Chronicles of a death foretold: dual sequential cell death checkpoints in TNF signaling

Abstract

The kinase RIP1 wears a coat of many colors during TNF receptor signaling and can regulate both activation of pro-survival NFkB and programmed cell death pathways. In this review, we outline how coating RIP1 with K63-linked ubiquitin chains forms a protective layer that prevents RIP1 from binding apoptotic regulators and serves as an early guard against cell death. Further on, binding of NFkB signaling components to the ubiquitin coat of RIP1 activates long-term pro-survival signaling and forms a more impenetrable suit of armor against cell death. If RIP1 is not decorated with ubiquitin chains it becomes an unstoppable harbinger of bad news: programmed cell death.

Figure 1

TNF induces rapid ubiquitination of RIP1. (A) Jurkat T cells were stimulated with 100 ng/ml human TNF for 5 minutes and TNFR1 was immunoprecipitated from the cytoplasmic protein fraction. The TNFR1 complex and a sample of the cytoplasmic lysate were probed by western blot for RIP1. The TNFR1 complex recruits the 75 kD RIP1 protein but the majority of the receptor-associated RIP1 undergoes modification consistent with ubiquitination (RIP1 Ub). (B) Jurkat T cells were stimulated with 100 ng/ml human TNF and cytoplasmic protein samples were probed by western blot for phosphorylated IκBα, total I㮫α and RIP1 as a loading control. Evidence of IKK activity is seen 5 minutes post-stimulation by the appearance of phosphorylated IκBα, co-incident with the polyubiquitination of receptor-associated RIP1 shown in (A). IκBα is then degraded but re-expression of this immediate early NFκB-inducible gene is not observable at the protein level until approximately one hour post stimulation. Therefore, the potentially pro-death RIP1 protein is bound to TNFR1 during the first hour of TNF stimulation before the appearance of NFκB-dependent gene products.

Figure 2

Ubiquitination of RIP1 is an early pro-survival checkpoint in TNF signaling. TNFR1 ligation triggers formation of a complex of TRADD, RIP1, TRAF2, cIAP1 and cIAP2 and the latter three enzymes catalyse the formation of K63-linked polyubiquitin chains on lysine 377 of RIP1. The K63-linked polyubiquitin chains form a docking site for proteins that contain a ubiquitin recognition motif such as TAB2, TAB3 and NEMO. These adaptor proteins recruit and activate the TAK1 and IKK complexes, which lead to phosphorylation and degradation of IκB. Formation of this complex on the ubiquitinated RIP1 prevents RIP1 from interacting with Caspase 8 and activating the apoptotic machinery. If the lysine 377 ubiquitin acceptor site of RIP1 is mutated or the E3 ligase enzymes cIAP1 and cIAP2 are degraded by treatment with SMAC mimetic or through TNFR2 ligation, non-ubiquitinated RIP1 rapidly forms a complex with Caspase 8 and turns on the apoptotic machinery. RIP1 and RIP3 are themselves substrates for Caspase 8, which may prevent them from triggering the alternate death pathway of programmed necrosis. If the activity of Caspase 8 is inhibited, RIP1 forms a complex with RIP3 and triggers cell death by necrosis, which is dependent on the kinase activity of RIP1 and RIP3. While binding of NEMO to ubiquitinated RIP1 is an important factor in this early pro-survival event, it remains unclear what contribution the binding of TAB2 and TAB3 or other proteins with a ubiquitin recognition domain might make in this pro-survival step. Similarly, it remains to be seen whether the TAK1 and IKK kinase complexes participate in a transcription-independent manner in this early pro-survival checkpoint, prior to long-term anti-death gene expression programs coming into effect.