Abstract 220: TNFα activates NF-κB through RIP1 ubiquitination-dependent and independent pathways

Abstract

Abstract TNFα-induced NF-κB activation has long been believed to depend on TRAF2/5- and cIAP1/2-mediated RIP1 ubiquitination. Recently, HOIP/HOIL-1-mediated linear ubiquitination of NEMO/IKKγ has emerged as a new player in TNFα-induced NF-κB activation. However, the mechanisms by which these proteins mediate NF-κB activation have become more complex than ever before due to an accumulation of conflicting results. Several independent studies even challenged the notion that these proteins ever play essential roles in NF-κB activation. Here, we report the identification of immediate and delayed phases of IKK activation in TNFα-treated cells. TNFα-induced immediate IKK activation requires ubiquitination of RIP1 catalyzed by cooperative actions of TRAF2, cIAP1/2, UbcH5 and Ubc13, and that in the absence of any one of these components; those remaining are only capable of mediating delayed IKK activation by recruiting the HOIP/HOIL-1 complex. In RIP1-deficient cells, TRAF2 undergoes increased auto-ubiquitination through K63-linkage, yet it is not capable of mediating immediate IKK activation. Notably, depletion of either HOIP in RIP1-deficient cells or both TRAF2 and cIAP1/2 in wild-type cells completely impairs IKK activation. In addition, deletion of the TRAF2 RING domain also severely reduces the immediate phase of IKK activation. These data suggest that two RING finger E3 ligases (TRAF2 and cIAP1) and two E2 enzymes (UbcH5 and Ubc13) cooperate to catalyze immediate RIP1 ubiquitination and IKK activation, and that, in the absence of RIP1, TRAF2 and cIAP1/2 recruit HOIP/HOIL-1 and mediate the delayed phase of IKK activation. TRAF5 has no substantial role in TNFα-induced NF-κB activation. Importantly, in embryonic hepatocytes, RIP1 is completely degraded, yet these cells are not sensitized to TNFα-triggered cell death because of delayed IKK activation. Thus, this study provides evidences for the existence of RIP1 ubiquitination-dependent and -independent pathways that mediate NF-κB activation, and thereby clarifies molecular details of this important signaling mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 220. doi:1538-7445.AM2012-220