Receptor-interacting protein 1 (RIP1; RIPK1) is a key regulator of multiple signaling pathways that mediate inflammatory responses and cell death. TNF-TNFR1 triggered signaling complex formation, subsequent NF-κB and MAPK activation and induction of cell death involve RIPK1 ubiquitination at several lysine residues including Lys376 and Lys115. Here we show that mutating the ubiquitination site K376 of RIPK1 (K376R) in mice activates cell death resulting in embryonic lethality. In contrast to Ripk1^K376R/K376R mice, Ripk1^K115R/K115R mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1^K376R/K376R embryos relied on RIPK1 kinase activity as administration of RIPK1 inhibitor GNE684 to pregnant heterozygous mice effectively blocked cell death and prolonged survival. Embryonic lethality of Ripk1^K376R/K376R mice was prevented by the loss of TNFR1, or by simultaneous deletion of caspase-8 and RIPK3. Interestingly, elimination of the wild-type allele from adult Ripk1^K376R/cko mice was tolerated. However, adult Ripk1^K376R/cko mice were exquisitely sensitive to TNF-induced hypothermia and associated lethality. Absence of the K376 ubiquitination site diminished K11-linked, K63-linked, and linear ubiquitination of RIPK1, and promoted the assembly of death-inducing cellular complexes, suggesting that multiple ubiquitin linkages contribute to the stability of the RIPK1 signaling complex that stimulates NF-κB and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-κB and MAPK signaling. Overall, our data indicate that selective impairment of RIPK1 ubiquitination can lower the threshold for RIPK1 activation by TNF resulting in cell death and embryonic lethality.

受体相互作用蛋白 1 (RIP1；RIPK1) 是介导炎症反应和细胞死亡的多种信号通路的关键调节因子。TNF-TNFR1 触发信号复合物形成，随后 NF-κB 和 MAPK 激活以及细胞死亡的诱导涉及 RIPK1 在几个赖氨酸残基（包括 Lys376 和 Lys115）处泛素化。在这里，我们发现，小鼠体内 RIPK1 (K376R) 泛素化位点 K376 的突变会激活细胞死亡，导致胚胎致死。与Ripk1 ^K376R/K376R小鼠相比，Ripk1 ^K115R/K115R小鼠成年后对 TNF 诱导的死亡表现出略高的反应性。在Ripk1 ^K376R/K376R胚胎中观察到的细胞死亡依赖于 RIPK1 激酶活性，因为对怀孕杂合小鼠施用 RIPK1 抑制剂 GNE684 可以有效阻止细胞死亡并延长生存期。通过缺失 TNFR1 或同时删除 caspase-8 和 RIPK3，可以防止Ripk1 ^K376R/K376R小鼠的胚胎致死。有趣的是，成年Ripk1 ^K376R/cko小鼠中野生型等位基因的消除是可以耐受的。然而，成年Ripk1 ^K376R/cko小鼠对 TNF 诱导的体温过低和相关致死极其敏感。K376 泛素化位点的缺失会减弱 RIPK1 的 K11 连接、K63 连接和线性泛素化，并促进死亡诱导细胞复合物的组装，表明多个泛素连接有助于刺激 NF- 的 RIPK1 信号复合物的稳定性。 κB 和 MAPK 激活。相反，K115 突变不会影响 RIPK1 泛素化或 TNF 刺激的 NF-κB 和 MAPK 信号传导。总体而言，我们的数据表明，RIPK1 泛素化的选择性损伤可以降低 TNF 激活 RIPK1 的阈值，从而导致细胞死亡和胚胎致死。