Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3 mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3 macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing.