Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death

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-kappa B 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 toRipk1(K376R/K376R)mice,Ripk1(K115R/K115R)mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed inRipk1(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 ofRipk1(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 adultRipk1(K376R/cko)mice was tolerated. However, adultRipk1(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-kappa B and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-kappa B 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.

Automated summary

Mutating the ubiquitination site K376 of RIPK1 in mice activates cell death, leading to embryonic lethality, while mutation at K115 has minimal impact on cell death. Embryonic lethality can be prevented by loss of TNFR1 or simultaneous deletion of Caspase-8 and RIPK3 genes. Elimination of the K376 ubiquitination site in adult mice is tolerated but results in sensitivity to TNF-induced lethality.