Identification of Critical Residues of the MyD88 Death Domain Involved in the Recruitment of Downstream Kinases
2009; Elsevier BV; Volume: 284; Issue: 41 Linguagem: Inglês
10.1074/jbc.m109.004465
ISSN1083-351X
AutoresMaria Loiarro, Grazia Gallo, Nicola Fantò, Rita De Santis, Paolo Carminati, Vito Ruggiero, Claudio Sette,
Tópico(s)Influenza Virus Research Studies
ResumoMyD88 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. To detect FLAG-MyD88 associated with Myc-IRAK1KD or Myc-IRAK4KD and GFP-MyD88-(27–72) associated with Myc-IRAK4KD, Myc-IRAK1KD, or Myc-MyD88, HEK293T cells were cultured in 6-cm diameter dishes and transfected by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For immnunofluorescence analysis, HeLa cells were cultured in 3-cm diameter dishes and transfected with constructs for GFP or GFP-MyD88-(27–72) expression by FuGENE 6 reagent (Roche Applied Science) according to the manufacturer's instructions. HEK293T cells were collected 20 h after transfection, washed in ice-cold PBS, and lysed in buffer containing 50 mm Hepes, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 20 mm β-glycerophosphate, 2 mm dithiothreitol, 1 mm Na3VO4, and protease inhibitors. After incubating for 10 min on ice, cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and cytosolic fractions were collected for immunoprecipitation. Cell extracts (1 mg of total proteins) were precleared by incubation for 1 h with protein G-Sepharose beads (Sigma) under constant shaking at 4 °C. After preclearing, cell extracts were incubated with 2 μg of mouse anti-FLAG M2 (Sigma) or rabbit anti-GFP antibodies (Molecular Probes) for 1 h under constant shaking at 4 °C. Concurrently, protein G-Sepharose beads or protein A-Sepharose beads were presaturated with 0.1% bovine serum albumin (Sigma) in PBS in the same conditions for 1 h. After incubation, the beads were washed twice with lysis buffer and then further incubated with cell extracts containing the antibodies for 1 h at 4 °C under constant shaking. Sepharose bead-bound immunocomplexes were washed three times in lysis buffer and eluted in SDS-PAGE sample buffer for Western blot analysis. Cell extracts or immunoprecipitated proteins were diluted in SDS sample buffer, as described above, and boiled for 5 min. Proteins were separated on 8–10% SDS-PAGE gel and transferred to polyvinylidene fluoride Immobilon-P membranes (Millipore S.p.A., Vimodrone, Italy) using a semidry blotting apparatus (Bio-Rad). Membranes were saturated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: mouse anti-Myc and rabbit anti-Erk2 (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-FLAG M2 and mouse anti-β tubulin (1:3000 and 1:1000 dilution, respectively; Sigma), and rabbit anti-GFP antibodies (1:1000 dilution; Molecular Probes). Secondary anti-mouse IgGs conjugated to horseradish peroxidase (Amersham Biosciences) were incubated with the membranes for 1 h at room temperature at a 1:10,000 dilution in PBS containing 0.1% Tween 20. Immunostained bands were detected by chemiluminescence (Santa Cruz Biotechnology). HeLa cells (1 × 105) were cultured in 12-well plates and transfected with 0.5 μg of an NF-κB-dependent luciferase reporter gene and Renilla luciferase reporter gene (4 ng) as an internal control together with the constructs for expression of MyD88 mutants or GFP-MyD88 fusion proteins by using the FuGENE 6 reagent according to the manufacturer's instructions. Twenty-four h after transfection, the cells were stimulated or not with 30 ng/ml IL-1β (R&D Systems, Minneapolis, MN) or 100 μg/ml poly(I-C) (Invivogen, San Diego, CA) for 6 h. After rinsing with PBS, the cells were harvested in two tubes and lysed separately. For biocounter luminometer analysis, the cells were lysed in 250 μl of passive lysis buffer (dual luciferase reporter assay system; Promega Italia S.r.l., Milan, Italy) for 15 min at room temperature. Cell lysates were cleared for 30 s by centrifugation at top speed in a refrigerated microcentrifuge and transferred to a fresh tube prior to reporter enzyme analysis. Ten μl of cell lysates were mixed with 100 μl of luciferase assay reagent II (Promega), and the NF-κB-firefly luciferase activity was determined using a biocounter luminometer. For the assessment of Renilla luciferase activity, 100 μl of Stop & Glo® reagent were added to the same sample. For all samples, the reporter data were normalized for transfection efficiency by dividing firefly luciferase activity by that of the Renilla luciferase. Data are expressed as mean -fold induction ± S.D. from a minimum of three separate experiments. To analyze the expression of the MyD88 mutants or GFP-MyD88 fusion proteins, HeLa cells were lysed in 30 μl of buffer (50 mm HEPES, pH 7.4, 15 mm MgCl2, 150 mm NaCl, 15 mm EGTA, 10% glycerol, 1% Triton X-100, protease inhibitor mixture (Sigma), 20 mm β-glycerophosphate, 2 mm dithiothreitol, 1 mm Na3VO4). Cells were centrifuged at 10,000 × g for 10 min, and the resulting supernatants were diluted in SDS sample buffer for Western blot analysis of levels of wild type or mutated MyD88 or GFP-MyD88 fusion proteins, respectively. HeLa cells were cultured in 3-cm diameter dishes and transfected with constructs for GFP or GFP-MyD88-(27–72) expression by FuGENE 6 reagent (Roche Applied Science) according to the manufacturer's instructions. Twenty h after transfection, HeLa cells expressing GFP or GFP-MyD88-(27–72) were stimulated with 20 ng/ml IL-1β for 20 min at 37 °C. The cells were fixed at room temperature for 10 min with 4% paraformaldehyde in PBS and permeabilized at room temperature for 30 min with 0.5% Tween 20, 10% goat serum, 1% bovine serum albumin in PBS. After two washes in PBS, the samples were incubated overnight at 4 °C with anti-p65 NF-κB antibodies (1:500 dilution in PBS with 1% goat serum; Santa Cruz Biotechnology). Cells were washed three times with PBS and incubated for 1 h at room temperature with secondary antibodies (1: 500 dilution in PBS; Jackson ImmunoResearch Laboratories). Hoechst dye (0.1 μg/ml; Sigma) was added during the last 10 min of incubation to stain the nuclei. Slides were mounted in Mowiol 4-88 reagent (Calbiochem). Densitometric analysis of the Western blots was performed using underexposed images from 3–5 experiments and analyzed by ImageQuant version 5.0 software. Statistical significance was determined using the two-tailed Student's t test. In order to delineate the relevant residues within the DD of MyD88 required for self-association and for interaction with downstream components of the pathway, we performed site-directed mutagenesis of selected residues. Previous mutagenesis studies carried out on FADD DD (20Jeong E.J. Bang S. Lee T.H. Park Y.I. Sim W.S. Kim K.S. J. Biol. Chem. 1999; 274: 16337-16342Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and Pelle DD (21Xiao T. Towb P. Wasserman S.A. Sprang S.R. Cell. 1999; 99: 545-555Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) highlighted the importance of polar/charged amino acids located on helices α2, α3, and α5 and loops α2-α3 and α4-α5 for the association with their partners Fas DD and Tube DD. For the alignment of human and mouse MyD88 and IRAK1 DD with homologous protein domains, we used the ClustalW algorithm (22Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55709) Google Scholar) (Fig. 1A). A structural alignment of the DD of homologous proteins was performed using the available crystallographic/NMR data and the Swiss-Pdb viewer (23Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9578) Google Scholar). The amino acids shown in boldface type (Fig. 1) in human MyD88 were substituted with alanine or asparagine (Arg81), and the effect on protein function was tested by assaying downstream activation of NF-κB caused by overexpression of MyD88 in the HeLa human cell line. Co-transfection of wild type or mutant MyD88 with a reporter construct containing NF-κB-responsive elements upstream of the firefly luciferase gene confirmed that wild type MyD88 triggers activation of NF-κB even in the absence of exogenous stimuli (Fig. 1B) (24Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. Di Marco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). On the other hand, several mutations strongly affected MyD88 function. The E52A/E53A, Y58A, and K95A mutations reduced NF-κB activation by almost 60% (Fig. 1B, gray bars), whereas the R81N mutation was slightly less effective. The other MyD88 mutants analyzed activated NF-κB to a similar extent as wild type MyD88. All of the MyD88 proteins tested were expressed at comparable levels (Fig. 1C). Remarkably, single substitution of Glu52 or Glu53 to alanine indicated that the first glutamic acid residue (Glu52) is the one required for full function of MyD88, whereas E53A behaved similarly to the wild type protein. These results indicate that residues Glu52 (predicted in helix α2), Tyr58 (predicted in helix α3), and Lys95 (predicted in helix α5) are required for MyD88 activity. Death domains have six anti-parallel α-helices, arranged in a Greek key structure (25Berglund H. Olerenshaw D. Sankar A. Federwisch M. McDonald N.Q. Driscoll P.C. J. Mol. Biol. 2000; 302: 171-188Crossref PubMed Scopus (83) Google Scholar). The DD can fold to offer different surfaces of homomeric interactions (26Park H.H. Logette E. Raunser S. Cuenin S. Walz T. Tschopp J. Wu H. Cell. 2007; 128: 533-546Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). In the case of MyD88, the DD self-associates and also binds 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, 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). To determine which of these functions were disrupted in the MyD88 loss-of-function mutants, we first tested their ability to recruit wild type MyD88 by a co-immunoprecipitation assay (28Loiarro M. Capolunghi F. Fantò N. Gallo G. Campo S. Arseni B. Carsetti R. Carminati P. De Santis R. Ruggiero V. Sette C. J. Leukocyte Biol. 2007; 82: 801-810Crossref PubMed Scopus (141) Google Scholar). HEK293T cells were co-transfected with FLAG-tagged wild type or mutated MyD88 and wild type Myc-tagged MyD88. Cell extracts were immunoprecipitated with anti-FLAG antibodies, and association with the wild type protein was tested by detecting Myc-MyD88 in the immunoprecipitates. Remarkably, none of the mutations impaired MyD88 self-association (Fig. 2, A and B), demonstrating that the Glu52, Tyr58, and Lys95 residues are not required for MyD88 self-association. Next, we tested whether the signaling-defective MyD88 mutants were impaired in their ability to associate with IRAK1 and IRAK4. Since the interaction between MyD88 and these kinases is rapid and transient, it can be reproducibly detected only by expressing kinase-dead IRAK1 and IRAK4 (IRAK1KD and IRAK4KD) (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, 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). Hence, wild type or mutant FLAG-tagged MyD88 proteins were co-expressed with either Myc-tagged IRAK1KD (Fig. 2C) or IRAK4KD (Fig. 2E), and cell extracts were immunoprecipitated with anti-FLAG antibodies. We observed a strong interaction between wild type MyD88 and IRAK1KD or IRAK4KD (lane 1 in Fig. 2, C and E). By contrast, MyD88E52A/E53A, MyD88E52A, and MyD88Y58A were impaired in the recruitment of both IRAK1KD (Fig. 2C) and IRAK4KD (Fig. 2E). In line with its ability to elicit NF-κB activation, MyD88E53A behaved like the wild type protein in the recruitment of both IRAKs (Fig. 2, C and E). Interestingly, mutation of the Lys95 residue exerted a different effect on MyD88 interaction with the two kinases. Indeed, whereas it strongly bound to IRAK1KD (Fig. 2, C and D), MyD88K95A interacted less efficiently than the wild type protein with IRAK4KD (Fig. 2, E and F). These findings highlight novel amino acid residues, within the DD of MyD88, that are crucial for the recruitment of IRAK1 and IRAK4 but not for its self-association. Our results suggested that residues 52–95 define a subregion of the MyD88 DD domain required for the interaction with IRAK1 and IRAK4. To determine whether expression of different regions of MyD88 DD affected IL1-R signal transduction, we produced a series of GFP-MyD88 fusion proteins containing portions of this domain, as schematically represented in Fig. 3A. We reasoned that these chimeric proteins might exert a dominant-negative effect on the endogenous MyD88 by titrating out IRAKs without triggering NF-κB activation. Using a reporter gene assay, we tested whether overexpressed GFP or GFP-MyD88 fusion proteins were able to interfere with IL-1-mediated activation of NF-κB. We found that GFP-MyD88-(27–72), GFP-MyD88-(30–66), and GFP-MyD88-(44–110) significantly reduced IL-1-dependent activation of NF-κB (by approximately 40, 30 and 25%, respectively; p < 0.01; Fig. 3B). Remarkably, the effect of the GFP-MyD88 DD proteins was specific, since they did not inhibit activation of NF-κB by the MyD88-independent (29Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4923) Google Scholar) poly(I-C)/TLR3 pathway (Fig. 3B). By contrast, GFP-MyD88-(27–45), MyD88-(40–72), and GFP-MyD88-(68–172) did not exert significant effects on either IL-1-dependent or poly(I-C)-dependent activation of NF-κB (Fig. 3B). Similar amounts of chimeric proteins were expressed in the samples (Fig. 3C). Since GFP-MyD88-(27–72) exerted the strongest inhibition of NF-κB transcriptional activity, which relies on its translocation from cytoplasm to the nucleus (30Ding G.J. Fischer P.A. Boltz R.C. Schmidt J.A. Colaianne J.J. Gough A. Rubin R.A. Miller D.K. J. Biol. Chem. 1998; 273: 28897-28905Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), we also tested whether overexpression of GFP-MyD88-(27–72) interfered with the subcellular localization of the p65/NF-κB protein. In non-stimulated cells, p65 was localized exclusively in the cytoplasm (Fig. 4, A and C). Stimulation with IL-1β for 20 min caused the translocation of p65 into the nucleus of GFP-transfected cells (Fig. 4B). However, in cells expressing GFP-MyD88-(27–72) (Fig. 4, G and H), we found that significant amounts of p65 remained in the cytoplasm after IL-1β treatment (indicated by the arrows in Fig. 4D). Taken together, these results suggest that GFP-MyD88-(27–72) attenuates MyD88-dependent activation of NF-κB, probably by exerting a dominant negative effect on MyD88 function in live cells.FIGURE 4GFP-MyD88-(27–72) interferes with the subcellular localization of the p65/NF-κB protein. Immunofluorescence analysis of the effect of GFP-MyD88-(27–72) on nuclear translocation of endogenous NF-κB p65. HeLa cells were transfected with GFP or GFP-MyD88-(27–72) (panels E and F and panels G and H, respectively). Twenty h after transfection, the cells were left untreated (panels A, E, I, and O and panels C, G, M, and Q) or treated with 20 ng/ml IL-1β for 20 min (panels B, F, L, and P and panels D, H, N, and R). Cells were fixed, blocked, and stained with anti-NF-κB p65 antibodies (red) and Hoechst (blue) for nuclear staining. Nuclear NF-κB p65 is observed 20 min after treatment with IL-1β in the cells expressing GFP, but it is held back in the cytoplasm in those expressing GFP-MyD88-(27–72) (indicated by an arrow in D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further investigate the inhibitory effect of GFP-MyD88-(27–72) on MyD88 signaling, we analyzed its ability to bind full-length MyD88 and IRAK1/4 by co-immunoprecipitation assays. GFP or GFP-MyD88-(27–72) were co-expressed in HEK293T cells with MyD88, IRAK1KD, or IRAK4KD. Western blot analysis showed that GFP-MyD88-(27–72) strongly interacted with IRAK1KD (Fig. 5A), whereas it bound very weakly to either IRAK4KD (Fig. 5A) or full-length MyD88 (Fig. 5A). The association of GFP-MyD88-(27–72) with IRAK1KD was comparable with that of full-length MyD88 (Fig. 5B), suggesting that the miniprotein folded correctly and maintained the original structure. This result suggests that this region of MyD88 is sufficient to associate with IRAK1. To test whether the inhibitory effect of GFP-MyD88-(27–72) was due to its ability to titrate out IRAK1, we performed co- immunoprecipitation assays between MyD88 and IRAK1KD in HEK293T cells expressing either GFP or GFP-MyD88-(27–72). As shown in Fig. 6A, GFP-MyD88-(27–72) was able to significantly inhibit (p < 0.01) IRAK1KD recruitment by MyD88 by ∼70%. This effect was specific, because in similar experiments, GFP-MyD88-(27–72) did not interfere with MyD88 self-association (Fig. 6B). To test whether inhibition of IRAK1 recruitment also affected the interaction of MyD88 with IRAK4, we co-expressed all components of the complex together with GFP or GFP-MyD88-(27–72). Co-immunoprecipitation assays of MyD88 with IRAK1KD and IRAK4KD in HEK293T cells showed that GFP-MyD88-(27–72) also prevents the recruitment of IRAK4 by MyD88 (Fig. 6C).FIGURE 6GFP-MyD88-(27–72) interferes with recruitment of IRAK1 and IRAK4 by MyD88. A, HEK293T cells were transfected with Myc-IRAK1KD alone (lane 1) or in combination with FLAG-MyD88 (lanes 2 and 3) in the presence of GFP (lanes 1 and 2) or GFP-MyD88-(27–72) (lane 3). Twenty h after transfection, the cells were collected, and the effect of either GFP or GFP-MyD88-(27–72) on the interaction of FLAG-MyD88 with Myc-IRAK1KD was evaluated by co-immunoprecipitation. Cell extracts were immunoprecipitated (IP) with anti-FLAG antibodies, and the immunoprecipitated proteins were then analyzed by Western blotting with either anti-FLAG or anti-Myc antibodies to detect association. GFP-MyD88-(27–72) strongly interferes with recruitment of IRAK1 by MyD88 (lane 3). Densitometric analysis of these results is depicted. GFP-MyD88-(27–72) significantly inhibited IRAK1KD recruitment by MyD88 (*, p < 0.01; n = 3). B, HEK293T cells were transfected with Myc-MyD88 alone (lane 1) or in combination with FLAG-MyD88 (lanes 2 and 3) in the presence of GFP (lanes 1 and 2) or GFP-MyD88-(27–72) (lane 3). Twenty h after transfection, the cells were harvested, and the effect of either GFP or GFP-MyD88-(27–72) on MyD88 self-association was assessed by co-immunoprecipitation. Cell extracts were immunoprecipitated with anti-FLAG antibodies, and immunoprecipitated proteins were analyzed by Western blotting with either anti-FLAG or the anti-Myc antibodies to detect MyD88 self-association. GFP-MyD88-(27–72) does not interfere with MyD88 self-association (lane 3). Densitometric analysis of these results is shown. C, HEK293T cells were transfected with Myc-IRAK1KD and Myc-IRAK4KD alone (lane 1) or in combination with FLAG-MyD88 (lanes 2 and 3) in the presence of
Referência(s)