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• W2613123103 abstract "•Phosphorylation of RIPK1 by MK2 acts as survival checkpoint in TNF signaling•TNF-induced activation of MK2 results in global RIPK1 phosphorylation•MK2-mediated phosphorylation suppresses RIPK1 kinase activation and cell death•Complex-II originates from RIPK1 in complex-I as well as cytosolic RIPK1 TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production. TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production. Tumor necrosis factor (TNF) is a major inflammatory cytokine that was first identified for its ability to induce rapid hemorrhagic necrosis of cancers (Balkwill, 2009Balkwill F. Tumour necrosis factor and cancer.Nat. Rev. Cancer. 2009; 9: 361-371Crossref PubMed Scopus (1287) Google Scholar). In response to insults and infection, TNF contributes to homeostasis by regulating inflammation, cell proliferation, differentiation, survival, and death (Walczak, 2011Walczak H. TNF and ubiquitin at the crossroads of gene activation, cell death, inflammation, and cancer.Immunol. Rev. 2011; 244: 9-28Crossref PubMed Scopus (183) Google Scholar). However, excessive or chronic engagement of TNFR1 can result in inflammatory diseases. Originally, it was considered that TNF contributes to such diseases by directly inducing the expression and production of inflammatory cytokines. However, recent evidence suggests that aberrant TNF-induced cell death may also contribute to the disease pathology (Gerlach et al., 2011Gerlach 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. et al.Linear ubiquitination prevents inflammation and regulates immune signalling.Nature. 2011; 471: 591-596Crossref PubMed Scopus (683) Google Scholar, Pasparakis and Vandenabeele, 2015Pasparakis M. Vandenabeele P. Necroptosis and its role in inflammation.Nature. 2015; 517: 311-320Crossref PubMed Scopus (1210) Google Scholar, Silke et al., 2015Silke J. Rickard J.A. Gerlic M. The diverse role of RIP kinases in necroptosis and inflammation.Nat. Immunol. 2015; 16: 689-697Crossref PubMed Scopus (342) Google Scholar). There are a number of different mechanisms to regulate TNF-induced cell death, including the formation of two distinct signaling complexes (Micheau and Tschopp, 2003Micheau O. Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1991) Google Scholar). Within minutes of stimulation, TNFR1 assembles complex-I by recruiting the adaptors TRADD and TRAF2, the kinase RIPK1, and the E3 ubiquitin (Ub) ligases cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2 (Silke, 2011Silke J. The regulation of TNF signalling: what a tangled web we weave.Curr. Opin. Immunol. 2011; 23: 620-626Crossref PubMed Scopus (89) Google Scholar, Ting and Bertrand, 2016Ting A.T. Bertrand M.J. More to Life than NF-κB in TNFR1 Signaling.Trends Immunol. 2016; 37: 535-545Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). cIAPs subsequently conjugate various types of Ub linkages to components of this complex, which in turn allows Ub-dependent recruitment of the kinase complex TAK1/TAB2/TAB3 and the E3 ligase linear Ub chain assembly complex (LUBAC, composed of HOIL-1/HOIP/Sharpin). LUBAC-mediated linear ubiquitylation of different components of this complex appears to stabilize or reinforce complex-I formation and promote TAK1-dependent activation of IKK2. Formation of complex-I causes activation of NF-κB and mitogen-activated protein kinases (MAPKs), which ultimately results in the production of cytokines and pro-survival proteins, such as cFLIP, that are necessary for a coordinated inflammatory response (Elliott et al., 2016Elliott P.R. Leske D. Hrdinka M. Bagola K. Fiil B.K. McLaughlin S.H. Wagstaff J. Volkmar N. Christianson J.C. Kessler B.M. et al.SPATA2 links CYLD to LUBAC, activates CYLD, and controls LUBAC signaling.Mol. Cell. 2016; 63: 990-1005Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, Hrdinka et al., 2016Hrdinka M. Fiil B.K. Zucca M. Leske D. Bagola K. Yabal M. Elliott P.R. Damgaard R.B. Komander D. Jost P.J. Gyrd-Hansen M. CYLD limits Lys63- and Met1-linked ubiquitin at receptor complexes to regulate innate immune signaling.Cell Rep. 2016; 14: 2846-2858Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Kupka et al., 2016Kupka S. De Miguel D. Draber P. Martino L. Surinova S. Rittinger K. Walczak H. SPATA2-mediated binding of CYLD to HOIP enables CYLD recruitment to signaling complexes.Cell Rep. 2016; 16: 2271-2280Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, Schlicher et al., 2016Schlicher L. Wissler M. Preiss F. Brauns-Schubert P. Jakob C. Dumit V. Borner C. Dengjel J. Maurer U. SPATA2 promotes CYLD activity and regulates TNF-induced NF-κB signaling and cell death.EMBO Rep. 2016; 17: 1485-1497Crossref PubMed Scopus (85) Google Scholar, Silke, 2011Silke J. The regulation of TNF signalling: what a tangled web we weave.Curr. Opin. Immunol. 2011; 23: 620-626Crossref PubMed Scopus (89) Google Scholar, Wagner et al., 2016Wagner S.A. Satpathy S. Beli P. Choudhary C. SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes.EMBO J. 2016; 35: 1868-1884Crossref PubMed Scopus (104) Google Scholar). TNF also initiates formation of an RIPK1-based cytoplasmic complex that chronologically appears after complex-I, and which can induce cell death. Therefore, this complex is frequently referred to as complex-II or the necrosome (Pasparakis and Vandenabeele, 2015Pasparakis M. Vandenabeele P. Necroptosis and its role in inflammation.Nature. 2015; 517: 311-320Crossref PubMed Scopus (1210) Google Scholar, Wang et al., 2008Wang L. Du F. Wang X. TNF-alpha induces two distinct caspase-8 activation pathways.Cell. 2008; 133: 693-703Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). Complex-II can kill by activating caspase-8 and apoptosis, or via RIPK3 and MLKL, which results in necroptosis. It is currently believed that a small fraction of RIPK1 dissociates from complex-I within 30 min to 3 hr, and together with TRADD, associates with the adaptor protein FADD and procaspase-8 to form complex-II (Micheau and Tschopp, 2003Micheau O. Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1991) Google Scholar). Whether TNF can induce lethal levels of complex-II is dependent on multiple checkpoints: cIAP- and LUBAC-mediated ubiquitylation of RIPK1 are decisive factors in limiting complex-II formation (Bertrand et al., 2008Bertrand 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: 689-700Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar, Gerlach et al., 2011Gerlach 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. et al.Linear ubiquitination prevents inflammation and regulates immune signalling.Nature. 2011; 471: 591-596Crossref PubMed Scopus (683) Google Scholar, Haas et al., 2009Haas T.L. Emmerich C.H. Gerlach B. Schmukle A.C. Cordier S.M. Rieser E. Feltham R. Vince J. Warnken U. Wenger T. et al.Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction.Mol. Cell. 2009; 36: 831-844Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). In the absence of either cIAPs or LUBAC, TNF fails to activate canonical NF-κB effectively, and consequently, cFLIP levels are insufficient to prevent caspase-8-mediated cell death. Under normal conditions, cFLIPL suppresses TNF-induced cell death by heterodimerizing with caspase-8. This inhibits formation of complex-II and the necrosome by cleaving RIPK1, RIPK3, and CYLD (Feng et al., 2007Feng S. Yang Y. Mei Y. Ma L. Zhu D.E. Hoti N. Castanares M. Wu M. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain.Cell. Signal. 2007; 19: 2056-2067Crossref PubMed Scopus (341) Google Scholar, Lin et al., 1999Lin Y. Devin A. Rodriguez Y. Liu Z.G. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis.Genes Dev. 1999; 13: 2514-2526Crossref PubMed Scopus (679) Google Scholar, O’Donnell et al., 2011O’Donnell M.A. Perez-Jimenez E. Oberst A. Ng A. Massoumi R. Xavier R. Green D.R. Ting A.T. Caspase 8 inhibits programmed necrosis by processing CYLD.Nat. Cell Biol. 2011; 13: 1437-1442Crossref PubMed Scopus (351) Google Scholar, Oberst et al., 2011Oberst A. Dillon C.P. Weinlich R. McCormick L.L. Fitzgerald P. Pop C. Hakem R. Salvesen G.S. Green D.R. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.Nature. 2011; 471: 363-367Crossref PubMed Scopus (909) Google Scholar). TAK1 and IKK2 also inhibit TNF-induced cell death. This has mainly been considered to be via induction of NF-κB and cFLIP; however, recent evidence suggests that they also regulate TNF killing independently of their role in NF-κB activation (Dondelinger et al., 2015Dondelinger Y. Jouan-Lanhouet S. Divert T. Theatre E. Bertin J. Gough P.J. Giansanti P. Heck A.J. Dejardin E. Vandenabeele P. Bertrand M.J. NF-κB-independent role of IKKα/IKKβ in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, Kondylis et al., 2015Kondylis V. Polykratis A. Ehlken H. Ochoa-Callejero L. Straub B.K. Krishna-Subramanian S. Van T.M. Curth H.M. Heise N. Weih F. et al.NEMO prevents steatohepatitis and hepatocellular carcinoma by inhibiting RIPK1 kinase activity-mediated hepatocyte apoptosis.Cancer Cell. 2015; 28: 582-598Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, O’Donnell et al., 2007O’Donnell M.A. Legarda-Addison D. Skountzos P. Yeh W.C. Ting A.T. Ubiquitination of RIP1 regulates an NF-kappaB-independent cell-death switch in TNF signaling.Curr. Biol. 2007; 17: 418-424Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, Vlantis et al., 2016Vlantis K. Wullaert A. Polykratis A. Kondylis V. Dannappel M. Schwarzer R. Welz P. Corona T. Walczak H. Weih F. et al.NEMO prevents RIP kinase 1-mediated epithelial cell death and chronic intestinal inflammation by NF-κB-dependent and -independent functions.Immunity. 2016; 44: 553-567Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In the absence of functional TAK1 or IKK, lethal levels of complex-II assemble despite RIPK1 ubiquitylation in complex-I (Dondelinger et al., 2013Dondelinger Y. Aguileta M.A. Goossens V. Dubuisson C. Grootjans S. Dejardin E. Vandenabeele P. Bertrand M.J. RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition.Cell Death Differ. 2013; 20: 1381-1392Crossref PubMed Scopus (216) Google Scholar, Dondelinger et al., 2015Dondelinger Y. Jouan-Lanhouet S. Divert T. Theatre E. Bertin J. Gough P.J. Giansanti P. Heck A.J. Dejardin E. Vandenabeele P. Bertrand M.J. NF-κB-independent role of IKKα/IKKβ in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, Legarda-Addison et al., 2009Legarda-Addison D. Hase H. O’Donnell M.A. Ting A.T. NEMO/IKKgamma regulates an early NF-kappaB-independent cell-death checkpoint during TNF signaling.Cell Death Differ. 2009; 16: 1279-1288Crossref PubMed Scopus (90) Google Scholar). Under these conditions, TNF-mediated, RIPK1-dependent apoptosis was shown to rely on the kinase activity of RIPK1 (Dondelinger et al., 2013Dondelinger Y. Aguileta M.A. Goossens V. Dubuisson C. Grootjans S. Dejardin E. Vandenabeele P. Bertrand M.J. RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition.Cell Death Differ. 2013; 20: 1381-1392Crossref PubMed Scopus (216) Google Scholar, Wang et al., 2008Wang L. Du F. Wang X. TNF-alpha induces two distinct caspase-8 activation pathways.Cell. 2008; 133: 693-703Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). It is unclear, however, whether TAK1 inhibits RIPK1 kinase activity directly, or indirectly via downstream kinases such as IKK2 (Dondelinger et al., 2015Dondelinger Y. Jouan-Lanhouet S. Divert T. Theatre E. Bertin J. Gough P.J. Giansanti P. Heck A.J. Dejardin E. Vandenabeele P. Bertrand M.J. NF-κB-independent role of IKKα/IKKβ in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). MAPK14 (p38α) and its substrate MAPKAPK2 (MK2) play essential roles in TNF-induced inflammatory cytokine production. Consequently, several pharmaceutical compounds have been developed to target these kinases in auto-inflammatory diseases (Genovese, 2009Genovese M.C. Inhibition of p38: has the fat lady sung?.Arthritis Rheum. 2009; 60: 317-320Crossref PubMed Scopus (160) Google Scholar). However, recently we proposed that the p38-MK2 axis also regulates TNF- and RIPK1-dependent SMAC-mimetic (SM)-induced cell death (Lalaoui et al., 2016Lalaoui N. Hänggi K. Brumatti G. Chau D. Nguyen N.Y. Vasilikos L. Spilgies L.M. Heckmann D.A. Ma C. Ghisi M. et al.Targeting p38 or MK2 enhances the anti-leukemic activity of Smac-mimetics.Cancer Cell. 2016; 29: 145-158Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These results therefore suggest that TAK1 mediates its pro-survival effect, at least in part, through activation of p38-MK2 (Sakurai, 2012Sakurai H. Targeting of TAK1 in inflammatory disorders and cancer.Trends Pharmacol. Sci. 2012; 33: 522-530Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). While it is now thought that many chronic inflammatory diseases are caused or exacerbated by aberrant cytokine-induced cell death, the molecular events that regulate this process are largely unknown. In this study, we demonstrate that RIPK1 is a bona fide substrate of MK2 in both human and mouse. We find that TNF-induced activation of MK2 selectively protects cells from RIPK1 kinase-dependent death. While MK2-mediated phosphorylation of RIPK1 at S321 (mouse) and S320 (human) has no effect on NF-κB activation, it selectively inhibits RIPK1 kinase-mediated formation of complex-II, induction of apoptosis, and necroptosis. Whereas loss of S321 phosphorylation sensitizes cells to TNF killing, introduction of an S321 to D phospho-mimetic knockin mutation partly protects from RIPK1-dependent cell death upon TNF stimulation. We find that MK2-mediated phosphorylation of RIPK1 at S321/S320 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Our data demonstrate that the TAK1 > p38 > MK2 kinase cascade directly limits the lethal potential of cytosolic and complex-I-associated RIPK1, thereby licensing TNF-induced transcription, mRNA stabilization, and increased translation of cytokines necessary for a coordinated inflammatory response. We have shown that inhibition of p38α, or its downstream kinase MK2, enhances the killing activity of the SM birinapant (Lalaoui et al., 2016Lalaoui N. Hänggi K. Brumatti G. Chau D. Nguyen N.Y. Vasilikos L. Spilgies L.M. Heckmann D.A. Ma C. Ghisi M. et al.Targeting p38 or MK2 enhances the anti-leukemic activity of Smac-mimetics.Cancer Cell. 2016; 29: 145-158Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Because SM kills cells by increasing the production of SM-induced TNF biosynthesis, and sensitizing cells to TNF-induced and RIPK1-mediated cell death, p38/MK2 might influence the sensitivity to TNF by influencing either or both of these processes. To distinguish between these scenarios, we treated bone marrow-derived macrophages (BMDMs) with SM and increasing concentrations of exogenous TNF and found that inhibition of MK2 sensitized BMDMs to TNF/SM (TS)-induced cell death in a dose-dependent manner, already 3 hr after treatment (Figure 1A). This suggests that inhibition of MK2 can sensitize cells to SM-induced killing independently of its role in inducing TNF biosynthesis (Gaestel, 2016Gaestel M. MAPK-activated protein kinases (MKs): novel insights and challenges.Front. Cell Dev. Biol. 2016; 3: 88Crossref PubMed Scopus (63) Google Scholar). To explore this further, we used primary mouse embryonic fibroblasts (MEFs) that do not produce autocrine TNF in response to SM (Vince et al., 2007Vince 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. et al.IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis.Cell. 2007; 131: 682-693Abstract Full Text Full Text PDF PubMed Scopus (888) Google Scholar) and hence are resistant to SM, caspase activation, and cell death (Figures 1B and 1C). Inhibition of MK2 sensitized primary MEFs to TS-induced caspase activation and cell death (Figures 1B, 1C, and S1A), and co-treatment with the RIPK1 kinase inhibitor GSK’963 (RIPK1i) reversed this sensitization (Figure 1C). Inhibition of MK2 also sensitized MEFs and human HT29 cells to RIPK1-dependent, TNF-induced necroptosis (Figures 1D and 1E). Consistent with the notion that the kinase activity of RIPK1 is required for TNF-induced cell death under these conditions, we found that primary MEFs and murine leukemic MLL-ENLs that express kinase dead RIPK1 were largely protected from TSM (TNF, SM, and MK2i)-induced death (Figures 1F and S1B). To exclude a potential off-target effect of the MK2i PF-3644022, we generated murine leukemic MLLENL Mk2−/− and found that the absence of MK2 highly sensitized those cells to TS-induced cell death (Figure S1C). MK2i also sensitized human breast cancer BT549 and MDA-MB-468 cells to TS (Figures 1G, 1H, S1D, and S1E), implying that MK2 inhibition sensitizes to TNF-induced cell death in general. Recent quantitative mass spectrometry analyses have identified TNF-induced phosphorylation of S320 of human RIPK1 (Degterev et al., 2008Degterev A. Hitomi J. Germscheid M. Ch’en I.L. Korkina O. Teng X. Abbott D. Cuny G.D. Yuan C. Wagner G. et al.Identification of RIP1 kinase as a specific cellular target of necrostatins.Nat. Chem. Biol. 2008; 4: 313-321Crossref PubMed Scopus (1468) Google Scholar, Krishnan et al., 2015Krishnan R.K. Nolte H. Sun T. Kaur H. Sreenivasan K. Looso M. Offermanns S. Krüger M. Swiercz J.M. Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate.Nat. Commun. 2015; 6: 6658Crossref PubMed Scopus (46) Google Scholar). Intriguingly, the motif surrounding S320 of human RIPK1 is evolutionarily conserved and conforms to the phosphorylation consensus motif of MK2, which is defined as Φ-X-R-X-(L/N)-pS/T-(I/V/F/L)-X, where Φ is a bulky hydrophobic residue (Figure 2A) (Cargnello and Roux, 2011Cargnello M. Roux P.P. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases.Microbiol. Mol. Biol. Rev. 2011; 75: 50-83Crossref PubMed Scopus (1860) Google Scholar). We therefore hypothesized that MK2 phosphorylates the serine within this conserved motif, and raised phospho-specific antibodies against P-S320 of human and P-S321 of mouse RIPK1 (Figure S2). Consistent with the notion that RIPK1 is phosphorylated at this motif by MK2, we found that TNF treatment of primary MEFs resulted in transient phosphorylation of RIPK1 at S321, which was blocked by pharmacological inhibition or genetic deletion of MK2 (Figures 2B and 2C). We found that phosphorylated RIPK1 migrates differently depending on the gel type used and was readily distinguishable from the un-phosphorylated form when lysates were separated on an 8% gel (Figure 2C). Similarly, TNF treatment induced RIPK1 phosphorylation of S321 in primary BMDMs in an MK2-dependent manner (Figure 2D). Likewise, human RIPK1 was phosphorylated at S320 in MDA-MB-468 cells (Figure 2E). MK2 is activated by p38α in response to many stimuli, including cytokines and bacterial infection (Cargnello and Roux, 2011Cargnello M. Roux P.P. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases.Microbiol. Mol. Biol. Rev. 2011; 75: 50-83Crossref PubMed Scopus (1860) Google Scholar). Consistent with the idea that RIPK1 is phosphorylated by MK2, stimuli that activated MK2, as measured by the appearance of phospho-MK2 (P-T222), also lead to phosphorylation of RIPK1 S321 (Figure 2F). LPS- and PGN-induced phosphorylation of S321 was longer lasting than the one triggered by TNF. To determine whether MK2 directly phosphorylated RIPK1, we conducted an in vitro kinase assay using recombinant MK2 and purified RIPK1. MK2 readily phosphorylated mouse and human RIPK1 on S320 and S321, respectively (Figure 2G). To dissect the signaling cascade that results in RIPK1 phosphorylation at S320/S321, we made use of pharmacologic inhibition and genetic mutation of components of the TNF receptor signaling complex. Phosphorylation of RIPK1 at S320/321 was dependent on the TAK1-p38α-MK2 kinase cascade because inhibition of either TAK1 or p38α, which block TNF-induced MK2 phosphorylation and activation (Figures 3A–3C), or inhibition of MK2 itself, abolished the appearance of P-S321 in primary MEFs and BMDMs, and of P-S320 in human breast cancer MDA-MB-468 cells (Figures 3A–3C). While pharmacological inhibition of IKK2 with TPCA-1 or BI605906 strongly inhibited IκBα degradation, as expected (Figures S3A and S3B), it did not prevent S320/321 phosphorylation in any of the three cell types tested (Figures 3A–3C, S3A, and S3B). Likewise, genetic deletion of NEMO, IKK1, or IKK2 did not interfere with TNF-induced phosphorylation of S320/S321 in mouse and human cells (Figures 3D and 3E). The IKK complex, therefore, does not appear to be involved in mediating phosphorylation of RIPK1 at these residues. Furthermore, treatment with an RIPK1 inhibitor did not interfere with S320/321 phosphorylation following TNF stimulation (Figures 3A–3C), implying that P-S320/321 is not an auto-phosphorylation event. Binding of TNF to TNFR1 results in activation of NF-κB and MAPKs, leading to transcriptional induction of pro-inflammatory cytokines as well as pro-survival genes such as cFLIP and cIAPs. Since defects in NF-κB are known to sensitize cells to TNF-induced cell death (Peltzer et al., 2016Peltzer N. Darding M. Walczak H. Holding RIPK1 on the ubiquitin leash in TNFR1 signaling.Trends Cell Biol. 2016; 26: 445-461Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), we examined whether inhibition of MK2 affected TNF-induced activation of NF-κB and MAPK. However, inhibition or deletion of MK2 had no effect on TNF-induced degradation of IκBα or phosphorylation of p65, JNK, or ERK in MEFs and BMDMs (Figures 4A and 4B ). Moreover, we found no evidence for defective ubiquitylation of RIPK1 in complex-I (Figure 4C), and UbiCRest (Ub chain restriction) analysis (Hospenthal et al., 2015Hospenthal M.K. Mevissen T.E. Komander D. Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest).Nat. Protoc. 2015; 10: 349-361Crossref PubMed Scopus (133) Google Scholar) of ubiquitylated RIPK1 in complex-I revealed no qualitative differences in Ub linkage types in the presence or absence of MK2i (Figure S4A). Intriguingly, only the non-ubiquitylated form of RIPK1 in complex-I was phosphorylated at S321 (Figure 4C). In contrast, phosphorylation at S166 of RIPK1 in complex-I readily occurs on ubiquitylated RIPK1 (Newton et al., 2016Newton K. Wickliffe K.E. Maltzman A. Dugger D.L. Strasser A. Pham V.C. Lill J.R. Roose-Girma M. Warming S. Solon M. et al.RIPK1 inhibits ZBP1-driven necroptosis during development.Nature. 2016; 540: 129-133Crossref PubMed Scopus (222) Google Scholar). Further, we found that RIPK1 was significantly more phosphorylated on S166 in Mk2−/− cells or in cells treated with MK2 inhibitors in response to TNF (Figures 4D and 4E), although the timing of S166 phosphorylation was unaffected by MK2 inhibition (Figures 4E and S4B). We found that P-S166 appeared after P-S321. Of note, the kinetics of P-S321 did not appear to change with SM, which prevents ubiquitylation of RIPK1, or SM+zVAD, which in addition inhibits caspases (Figure S4B). Together, these results suggest that MK2-mediated RIPK1 S321 phosphorylation occurs in an IAP- and Ub modification-independent manner. While P-S321 RIPK1 in complex-I is not ubiquitylated, this phosphorylation does not prevent normal levels of ubiquitylated RIPK1 from being generated in this complex. Further, our data support the notion that P-S321 suppresses RIPK1 S166 auto-phosphorylation. Remarkably, P-S321 RIPK1 was present in both complex-I and the complex-I immuno-depleted fraction (lysates post-FLAG immunoprecipitation) after only 5 min of TNF stimulation (Figure 4C), suggesting that cytosolic RIPK1 is phosphorylated by MK2. To conclusively test whether recruitment of RIPK1 to complex-I was dispensable for S321 phosphorylation, we reconstituted wild-type (WT) and Ripk1−/− MEFs with an RIPK1 mutant that lacks the death domain (ΔDD). This mutant is not recruited to complex-I and, therefore, cannot become ubiquitylated (Figures S4C–S4E). Even though RIPK1-ΔDD was not recruited to complex-I, it was readily phosphorylated at S321 (Figure 4F). Together, these data demonstrate that TNF activates MK2, which in turn rapidly phosphorylates non-ubiquitylated RIPK1 in complex-I and the cytosol. Thus far, our data suggest that MK2 inhibition neither affects TNF-induced recruitment of RIPK1 into complex-I nor limits activation of NF-κB/MAPK pathways, yet increases phosphorylation of RIPK1 on S166 and sensitizes cells to TNF-induced death. This, therefore, suggests a role for MK2 in regulating RIPK1 and complex-II formation. Consistent with this, we found that loss of MK2 dramatically enhanced TNF-induced association of RIPK1, FADD, and active caspase-8 (Figure 5A). Pharmacological inhibition of MK2 similarly increased complex-II formation and activation in response to TS (Figures 5B and S5A). These data suggested that more RIPK1 was available for recruitment into complex-II and prompted us to monitor the levels of ubiquitylated RIPK1 in the presence and absence of active MK2 post-TNF stimulation. Using tandem Ub binding entities (TUBEs) (Hjerpe et al., 2009Hjerpe R. Aillet F. Lopitz-Otsoa F. Lang V. England P. Rodriguez M.S. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities.EMBO Rep. 2009; 10: 1250-1258Crossref PubMed Scopus (342) Google Scholar), which allow isolation of polyubiquitylated proteins, we purified all ubiquitylated proteins over a TNF time course and probed with an anti-RIPK1 antibody. Using the non-specific deubiquitylating enzyme (DUB), USP21, to confirm ubiquitylation, we found that in WT cells, the levels of ubiquitylated RIPK1 increased within 15 min of TNF stimulation, and then steadily decreased over 3 hr of TNF treatment (Figure 5C). Upon MK2 inhibition, the levels of ubiquitylated RIPK1 were more prominent at the earliest times following TNF stimulation. TNF-induced accumulation of RIPK1 in the ubiquitylated fraction correlated with a significant increase in formation of complex-II an" @default.
• W2613123103 created "2017-05-19" @default.
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• W2613123103 date "2017-06-01" @default.
• W2613123103 modified "2023-10-14" @default.
• W2613123103 title "MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death" @default.
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• W2613123103 doi "https://doi.org/10.1016/j.molcel.2017.05.003" @default.
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