Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

Xingyan Li,Mengmeng Zhang,Bing Shan,Wei Liang,Heling Pan,Lihui Qian,Yanxia Li,Junying Yuan,Linyu Shi,Ganquan Li,Xinyue Huang,Hong Zhu,Xiaojuan Lu

doi:10.1038/s41467-020-19935-y

Xingyan Li, Mengmeng Zhang + Show 11 more

Open Access

https://doi.org/10.1038/s41467-020-19935-y

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Abstract

RIPK1 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, Ripk1K612R/K612R mutation sensitizes cells to necroptosis and caspase-1 activation in response to TLRs signaling. Ripk1K612R/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.

Highlights

Receptor interacting protein kinase 1 (RIPK1) is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation
We identify that K627 in human RIPK1 and its conserved residue K612 in murine RIPK1 in the DD are important for mediating RIPK1 ubiquitination, homodimerization as well as heterodimerization of RIPK1 with TNFR1, TRADD, and FADD
Among the ubiquitinating site mutants screened, we found that only RIPK1 K612R complemented mouse embryonic fibroblast (MEFs) showed significant resistance to both RIPK1 kinase dependent apoptosis (RDA) induced by TNFα/SM-164 or TNFα/5Z-7 and necroptosis induced by the additional presence of pan caspase inhibitor zVAD.fmk (Supplementary Fig. 1a)

Summary

Introduction

RIPK1 is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation. We show that the interaction of RIPK1 with FADD contributes to suppressing the activation of RIPK3 mediated by TLRs signaling. In TNFα stimulated cells, RIPK1 and TRADD, an adapter protein with a DD, bind with the DD in the intracellular domain of TNFR1 to form a key signaling complex (TNF-RSC/complex I)[11]. The activation of RIPK1 is regulated by complex ubiquitination modifications, and the interaction with multiple DD-containing proteins, including TRADD, FADD, and TNFR1 as well as itself.

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