Caspase cleavage of RIPK3 after Asp333 is dispensable for mouse embryogenesis
Kim Newton, Katherine E. Wickliffe, Allie Maltzman, Debra L. Dugger, Joshua D. Webster, Hongyan Guo, Vishva M. Dixit
Abstract
Abstract The proteolytic activity of caspase-8 suppresses lethal RIPK1-, RIPK3- and MLKL-dependent necroptosis during mouse embryogenesis. Caspase-8 is reported to cleave RIPK3 in addition to the RIPK3-interacting kinase RIPK1, but whether cleavage of RIPK3 is crucial for necroptosis suppression is unclear. Here we show that caspase-8-driven cleavage of endogenous mouse RIPK3 after Asp 333 is dependent on downstream caspase-3. Consistent with RIPK3 cleavage being a consequence of apoptosis rather than a critical brake on necroptosis, Ripk3 D333A/D333A knock-in mice lacking the Asp 333 cleavage site are viable and develop normally. Moreover, in contrast to mice lacking caspase-8 in their intestinal epithelial cells, Ripk3 D333A/D333A mice do not exhibit increased sensitivity to high dose tumor necrosis factor (TNF). Ripk3 D333A/D333A macrophages died at the same rate as wild-type (WT) macrophages in response to TNF plus cycloheximide, TNF plus emricasan, or infection with murine cytomegalovirus (MCMV) lacking M36 and M45 to inhibit caspase-8 and RIPK3 activation, respectively. We conclude that caspase cleavage of RIPK3 is dispensable for mouse development, and that cleavage of caspase-8 substrates, including RIPK1, is sufficient to prevent necroptosis.