RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1\u03b3 promote apoptosis and necroptosis

Jingchun Du,Shuzhen Liu,Zhigao Wang,Ashwani Kumar,Hua Liu,Yougui Xiang,Chao Xing

doi:10.1038/s41467-021-27367-5

Abstract

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. Many sites on RIPK1, including serine 25, are phosphorylated to inhibit its kinase activity and cell death. How these inhibitory phosphorylation sites are dephosphorylated is poorly understood. Using a sensitized CRISPR whole-genome knockout screen, we discover that protein phosphatase 1 regulatory subunit 3G (PPP1R3G) is required for RIPK1-dependent apoptosis and type I necroptosis. Mechanistically, PPP1R3G recruits its catalytic subunit protein phosphatase 1 gamma (PP1γ) to complex I to remove inhibitory phosphorylations of RIPK1. A PPP1R3G mutant which does not bind PP1γ fails to rescue RIPK1 activation and cell death. Furthermore, chemical prevention of RIPK1 inhibitory phosphorylations or mutation of serine 25 of RIPK1 to alanine largely restores cell death in PPP1R3G-knockout cells. Finally, Ppp1r3g−/− mice are protected from tumor necrosis factor-induced systemic inflammatory response syndrome, confirming the important role of PPP1R3G in regulating apoptosis and necroptosis in vivo.

Highlights

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death
We identified a phosphatase subunit protein phosphatase 1 regulatory subunit 3G (PPP1R3G) that was essential for T/S/Z-induced necroptosis from a sensitized whole-genome CRISPR knockout screen
Loss of PPP1R3G blocked T/S-induced RIPK1-dependent apoptosis (RDA) and T/S/Z-induced type I necroptosis, both of which depend on RIPK1 kinase activity (Fig. 2a–f)

Summary

Introduction

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. TNF engages its membrane receptor TNFR1 to induce the formation of complex I, consisting of TRADD (TNFR1-associated death domain protein) and RIPK1 (receptor-interacting protein kinase 1), as well as E3 ubiquitin ligases, such as TRAF2/5 (TNF receptor-associated factor 2 and 5), cIAP1/2 (cellular inhibitor of apoptosis 1 and 2) and LUBAC (linear ubiquitin chain assembly complex)[8] These E3 ligases catalyze many different types of ubiquitination of RIPK1 which leads to the recruitment of TAK1 (transforming growth factor-β-activating kinase 1) as well as a protein complex including NEMO (NF-κB essential modulator) and IKKα/IKKβ (inhibitor of the NF-κB kinase α/β), to activate NF-κB signaling and cell survival[9,10,11,12,13]. Little is known about how these inhibitory phosphorylation sites are dephosphorylated

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Conclusion