Identification of threonine 66 as a functionally critical residue of the interleukin-1 receptor-associated kinase.
We have mutated a conserved residue of the death domain of the interleukin-1 (IL-1) receptor-associated kinase (IRAK), threonine 66. The substitution of Thr-66 with alanine or glutamate prevented spontaneous activation of NF-kappaB by overexpressed IRAK but enhanced IL-1-induced activation of the factor. Like the kinase-inactivating mutation, K239S, the T66A and T66E mutations interfered with the ability of IRAK to autophosphorylate and facilitated the interactions of IRAK with TRAF6 and with the IL-1 receptor accessory protein, AcP. Wild-type IRAK constructs tagged with fluorescent proteins formed complexes that adopted a punctate distribution in the cytoplasm. The Thr-66 mutations prevented the formation of these complexes. Measurements of fluorescence resonance energy transfer among fluorescent constructs showed that the Thr-66 mutations abolished the capacity of IRAK to dimerize. In contrast, the K239S mutation did not inhibit dimerization of IRAK as evidenced by fluorescence resonance energy transfer measurements, even though microscopy showed that it prevented the formation of punctate complexes. Our results show that Thr-66 plays a crucial role in the ability of IRAK to form homodimers and that its kinase activity regulates its ability to form high molecular weight complexes. These properties in turn determine key aspects of the signaling function of IRAK.
Division of Genomic Medicine, University of Sheffield, M Floor, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom.
Kehinde
K
Ross
Lin
L
Yang
Steve
S
Dower
Filippo
F
Volpe
Francois
F
Guesdon
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D049452:Green Fluorescent Proteins; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D010446:Peptide Fragments / Q000737:chemistry; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D017472:Receptors, Interleukin-1 / Q000737:chemistry / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology; D013912:Threonine; D014162:Transfection
D008164:Luminescent Proteins; D010446:Peptide Fragments; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; D011494:Protein Kinases; D053592:Interleukin-1 Receptor-Associated Kinases