Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Caspase 8 inhibits programmed necrosis by processing CYLD

Nature Cell Biology volume 13, pages 1437–1442 (2011)Cite this article

Subjects

Abstract

Caspase 8 initiates apoptosis downstream of TNF death receptors by undergoing autocleavage and processing the executioner caspase 3 (ref. 1). However, the dominant function of caspase 8 is to transmit a pro-survival signal that suppresses programmed necrosis (or necroptosis) mediated by RIPK1 and RIPK3 (refs 2, 3, 4, 5, 6) during embryogenesis and haematopoiesis7,8,9. Suppression of necrotic cell death by caspase 8 requires its catalytic activity but not the autocleavage essential for apoptosis10; however, the key substrate processed by caspase 8 to block necrosis has been elusive. A key substrate must meet three criteria: it must be essential for programmed necrosis; it must be cleaved by caspase 8 in situations where caspase 8 is blocking necrosis; and mutation of the caspase 8 processing site on the substrate should convert a pro-survival response to necrotic death without the need for caspase 8 inhibition. We now identify CYLD as a substrate for caspase 8 that satisfies these criteria. Following TNF stimulation, caspase 8 cleaves CYLD to generate a survival signal. In contrast, loss of caspase 8 prevented CYLD degradation, resulting in necrotic death. A CYLD substitution mutation at Asp 215 that cannot be cleaved by caspase 8 switches cell survival to necrotic cell death in response to TNF.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: CYLD is essential for necrosis.
Figure 2: CYLD is a substrate for proteolysis by caspase 8.
Figure 3: Caspase 8 processes CYLD at aspartic acid 215.
Figure 4: CYLDD215A triggers programmed necrosis in the absence of caspase inhibitors.
Figure 5: CYLDD215A switches the pro-survival NEMO–RIPK1 complex to the pro-necrotic RIPK1–FADD complex.

Similar content being viewed by others

References

  1. Brenner, D. & Mak, T. W. Mitochondrial cell death effectors. Curr. Opin. Cell Biol. 21, 871–877 (2009).

    Article  CAS  Google Scholar 

  2. Chan, F. K. et al. A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J. Biol. Chem. 278, 51613–51621 (2003).

    Article  CAS  Google Scholar 

  3. Degterev, A. et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. Chem. Biol. 1, 112–119 (2005).

    Article  CAS  Google Scholar 

  4. Cho, Y. S. et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137, 1112–1123 (2009).

    Article  CAS  Google Scholar 

  5. He, S. et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. Cell 137, 1100–1111 (2009).

    Article  CAS  Google Scholar 

  6. Zhang, D. W. et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325, 332–336 (2009).

    Article  CAS  Google Scholar 

  7. Oberst, A. et al. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471, 363–367 (2011).

    Article  CAS  Google Scholar 

  8. Ch’en, I. L., Tsau, J. S., Molkentin, J. D., Komatsu, M. & Hedrick, S. M. Mechanisms of necroptosis in T cells. J. Exp. Med. 208, 633–641 (2011).

    Article  Google Scholar 

  9. Zhang, H. et al. Functional complementation between FADD and RIP1 in embryos and lymphocytes. Nature 471, 373–376 (2011).

    Article  CAS  Google Scholar 

  10. Kang, T. B. et al. Mutation of a self-processing site in caspase-8 compromises its apoptotic but not its nonapoptotic functions in bacterial artificial chromosome-transgenic mice. J. Immunol. 181, 2522–2532 (2008).

    Article  CAS  Google Scholar 

  11. Hitomi, J. et al. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135, 1311–1323 (2008).

    Article  CAS  Google Scholar 

  12. Li, M. & Beg, A. A. Induction of necrotic-like cell death by tumor necrosis factor alpha and caspase inhibitors: novel mechanism for killing virus-infected cells. J. Virol. 74, 7470–7477 (2000).

    Article  CAS  Google Scholar 

  13. Wang, L., Du, F. & Wang, X. TNF-α induces two distinct caspase-8 activation pathways. Cell 133, 693–703 (2008).

    Article  CAS  Google Scholar 

  14. He, K. L. & Ting, A. T. Essential role for IKKγ/NEMO in TCR-induced IL-2 expression in Jurkat T cells. Eur. J. Immunol. 33, 1917–1924 (2003).

    Article  CAS  Google Scholar 

  15. Wolf, B. B., Schuler, M., Echeverri, F. & Green, D. R. Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J. Biol. Chem. 274, 30651–30656 (1999).

    Article  CAS  Google Scholar 

  16. Holler, N. et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1, 489–495 (2000).

    Article  CAS  Google Scholar 

  17. Degterev, A. et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat. Chem. Biol. 4, 313–321 (2008).

    Article  CAS  Google Scholar 

  18. Wright, A. et al. Regulation of early wave of germ cell apoptosis and spermatogenesis by deubiquitinating enzyme CYLD. Dev. Cell 13, 705–716 (2007).

    Article  CAS  Google Scholar 

  19. Ea, C. K., Deng, L., Xia, Z. P., Pineda, G. & Chen, Z. J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006).

    Article  CAS  Google Scholar 

  20. Wu, C. J., Conze, D. B., Li, T., Srinivasula, S. M. & Ashwell, J. D. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-κB activation. Nat. Cell Biol. 8, 398–406 (2006).

    Article  CAS  Google Scholar 

  21. O’Donnell, M. A., Legarda, D., Skountzos, P., Yeh, W. C. & Ting, A. T. Ubiquitination of RIP1 regulates an NF-κB-independent cell-death switch in TNF signaling. Curr. Biol. 17, 418–424 (2007).

    Article  Google Scholar 

  22. Legarda-Addison, D., Hase, H., O’Donnell, M. A. & Ting, A. T. NEMO/IKKγ regulates an early NF-κB-independent cell-death checkpoint during TNF signaling. Cell Death Differ. 16, 1279–1288 (2009).

    Article  CAS  Google Scholar 

  23. O’Donnell, M. A. & Ting, A. T. Chronicles of a death foretold: Dual sequential cell death checkpoints in TNF signaling. Cell Cycle 9, 1065–1071 (2010).

    Article  Google Scholar 

  24. O’Donnell, M. A. & Ting, A. T. RIP1 comes back to life as a cell death regulator in TNFR1 signaling. FEBS J. 278, 877–887 (2011).

    Article  Google Scholar 

  25. Massoumi, R., Chmielarska, K., Hennecke, K., Pfeifer, A. & Fassler, R. Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-κB signaling. Cell 125, 665–677 (2006).

    Article  CAS  Google Scholar 

  26. Peters, M. E. Programmed cell death: Apoptosis meets necrosis. Nature 471, 310–312 (2011).

    Article  Google Scholar 

  27. Brouckaert, G. et al. Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production. Mol. Biol. Cell 15, 1089–1100 (2004).

    Article  CAS  Google Scholar 

  28. Hirt, U. A. & Leist, M. Rapid, noninflammatory and PS-dependent phagocytic clearance of necrotic cells. Cell Death Differ. 10, 1156–1164 (2003).

    Article  CAS  Google Scholar 

  29. van Delft, M. F., Smith, D. P., Lahoud, M. H., Huang, D. C. & Adams, J. M. Apoptosis and non-inflammatory phagocytosis can be induced by mitochondrial damage without caspases. Cell Death Differ. 17, 821–832 (2010).

    Article  CAS  Google Scholar 

  30. Waring, P., Lambert, D., Sjaarda, A., Hurne, A. & Beaver, J. Increased cell surface exposure of phosphatidylserine on propidium iodide negative thymocytes undergoing death by necrosis. Cell Death Differ. 6, 624–637 (1999).

    Article  CAS  Google Scholar 

  31. Friedman, C. S. et al. The tumour suppressor CYLD is a negative regulator of RIG-I-mediated antiviral response. EMBO Rep. 9, 930–936 (2008).

    Article  CAS  Google Scholar 

  32. Su, A. I. et al. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl Acad. Sci. USA 101, 6062–6067 (2004).

    Article  CAS  Google Scholar 

  33. Saldanha, A. J. Java Treeview-extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are indebted to a number of colleagues for providing reagents used in these studies. We thank X. Wang (UT Southwestern, Dallas, Texas, USA) for his gift of SMAC mimetics and human RIPK3 antibody, M. Kelliher (UMASS, Worcester, Massachusetts, USA) for R i p k 1+/+ and R i p k 1−/− MEFs, J. Chipuk (Mount Sinai School of Medicine, New York, USA) for suggesting the use of MCF7 cells and S. Aaronson (Mount Sinai School of Medicine, New York, USA) for providing these cells. This work was supported by National Institutes of Health grants AI052417 (A.T.T.) and AI44828 (D.R.G.). M.A.O’D. is a recipient of a Research Fellowship Award from the Crohn’s and Colitis Foundation of America. A.T.T. is a recipient of the Irma T. Hirschl Career Scientist Award.

Author information

Author notes

  1. Eva Perez-Jimenez

    Present address: Present address: Laboratory of Cellular Biology, Centro de Investigacion Principe Felipe, Valencia 46012, Spain,

  2. Marie Anne O’Donnell and Eva Perez-Jimenez: These authors contributed equally to this work

Authors and Affiliations

  1. Immunology Institute, Mount Sinai School of Medicine, New York, New York 10029, USA

    Marie Anne O’Donnell, Eva Perez-Jimenez & Adrian T. Ting

  2. Department of Immunology, St Jude’s Children’s Research Hospital, Memphis, Tennessee 38105, USA

    Andrew Oberst & Douglas R. Green

  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    Aylwin Ng & Ramnik Xavier

  4. Department of Laboratory Medicine, Clinical Research Center, Lund University, SE-205 Malmo, Sweden

    Ramin Massoumi

Authors
  1. Marie Anne O’Donnell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eva Perez-Jimenez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew Oberst
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aylwin Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ramin Massoumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ramnik Xavier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Douglas R. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Adrian T. Ting
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.A.O’D. and E.P-J. designed and conducted the experiments. A.O. designed and carried out the Casp8 and Cyld siRNA experiments in L929 cells. A.N. and R.X. carried out the bioinformatics analyses and wrote the corresponding section of the manuscript. A.O. and D.R.G. provided C a s p 8+/+ and C a s p 8−/− MEFs on both the R i p k 3+/+ and R i p k 3−/− backgrounds and helpful discussions of the manuscript. R.M. provided the C y l d−/− MEFs. M.A.O’D. and A.T.T. wrote the manuscript. A.T.T. directed the studies.

Corresponding authors

Correspondence to Marie Anne O’Donnell or Adrian T. Ting.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2868 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

O’Donnell, M., Perez-Jimenez, E., Oberst, A. et al. Caspase 8 inhibits programmed necrosis by processing CYLD. Nat Cell Biol 13, 1437–1442 (2011). https://doi.org/10.1038/ncb2362

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2362

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing