Cooke, Emma-Louise; (2001) Structure-function analysis of the interleukin-1 receptor associated kinase (IRAK-1) in IL-1beta signal transduction to NFkappaB. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The IL-1 receptor-associated kinase (IRAK1) is essential for IL-1-stimulated NFκB -dependent gene activation. To study the role of IRAK-1 in IL-1β 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 NFκB responsive reporter in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unable to activate NFκB 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-1β and TNFα-induced NFκB 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-1β and TNFα-induced effects. Expression of an IRAK-1 variant lacking only the C-terminal domain preferentially inhibited IL-1β versus TNFα-induced NFκB 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. We have demonstrated that endogenous IRAK-1 becomes phosphorylated upon IL-1β treatment and can be detected along with NEMO and IKKγ in high molecular weight complexes of 600-800kDa. Moreover, IRAK-1 could be detected in NEMO immunoprecipitates from IL-1β stimulated cells, suggesting that NEMO recruits IRAK-1 into the IKB kinase complex. Additionally, we have identified proteins that potentially interact with the C-terminus of IRAK-1 by utilising the yeast 2-hybrid system. A novel protein, which we called IRIP (IRAK-1-interacting protein) was identified. Bioinformatic analysis suggests that IRIP is a member of a family of ecto-nucleotide pyrophosphatase/phosphodiesterases and is likely to be the human homologue of a yeast protein called GPI7, which is involved in the addition of side chains to GPI anchors. It remains to be established whether IRIP has phosphodiesterase/pyrophosphatase activity and also what role this protein may play in IL-1 signal transduction or cell regulation.

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