The role of RIPK1 auto-phosphorylation in cell death and inflammation

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RIPK1 has emerged as an important regulator of cell death and inflammation. RIPK1 controls signalling downstream of cytokine and innate immune receptors, such as TNFR1, TLR3 and TLR4, via kinase activity-dependent and -independent functions. While its kinase activity has been shown to induce cell death by activating caspase-8-mediated apoptosis as well as RIPK3-MLKL-mediated necroptosis, RIPK1 also exhibits kinase activity-independent scaffolding functions that contribute to the activation of NF-B and MAPK signalling and inhibit cell death. Studies in mouse models showed that RIPK1 kinase activity-mediated cell death is a potent driver of inflammatory and degenerative pathologies, suggesting that RIPK1 kinase inhibitors may be beneficial for the treatment of human inflammatory diseases. However, the mechanisms by which RIPK1 kinase activity induces cell death and inflammation remain poorly understood. RIPK1 autophosphorylation is thought to be critical for cell death induction, possibly by regulating the recruitment of RIPK1 in diverse signalling complexes. Yet, it is unclear if RIPK1 has additional substrates that contribute to cell death or if autophosphorylation is sufficient for activation of cell death signalling. Here we aim to study the role of RIPK1 autophosphorylation in cell death and inflammation using genetically modified mouse models. Specifically, we will use knock-in mice expressing endogenous RIPK1 with mutations abrogating or mimicking phosphorylation at serine residues S161 and S166 to assess how phosphorylation of these sites affects RIPK1 signalling. We will employ in vitro experiments in primary cells from these mice to investigate the impact of the mutations on RIPK1-dependent signalling, combined with biochemical assays addressing the underlying molecular mechanisms. In addition, we will perform studies in mouse models of inflammatory diseases that are triggered by RIPK1 kinase activity-dependent processes, in order to address the role of RIPK1 autophosphorylation in the regulation of cell death and inflammation in vivo. By shedding light on the mechanisms by which RIPK1 kinase activity induces cell death and inflammation in vitro and in vivo, these experiments will not only contribute to deciphering the complex regulation of cell death and inflammatory pathways in innate immune signalling but will also lead to the better understanding of the pathogenesis of inflammatory diseases.