Tartaglia L.A. Ayres T.M. Wong G.H. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1168) Google Scholar, 19Feinstein E. Kimchi A. Wallach D. Boldin M. Varfolomeev E. Trends Biochem. Sci. 1995; 20: 342-344Abstract Full Text PDF PubMed Scopus (271) Google Scholar). In analogy with other DD-containing proteins, this domain in MyD88 is also involved in the formation of homomeric and heteromeric interactions. Herein, we have undertaken an alanine-scanning mutational analysis to identify amino acids that are required for downstream signaling and might participate in the homomeric and heteromeric interactions. Our studies revealed that MyD88E52A and MyD88Y58A mutants are strongly impaired in the recruitment of both IRAK1 and IRAK4, whereas the MyD88K95A mutant is deficient in recruiting IRAK4. These findings identify residues within the DD of MyD88 crucially involved in the formation of higher order complexes containing IRAK1 and IRAK4 and required for the propagation of the TLR/IL1-R signaling pathways. Expression vectors for FLAG-tagged MyD88 or Myc-tagged MyD88, Myc-tagged IRAK1-kinase-dead (IRAK1KD) (K239S) and Myc-tagged IRAK4KD (K213A/K214A) were constructed by inserting PCR-generated cDNA fragments in the mammalian expression vectors p3X-FLAG and pCDNA3-N2-Myc, respectively. All site-directed mutations were inserted by PCR using oligonucleotides containing the mutated residue. Constructs for GFP-MyD88 fusion protein expression were obtained by subcloning cDNA encoding each protein into pEGFP-c1 vector. All constructs were confirmed by Cycle Sequencing (BMR Genomics, Padua, Italy). For the NF-κB reporter assays, the NF-κB luciferase and Renilla luciferase constructs were used according to the manufacturer's instructions (Promega Italia S.r.l., Milan, Italy). The human embryonic kidney (HEK) 293T and HeLa cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) and grown at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. For co-immunoprecipitation of FLAG-MyD88/Myc-MyD88, HEK293T cells were first cultured in 10-cm diameter dishes and then transfected by the calcium phosphate method with 4–5 μg of the appropriate plasmids. with or and GFP-MyD88-(27–72) with or HEK293T cells were cultured in diameter dishes and transfected by (Invitrogen) according to the manufacturer's For HeLa cells were cultured in diameter dishes and transfected with constructs for or GFP-MyD88-(27–72) expression by according to the manufacturer's HEK293T cells were in and in containing and for on cell were at for at and were for of were by for with protein at cell were with μg of mouse or for at protein or protein were with bovine serum in in the for the were with and then with cell containing the for at °C were in and in for or proteins were in as and for were on and to using a were with 5% in containing for at and at °C with the mouse and mouse and mouse and and to were with the for at at a in containing were by HeLa cells were cultured in and transfected with μg of an luciferase reporter and Renilla luciferase reporter as an with the constructs for expression of MyD88 mutants or GFP-MyD88 fusion proteins by using the according to the manufacturer's the cells were or not with or for with the cells were in and For the cells were in of luciferase reporter Italia S.r.l., Milan, for at were for by at in a and to a to reporter of cell were with of luciferase and the luciferase was using a For the of Renilla luciferase of were to the For all the reporter were for by luciferase by that of the Renilla are as induction a of the expression of the MyD88 mutants or GFP-MyD88 fusion proteins, HeLa cells were in of 10% were at for and the were in for analysis of of or mutated MyD88 or GFP-MyD88 fusion proteins, respectively. HeLa cells were cultured in diameter dishes and transfected with constructs for or GFP-MyD88-(27–72) expression by according to the manufacturer's HeLa cells or GFP-MyD88-(27–72) were with for at 37 The cells were at for with in and at for with 10% bovine serum in in the were at °C with NF-κB in with were with and for at with in was during the of to the were in analysis of the was performed using experiments and analyzed by was using the In order to the residues within the DD of MyD88 required for and for interaction with downstream of the we performed site-directed mutagenesis of mutagenesis studies on FADD DD E.J. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and Pelle DD P. Cell. 1999; 99: Full Text Full Text PDF PubMed Scopus Google Scholar) the of amino acids located on and and and for the with their DD and For the of human and mouse MyD88 and IRAK1 DD with homologous protein we used the Res. 1994; PubMed Scopus Google Scholar) of the DD of homologous proteins was performed using the available and the N. 1997; PubMed Scopus Google Scholar). The amino acids shown in in human MyD88 were with or and the effect on protein was by downstream activation of NF-κB by overexpression of MyD88 in the HeLa human cell of or mutant MyD88 with a reporter containing of the luciferase confirmed that MyD88 activation of NF-κB in the of stimuli K. F. C. H. P. F. L. Tschopp J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the other mutations strongly affected MyD88 The and mutations NF-κB activation by whereas the mutation was The other MyD88 mutants analyzed activated NF-κB to a as All of the MyD88 proteins were at of Glu52 or to that the first residue is the required for of MyD88, whereas to the These that residues Glu52 in Tyr58 in and in are required for MyD88 domains have in a H. D. A. M. J. Mol. Biol. PubMed Scopus Google Scholar). The DD to of homomeric H.H. E. S. S. Tschopp J. Wu H. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of MyD88, the DD and also to the DDs of IRAKs (7Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (982) Google Scholar, 8Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 5567-5572Crossref PubMed Scopus (540) Google Scholar). of were in the MyD88 we first their to recruit MyD88 by a co-immunoprecipitation M. F. N. G. S. B. R. P. R. C. J. Biol. 2007; PubMed Scopus Google Scholar). HEK293T cells were with FLAG-tagged or mutated MyD88 and Myc-tagged were with and with the protein was by in the of the mutations impaired MyD88 and that the and residues are not required for MyD88 we the MyD88 mutants were impaired in their to with IRAK1 and IRAK4. Since the interaction between MyD88 and kinases is and be only by IRAK1 and IRAK4 and H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 5567-5572Crossref PubMed Scopus (540) Google Scholar). or mutant FLAG-tagged MyD88 proteins were with either Myc-tagged or IRAK4KD and cell were with We a interaction between MyD88 and or IRAK4KD in and By and MyD88Y58A were impaired in the recruitment of both and IRAK4KD In with its to elicit NF-κB activation, the protein in the recruitment of both IRAKs and mutation of the residue exerted a effect on MyD88 interaction with the whereas strongly to and MyD88K95A the protein with IRAK4KD and These findings novel amino residues, within the DD of MyD88, that are crucial for the recruitment of IRAK1 and IRAK4 but not for its Our that residues a of the MyD88 DD domain required for the interaction with IRAK1 and IRAK4. expression of of MyD88 DD affected signal we a of GFP-MyD88 fusion proteins containing of this domain, as in We that proteins might a effect on the MyD88 by titrating IRAKs NF-κB activation. a reporter we or GFP-MyD88 fusion proteins were to with activation of We found that and activation of NF-κB and the effect of the GFP-MyD88 DD proteins was not activation of NF-κB by the MyD88-independent L. R. R.A. PubMed Scopus Google Scholar) pathway By and not on either or activation of NF-κB of proteins were in the Since GFP-MyD88-(27–72) exerted the of NF-κB on its to the A. R.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), we also overexpression of GFP-MyD88-(27–72) interfered with the of the In was in the and with for the of into the of cells in cells GFP-MyD88-(27–72) and we found that of in the by the in that GFP-MyD88-(27–72) activation of by a effect on MyD88 in with the of the analysis of the effect of GFP-MyD88-(27–72) on of NF-κB HeLa cells were transfected with or GFP-MyD88-(27–72) and and and the cells were and and and or with for and and and were and with and for NF-κB is with in the cells but is in the in GFP-MyD88-(27–72) by an in the effect of GFP-MyD88-(27–72) on MyD88 signaling, we analyzed its to MyD88 and by co-immunoprecipitation or GFP-MyD88-(27–72) were in HEK293T cells with MyD88, or analysis showed that GFP-MyD88-(27–72) strongly with whereas to either IRAK4KD or MyD88 The of GFP-MyD88-(27–72) with was with that of MyD88 suggesting that the and the that this region of MyD88 is to with the effect of GFP-MyD88-(27–72) was due to its to we performed between MyD88 and in HEK293T cells either or shown in GFP-MyD88-(27–72) was to recruitment by MyD88 by effect was in GFP-MyD88-(27–72) not with MyD88 of IRAK1 recruitment also affected the interaction of MyD88 with IRAK4, we all of the complex with or of MyD88 with and IRAK4KD in HEK293T cells showed that GFP-MyD88-(27–72) also the recruitment of IRAK4 by MyD88 with recruitment of IRAK1 and IRAK4 by HEK293T cells were transfected with or in with and in the of and or GFP-MyD88-(27–72) the cells were and the effect of either or GFP-MyD88-(27–72) on the interaction of with was by were with and the proteins were then analyzed by with either or to GFP-MyD88-(27–72) strongly with recruitment of IRAK1 by MyD88 analysis of is GFP-MyD88-(27–72) recruitment by MyD88 HEK293T cells were transfected with or in with and in the of and or GFP-MyD88-(27–72) the cells were and the effect of either or GFP-MyD88-(27–72) on MyD88 was by were with and proteins were analyzed by with either or the to MyD88 GFP-MyD88-(27–72) not with MyD88 analysis of is HEK293T cells were transfected with and or in with and in the of and or GFP-MyD88-(27–72) the cells were and the effect of either or GFP-MyD88-(27–72) on the interaction of with and was by were with and the proteins were then analyzed by with either or to GFP-MyD88-(27–72) strongly with recruitment of IRAKs by MyD88 GFP-MyD88-(27–72) signaling through of also with the activation of NF-κB this is H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, X. M. Z. Stark G.R. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). a reporter we that overexpression of or IRAK1 NF-κB activation in both of GFP-MyD88-(27–72) strongly NF-κB activation, whereas not of or GFP-MyD88-(27–72) and or were in the In and IRAK4 were to NF-κB activation in HEK293T cells not These that the region of the MyD88 DD between residues and is required for the recruitment of IRAK1 and for the activation of the downstream pathway. MyD88 is an adaptor protein that a crucial role in (10Janssens S. Beyaert R. Trends Biochem. Sci. 2002; 27: 474-482Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). MyD88 is required for the intracellular signaling triggered by of Although is that MyD88 by recruiting the kinases IRAK1 and IRAK4 through interaction between their respective the specific residues involved in have not been Herein, we residues that are required for the interaction of MyD88 with IRAK1 IRAK4 and strongly activation of Our that the region of the DD of MyD88 of residues and is required for the recruitment of Moreover, we that overexpression of this region in cells IRAK1 and signaling. the other mutation of in the only recruitment of IRAK4. the first of in the DD of MyD88 that with downstream of its signaling pathway. on DD and DD has demonstrated that the DDs of that or complexes H.H. E. S. S. Tschopp J. Wu H. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). on C. Trends Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar), homomeric and heteromeric were and and and and H.H. E. S. S. Tschopp J. Wu H. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). By analogy with DD-containing proteins, MyD88 might higher order in each domain be in of interactions, These either or to a multimeric complex competent for signaling. By the of interaction for complex H.H. E. S. S. Tschopp J. Wu H. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), the residues Glu52 and Tyr58 of MyD88 are in a with a the residues and in in complex their the region the residues interact with a of MyD88 or IRAK4. that in the of MyD88, this recruits is by the that mutations in residues strongly the recruitment of IRAKs but not MyD88 Moreover, a mini-MyD88 protein residues with IRAK1 but not with IRAK4. Although GFP-MyD88-(27–72) is an appeared to its to with IRAK1 was with that of Our co-immunoprecipitation experiments that residue of MyD88 is required for the between MyD88 and IRAK4. By the in the residues of and to are and and in the of and of are to and in in a heteromeric interaction with the residue of is located at the of the H.H. E. S. S. Tschopp J. Wu H. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). on findings and we might that heteromeric also via an comprising the that the first residues of MyD88 are required for the interaction with IRAK4. region the DD and ID and a of the TIR domain (6Burns K. Janssens S. Brissoni B. Olivos N. Beyaert R. Tschopp J. J. Exp. Med. 2003; 197: 263-268Crossref PubMed Scopus (412) Google Scholar). the DD the ID was for this suggesting that the region is of residues both the DD and the ID or that the ID a in the of the DD that is required for with IRAK4 (6Burns K. Janssens S. Brissoni B. Olivos N. Beyaert R. Tschopp J. J. Exp. Med. 2003; 197: 263-268Crossref PubMed Scopus (412) Google Scholar). Our are with both in the and that is of the residues in the DD that are crucial for the recruitment of IRAK4. Since is by a in mouse MyD88, is that the of the rather its is for this of complexes be required to the of interaction between this was in was that with MyD88 mutations in the DD or TIR domain of the protein H. C. Z. R. H. M. P. J. N. M. C. L. E. M. C. J. J. M. H.H. L. H. A. L. C. J. L. Li X. D. A. Science. PubMed Scopus Google Scholar). of the TIR mutations mutation was within the that we showed to be critically involved in of the TIR domains of MyD88 M. C. G. A. N. D. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and H. C. Z. R. H. M. P. J. N. M. C. L. E. M. C. J. J. M. H.H. L. H. A. L. C. J. L. Li X. D. A. Science. PubMed Scopus Google Scholar) have also found that a MyD88 MyD88 due to defective IRAK4 recruitment. Although not IRAK1 is present that MyD88E52A is defective in the recruitment of both IRAK1 and IRAK4. Moreover, the region of residues with IRAK1 and not IRAK4. is that mutation of Glu52 also the region of interaction with IRAK4, due to their in an heteromeric to that in the in H.H. E. S. S. Tschopp J. Wu H. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). Since mutagenesis has a MyD88 mutation found in is that Tyr58 and are residues defective MyD88 signaling and might the for of residues in affected by other The is on of MyD88 and on site-directed mutagenesis of residues involved in interactions. Although of experiments are performed in transfected cells proteins, that the that be with this are a using the with the TIR domain of MyD88 has to identify a region of residues that are required for MyD88 M. C. G. A. N. D. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These were then confirmed by other S. J. A. Jr., J. Proc. Natl. Acad. Sci. U.S.A. 2003; PubMed Scopus Google Scholar). a amino acids MyD88 also in cells M. C. G. A. N. D. P. J. 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