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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.

Pubmed ID: 14625308 RIS Download

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