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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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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