The kinase RIPK1 received a lot of attention in the past decade due to its ability to transduce a death signal whose consequences have been implicated in the pathogenesis of various diseases. While these findings motivated the recent use of small molecule RIPK1 kinase inhibitors in human clinical trials, how active RIPK1 transmits the lethal signal has remained an important and fundamental unanswered question is the field. Indeed, no lethal substrate of RIPK1 has been identified to date. In our research group, we previously performed a phosphoproteomic screen to identify potential RIPK1 substrates. In this project proposal, my aim is to validate the obtained hits as bona fide RIPK1 substrates and to uncover how they contribute to cell death induction. To reach this objective, I propose a complementary approach combining experiments performed at the biochemical, cellular and in vivo levels. This involves the generation of substrate deficient cells using the CRISPR/cas9 technology and their reconstitution with phospho-mutated versions of the substrate, the obtention of a phospho-specific antibody against the substrate, and the generation of a transgenic mouse line expressing the phosphodeficient version of the substrate. The importance of the phosphorylation event will be studied in the physiological context of Yersinia infection and in the pathological context of TNF-driven acute and chronic inflammatory pathologies, different established in vivo models of RIPK1 cytotoxicity.