Functional analysis of the interleukin-1-receptor-associated kinase (IRAK-1) in interleukin-1 beta-stimulated nuclear factor kappa B (NF-kappa B) pathway activation: IRAK-1 associates with the NF-kappa B essential modulator (NEMO) upon receptor stimulation

Biochem J. 2001 Oct 15;359(Pt 2):403-10. doi: 10.1042/0264-6021:3590403.

Abstract

The interleukin-1 (IL-1)-receptor-associated kinase (IRAK-1) is essential for IL-1-stimulated nuclear factor kappa B (NF-kappa B) activation. To study the role of IRAK-1 in IL-1 beta signalling, we have generated a set of IRAK-1 variants that express distinct domains of IRAK-1 either alone or in combination and have examined their effects on an NF-kappa B-responsive reporter in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unable to activate NF-kappa B in the absence of cytokine stimulation. However, an IRAK-1 variant lacking only the N-terminal domain retained the ability of the full-length protein to potentiate both IL-1 beta and tumour necrosis factor alpha (TNF alpha)-induced NF-kappa B activation. In contrast, expression of the N-terminus or the C-terminus of IRAK-1, or a fusion protein incorporating both domains, inhibited both IL-1 beta- and TNF alpha-induced effects. Expression of an IRAK-1 variant lacking only the C-terminal domain preferentially inhibited IL-1 beta versus TNF alpha-induced NF-kappa B activation. These data suggest that the C-terminal domain may link IRAK-1 to downstream signalling components common to both the IL-1 and TNF pathways. Furthermore, we have demonstrated that endogenous IRAK-1 becomes phosphorylated upon IL-1 beta treatment and can be detected along with NF-kappa B essential modulator (NEMO) and I kappa B kinase beta (IKK beta) in high-molecular-mass complexes of 600-800 kDa. Moreover, IRAK-1 could be detected in NEMO immunoprecipitates from IL-1 beta-stimulated cells. We conclude that IRAK-1 mediates IL-1 beta signal transduction through a ligand-dependent association of IRAK-1 with the IKK complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Genes, Reporter
  • HeLa Cells
  • Humans
  • I-kappa B Kinase
  • In Vitro Techniques
  • Interleukin-1 / metabolism
  • Interleukin-1 / pharmacology*
  • Interleukin-1 Receptor-Associated Kinases
  • NF-kappa B / metabolism*
  • Protein Kinases / chemistry
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Receptors, Interleukin-1 / drug effects
  • Receptors, Interleukin-1 / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Sequence Deletion
  • Signal Transduction
  • Tumor Necrosis Factor-alpha / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Interleukin-1
  • NF-kappa B
  • Receptors, Interleukin-1
  • Recombinant Proteins
  • Tumor Necrosis Factor-alpha
  • Protein Kinases
  • Interleukin-1 Receptor-Associated Kinases
  • Protein-Serine-Threonine Kinases
  • CHUK protein, human
  • I-kappa B Kinase
  • IKBKB protein, human
  • IKBKE protein, human