Abstract
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 Ripk1K376R/K376R mice, Ripk1K115R/K115R mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1K376R/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 Ripk1K376R/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 Ripk1K376R/cko mice was tolerated. However, adult Ripk1K376R/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.
Highlights
Ubiquitination of RIPK1 at lysine 376 occurs in the TNFR1-associated complex I and is linked to activation of NF-κB and MAPK signaling [12, 14]
Both cleaved caspase-3 and phosphorylated RIPK3 were detected in the K376 of RIPK1 (K376R) placenta, suggesting activation of apoptotic and necroptotic cell death pathways (Figs. 1e and S1C, D)
There was an overall decrease in K11-linked, K63-linked, and linear ubiquitination on RIPK1(K376R) when compared with WT RIPK1 (Figs. 6e and S6C). These findings suggest that K376 of RIPK1 could be a site for ubiquitination by multiple linkages, all of them likely contributing to TNF stimulated NF-κB and MAPK signaling as well as complex I stability
Summary
A RIPK1 (K377R) mutant showed reduced ubiquitination and impaired recruitment of RIPK1 to the TNFR1 receptor complex, leading to decreased NF-κB activation [12, 14] Another reported ubiquitination site on RIPK1, K115, is ubiquitinated during TNF-induced necroptotic cell death [15,16,17]. Complex II is a cytosolic complex consisting of caspase-8, FADD (Fasassociated protein with death domain), FLIP (FLICE inhibitory protein) and RIPK1 [20, 26] This complex can activate caspase-3 and -7 resulting in apoptosis, but will recruit RIPK3 to form complex IIb if caspases are inhibited or absent, leading to necroptosis [1, 2]. Our data suggest that selective impairment of RIPK1 ubiquitination promotes activation of RIPK1, cell death, and embryonic lethality
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