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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

Authors

  • Kollewe C
  • Mackensen AC
  • Neumann D
  • Knop J
  • Cao P
  • Li S
  • Wesche H
  • Martin MU

Journal

The Journal of biological chemistry

Publication Data

February 13, 2004

Associated Grants

None

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