Abstract
The linear ubiquitin chain assembly complex (LUBAC) regulates NF-κB activation by modifying proteins with linear (M1-linked) ubiquitination chains. Although LUBAC also regulates the apoptosis pathway, the precise mechanism by which LUBAC regulates apoptosis remains not fully defined. Here, we report that LUBAC-mediated M1-linked ubiquitination of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic molecule, contributes to tumor necrosis factor (TNF) α-induced apoptosis. We found that deficiency of RNF31, the catalytic subunit of the LUBAC complex, promoted cFLIP degradation in a proteasome-dependent manner. Moreover, we observed RNF31 directly interact with cFLIP, and LUBAC further conjugated M1-linked ubiquitination chains at Lys-351 and Lys-353 of cFLIP to stabilize cFLIP, thereby protecting cells from TNFα-induced apoptosis. Together, our study identifies a new substrate of LUBAC and reveals a new molecular mechanism through which LUBAC regulates TNFα-induced apoptosis via M1-linked ubiquitination.
Highlights
The linear ubiquitin chain assembly complex (LUBAC) regulates nuclear factor (NF)-B activation by modifying proteins with linear (M1linked) ubiquitination chains
After tumor necrosis factor (TNF)␣ plus cycloheximide (CHX) stimulation, RNF31-silenced cells were highly sensitive to apoptosis, which were indicated by cleavage of caspase 3/8 and PARP (Fig. 1A and Fig. S1A)
The results showed that cellular FLICE-like inhibitory protein (cFLIP) KO cells reconstituted with cFLIP-K351R/K353R double mutations were more sensitive to TNF␣/CHX-induced apoptosis than cells reconstituted with other cFLIP mutants, but comparable with cells expressing the cFLIP mutant with all six lysine residues converted to arginine (Fig. S3D), indicating that Lys-351 and Lys-353 are the main M1-linked ubiquitination sites contributing to the protection of cells from TNF␣-induced apoptosis
Summary
Deficient mouse embryonic fibroblasts were highly susceptible to TNF␣-induced cell death [20], and cell death-dependent skin inflammation was observed in Sharpin-deficient mice [26, 27]. The exact molecular mechanism of M1-linked ubiquitination in TNF␣-mediated cell death remains largely unknown. We identified cFLIP as a new substrate of LUBAC, and found that LUBAC conjugates M1-linked ubiquitination to cFLIP, and stabilizes it to protect cells from TNF␣-induced apoptosis. These findings provide molecular insight into the regulatory mechanism of apoptosis by M1-linked ubiquitination, and modulating this mechanism may be a novel therapeutic approach for diseases oriented from deregulation of cell death
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