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

Activation of 'initiator' ( or 'apical') caspases- 2, - 8 or - 9 ( refs 1 - 3) 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.