Editors' ChoiceCell Biology

Preventing Necrosis by Cleaving a Deubiquitinase

Science Signaling  13 Dec 2011:
Vol. 4, Issue 203, pp. ec344
DOI: 10.1126/scisignal.4203ec344

During development, caspase 8 blocks necrosis induced by tumor necrosis factor (TNF) in a manner that requires its catalytic activity. O’Donnell et al. identified the deubiquitinase CYLD as the substrate for caspase 8 whose cleavage blocks necrosis. In mouse embryo fibroblasts (MEFs) deficient in Cyld (Cyld–/–), TNF treatment did not induce necrosis, an effect that was abrogated by reconstitution with exogenous FLAG-tagged CYLD. TNF stimulation reduced the overall abundance of FLAG-CYLD and generated a 25-kD FLAG-tagged fragment, and a 25-kD fragment of CYLD was also present in untransfected MEFs. Generation of this CYLD fragment was blocked by pharmacological inhibition of caspase 8 and was not detected in cells overexpressing a catalytically inactive form of caspase 8, in cells deficient in Casp8, or in cells expressing CrmA, a viral protein that inhibits caspase 8. In vitro assays confirmed that CYLD was processed by caspase 8 to generate an N-terminal 25-kD fragment, a cleavage event that was prevented by mutation of Asp215 to Ala (D215A) in a putative caspase 8 cleavage site motif in CYLD. The 25-kD fragment of CYLD was not produced in Cyld–/– MEFs reconstituted with CYLDD215A, and these MEFs underwent necrosis in response to TNF. Deubiquitination of the kinase RIP1 by CYLD elicits ubiquitination of the kinase RIPK1, thereby causing RIPK1 to be recruited to FADD-containing, pro-death protein complexes, whereas preventing the deubiquitination of RIP1 results in the association of RIPK1 with NEMO-containing, prosurvival protein complexes. In TNF-treated Cyld–/– MEFs reconstituted with wild-type CYLD, ubiquitination of RIPK1 was greater, and more RIPK1 associated with NEMO and less RIPK1 associated with FADD compared with MEFs reconstituted with CYLDD215A. Thus, cleavage of CYLD by caspase 8 is required to prevent necrosis in response to TNF.

M. A. O’Donnell, E. Perez-Jimenez, A. Oberst, A. Ng, R. Massoumi, R. Xavier, D. R. Green, A. T. Ting, Caspase 8 inhibits programmed necrosis by processing CYLD. Nat. Cell Biol. 13, 1437–1442 (2011). [PubMed]