Abstract
Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.
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Abbreviations
- CHX (C):
-
cycloheximide
- cIAP:
-
cellular inhibitor of apoptosis protein
- CYLD:
-
cylindromatosis
- FADD:
-
Fas-associated death domain
- IFN:
-
interferon
- MEF:
-
mouse-embryo fibroblast
- MLKL:
-
mixed lineage kinase domain-like
- Nec-1 (N):
-
necrostatin-1
- NF-κB:
-
nuclear factor-κB
- RIPK1:
-
receptor interacting protein kinase 1
- RIPK3:
-
receptor interacting protein kinase 3
- TNFα (T):
-
tumor necrosis factor α
- TRAF2:
-
TNF receptor-associated factor 2
- Ub:
-
ubiquitin
- Z-VAD-fmk (Z):
-
benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone
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Acknowledgements
We thank Eugene Varfolomeev and Domagoj Vucic for TRAF2 and cIAP1 KO MEFs, and Kim Newton and Vishva Dixit for TRAF2 and RIPK3 KO mice and for advice and comments. We also thank Genentech’s mouse-facility staff for their support.
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Petersen, S., Chen, T., Lawrence, D. et al. TRAF2 is a biologically important necroptosis suppressor. Cell Death Differ 22, 1846–1857 (2015). https://doi.org/10.1038/cdd.2015.35
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DOI: https://doi.org/10.1038/cdd.2015.35
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