Mind Bomb Regulates Cell Death during TNF Signaling by Suppressing RIPK1’s Cytotoxic Potential

Abstract

SummaryTumor necrosis factor (TNF) is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2) regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2’s E3 activity or RIPK1’s ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF.

Targeting triple-negative breast cancers with the Smac-mimetic birinapant

“…Taken together, our findings suggest that most ER + tumors, in contrast to TNBC, are deficient in their ability to form and/or activate complex II. Recent reports showed that complex II activity can be limited by the kinases IKKα, β, ε, p38, MK2, TBK1, or by the E3 ligase MIB2 [35][36][37][38][39][40][41][42]. These proteins restrain the cytotoxic activity of RIPK1 upon Smacmimetic treatment [35,36].…”

“…Recent reports showed that complex II activity can be limited by the kinases IKKα, β, ε, p38, MK2, TBK1, or by the E3 ligase MIB2 [35][36][37][38][39][40][41][42]. These proteins restrain the cytotoxic activity of RIPK1 upon Smacmimetic treatment [35,36]. Interestingly, the level of IKKα/β transcripts was significantly lower in TNBC compared with ER + tumors in TCGA samples, which may in part account for the ability of TNBC cells to activate the cytotoxic activity of RIPK1 ( Supplementary Fig.…”

RIPK1‐mediated immunogenic cell death promotes anti‐tumour immunity against soft‐tissue sarcoma

“…DEVDase assay was performed as previously described (Feltham et al , ). In brief, cells were plated in 96‐well plates and retro siRNA transfection was performed for 40 h. After treatment, medium was removed and 1% DISC lysis buffer was added to each well.…”

“…Complex‐I/II purification was essentially performed as previously described (Feltham et al , ). In brief, cells were seeded in 15 cm dishes and treated as indicated using pre‐warmed media containing 3xFLAG‐hTNF (0.8 μg/ml).…”

“…In healthy cells, TNFR1 activation results in rapid post‐translational modification of RIPK1 at the receptor complex, which actively suppresses the cytotoxic potential of RIPK1. While the E3‐Ub ligases cIAP1, cIAP2, MIB2 and LUBAC conjugate inhibitory ubiquitin (Ub) chains to RIPK1 (Feltham & Silke, ; Annibaldi & Meier, ; Feltham et al , ), multiple kinases (e.g. IKK, MK2 and TBK1) inactivate RIPK1 via direct phosphorylation (Dondelinger et al , , ; Jaco et al , ; Lafont et al , ).…”