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 (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.
Loiarro
Maria
M
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.
Gallo
Grazia
G
Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
Fantò
Nicola
N
Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
De Santis
Rita
R
Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
Carminati
Paolo
P
Department of Immunology (Building LABIO), Sigma-tau Industrie Farmaceutiche Riunite S.p.A, Via Pontina km 30.400, 00040 Pomezia (RM), Italy.
Ruggiero
Vito
V
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.
Sette
Claudio
C
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.
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
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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