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

The interleukin-1 (IL-1)-receptor-associated kinase (IRAK-1) is essential for IL-1-stimulated nuclear factor kappaB (NF-kappaB ) 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-kappaB -responsive reporter in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unable to activate NF-kappaB 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 a (TNF alpha)-induced NF-kappaB 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-kappaB 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-kappaB essential modulator (NEMO) and I kappaB kinase (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.