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.
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.
Cooke
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Department of Molecular Pathology, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1P 6DB, UK.
Uings
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Xia
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Woo
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Ray
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D001665:Binding Sites; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000378:metabolism / Q000494:pharmacology*; D053592:Interleukin-1 Receptor-Associated Kinases; D016328:NF-kappa B / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017472:Receptors, Interleukin-1 / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
D007375:Interleukin-1; D016328:NF-kappa B; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D011494:Protein Kinases; D053592:Interleukin-1 Receptor-Associated Kinases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human