Regulation of RIPK1 Dependent Apoptosis and Necroptosis

Abstract

RIPK1 is a Ser/Thr kinase critically involved in mediating multiple signaling pathways activated by TNFa. When cells are stimulated by TNFa, RIPK1 is rapidly recruited to the intracellular domain of TNFR1 to form the TNF-RSC (TNF receptor signaling complex). When survival signals are limited, RIPK1 transitions to form a pro-apoptotic cytosolic complex, independently of its kinase activity. Under apoptosis-deficient conditions, RIPK1 may be activated to mediate necroptosis. On the other hand, when TNF-RSC members TAK1 or cIAP1/2 are inhibited, cells may activate a form of apoptosis mediated by RIPK1 kinase activity, termed RIPK1 dependent apoptosis, or RDA. Most studies of RIPK1 focus on its role in necroptosis, while the mechanism that mediates the activation of RIPK1 in RDA is not well understood. Here, I investigated and compared the mechanism of RIPK1 activation in RDA, apoptosis, and necroptosis and identified a unique RIPK1 activation profile in RDA. To characterize the regulation of this unique profile, I carried out a targeted siRNA screen of post-translational modification genes, including kinases, phosphatases, ubiquitin ligases and deubiquitinating enzymes for protectors and sensitizers of RDA. I identified a set of genes which selectively promoted RDA, but not TNFa/cycloheximide-induced apoptosis or necroptosis. In particular, I identified a novel E3 ligase which was recruited to the TNF-RSC to promote the modification and activation of RIPK1 in RDA. Further, I demonstrated that several known TNF-RSC components and a novel E3 ligase play a role in limiting RDA. Finally, my data revealed the existence of two distinct necroptosis initiation pathways by TNFa. Taken together, my study provides important insights into the mechanism of RIPK1 activation in RDA, and identifies a set of enzymes that uniquely modulate RIPK1 activity in RDA but not necroptosis.