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Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly

Abstract

The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.

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References

  1. 1.

    Krammer PH, Arnold R, Lavrik IN. Life and death in peripheral T cells. Nat Rev Immunol. 2007;7:532–42.

    CAS  Article  Google Scholar 

  2. 2.

    Lavrik IN, Krammer PH. Regulation of CD95/Fas signaling at the DISC. Cell Death Differ. 2012;19:36–41.

    CAS  Article  Google Scholar 

  3. 3.

    Dickens LS, Boyd RS, Jukes-Jones R, Hughes MA, Robinson GL, Fairall L, et al. A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death. Mol Cell. 2012;47:291–305.

    CAS  Article  Google Scholar 

  4. 4.

    Fu TM, Li Y, Lu A, Li Z, Vajjhala PR, Cruz AC, et al. Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex. Mol Cell. 2016;64:236–50.

    CAS  Article  Google Scholar 

  5. 5.

    Schleich K, Warnken U, Fricker N, Ozturk S, Richter P, Kammerer K, et al. Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model. Mol Cell. 2012;47:306–19.

    CAS  Article  Google Scholar 

  6. 6.

    Ozturk S, Schleich K, Lavrik IN. Cellular FLICE-like inhibitory proteins (c-FLIPs): fine-tuners of life and death decisions. Exp Cell Res. 2012;318:1324–31.

    CAS  Article  Google Scholar 

  7. 7.

    Golks A, Brenner D, Fritsch C, Krammer PH, Lavrik IN. c-FLIPR, a new regulator of death receptor-induced apoptosis. J Biol Chem. 2005;280:14507–13.

    CAS  Article  Google Scholar 

  8. 8.

    Chang DW, Xing Z, Pan Y, Algeciras-Schimnich A, Barnhart BC, Yaish-Ohad S, et al. c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J. 2002;21:3704–14.

    CAS  Article  Google Scholar 

  9. 9.

    Micheau O, Thome M, Schneider P, Holler N, Tschopp J, Nicholson DW, et al. The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex. J Biol Chem. 2002;277:45162–71.

    CAS  Article  Google Scholar 

  10. 10.

    Peter ME. The flip side of FLIP. Biochemical J. 2004;382:e1–3.

    CAS  Article  Google Scholar 

  11. 11.

    Thome M, Tschopp J. Regulation of lymphocyte proliferation and death by FLIP. Nat Rev Immunol. 2001;1:50–58.

    CAS  Article  Google Scholar 

  12. 12.

    Yu JW, Jeffrey PD, Shi Y. Mechanism of procaspase-8 activation by c-FLIPL. Proc Natl Acad Sci USA. 2009;106:8169–74.

    CAS  Article  Google Scholar 

  13. 13.

    Fricker N, Beaudouin J, Richter P, Eils R, Krammer PH, Lavrik IN. Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL. J Cell Biol. 2010;190:377–89.

    CAS  Article  Google Scholar 

  14. 14.

    Hughes MA, Powley IR, Jukes-Jones R, Horn S, Feoktistova M, Fairall L, et al. Co-operative and hierarchical binding of c-FLIP and caspase-8: a unified model defines how c-flip isoforms differentially control cell fate. Mol Cell. 2016;61:834–49.

    CAS  Article  Google Scholar 

  15. 15.

    Schleich K, Buchbinder JH, Pietkiewicz S, Kahne T, Warnken U, Ozturk S, et al. Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain. Cell Death Differ. 2016;23:681–94.

    CAS  Article  Google Scholar 

  16. 16.

    Scaffidi C, Schmitz I, Krammer PH, Peter ME. The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem. 1999;274:1541–8.

    CAS  Article  Google Scholar 

  17. 17.

    Hwang EY, Jeong MS, Park SY, Jang SB. Evidence of complex formation between FADD and c-FLIP death effector domains for the death inducing signaling complex. BMB Rep. 2014;47:488–93.

    Article  Google Scholar 

  18. 18.

    Ueffing N, Keil E, Freund C, Kuhne R, Schulze-Osthoff K, Schmitz I. Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment. Cell Death Differ. 2008;15:773–82.

    CAS  Article  Google Scholar 

  19. 19.

    Majkut J, Sgobba M, Holohan C, Crawford N, Logan AE, Kerr E, et al. Differential affinity of FLIP and procaspase 8 for FADD’s DED binding surfaces regulates DISC assembly. Nat Commun. 2014;5:3350.

    CAS  Article  Google Scholar 

  20. 20.

    Golks A, Brenner D, Schmitz I, Watzl C, Krueger A, Krammer PH, et al. The role of CAP3 in CD95 signaling: new insights into the mechanism of procaspase-8 activation. Cell Death Differ. 2006;13:489–98.

    CAS  Article  Google Scholar 

  21. 21.

    Hoffmann JC, Pappa A, Krammer PH, Lavrik IN. A new C-terminal cleavage product of procaspase-8, p30, defines an alternative pathway of procaspase-8 activation. Mol Cell Biol. 2009;29:4431–40.

    CAS  Article  Google Scholar 

  22. 22.

    Juo P, Kuo CJ, Yuan J, Blenis J. Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr Biol. 1998;8:1001–8.

    CAS  Article  Google Scholar 

  23. 23.

    Sprick MR, Rieser E, Stahl H, Grosse-Wilde A, Weigand MA, Walczak H. Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8. EMBO J. 2002;21:4520–30.

    CAS  Article  Google Scholar 

  24. 24.

    Lavrik IN. Systems biology of death receptor networks: live and let die. Cell Death Dis. 2014;5:e1259.

    CAS  Article  Google Scholar 

  25. 25.

    Bentele M, Lavrik I, Ulrich M, Stosser S, Heermann DW, Kalthoff H, et al. Mathematical modeling reveals threshold mechanism in CD95-induced apoptosis. J Cell Biol. 2004;166:839–51.

    CAS  Article  Google Scholar 

  26. 26.

    Warnken U, Schleich K, Schnolzer M, Lavrik I. Quantification of high-molecular weight protein platforms by AQUA mass spectrometry as exemplified for the CD95 death-inducing signaling complex (DISC). Cells. 2013;2:476–95.

    CAS  Article  Google Scholar 

  27. 27.

    Schilling B, Rardin MJ, MacLean BX, Zawadzka AM, Frewen BE, Cusack MP, et al. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation. Mol Cell Proteom. 2012;11:202–14.

    CAS  Article  Google Scholar 

  28. 28.

    Chaudhury S, Berrondo M, Weitzner BD, Muthu P, Bergman H, Gray JJ. Benchmarking and analysis of protein docking performance in Rosetta v3.2. PLoS ONE. 2011;6:e22477.

    CAS  Article  Google Scholar 

  29. 29.

    Lavrik I, Golks A, Krammer PH. Death receptor signaling. J Cell Sci. 2005;118:265–7.

    CAS  Article  Google Scholar 

  30. 30.

    Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME. Molecular ordering of the initial signaling events of CD95. Mol Cell Biol. 2002;22:207–20.

    CAS  Article  Google Scholar 

  31. 31.

    Algeciras-Schimnich A, Peter ME. Actin dependent CD95 internalization is specific for Type I cells. FEBS Lett. 2003;546:185–8.

    CAS  Article  Google Scholar 

  32. 32.

    Horn S, Hughes MA, Schilling R, Sticht C, Tenev T, Ploesser M, et al. Caspase-10 negatively regulates caspase-8-mediated cell death, switching the response to CD95L in favor of NF-kappaB activation and cell survival. Cell Rep. 2017;19:785–97.

    CAS  Article  Google Scholar 

  33. 33.

    Neumann L, Pforr C, Beaudouin J, Pappa A, Fricker N, Krammer PH, et al. Dynamics within the CD95 death-inducing signaling complex decide life and death of cells. Mol Syst Biol. 2010;6:352.

    Article  Google Scholar 

  34. 34.

    Schmidt JH, Pietkiewicz S, Naumann M, Lavrik IN. Quantification of CD95-induced apoptosis and NF-kappaB activation at the single cell level. J Immunol Methods. 2015;423:12–17.

    CAS  Article  Google Scholar 

  35. 35.

    Pietkiewicz S, Eils R, Krammer PH, Giese N, Lavrik IN. Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network. Exp Cell Res. 2015;339:1–9.

    CAS  Article  Google Scholar 

  36. 36.

    Pietkiewicz S, Schmidt JH, Lavrik IN. Quantification of apoptosis and necroptosis at the single cell level by a combination of Imaging Flow Cytometry with classical Annexin V/propidium iodide staining. J Immunol Methods. 2015;423:99–103.

    CAS  Article  Google Scholar 

  37. 37.

    MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26:966–8.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We acknowledge Volkswagen Foundation (VW 90315), Wilhelm Sander-Stiftung (2017.008.01), Center of dynamic systems (CDS), funded by the EU-program ERDF (European Regional Development Fund) and DFG (LA 2386) for supporting our work.

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Correspondence to Inna N. Lavrik.

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Hillert, L.K., Ivanisenko, N.V., Espe, J. et al. Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly. Oncogene 39, 1756–1772 (2020). https://doi.org/10.1038/s41388-019-1100-3

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