Abstract
Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.
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References
Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–57.
Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 2012;19:107–20.
Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH, et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 1995;14:5579–88.
Thome M, Schneider P, Hofmann K, Fickenscher H, Meinl E, Neipel F, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature. 1997;386:517–21.
Algeciras-Schimnich A, Griffith TS, Lynch DH, Paya CV. Cell cycle-dependent regulation of FLIP levels and susceptibility to Fas-mediated apoptosis. J Immunol. 1999;162:5205–11.
Mert U, Sanlioglu AD. Intracellular localization of DR5 and related regulatory pathways as a mechanism of resistance to TRAIL in cancer. Cell Mol Life Sci. 2016;74:245–55. https://doi.org/10.1007/s00018-016-2321-z
Wagner KW, Punnoose EA, Januario T, Lawrence DA, Pitti RM, Lancaster K, et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med. 2007;13:1070–7.
McDonald ER 3rd, El-Deiry WS. Suppression of caspase-8- and -10-associated RING proteins results in sensitization to death ligands and inhibition of tumor cell growth. Proc Natl Acad Sci USA. 2004;101:6170–5.
Cursi S, Rufini A, Stagni V, Condo I, Matafora V, Bachi A, et al. Src kinase phosphorylates Caspase-8 on Tyr380: A novel mechanism of apoptosis suppression. EMBO J. 2006;25:1895–905.
Scaffidi C, Volkland J, Blomberg I, Hoffmann I, Krammer PH, Peter ME. Phosphorylation of FADD/ MORT1 at serine 194 and association with a 70-kDa cell cycle-regulated protein kinase. J Immunol. 2000;164:1236–42.
Rochat-Steiner V, Becker K, Micheau O, Schneider P, Burns K, Tschopp J. FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation. J Exp Med. 2000;192:1165–74.
Alappat EC, Feig C, Boyerinas B, Volkland J, Samuels M, Murmann AE, et al. Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities. Mol Cell. 2005;19:321–32.
Lee EW, Kim JH, Ahn YH, Seo J, Ko A, Jeong M, et al. Ubiquitination and degradation of the FADD adaptor protein regulate death receptor-mediated apoptosis and necroptosis. Nat Commun. 2012;3:978.
Alappat EC, Volkland J, Peter ME. Cell cycle effects by C-FADD depend on its C-terminal phosphorylation site. J Biol Chem. 2003;278:41585–8.
Hua ZC, Sohn SJ, Kang C, Cado D, Winoto A. A function of Fas-associated death domain protein in cell cycle progression localized to a single amino acid at its C-terminal region. Immunity. 2003;18:513–21.
Ballinger CA, Connell P, Wu Y, Hu Z, Thompson LJ, Yin LY, et al. Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol Cell Biol. 1999;19:4535–45.
Murata S, Minami Y, Minami M, Chiba T, Tanaka K. CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Rep. 2001;2:1133–8.
Paul I, Ghosh MK. A CHIPotle in physiology and disease. Int J Biochem Cell Biol. 2015;58:37–52.
Esser C, Scheffner M, Hohfeld J. The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation. J Biol Chem. 2005;280:27443–8.
Kim C, Yun N, Lee J, Youdim MB, Ju C, Kim WK, et al. Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death. Cell Death Differ. 2016;23:333–46.
Seo J, Lee EW, Sung H, Seong D, Dondelinger Y, Shin J, et al. CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3. Nat Cell Biol. 2016;18:291–302.
Fulda S, Meyer E, Debatin KM. Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression. Cancer Res. 2000;60:3947–56.
Stanger BZ, Leder P, Lee TH, Kim E, Seed B. RIP: A novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell. 1995;81:513–23.
Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000;1:489–95.
Morgan MJ, Kim YS, Liu ZG. Membrane-bound Fas ligand requires RIP1 for efficient activation of caspase-8 within the death-inducing signaling complex. J Immunol. 2009;183:3278–84.
Geserick P, Hupe M, Moulin M, Wong WW, Feoktistova M, Kellert B, et al. Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment. J Cell Biol. 2009;187:1037–54.
Bellail AC, Olson JJ, Yang X, Chen ZJ, Hao C. A20 ubiquitin ligase-mediated polyubiquitination of RIP1 inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma. Cancer Discov. 2012;2:140–55.
Xu W, Marcu M, Yuan X, Mimnaugh E, Patterson C, Neckers L. Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu. Proc Natl Acad Sci USA. 2002;99:12847–52.
Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell. 1995;81:505–12.
Henry CM, Martin SJ. Caspase-8 acts in a non-enzymatic role as a scaffold for assembly of a pro-inflammatory “FADDosome” complex upon TRAIL stimulation. Mol Cell. 2017;65:715–29. e715
Scott FL, Stec B, Pop C, Dobaczewska MK, Lee JJ, Monosov E, et al. The Fas-FADD death domain complex structure unravels signalling by receptor clustering. Nature. 2009;457:1019–22.
Wang L, Yang JK, Kabaleeswaran V, Rice AJ, Cruz AC, Park AY, et al. The Fas-FADD death domain complex structure reveals the basis of DISC assembly and disease mutations. Nat Struct Mol Biol. 2010;17:1324–9.
Wu-Baer F, Lagrazon K, Yuan W, Baer R. The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin. J Biol Chem. 2003;278:34743–6.
Morris JR, Solomon E. BRCA1: BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair. Hum Mol Genet. 2004;13:807–17.
Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, Marsischky G, et al. A proteomics approach to understanding protein ubiquitination. Nat Biotechnol. 2003;21:921–6.
Ben-Saadon R, Zaaroor D, Ziv T, Ciechanover A. The polycomb protein Ring1B generates self atypical mixed ubiquitin chains required for its in vitro histone H2A ligase activity. Mol Cell. 2006;24:701–11.
Srivastava D, Chakrabarti O. Mahogunin-mediated alpha-tubulin ubiquitination via noncanonical K6 linkage regulates microtubule stability and mitotic spindle orientation. Cell Death Dis. 2014;5:e1064.
Durcan TM, Tang MY, Perusse JR, Dashti EA, Aguileta MA, McLelland GL, et al. USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin. EMBO J. 2014;33:2473–91.
Ohta T, Sato K, Wu W. The BRCA1 ubiquitin ligase and homologous recombination repair. FEBS Lett. 2011;585:2836–44.
Hospenthal MK, Freund SM, Komander D. Assembly, analysis and architecture of atypical ubiquitin chains. Nat Struct Mol Biol. 2013;20:555–65.
Shimamoto S, Kubota Y, Yamaguchi F, Tokumitsu H, Kobayashi R. Ca2+/S100 proteins act as upstream regulators of the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein). J Biol Chem. 2013;288:7158–68.
Lees MJ, Peet DJ, Whitelaw ML. Defining the role for XAP2 in stabilization of the dioxin receptor. J Biol Chem. 2003;278:35878–88.
Alberti S, Demand J, Esser C, Emmerich N, Schild H, Hohfeld J. Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome. J Biol Chem. 2002;277:45920–7.
Arndt V, Daniel C, Nastainczyk W, Alberti S, Hohfeld J. BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP. Mol Biol Cell. 2005;16:5891–5900.
Kalia LV, Kalia SK, Chau H, Lozano AM, Hyman BT, McLean PJ. Ubiquitinylation of alpha-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is regulated by Bcl-2-associated athanogene 5 (BAG5). PLoS ONE. 2011;6:e14695.
Kim HT, Kim KP, Uchiki T, Gygi SP, Goldberg AL. S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains. EMBO J. 2009;28:1867–77.
Scaglione KM, Zavodszky E, Todi SV, Patury S, Xu P, Rodriguez-Lebron E, et al. Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP. Mol Cell. 2011;43:599–612.
Jeong M, Lee EW, Seong D, Seo J, Kim JH, Grootjans S, et al. USP8 suppresses death receptor-mediated apoptosis by enhancing FLIPL stability. Oncogene. 2017;36:458–70.
Acknowledgements
This research was supported by a grant from the Ministry of Science, ICT, and Future Planning (2015R1A3A2066581) (Jaewhan S.) and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) Funded by the Ministry of Education (2017R1A6A3A11035262) (Jinho S.). Additionally, this research was partly supported by the BK21 Plus project of the National Research Foundation of Korea Grant (Jinho S., D.S., M.J., and Y.W.N.), and C.L. acknowledges institutional support by KIST.
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Seo, J., Lee, EW., Shin, J. et al. K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death. Oncogene 37, 4994–5006 (2018). https://doi.org/10.1038/s41388-018-0323-z
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DOI: https://doi.org/10.1038/s41388-018-0323-z
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