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.

  • Article
  • Published:

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

Nature Cell Biology volume 2, pages 469–475 (2000)Cite this article

Abstract

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

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: Recombinant Hsp70, but not Hsp70AAAA, inhibits caspase processing in vitro.
Figure 2: Hsp70 inhibits activation of the recombinant apoptosome.
Figure 3: Hsp70 directly and selectively associates with Apaf-1 but not with other apoptosome components.
Figure 4: Apaf-1 is the functional target of Hsp70 for inhibition of caspase activation.
Figure 5: Hsp70 does not compete with procaspase-9 for binding to the Apaf-1 CARD.
Figure 6: Hsp70 inhibits dATP-dependent caspase activation by blocking recruitment of procaspase-9 to the apoptosome.
Figure 7: Figure 7 Hsp70AAAA does not inhibit recruitment of procaspases to the apoptosome or in vitro caspase processing.

Similar content being viewed by others

References

  1. Wolf, B. & Green, D. R. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J. Biol. Chem. 274, 20049–20052 (1999).

    Article  CAS  Google Scholar 

  2. Liu, X., Kim, C. N., Yang, J., Jemmerson, R. & Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147–157 (1996).

    Article  CAS  Google Scholar 

  3. Zou, H., Henzel, W. J., Liu, X., Lutschg, A. & Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413 (1997).

    Article  CAS  Google Scholar 

  4. Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T. & Alnemri, E. S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell 1, 949–957 (1998).

    Article  CAS  Google Scholar 

  5. Zou, H., Li, Y., Liu, X. & Wang, X. An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11549–11556 (1999).

    Article  CAS  Google Scholar 

  6. Cain, K. et al. Apaf-1 oligomerizes into biologically active ~700-kDa and inactive ~1.4-MDa apoptosome complexes. J. Biol. Chem. 275, 6067–6070 (2000).

    Article  CAS  Google Scholar 

  7. Cain, K., Brown, D. G., Langlais, C. & Cohen, G. M. Caspase activation involves the formation of the aposome, a large (~700 kDa) caspase-activating complex. J. Biol. Chem. 274, 22686–22692 (1999).

    Article  CAS  Google Scholar 

  8. Hu, Y., Benedict, M. A., Ding, L. & Nunez, G. Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase- 9 activation and apoptosis. EMBO J. 18, 3586–3595 (1999).

    Article  CAS  Google Scholar 

  9. Lindquist, S. & Craig, E. A. The heat-shock proteins. Annu. Rev. Genet. 22, 631–677 (1988).

    Article  CAS  Google Scholar 

  10. Gething, M. J. & Sambrook, J. Protein folding in the cell. Nature 355, 33–45 (1992).

    Article  CAS  Google Scholar 

  11. Parsell, D. A., Taulien, J. & Lindquist, S. The role of heat-shock proteins in thermotolerance. Phil. Trans. R. Soc. Lond. B 339, 279–285 (1993).

    Article  CAS  Google Scholar 

  12. Parsell, D. A. & Lindquist, S. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27, 437–496 (1993).

    Article  CAS  Google Scholar 

  13. Milarski, K. L. & Morimoto, R. I. Expression of human HSP70 during the synthetic phase of the cell cycle. Proc. Natl Acad. Sci. USA 83, 9517–9521 (1986).

    Article  CAS  Google Scholar 

  14. Samali, A. & Orrenius, S. Heat shock proteins: regulators of stress response and apoptosis. Cell Stress Chaperones 3, 228–236 (1998).

    Article  CAS  Google Scholar 

  15. Jaattela, M. Escaping cell death: survival proteins in cancer. Exp. Cell Res. 248, 30–43 (1999).

    Article  CAS  Google Scholar 

  16. Freeman, B. C., Myers, M. P., Schumacher, R. & Morimoto, R. I. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO J. 14, 2281–2292 (1995).

    Article  CAS  Google Scholar 

  17. Kluck, R. M., Bossy-Wetzel, E., Green, D. R. & Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136 (1997).

    Article  CAS  Google Scholar 

  18. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 (1997).

    Article  CAS  Google Scholar 

  19. Stennicke, H. R. et al. Caspase-9 can be activated without proteolytic processing. J. Biol. Chem. 274, 8359–8362 (1999).

    Article  CAS  Google Scholar 

  20. Benedict, M. A., Hu, Y., Inohara, N. & Nunez, G. Expression and functional analysis of Apaf-1 isoforms. Extra WD-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9. J. Biol. Chem. 275, 8461–8468 (2000).

    Article  CAS  Google Scholar 

  21. Hahn, G. M. & Li, G. C. Thermotolerance and heat shock proteins in mammalian cells. Radiat. Res. 92, 452–457 (1982).

    Article  CAS  Google Scholar 

  22. Mosser, D. D. & Martin, L. H. Induced thermotolerance to apoptosis in a human T lymphocyte cell line. J. Cell Physiol. 151, 561–570 (1992).

    Article  CAS  Google Scholar 

  23. Mosser, D. D., Caron, A. W., Bourget, L., Denis-Larose, C. & Massie, B. Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol. Cell Biol. 17, 5317–5327 (1997).

    Article  CAS  Google Scholar 

  24. Samali, A. & Cotter, T. G. Heat shock proteins increase resistance to apoptosis. Exp. Cell Res. 223, 163–170 (1996).

    Article  CAS  Google Scholar 

  25. Simon, M. M. et al. Heat shock protein 70 overexpression affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of cytokine release. J. Clin. Invest. 95, 926–933 (1995).

    Article  CAS  Google Scholar 

  26. Jaattela, M., Wissing, D., Bauer, P. A. & Li, G. C. Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. EMBO J. 11, 3507–3512 (1992).

    Article  CAS  Google Scholar 

  27. Liossis, S. N., Ding, X. Z., Kiang, J. G. & Tsokos, G. C. Overexpression of the heat shock protein 70 enhances the TCR/CD 3- and Fas/Apo-1/CD 95-mediated apoptotic cell death in Jurkat T cells. J. Immunol. 158, 5668–5675 (1997).

    CAS  PubMed  Google Scholar 

  28. Creagh, E. M. & Cotter, T. G. Selective protection by hsp70 against cytotoxic drug-, but not Fas- induced T-cell apoptosis. Immunology 97, 36–44 (1999).

    Article  CAS  Google Scholar 

  29. Mehlen, P., Schulze-Osthoff, K. & Arrigo, A. P. Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death. J. Biol. Chem. 271, 16510–16514 (1996).

    Article  CAS  Google Scholar 

  30. Li, C-Y., Lee, J-S., Ko, Y-G., Kim, J-I., & Seo, J-S. Hsp70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation. J. Biol. Chem. (in the press).

  31. Jaattela, M., Wissing, D., Kokholm, K., Kallunki, T. & Egeblad, M. Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J. 17, 6124–6134 (1998).

    Article  CAS  Google Scholar 

  32. Gabai, V. L. et al. Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J. Biol. Chem. 272, 18033–18037 (1997).

    Article  CAS  Google Scholar 

  33. Patriarca, E. J. & Maresca, B. Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae. Exp. Cell. Res. 190, 57–64 (1990).

    Article  CAS  Google Scholar 

  34. Polla, B. S. et al. Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc. Natl Acad. Sci. USA 93, 6458–6463 (1996).

    Article  CAS  Google Scholar 

  35. Evan, G.I. et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119–128 (1992).

    Article  CAS  Google Scholar 

  36. Juin, P., Hueber, A. O., Littlewood, T. & Evan, G. c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release. Genes Dev. 13, 1367–1381 (1999).

    Article  CAS  Google Scholar 

  37. Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. A. & Gruss, P. Apaf-1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727–737 (1998).

    Article  CAS  Google Scholar 

  38. Yoshida, H. et al. Apaf-1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750 (1998).

    Article  CAS  Google Scholar 

  39. Soengas, M. S. et al. Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science 284, 156–159 (1999).

    Article  CAS  Google Scholar 

  40. Xanthoudakis, S. et al. Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis. EMBO J. 18, 2049–2056 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  42. Rodriguez, J. & Lazebnik, Y. Caspase-9 and APAF-1 form an active holoenzyme. Genes Dev. 13, 3179–3184 (1999).

    Article  CAS  Google Scholar 

  43. Wolf, B.B. et al. Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation. Blood 94, 1683–1692 (1999).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank G. Nunez and Y. Lazebnik for reagents, and C. Langlais and D. Brown for technical assistance. This work was supported by grants AI40646 and AI47891 from the National Institutes of Health.

Correspondence and requests for materials should be addressed to D.R.G.

Author information

Authors and Affiliations

  1. La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, 92121, USA, California

    Helen M. Beere, Beni B. Wolf, Artin Mahboubi, Tomomi Kuwana & Douglas R. Green

  2. Biotechnology Research Institute, National Research Council of Canada, Montreal,, H4P 2R2, Quebec, Canada

    Dick D. Mosser

  3. Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Drummond Street, Montreal, H3G 1YG, Quebec, Canada

    Pankaj Tailor

  4. MRC Toxicology Unit, Hodgkin Building, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK

    Kelvin Cain & Gerald M. Cohen

  5. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 North Campus Drive, Evanston, 60208, Illinois , USA

    Richard I. Morimoto

Authors
  1. Helen M. Beere
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Beni B. Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kelvin Cain
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dick D. Mosser
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Artin Mahboubi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tomomi Kuwana
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Pankaj Tailor
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Richard I. Morimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gerald M. Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Douglas R. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Beere, H., Wolf, B., Cain, K. et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2, 469–475 (2000). https://doi.org/10.1038/35019501

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35019501

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