title: Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases.
abstract: 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 (MyD88(E52A)) and 58 (MyD88(Y58A)) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88(K95A)) 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 Glu(52) and Tyr(58) residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-kappaB 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-kappaB 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.
authors.affiliation: Department of Public Health and Cell Biology, University of Rome "Tor Vergata," 00133 Rome, Italy; Fondazione Santa Lucia di Roma è Uno Degli Istituti di Ricovero e Cura a Carattere Scientifico, Laboratory of Neuroembryology, 00143 Rome, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Loiarro
authors.affiliation: Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
authors.forename: Grazia
authors.initials: G
authors.lastname: Gallo
authors.affiliation: Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Fantò
authors.affiliation: Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: De Santis
authors.affiliation: Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Carminati
authors.affiliation: Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy. Electronic address: vito.ruggiero@sigma-tau.it.
authors.forename: Vito
authors.initials: V
authors.lastname: Ruggiero
authors.affiliation: Department of Public Health and Cell Biology, University of Rome "Tor Vergata," 00133 Rome, Italy; Fondazione Santa Lucia di Roma è Uno Degli Istituti di Ricovero e Cura a Carattere Scientifico, Laboratory of Neuroembryology, 00143 Rome, Italy. Electronic address: claudio.sette@uniroma2.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Sette
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D053594:Myeloid Differentiation Factor 88 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D051196:Toll-Like Receptor 3 / Q000235:genetics / Q000378:metabolism
chemical_list: D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D020033:Protein Isoforms; D016271:Proto-Oncogene Proteins c-myc; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D051196:Toll-Like Receptor 3; D053592:Interleukin-1 Receptor-Associated Kinases
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