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 f or 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 comb ination and have examined their effects on an NF-kappaB -responsive reporte r in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unabl e 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 fa ctor 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 incorporat ing both domains, inhibited both IL-1 beta- and TNF alpha -induced effects. Expression of an IRAK-1 variant lacking only the C-terminal domain prefere ntially inhibited IL-1 beta versus TNF alpha -induced NF-kappaB activation. These data suggest that the C-terminal domain may link IRAK-1 to downstrea m signalling components common to both the IL-1 and TNF pathways. Furthermo re, we have demonstrated that endogenous IRAK-1 becomes phosphorylated upon IL-1 beta treatment and can be detected along with NF-kappaB essential mod ulator (NEMO) and I kappaB kinase (IKK beta) in high-molecular-mass complex es of 600-800 kDa. Moreover, IRAK-1 could be detected in NEMO immunoprecipi tates 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.