Sequential autophosphorylation steps in the interleukin-1 receptor-associated kinase-1 regulate its availability as an adapter in interleukin-1 signaling.
The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, ... the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr209. By fluorescence energy transfer experiments, we demonstrate that Thr209 phosphorylation results in a conformational change of the kinase domain, permitting further phosphorylations to take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1. Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity. Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain and kinase domain. Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. This identifies IRAK-1 as a novel type of adapter protein, which employs its own kinase activity to introduce negative charges adjacent to the protein interaction domain, which anchors IRAK-1 at the active receptor complex. Thus, IRAK-1 regulates its own availability as an adapter molecule by sequential autophosphorylation.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Antigens, Differentiation, Carrier Proteins, Cell Line, Cloning, Molecular, Dimerization, Fluorescence Resonance Energy Transfer, Genetic Vectors, Humans, Immunoblotting, Interleukin-1, Interleukin-1 Receptor-Associated Kinases, Intracellular Signaling Peptides and Proteins, Mass Spectrometry, Models, Biological, Molecular Sequence Data, Myeloid Differentiation Factor 88, Phosphorylation, Precipitin Tests, Protein Conformation, Protein Kinases, Protein Structure, Tertiary, Receptors, Immunologic, Receptors, Interleukin-1, Signal Transduction, Spectrometry, Mass, Electrospray Ionization, Threonine, Time Factors, Transfection
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Antigens, Differentiation, Carrier Proteins, Cell Line, Cloning, Molecular, Dimerization, Fluorescence Resonance Energy Transfer, Genetic Vectors, Humans, Immunoblotting, Interleukin-1, Interleukin-1 Receptor-Associated Kinases, Intracellular Signaling Peptides and Proteins, Mass Spectrometry, Models, Biological, Molecular Sequence Data, Myeloid Differentiation Factor 88, Phosphorylation, Precipitin Tests, Protein Conformation, Protein Kinases, Protein Structure, Tertiary, Receptors, Immunologic, Receptors, Interleukin-1, Signal Transduction, Spectrometry, Mass, Electrospray Ionization, Threonine, Time Factors, Transfection
J. Biol. Chem.
Date: Feb. 13, 2004
PubMed ID: 14625308
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