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In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis

Abstract

Activation of 'initiator' (or 'apical') caspases-2, -8 or -9 (refs 13) is crucial for induction of apoptosis. These caspases function to activate executioner caspapses that, in turn, orchestrate apoptotic cell death. Here, we show that a cell-permeable, biotinylated pan-caspase inhibitor (bVAD–fmk) both inhibited and 'trapped' the apical caspase activated when apoptosis was triggered. As expected, only caspase-8 was trapped in response to ligation of death receptors, whereas only caspase-9 was trapped in response to a variety of other apoptosis-inducing agents. Caspase-2 was exclusively activated in heat shock-induced apoptosis. This activation of caspase-2 was also observed in cells protected from heat-shock-induced apoptosis by Bcl-2 or Bcl-xL. Reduced sensitivity to heat-shock-induced death was observed in caspase-2−/− cells. Furthermore, cells lacking the adapter molecule RAIDD failed to activate caspase-2 after heat shock treatment and showed resistance to apoptosis in this setting. This approach unambiguously identifies the apical caspase activated in response to apoptotic stimuli, and establishes caspase-2 as a proximal mediator of heat shock-induced apoptosis.

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Figure 1: bVAD–fmk associates with active caspases during apoptosis.
Figure 2: Initiator-caspase activation after different modes of apoptosis induction.
Figure 3: Bcl-2 and Bcl-XL block heat-shock-induced apoptosis, but not activation, of caspase-2.
Figure 4: Caspase-2−/− cells show resistance to heat shock-induced death.
Figure 5: RAIDD is required for heat-shock-induced activation of caspase-2.

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References

  1. Renatus, M. et al. Dimer formation drives the activation of the cell death protease caspase 9. Proc. Natl Acad. Sci. USA 98, 14250–14255 (2001).

    Article  CAS  Google Scholar 

  2. Boatright, K. M. et al. A unified model for apical caspase activation. Molecular Cell 11, 1–20 (2003).

    Article  Google Scholar 

  3. Baliga, B. C, Read, S. H, Kumar, S. The biochemical mechanism of caspase-2 activation. Cell Death Differ. 11, 1234–1241 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Boatright, K. M. & Salvesen, G. S. Mechanisms of caspase activation. Curr. Opin. Cell Biol. 15, 725–731 (2003).

    Article  CAS  Google Scholar 

  6. Chang, D. W. et al. Interdimer processing mechanism of procaspase-8 activation. EMBO J. 22, 4132–4142 (2003).

    Article  CAS  Google Scholar 

  7. Scaffidi, C. et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17, 1675–1687 (1998).

    Article  CAS  Google Scholar 

  8. Boatright, K. M. et al. Activation of caspases-8 and -10 by FLIP(L). Biochem. J. 382, 651–657 (2004).

    Article  CAS  Google Scholar 

  9. Zheng, T. S., Hunot, S., Kuida, K. & Flavell, R. A. Caspase knockouts: matters of life and death. Cell Death Differ. 6, 1043–1053 (1999).

    Article  CAS  Google Scholar 

  10. Duan, H. & Dixit, V. M. RAIDD is a new 'death' adaptor molecule. Nature 385, 86–89 (1997).

    Article  CAS  Google Scholar 

  11. Bergeron, L. et al. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev. 12, 1304–1314 (1998).

    Article  CAS  Google Scholar 

  12. Lassus, P., Opitz-Araya, X. & Lazebnik, Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 297, 1352–1354 (2002).

    Article  CAS  Google Scholar 

  13. Lin, C. F. et al. Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramide and etoposide-induced apoptosis. J. Biol. Chem. 279, 40755–40761 (2004).

    Article  CAS  Google Scholar 

  14. Tinel, A. & Tschopp, J. The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 304, 843–846 (2004).

    Article  CAS  Google Scholar 

  15. Troy, C. M., Stefanis, L., Greene, L. A. & Shelanski, M. L. Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells. J. Neurosci. 17, 1911–1918 (1997).

    Article  CAS  Google Scholar 

  16. Troy, C. M. & Shelanski, M. L. Caspase-2 redux. Cell Death Differ. 10, 101–107 (2003).

    Article  CAS  Google Scholar 

  17. Ekert, P. G., Silke, J. & Vaux, D. L. Caspase inhibitors. Cell Death Differ. 6, 1081–1086 (1999).

    Article  CAS  Google Scholar 

  18. Kasibhatla, S. et al. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-κB and AP-1. Mol. Cell 1, 543–551 (1998).

    Article  CAS  Google Scholar 

  19. Petak, I. et al. Fas-dependent and -independent mechanisms of cell death following DNA damage in human colon carcinoma cells. Cancer Res. 60, 2643–2650 (2000).

    CAS  PubMed  Google Scholar 

  20. Brunner, T. et al. Expression of Fas ligand in activated T cells is regulated by c-Myc. J. Biol. Chem. 275, 9767–9772 (2000).

    Article  CAS  Google Scholar 

  21. Robertson, J. D. et al. Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J. Biol. Chem. 277, 29803–29809 (2002).

    Article  CAS  Google Scholar 

  22. Lin, C. F. et al. Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation. J. Biol. Chem. 280, 23758–23765 (2005).

    Article  CAS  Google Scholar 

  23. Lassus, P., Opitz-Araya, X. & Lazebnik, Y. Corrections and clarifications. Science 306, 1683 (2004).

    Article  CAS  Google Scholar 

  24. Cuende, E. et al. Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells. EMBO J. 12, 1555–1560 (1993).

    Article  CAS  Google Scholar 

  25. 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 

  26. Yang, J. et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129–1132 (1997).

    Article  CAS  Google Scholar 

  27. Mosser, D. D. et al. The chaperone function of hsp70 is required for protection against stress-induced apoptosis. Mol. Cell Biol. 20, 7146–7159 (2000).

    Article  CAS  Google Scholar 

  28. Cippitelli, M. et al. Hyperthermia enhances CD95-ligand gene expression in T lymphocytes. J. Immunol. 174, 223–232 (2005).

    Article  CAS  Google Scholar 

  29. Berube, C. et al. Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein RAIDD. Proc. Natl Acad. Sci. USA 102, 14314–14320 (2005).

    Article  CAS  Google Scholar 

  30. Sohn, D., Schulze-Osthoff, K. & Janicke, R. U. Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during drug-induced apoptosis. J. Biol. Chem. 280, 5267–5273 (2005).

    Article  CAS  Google Scholar 

  31. Shin, S. et al. Caspase-2 primes cancer cells for TRAIL-mediated apoptosis by processing procaspase-8. EMBO J. 24, 3532–3542 (2005).

    Article  CAS  Google Scholar 

  32. Baliga, B. C. et al. Role of prodomain in importin-mediated nuclear localization and activation of caspase-2. J. Biol. Chem. 278, 4899–4905 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Melissa O'Leary for her excellent technical assistance and Drs Guy Salvesen, Michael Pinkoski and Christine Bonzon for helpful discussion. We are grateful to Drs Guy Salvesen, Carol Troy, Seamus Martin, Marcus Peter, Andreas Strasser and Vanessa Marsden for their generous contribution of reagents. These studies were supported by the National Institutes of Health grants (AI40646, AI44828, AI47891, AI58422).

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Correspondence to Helen M. Beere or Douglas R. Green.

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Supplementary figures S1, S2 and S3 plus Supplementary Methods (PDF 168 kb)

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Tu, S., McStay, G., Boucher, LM. et al. In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis. Nat Cell Biol 8, 72–77 (2006). https://doi.org/10.1038/ncb1340

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