Interleukin-1 (IL-1) is a proinflammatory cytokine that elicits its pleiotropic effects through activation of the transcription factors NF-kappaB and AP-1. Binding of IL-1 to its receptor results in rapid assembly of a membrane-proximal signalling complex that consists of two different receptor chains (IL-1Rs), IL-1RI and IL-1RAcP, the adaptor protein MyD88, the serine/threonine kinase IRAK and a new protein, which we have named Tollip. Here we show that, before IL-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with IL-1RAcP. Co-recruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and IL-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the IL-1R signalling pathway.

Following interleukin-1 (ILl) stimulation, an ILl receptor associated kinase (IRAK) is rapidly recruited to the receptor complex. However, it is not understood if IRAK is able to interact directly with the intracellular portion of the IL1-RI or if its recruitment is mediated by a different molecule. Using the yeast two-hybrid system, we have analysed possible proteinprotein interactions between IRAK, IL1-RI and ILl-RAcP. We found that IRAK is able to interact with the equivalent cytoplasmic region of the ILl-RAcP but is unable to interact with the cytoplasmic region of the IL1-RI. Immunoprecipitation of the ILl-RAcP followed by Western blot analysis using anti-IRAK antibodies revealed that IRAK co-precipitated with the ILl-RAcP. We propose that, in non-stimulated cells, IRAK is bound to the ILl-RAcP and therefore, following ILl stimulation, both molecules are recruited simultaneously to the IL1-RI complex.