The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.

Bill Lyons Informatics Centre UCL Cancer Institute University College London London UK

Centre for Cell Death Cancer and Inflammation UCL Cancer Institute University College London London UK

Division of Nephrology Department of Internal Medicine 3 University Hospital Carl Gustav Carus at the Technical University Dresden Dresden Germany

Laboratory of Adaptive Immunity Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Proteomics Research Core Facility UCL Cancer Institute University College London London UK

Komentář v

Nat Cell Biol. 2018 Dec;20(12):1330-1331. doi: 10.1038/s41556-018-0239-4. PubMed

Zobrazit více v PubMed

Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell. 2003;114:181–190. PubMed

Kalliolias GD, Ivashkiv LB. TNF biology, pathogenic mechanisms and emerging therapeutic strategies. Nature reviews Rheumatology. 2016;12:49–62. PubMed PMC

Brenner D, Blaser H, Mak TW. Regulation of tumour necrosis factor signalling: live or let die. Nature reviews Immunology. 2015;15:362–374. PubMed

Hrdinka M, Gyrd-Hansen M. The Met1-Linked Ubiquitin Machinery: Emerging Themes of (De)regulation. Mol Cell. 2017;68:265–280. PubMed

Shimizu Y, Taraborrelli L, Walczak H. Linear ubiquitination in immunity. Immunological reviews. 2015;266:190–207. PubMed PMC

Kirisako T, et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 2006;25:4877–4887. PubMed PMC

Ikeda F, et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-kappaB activity and apoptosis. Nature. 2011;471:637–641. PubMed PMC

Gerlach B, et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature. 2011;471:591–596. PubMed

Tokunaga F, et al. SHARPIN is a component of the NF-kappaB-activating linear ubiquitin chain assembly complex. Nature. 2011;471:633–636. PubMed

Peltzer N, et al. HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death. Cell reports. 2014;9:153–165. PubMed

Emmerich CH, et al. Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains. Proc Natl Acad Sci U S A. 2013;110:15247–15252. PubMed PMC

Peltzer N, et al. LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis. Nature. 2018;557:112–117. PubMed PMC

Haas TL, 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;36:831–844. PubMed

Draber P, et al. LUBAC-Recruited CYLD and A20 Regulate Gene Activation and Cell Death by Exerting Opposing Effects on Linear Ubiquitin in Signaling Complexes. Cell reports. 2015;13:2258–2272. PubMed PMC

Kupka S, Reichert M, Draber P, Walczak H. Formation and removal of poly-ubiquitin chains in the regulation of tumor necrosis factor-induced gene activation and cell death. FEBS J. 2016;283:2626–2639. PubMed

Rahighi S, et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation. Cell. 2009;136:1098–1109. PubMed

Dondelinger Y, et al. NF-kappaB-Independent Role of IKKalpha/IKKbeta in Preventing RIPK1 Kinase-Dependent Apoptotic and Necroptotic Cell Death during TNF Signaling. Mol Cell. 2015;60:63–76. PubMed

Menon MB, et al. p38MAPK/MK2-dependent phosphorylation controls cytotoxic RIPK1 signalling in inflammation and infection. Nature cell biology. 2017;19:1248–1259. PubMed

Jaco I, et al. MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death. Mol Cell. 2017 PubMed PMC

Dondelinger Y, et al. MK2 phosphorylation of RIPK1 regulates TNF-mediated cell death. Nature cell biology. 2017;19:1237–1247. PubMed

Kotlyarov A, et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nature cell biology. 1999;1:94–97. PubMed

Kanayama A, et al. TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell. 2004;15:535–548. PubMed

Wang C, et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature. 2001;412:346–351. PubMed

Adhikari A, Xu M, Chen ZJ. Ubiquitin-mediated activation of TAK1 and IKK. Oncogene. 2007;26:3214–3226. PubMed

Wu CJ, Conze DB, Li T, Srinivasula SM, Ashwell JD. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected] Nature cell biology. 2006;8:398–406. PubMed

Salmeron A, et al. Direct phosphorylation of NF-kappaB1 p105 by the IkappaB kinase complex on serine 927 is essential for signal-induced p105 proteolysis. The Journal of biological chemistry. 2001;276:22215–22222. PubMed

Tojima Y, et al. NAK is an IkappaB kinase-activating kinase. Nature. 2000;404:778–782. PubMed

Peters RT, Liao SM, Maniatis T. IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex. Mol Cell. 2000;5:513–522. PubMed

Helgason E, Phung QT, Dueber EC. Recent insights into the complexity of Tank-binding kinase 1 signaling networks: the emerging role of cellular localization in the activation and substrate specificity of TBK1. FEBS Lett. 2013;587:1230–1237. PubMed

Hemmi H, et al. The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection. The Journal of experimental medicine. 2004;199:1641–1650. PubMed PMC

Perry AK, Chow EK, Goodnough JB, Yeh WC, Cheng G. Differential requirement for TANK-binding kinase-1 in type I interferon responses to toll-like receptor activation and viral infection. The Journal of experimental medicine. 2004;199:1651–1658. PubMed PMC

Kupka S, et al. SPATA2-Mediated Binding of CYLD to HOIP Enables CYLD Recruitment to Signaling Complexes. Cell reports. 2016;16:2271–2280. PubMed PMC

Komander D, et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO reports. 2009;10:466–473. PubMed PMC

Laplantine E, et al. NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain. EMBO J. 2009;28:2885–2895. PubMed PMC

Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ. Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell. 2006;22:245–257. PubMed

Hadian K, et al. NF-kappaB essential modulator (NEMO) interaction with linear and lys-63 ubiquitin chains contributes to NF-kappaB activation. The Journal of biological chemistry. 2011;286:26107–26117. PubMed PMC

Chau TL, et al. Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated? Trends in biochemical sciences. 2008;33:171–180. PubMed

Kishore N, et al. IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2: comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2. The Journal of biological chemistry. 2002;277:13840–13847. PubMed

Lafont E, et al. The linear ubiquitin chain assembly complex regulates TRAIL-induced gene activation and cell death. Embo j. 2017 PubMed PMC

Fitzgerald KA, et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nature immunology. 2003;4:491–496. PubMed

Sharma S, et al. Triggering the interferon antiviral response through an IKK-related pathway. Science (New York, N.Y.) 2003;300:1148–1151. PubMed

Clement JF, Meloche S, Servant MJ. The IKK-related kinases: from innate immunity to oncogenesis. Cell research. 2008;18:889–899. PubMed

Clark K, et al. Novel cross-talk within the IKK family controls innate immunity. The Biochemical journal. 2011;434:93–104. PubMed

Schmidt-Supprian M, et al. NEMO/IKK gamma-deficient mice model incontinentia pigmenti. Mol Cell. 2000;5:981–992. PubMed

Clark K, Plater L, Peggie M, Cohen P. Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation. The Journal of biological chemistry. 2009;284:14136–14146. PubMed PMC

Feng S, et al. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cellular signalling. 2007;19:2056–2067. PubMed

Lin Y, Devin A, Rodriguez Y, Liu ZG. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes & development. 1999;13:2514–2526. PubMed PMC

O'Donnell MA, et al. Caspase 8 inhibits programmed necrosis by processing CYLD. Nature cell biology. 2011;13:1437–1442. PubMed PMC

Duprez L, et al. RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome. Immunity. 2011;35:908–918. PubMed

Polykratis A, et al. Cutting edge: RIPK1 Kinase inactive mice are viable and protected from TNF-induced necroptosis in vivo. Journal of immunology (Baltimore, Md. : 1950) 2014;193:1539–1543. PubMed PMC

Pomerantz JL, Baltimore D. NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase. Embo j. 1999;18:6694–6704. PubMed PMC

Wagner SA, 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;35:1868–1884. PubMed PMC

Chariot A, et al. Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases. The Journal of biological chemistry. 2002;277:37029–37036. PubMed

Dynek JN, et al. c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. Embo j. 2010;29:4198–4209. PubMed PMC

Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8 activation pathways. Cell. 2008;133:693–703. PubMed

Peltzer N, Darding M, Walczak H. Holding RIPK1 on the Ubiquitin Leash in TNFR1 Signaling. Trends Cell Biol. 2016;26:445–461. PubMed

Annibaldi A, Meier P. Checkpoints in TNF-Induced Cell Death: Implications in Inflammation and Cancer. Trends in molecular medicine. 2017 PubMed

Ting AT, Bertrand MJ. More to Life than NF-kappaB in TNFR1 Signaling. Trends in immunology. 2016;37:535–545. PubMed PMC

Vanden Berghe T, Kalai M, van Loo G, Declercq W, Vandenabeele P. Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis. The Journal of biological chemistry. 2003;278:5622–5629. PubMed

Xu D, et al. TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during Development and in Aging. Cell. 2018;174:1477–1491.e1419. PubMed PMC

Rudolph D, et al. Severe liver degeneration and lack of NF-kappaB activation in NEMO/IKKgamma-deficient mice. Genes & development. 2000;14:854–862. PubMed PMC

Legarda-Addison D, Hase H, O'Donnell MA, Ting AT. NEMO/IKKgamma regulates an early NF-kappaB-independent cell-death checkpoint during TNF signaling. Cell death and differentiation. 2009;16:1279–1288. PubMed PMC

Maubach G, Schmadicke AC, Naumann M. NEMO Links Nuclear Factor-kappaB to Human Diseases. Trends in molecular medicine. 2017;23:1138–1155. PubMed

Udalova I, Monaco C, Nanchahal J, Feldmann M. Anti-TNF Therapy. Microbiology spectrum. 2016;4 PubMed

Zhao W. Negative regulation of TBK1-mediated antiviral immunity. FEBS Lett. 2013;587:542–548. PubMed PMC

Thurston TL, Ryzhakov G, Bloor S, von Muhlinen N, Randow F. The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nature immunology. 2009;10:1215–1221. PubMed

Pilli M, et al. TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity. 2012;37:223–234. PubMed PMC

Ahmad L, Zhang SY, Casanova JL, Sancho-Shimizu V. Human TBK1: A Gatekeeper of Neuroinflammation. Trends in molecular medicine. 2016;22:511–527. PubMed PMC

Verhelst K, Verstrepen L, Carpentier I, Beyaert R. IkappaB kinase epsilon (IKKepsilon): a therapeutic target in inflammation and cancer. Biochemical pharmacology. 2013;85:873–880. PubMed PMC

Vizcaino JA, et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 2016;44:11033. PubMed PMC

TBK1-associated adapters TANK and AZI2 protect mice against TNF-induced cell death and severe autoinflammatory diseases

ABIN1 is a negative regulator of effector functions in cytotoxic T cells

Systematic analysis of the IL-17 receptor signalosome reveals a robust regulatory feedback loop