The interleukin-1 (IL-1)-receptor-associated kinase (IRAK-1) is essential for IL-1-stimulated nuclear factor κB (NF-κB) 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 NF-κB-responsive reporter in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unable to activate NF-κB 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 tumour necrosis factor α (TNFα)-induced NF-κB 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 NF-κB 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-1β treatment and can be detected along with NF-κB essential modulator (NEMO) and IκB kinase β (IKKβ) in high-molecular-mass complexes of 600–800kDa. Moreover, IRAK-1 could be detected in NEMO immunoprecipitates from IL-1β-stimulated cells. We conclude that IRAK-1 mediates IL-1β signal transduction through a ligand-dependent association of IRAK-1 with the IKK complex.
Abbreviations used: IL-1, interleukin-1; TNF, tumour necrosis factor; IL-1RI, type 1 interleukin 1 receptor; TNFR1, type 1 TNF receptor; IL-1RAcP, interleukin-1 receptor accessory protein; IRAK, interleukin-1-receptor-associated kinase; NEMO, Nuclear factor κB (NF-κB) essential modulator; IKK, IκB kinase; RIP, receptor-interacting protein; sAP, secreted alkaline phosphatase; NLS, nuclear localization signal; SODD, silencer of death domains; TRADD, TNF-receptor-associated death domain protein; TK, thymidine kinase; RSV, Rous sarcoma virus; βlac, β-lactamase; TAK-1, TGFβ activated kinase 1.