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• W2898593788 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. 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. Tumor necrosis factor (TNF) 3The abbreviations used are: TNFαtumor necrosis factor αLUBAClinear ubiquitin chain assembly complexcFLIPcellular FLICE-like inhibitory proteinRIPK1/3receptor-interacting serine/threonine-protein kinase 1/3TRAFsTNF receptor-associated factorsIKKIκB kinaseFADDFAS-associated death domain proteinBCL2B-cell lymphoma 2cIAPinhibitor of apoptosis proteinCHXcycloheximideRBRRING-IBR-RING domainDED1death effector domain 1shRNAshort hairpin RNAPARPpoly(ADP-ribose) polymeraseFMKfluoromethyl ketoneZbenzyloxycarbonylFBSfetal bovine serum.α is a cytokine that plays roles in various cellular processes, such as proliferation, differentiation, and death. It has been reported that it mainly activates two different signaling pathways: nuclear factor (NF)-κB activation and cell death (1Baud V. Karin M. Signal transduction by tumor necrosis factor and its relatives.Trends Cell Biol. 2001; 11 (11514191): 372-37710.1016/S0962-8924(01)02064-5Abstract Full Text Full Text PDF PubMed Scopus (1373) Google Scholar, 2Ofengeim D. Yuan J. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death.Nat. Rev. Mol. Cell Biol. 2013; 14 (24129419): 727-73610.1038/nrm3683Crossref PubMed Scopus (392) Google Scholar). Once TNFα binds to its receptor, signaling molecules such as the TNF receptor 1-associated DEATH domain, receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and TNF receptor-associated factors (TRAFs) are recruited and form the receptor-signaling complex. This complex induces the activation of the IκB kinase (IKK) complex, which further induces the degradation of inhibitor of κB-α (IκB-α) and translocation of NF-κB transcriptional factors, sequentially (3Brenner D. Blaser H. Mak T.W. Regulation of tumour necrosis factor signalling: live or let die.Nat. Rev. Immunol. 2015; 15 (26008591): 362-37410.1038/nri3834Crossref PubMed Scopus (591) Google Scholar). At the same time, RIPK1/FAS-associated death domain (FADD)-caspase 8 complex is assembled to activate apoptosis (4Wajant H. Pfizenmaier K. Scheurich P. Tumor necrosis factor signaling.Cell Death Differ. 2003; 10 (12655295): 45-6510.1038/sj.cdd.4401189Crossref PubMed Scopus (1890) Google Scholar, 5Micheau O. Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell. 2003; 114 (12887920): 181-19010.1016/S0092-8674(03)00521-XAbstract Full Text Full Text PDF PubMed Scopus (1990) Google Scholar). When caspase 8 activity is inhibited, RIPK1 can form a complex with RIPK3 to trigger necroptosis (6Cho Y.S. Challa S. Moquin D. Genga R. Ray T.D. Guildford M. Chan F.K. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.Cell. 2009; 137 (19524513): 1112-112310.1016/j.cell.2009.05.037Abstract Full Text Full Text PDF PubMed Scopus (1689) Google Scholar, 7He S. Wang L. Miao L. Wang T. Du F. Zhao L. Wang X. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α.Cell. 2009; 137 (19524512): 1100-111110.1016/j.cell.2009.05.021Abstract Full Text Full Text PDF PubMed Scopus (1618) Google Scholar8Zhang D.W. Shao J. Lin J. Zhang N. Lu B.J. Lin S.C. Dong M.Q. Han J. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis.Science. 2009; 325 (19498109): 332-33610.1126/science.1172308Crossref PubMed Scopus (1419) Google Scholar). Because the cell-death process is critical for homeostasis, it is tightly regulated by various inhibitory mechanisms. For example, cellular FLICE-like inhibitory protein (cFLIP) interacts and forms a heterodimer with caspase 8, inhibiting activation of caspase 8 and apoptosis signaling (9Budd R.C. Yeh W.C. Tschopp J. cFLIP regulation of lymphocyte activation and development.Nat. Rev. Immunol. 2006; 6 (16498450): 196-20410.1038/nri1787Crossref PubMed Scopus (225) Google Scholar). In addition, B-cell lymphoma 2 (BCL2) family proteins and inhibitor of apoptosis proteins (cIAPs) directly and indirectly suppress caspases activation (10Karin M. Lin A. NF-κB at the crossroads of life and death.Nat. Immunol. 2002; 3 (11875461): 221-22710.1038/ni0302-221Crossref PubMed Scopus (2445) Google Scholar, 11Vince J.E. Wong W.W. Khan N. Feltham R. Chau D. Ahmed A.U. Benetatos C.A. Chunduru S.K. Condon S.M. McKinlay M. Brink R. Leverkus M. Tergaonkar V. Schneider P. Callus B.A. et al.IAP antagonists target cIAP1 to induce TNFα-dependent apoptosis.Cell. 2007; 131 (18022363): 682-69310.1016/j.cell.2007.10.037Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar12Bertrand M.J. Milutinovic S. Dickson K.M. Ho W.C. Boudreault A. Durkin J. Gillard J.W. Jaquith J.B. Morris S.J. Barker P.A. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination.Mol. Cell. 2008; 30 (18570872): 689-70010.1016/j.molcel.2008.05.014Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). tumor necrosis factor α linear ubiquitin chain assembly complex cellular FLICE-like inhibitory protein receptor-interacting serine/threonine-protein kinase 1/3 TNF receptor-associated factors IκB kinase FAS-associated death domain protein B-cell lymphoma 2 inhibitor of apoptosis protein cycloheximide RING-IBR-RING domain death effector domain 1 short hairpin RNA poly(ADP-ribose) polymerase fluoromethyl ketone benzyloxycarbonyl fetal bovine serum. Ubiquitination is a key posttranslational modification (PTM) in TNFα-induced signaling. For example, Lys-48–linked ubiquitination regulates degradation of IκBα (13Ghosh S. May M.J. Kopp E.B. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses.Annu. Rev. Immunol. 1998; 16 (9597130): 225-26010.1146/annurev.immunol.16.1.225Crossref PubMed Scopus (4597) Google Scholar) and FADD (14Lee E.W. Kim J.H. Ahn Y.H. Seo J. Ko A. Jeong M. Kim S.J. Ro J.Y. Park K.M. Lee H.W. Park E.J. Chun K.H. Song J. Ubiquitination and degradation of the FADD adaptor protein regulate death receptor-mediated apoptosis and necroptosis.Nat. Commun. 2012; 3 (22864571): 97810.1038/ncomms1981Crossref PubMed Scopus (84) Google Scholar), and Lys-63–linked ubiquitination of NEMO, RIPK1, and TRAFs triggers the functional activation of these molecules (15Skaug B. Jiang X. Chen Z.J. The role of ubiquitin in NF-κB regulatory pathways.Annu. Rev. Biochem. 2009; 78 (19489733): 769-79610.1146/annurev.biochem.78.070907.102750Crossref PubMed Scopus (410) Google Scholar), which further recruit downstream molecules. Recently, researchers discovered M1-linked ubiquitination to be a novel type of ubiquitination involved in the TNFα signaling pathway, especially NF-κB signaling (16Iwai K. Tokunaga F. Linear polyubiquitination: a new regulator of NF-κB activation.EMBO Rep. 2009; 10 (19543231): 706-71310.1038/embor.2009.144Crossref PubMed Scopus (184) Google Scholar). To date, linear ubiquitin chain assembly complex (LUBAC) was identified as the only ligase complex for M1-linked ubiquitination. LUBAC has three components: RNF31 (also named as HOIP), HOIL-1(also named as HOIL-1L or RBCK1), and Sharpin. In particular, RNF31 has a major role in activation of NF-κB signaling by conjugating M1-linked ubiquitination chains with NEMO and RIPK1, whereas HOIL-1 and Sharpin are involved in the functional activation of RNF31 (17Iwai K. Fujita H. Sasaki Y. Linear ubiquitin chains: NF-κB signalling, cell death and beyond.Nat. Rev. Mol. Cell Biol. 2014; 15 (25027653): 503-50810.1038/nrm3836Crossref PubMed Scopus (139) Google Scholar). Genetic studies demonstrated that defects in LUBAC components attenuate TNFα-induced NF-κB activation and gene expression (18Gerlach B. Cordier S.M. Schmukle A.C. Emmerich C.H. Rieser E. Haas T.L. Webb A.I. Rickard J.A. Anderton H. Wong W.W. Nachbur U. Gangoda L. Warnken U. Purcell A.W. Silke J. Walczak H. Linear ubiquitination prevents inflammation and regulates immune signalling.Nature. 2011; 471 (21455173): 591-59610.1038/nature09816Crossref PubMed Scopus (683) Google Scholar, 19Ikeda F. Deribe Y.L. Skånland S.S. Stieglitz B. Grabbe C. Franz-Wachtel M. van Wijk S.J. Goswami P. Nagy V. Terzic J. Tokunaga F. Androulidaki A. Nakagawa T. Pasparakis M. Iwai K. et al.SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis.Nature. 2011; 471 (21455181): 637-64110.1038/nature09814Crossref PubMed Scopus (548) Google Scholar20Tokunaga F. Nakagawa T. Nakahara M. Saeki Y. Taniguchi M. Sakata S. Tanaka K. Nakano H. Iwai K. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex.Nature. 2011; 471 (21455180): 633-63610.1038/nature09815Crossref PubMed Scopus (475) Google Scholar). In addition to TNFα, LUBAC regulates interleukin-1, CD40-, lymphotoxin β-, Toll-like receptor-, T cell receptor-, and nucleotide-binding oligomerization domain containing 2 (NOD2)-mediated cellular events through regulating the NF-κB pathway (21Yang Y.K. Yang C. Chan W. Wang Z. Deibel K.E. Pomerantz J.L. Molecular determinants of scaffold-induced linear ubiquitinylation of B cell lymphoma/leukemia 10 (Bcl10) during T cell receptor and oncogenic caspase recruitment domain-containing protein 11 (CARD11) signaling.J. Biol. Chem. 2016; 291 (27777308): 25921-2593610.1074/jbc.M116.754028Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22Teh C.E. Lalaoui N. Jain R. Policheni A.N. Heinlein M. Alvarez-Diaz S. Sheridan J.M. Rieser E. Deuser S. Darding M. Koay H.F. Hu Y. Kupresanin F. O'Reilly L.A. Godfrey D.I. et al.Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis.Nat. Commun. 2016; 7 (27857075)1335310.1038/ncomms13353Crossref PubMed Scopus (38) Google Scholar23Rieser E. Cordier S.M. Walczak H. Linear ubiquitination: a newly discovered regulator of cell signalling.Trends Biochem. Sci. 2013; 38 (23333406): 94-10210.1016/j.tibs.2012.11.007Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Recent studies reported M1-linked ubiquitination to have an NF-κB-independent function as well (24Park Y. Jin H.S. Lopez J. Lee J. Liao L. Elly C. Liu Y.C. SHARPIN controls regulatory T cells by negatively modulating the T cell antigen receptor complex.Nat. Immunol. 2016; 17 (26829767): 286-29610.1038/ni.3352Crossref PubMed Scopus (46) Google Scholar, 25Rodgers M.A. Bowman J.W. Fujita H. Orazio N. Shi M. Liang Q. Amatya R. Kelly T.J. Iwai K. Ting J. Jung J.U. The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation.J. Exp. Med. 2014; 211 (24958845): 1333-134710.1084/jem.20132486Crossref PubMed Scopus (173) Google Scholar). Additionally, biochemical and mouse developmental data have suggested that LUBAC has a role in apoptosis signaling. For example, Sharpin-deficient mouse embryonic fibroblasts were highly susceptible to TNFα-induced cell death (20Tokunaga F. Nakagawa T. Nakahara M. Saeki Y. Taniguchi M. Sakata S. Tanaka K. Nakano H. Iwai K. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex.Nature. 2011; 471 (21455180): 633-63610.1038/nature09815Crossref PubMed Scopus (475) Google Scholar), and cell death-dependent skin inflammation was observed in Sharpin-deficient mice (26Kumari S. Redouane Y. Lopez-Mosqueda J. Shiraishi R. Romanowska M. Lutzmayer S. Kuiper J. Martinez C. Dikic I. Pasparakis M. Ikeda F. Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis.Elife. 2014; 3e0342210.7554/eLife.03422Crossref Scopus (131) Google Scholar, 27Rickard J.A. Anderton H. Etemadi N. Nachbur U. Darding M. Peltzer N. Lalaoui N. Lawlor K.E. Vanyai H. Hall C. Bankovacki A. Gangoda L. Wong W.W. Corbin J. Huang C. et al.TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice.Elife. 2014; 3e0346410.7554/eLife.03464Crossref Scopus (194) Google Scholar). RNF31 or HOIL-1-knockout (KO) mice are embryonic lethal due to massive cell death (28Peltzer N. Rieser E. Taraborrelli L. Draber P. Darding M. Pernaute B. Shimizu Y. Sarr A. Draberova H. Montinaro A. Martinez-Barbera J.P. Silke J. Rodriguez T.A. Walczak H. HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death.Cell Rep. 2014; 9 (25284787): 153-16510.1016/j.celrep.2014.08.066Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 29Peltzer N. Darding M. Montinaro A. Draber P. Draberova H. Kupka S. Rieser E. Fisher A. Hutchinson C. Taraborrelli L. Hartwig T. Lafont E. Haas T.L. Shimizu Y. Böiers C. et al.LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.Nature. 2018; 557 (29695863): 112-11710.1038/s41586-018-0064-8Crossref PubMed Scopus (125) Google Scholar30Fujita H. Tokunaga A. Shimizu S. Whiting A.L. Aguilar-Alonso F. Takagi K. Walinda E. Sasaki Y. Shimokawa T. Mizushima T. Ohki I. Ariyoshi M. Tochio H. Bernal F. Shirakawa M. Iwai K. Cooperative domain formation by homologous motifs in HOIL-1L and SHARPIN plays a crucial role in LUBAC stabilization.Cell Rep. 2018; 23 (29694895): 1192-120410.1016/j.celrep.2018.03.112Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, the exact molecular mechanism of M1-linked ubiquitination in TNFα-mediated cell death remains largely unknown. Because the deregulation of cell survival and death is the main cause of many diseases such as cancer and autoimmune disease, the clear understanding of cell death regulation is critical for the therapeutic strategies to control these diseases. In the present study, we determined the molecular mechanism of RNF31 in the regulation of apoptosis pathway. 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 TNFα-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. Previous studies have shown that loss of LUBAC components could promote TNFα-induced apoptosis (18Gerlach B. Cordier S.M. Schmukle A.C. Emmerich C.H. Rieser E. Haas T.L. Webb A.I. Rickard J.A. Anderton H. Wong W.W. Nachbur U. Gangoda L. Warnken U. Purcell A.W. Silke J. Walczak H. Linear ubiquitination prevents inflammation and regulates immune signalling.Nature. 2011; 471 (21455173): 591-59610.1038/nature09816Crossref PubMed Scopus (683) Google Scholar, 28Peltzer N. Rieser E. Taraborrelli L. Draber P. Darding M. Pernaute B. Shimizu Y. Sarr A. Draberova H. Montinaro A. Martinez-Barbera J.P. Silke J. Rodriguez T.A. Walczak H. HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death.Cell Rep. 2014; 9 (25284787): 153-16510.1016/j.celrep.2014.08.066Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 29Peltzer N. Darding M. Montinaro A. Draber P. Draberova H. Kupka S. Rieser E. Fisher A. Hutchinson C. Taraborrelli L. Hartwig T. Lafont E. Haas T.L. Shimizu Y. Böiers C. et al.LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.Nature. 2018; 557 (29695863): 112-11710.1038/s41586-018-0064-8Crossref PubMed Scopus (125) Google Scholar). But it is unclear whether LUBAC directly regulates apoptosis. First, we generated stable HeLa cells in which RNF31 was silenced using a short hairpin RNA (shRNA) system and compared them with control HeLa cells. After TNFα 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). Smac is a mitochondrial protein that can be released into the cytosol to promote caspase activation in the cytochrome c/Apaf-1/caspase 9 pathway (31Du C. Fang M. Li Y. Li L. Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition.Cell. 2000; 102 (10929711): 33-4210.1016/S0092-8674(00)00008-8Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). TNFα plus Smac mimetics stimulation also triggered more apoptosis in RNF31-silenced cells (Fig. 1B and Fig. S1B). To elucidate the molecular mechanism by which RNF31 regulates the apoptosis pathway, we examined the levels of anti-apoptotic molecules in TNFα-treated HeLa cells. RNF31 silencing did not alter the level of most of anti-apoptotic molecules, including BCL2 family proteins (BCLxL, BCL2, and NOXA), MCL-1 and cIAP2 (Fig. 1C), but we found that the level of cFLIP decreased markedly in RNF31-silenced HeLa cells upon treatment with TNFα and CHX (Fig. 1D and Fig. S1C). To confirm this result, we generated RNF31 knockout cells by CRISPR-Cas9 technique. After TNFα and Smac mimetics stimulation, cFLIP also degraded faster in RNF31 knockout cells (Fig. 1E and Fig. S1D). Above all, deficiency of RNF31 could promote TNFα-induced apoptosis and accelerate the degradation of cFLIP. To determine whether the rapid decrease of cFLIP in RNF31-deficient cells depended on activation of TNFα signaling, we treated control and RNF31-silenced HeLa cells with CHX only. We observed a similar pattern of decreased cFLIP in control and RNF31-silenced cells upon CHX treatment (Fig. 2A), indicating that loss of RNF31 did not alter the basal turnover rate for cFLIP. Previous studies reported that treatment with the proteasome inhibitor bortezomib reduced the severity of skin problems in Sharpin-deficient mice (32Liang Y. Seymour R.E. Sundberg J.P. Inhibition of NF-κB signaling retards eosinophilic dermatitis in SHARPIN-deficient mice.J. Invest. Dermatol. 2011; 131 (20811394): 141-14910.1038/jid.2010.259Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), suggesting that deficiency of LUBAC could result in degradation of some molecules. cFLIP could be ubiquitinated and led to proteasome-dependent degradation (33Poukkula M. Kaunisto A. Hietakangas V. Denessiouk K. Katajamäki T. Johnson M.S. Sistonen L. Eriksson J.E. Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.J. Biol. Chem. 2005; 280 (15886205): 27345-2735510.1074/jbc.M504019200Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 34Wilkie-Grantham R.P. Matsuzawa S. Reed J.C. Novel phosphorylation and ubiquitination sites regulate reactive oxygen species-dependent degradation of anti-apoptotic c-FLIP protein.J. Biol. Chem. 2013; 288 (23519470): 12777-1279010.1074/jbc.M112.431320Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Furthermore, cFLIP also was identified as a substrate of caspase 8 and can be cleaved by activated caspase 8 during apoptosis (9Budd R.C. Yeh W.C. Tschopp J. cFLIP regulation of lymphocyte activation and development.Nat. Rev. Immunol. 2006; 6 (16498450): 196-20410.1038/nri1787Crossref PubMed Scopus (225) Google Scholar). So, we pre-treated HeLa cells with caspase inhibitor Z-VAD-FMK or the proteasome inhibitor MG132 to determine whether the decreased cFLIP resulted from caspase activity or occurred in a degradation-dependent manner. We found that pre-treatment with Z-VAD-FMK fully inhibited cleavage of PARP and caspase 8 but only slightly restored the level of cFLIP (Fig. 2B and Fig. S2A). However, pre-treatment with MG132 fully blocked the decrease of cFLIP, cleavage of PARP and caspase 8(Fig. 2B and Fig. S2A), suggesting that the TNFα/CHX-induced decrease of cFLIP mainly results from the proteasome-dependent degradation. To confirm the role of proteasome-mediated degradation in the increased sensitivity of RNF31-silenced cells to apoptosis, we pre-treated control and RNF31-silenced HeLa cells with MG132 and then induced apoptosis with stimulation of TNFα plus CHX. Pre-treatment with MG132 completely blocked the decrease of cFLIP in RNF31-silenced cells (Fig. 2C and Fig. S2B), and the apoptosis sensitization in RNF31-silencing cells was also fully blocked (Fig. 2C), indicating that the sensitization of RNF31-silencing cells to apoptosis is mainly mediated by a proteasome-dependent degradation pathway. Moreover, in RNF31 knockout cells, degradation of cFLIP is fully suppressed by MG132 treatment, but not by Z-VAD-FMK treatment (Fig. 2D and Fig. S2C). Taken together, these data suggested that deficiency of RNF31 promotes cFLIP degradation in a proteasome-dependent manner. Next, we examined whether RNF31 have a direct interaction with cFLIP. We expressed Myc-tagged RNF31 together with FLAG-tagged cFLIP in HEK293T cells, and found that RNF31 were co-precipitated with cFLIP (Fig. 3A and Fig. S2D). In addition, stimulation of HeLa cells stably expressing FLAG-tagged cFLIP with TNFα led to inducible interaction between cFLIP and LUBAC components, including RNF31 and Sharpin (Fig. 3B). Similarly, TNFα induces endogenous interaction between RNF31 and cFLIP (Fig. 3C). RNF31 and cFLIP are composed of multiple domains (Fig. 3D). Therefore, we determined which domains in cFLIP and RNF31 are responsible for their interaction with each other. The domain mapping by expressing full-length cFLIP together with different RNF31 domains indicated that the RING-IBR-RING (RBR) domain of RNF31 is essential for its binding with cFLIP (Fig. 3E). Furthermore, a co-immunoprecipitation experiment with full-length RNF31 and constructs encoding different cFLIP domains demonstrated that death effector domain 1 (DED1) of cFLIP is critical for the interaction between RNF31 and cFLIP (Fig. 3F). Taken together, these data suggested that the RBR domain of RNF31 and DED1 domain of cFLIP are responsible for their interaction. RNF31 directly interact with cFLIP, indicating that cFLIP may be a substrate of LUBAC components. To test our hypothesis, we performed an in vitro ubiquitination assay to determine whether cFLIP is a substrate of LUBAC. We incubated recombinant cFLIP with or without recombinant LUBAC, E1, E2, and lysine KO ubiquitin (all lysine residues are mutated to arginine), and then detected M1-linked ubiquitination of cFLIP. The results showed LUBAC conjugates M1-linked ubiquitination chains to cFLIP (Fig. 4A). However, catalytically dead mutant C885S of RNF31 failed to induce M1-linked ubiquitination chains to cFLIP (Fig. 4A), suggesting that the catalytic activity of RNF31 is critical for the M1-linked ubiquitination of cFLIP. Besides, expression of LUBAC promoted M1-linked ubiquitination of cFLIP as well as the known substrate RIPK1 in HEK293T cells (Fig. 4B). Because cFLIP degrades rapidly in RNF31-deficient cells through a proteasome-dependent manner, we hypothesized that M1-linked ubiquitination stabilizes cFLIP by competing with Lys-48–linked ubiquitination, thereby suppressing proteasome-mediated degradation of cFLIP. We detected the Lys-48–linked ubiquitination of cFLIP in control and RNF31-silenced HeLa cells introduced with FLAG-cFLIP, and observed that silencing of RNF31 promoted Lys-48–linked ubiquitination of cFLIP at the endogenous level upon treatment with TNFα and CHX (Fig. 4C and Fig. S2E). These results were consistent with the results of MG132 treatment, and suggested that M1-linked ubiquitination of cFLIP can compete with Lys-48 ubiquitination to stabilize cFLIP. To determine which residues in cFLIP are the sites for RNF31-mediated ubiquitination and contribute to the regulation of apoptosis, we performed LC and MS to find the sites of M1-linked ubiquitination on cFLIP by LUBAC (Fig. 5A). We co-expressed FLAG-tagged cFLIP with LUBAC complex in HEK293T cells, and expression of cFLIP alone as control. Using FLAG antibody-conjugated beads to pull down the cFLIP protein, and then detected ubiquitination sites on cFLIP by mass spectrometry. Finally, six candidate ubiquitination sites that were dependent on the expression of LUBAC were detected (Lys-49, Lys-69, Lys-154, Lys-157, Lys-351, and Lys-353) (Fig. 5A). Because the above data suggested that M1-linked ubiquitination of cFLIP can protect cells from TNFα-induced apoptosis, we hypothesized that mutations of these M1-linked ubiquitination sites on cFLIP should promote TNFα-induced apoptosis. We replaced those identified lysine residues with arginine (K49R, K69R, K154R, K157R, K351R, K353R) in different combinations, and expressed these cFLIP mutants in cFLIP KO Jurkat cells generated by the CRISPR-Cas9 method (Fig. S3, A–C). We then monitored the sensitivity of these cells to TNFα/CHX-induced apoptosis. The results showed that cFLIP KO cells reconstituted with cFLIP-K351R/K353R double mutations were more sensitive to TNFα/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 (simplify to K6R) (Fig. S3D), indicating that Lys-351 and Lys-353 are the main M1-linked ubiquitination sites contributing to the protection of cells from TNFα-induced apoptosis. To further determine which M1-linked ubiquitination site in cFLIP is functionally important for preventing TNFα-induced apoptosis, we generated cFLIP mutants with K351R or K353R alone and expressed these mutants in cFLIP KO cells. We found that cells expressing K351R or K353R enhance both the TNFα/Smac mimetics and TNFα/CHX-induced apoptosis compared with WT cFLIP-reconstituted cells (Fig. 5, B and C, and Fig. S3, E and F). These data indicated that Lys-351 and Lys-353 may play a redundant role in protecting cells from TNFα-induced apoptosis. Consistent with the annexin V staining, we found that more and earlier cleavage of PARP and caspase 8 in cFLIP KO cells reconstituted with K351R/K353R double mutant (Fig. 5D), and mutated cFLIP also degraded faster than WT cFLIP (Fig. 5D). Finally, we monitored the NF-κB activation in K351R/K353R double-mutated cells. The results showed that K351R/K353R mutation of cFLIP did not affect TNFα-induced NF-κB activation, indicated by phosphorylation of IκBα, degradation of IκBα, and translocated p65 in the nucleus (Fig. 5E). These results suggested that linear ubiquitination of cFLIP protected the cell from TNFα-induced apoptosis through a NF-κB-independent manner. To further confirm whether Lys-351 and Lys-353 are the main M1-linked ubiquitination sites of cFLIP, we performed the ubiquitin assay in HEK293T cells. The results also showed that mutation of K351R/K353R significantly blocked M1-linked ubiquitination of cFLIP compared with WT cFLIP (Fig. 6A). Because we proposed that M1-linked ubiquitination of cFLIP could compete Lys-48–linked ubiquitination, we detected M1-ubiquitination and Lys-48–linked ubiquitination of WT and mutated cFLIP in reconstituted Jurkat cells. After TNFα/CHX treatment, the M1-linked ubiquitination of cFLIP was significantly inhibited in cFLIP-K351R/K353R cells, but Lys-48–linked ubiquitination was significantly increased (Fig. 6B). Together, these results indicate that M1-linked ubiquitination of cFLIP on Lys-351 and Lys-353 competes with Lys-48-linked ubiquitination, which contributes to protect cells from TNFα-induced apoptosis (Fig. 7).Figure 7Proposed model of LUBAC-mediated ubiquitination on cFLIP in the TNFα receptor signaling pathway. In normal conditions, linear ubiquitination of cFLIP induced by LUBAC inhibits Lys-48–linked ubiquitination of cFLIP, thereby suppressing caspase 8 activity and apoptosis process. When LUBAC is deficient, cFLIP cannot be ubiquitinated by LUBAC. Instead, cFLIP degrades through Lys-48–linked ubiquitination in a dependent manner, thereby promoting apoptosis process.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In summary, we found that cFLIP was a new substrate of M1-linked ubiquitination and identified the target sites of this modification, reve" @default.
• W2898593788 created "2018-11-02" @default.
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• W2898593788 date "2018-12-01" @default.
• W2898593788 modified "2023-10-17" @default.
• W2898593788 title "Linear ubiquitination of cFLIP induced by LUBAC contributes to TNFα-induced apoptosis" @default.
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• W2898593788 doi "https://doi.org/10.1074/jbc.ra118.005449" @default.
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