iProXProteomicsJunying YuanHomo SapiensMus Musculushttp://www.iprox.org/page/project.html?id=IPX0002548000junying_yuan@sioc.ac.cnShanghai institute of Organic Chemistry,Chinese academy of sciencesiProXRIPK1 is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation. RIPK1 activation is known to be regulated by its DD-mediated interaction and ubiquitination, though underlying mechanisms remain incompletely understood. Here we show that K627 in human RIPK1-DD and its equivalent K612 in murine RIPK1-DD is a key ubiquitination site that regulates the overall ubiquitination pattern of RIPK1 and its DD-mediated interactions with other DD-containing proteins. K627R/K612R mutation inhibits the activation of RIPK1 and blocks both apoptosis and necroptosis mediated by TNFR1 signaling. However, Ripk1^K612R/K612R mutation sensitizes cells to necroptosis and caspase-1 activation in response to TLRs signaling. Ripk1^K612R/K612R mice are viable, but develop age-dependent reduction of RIPK1 expression, spontaneous intestinal inflammation and splenomegaly, which can be rescued by antibiotic treatment and partially by Ripk3 deficiency. Furthermore, we show that the interaction of RIPK1 with FADD contributes to suppressing the activation of RIPK3 mediated by TLRs signaling. Our study demonstrates the distinct roles of K612 ubiquitination in mRIPK1/K627 ubiquitination in hRIPK1 in regulating its pro-death kinase activity in response to TNFα and pro-survival activity in response to TLRs signaling.Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners.Li Xingyan X, Zhang Mengmeng M, Huang Xinyue X, Liang Wei W, Li Ganquan G, Lu Xiaojuan X, Li Yanxia Y, Pan Heling H, Shi Linyu L, Zhu Hong H, Qian Lihui L, Shan Bing B, Yuan Junying JfalseDistinct functions of RIPK1 ubiquitination in regulating its activation mediated by TNFR1 and TLRs signalingWe identified and quantified ubiquitination sites of WT RIPK1 and K627R mutant in TNF signaling pathway and 293T overexpression systemFri Oct 23 00:00:00 BST 2020PXD02215710090960633311474