Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1’s cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity.

肿瘤坏死因子 (TNF) 可以驱动炎症、细胞存活和死亡。虽然泛素化、磷酸化和核因子 κB (NF-κB) 依赖性检查点抑制 TNF 的细胞毒性潜力，但尚不清楚泛素化是否可以直接抑制 TNF 诱导的死亡。在这里，我们表明泛素化不仅通过激活下游激酶和 NF-kB 转录反应，而且通过泛素依赖性失活直接抑制 RIPK1 激酶活性来调节 RIPK1 的细胞毒性潜力。我们发现细胞凋亡抑制剂 (cIAP)1 的泛素相关 (UBA) 结构域是 RIPK1 的最佳泛素-赖氨酸占据和 K48 泛素化所必需的。独立于 IKK 和 MK2，cIAP1 介导和 UBA 辅助泛素化抑制 RIPK1 激酶自动激活，此外，标记为蛋白酶体降解。在没有 cIAP1 的功能性 UBA 结构域的情况下，更活跃的 RIPK1 激酶响应 TNF 积累，导致 RIPK1 激酶介导的细胞死亡和全身炎症反应综合征。这些结果揭示了 cIAP 介导的泛素化在控制 RIPK1 激酶活性和防止 TNF 介导的细胞毒性中的直接作用。