It has recently been shown that Interleukin-1 receptor, type 1, an essential regulator of inflammation and inate immunity, undergoes regulated intramembrane proteolysis (RIP). Although IL-1R1-mediated intracellular signalling has been well Studied, very little is known about how RIP of IL-1R1 is modulated. In this study, by using wild-type TRAF6 and TRAF6 mutants that are defective in its ubiquitin ligase activity, we show for the first time that TRAF6 induces ubiquitination of IL-1R1. We further demonstrate that of all TRAF family members examined, TRAF6 preferentially ubiquitinates IL-1R1. Moreover, we show that TRAF6 ubiquitin ligase activity and ubiquitination of IL-1R1 are positively correlated with IL-1R1 ectodomain shedding and subsequent gamma-secretase cleavage. Our results indicate that TRAF6-mediated ubiquitination of IL-1R1 has a decisive role in IL-1R1 signalling and propose a molecular mechanism whereby TRAF6 promotes ubiquitination and RIP of IL-1R1 through its ubiquitin ligase activity. (C) 2009 Elsevier Inc. All rights reserved.