RIPK1 ubiquitination regulates its kinase-independent function in development and inflammation.

Abstract

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of cell death and inflammation, with its activation modulated by diverse posttranslational modifications. While ubiquitination of RIPK1 at lysine 376 (K376) has been shown to inhibit apoptosis and necroptosis both in vitro and in vivo, its role in inflammation remains undefined. In this study, we introduced a kinase-dead D138N mutation into <i>Ripk1^K376R/K376R</i> mice. Notably, <i>Ripk1^{K376R,D138N/K376R,D138N}</i> mice rescued the embryonic lethality observed in <i>Ripk1^K376R/K376R</i> mice, but developed systemic inflammation. Remarkably, this inflammation was significantly alleviated by codeletion of <i>Caspase-1/11</i>, but not <i>Trif</i>, indicating a critical role for inflammasome activation. Mechanistically, loss of ubiquitination at the K376 residue of RIPK1 promotes kinase activity-dependent cell death, which underlies the lethality of <i>Ripk1^K376R/K376R</i> mice. Importantly, the K376R mutation also drives RIPK1 kinase-independent inflammatory responses by triggering intrinsic NLRP3 inflammasome activation and downstream IL-1β secretion. Furthermore, we found that RIPK1 promotes this process through a RIPK3-dependent mechanism. Consistently, deletion of <i>Ripk3</i>-but not <i>Mlkl</i>-ameliorated this inflammation, highlighting a necroptosis-independent inflammatory axis. Together, our findings demonstrate that the RIPK1^K376R mutant not only induces kinase activity-dependent cell death during embryogenesis but also promotes kinase-independent, scaffold-driven inflammation in adults via RIPK3-mediated metabolic reprogramming that activates the NLRP3 inflammasome.