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LETTERSIn situ trapping of activated initiator caspases reveals a
role for caspase-2 in heat shock-induced apoptosis Shine Tu1, Gavin P. McStay1,2, Louis-Martin Boucher3, Tak Mak3, Helen M. Beere1,2,4,5 and Douglas R. Green1,2,4,5Activation 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 cellpermeable, biotinylated pan-caspase inhibitor (bVADfmk)
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 heatshock-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.The cleavage and activation of the executioner caspases-3 and -7 to
orchestrate apoptosis is usually mediated by the apical or initiator caspases, caspases-2, -8 and -9 (and possibly -10)13. In marked contrast to
the executioner caspases, the inactive proforms of these initiator caspases
are monomeric and do not require activation by proteolytic cleavage1,2,4.
Instead, activation is generally mediated by the binding of adapter molecules to protein-interaction regions in their long pro-domains to induce
caspase dimerization and assembly of active protease sites5. This allows the
activated initiator caspases to undergo autocleavage, which may stabilize
the resulting active forms2,3,6. Thus, cleavage does not necessarily indicate
activation of an initiator caspase, and is therefore not a definitive method
for identifying the caspase(s) responsible for triggering apoptosis.Different apoptotic stimuli engage different initiator caspases, thus
defining specific apoptotic pathways. For example, caspase-8 (and probably caspase-10) are activated by ligation of death receptors7,8,
whereas many other stressors engage the mitochondrial pathway and
caspase-9 (ref. 9). A role for caspase-2 as an initiator caspase has been
suggested in several settings, including death receptor ligation10, endoplasmic reticulum stress11, genotoxic stress1214 and...