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.
Tu
Shine
S
Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121, USA.
McStay
Gavin P
GP
Boucher
Louis-Martin
LM
Mak
Tak
T
Beere
Helen M
HM
Green
Douglas R
DR
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053420:CRADD Signaling Adaptor Protein; D053143:Caspase 2; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006358:Hot Temperature; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism; D019014:fas Receptor / Q000528:radiation effects
D048868:Adaptor Proteins, Signal Transducing; D053420:CRADD Signaling Adaptor Protein; D061945:Caspase Inhibitors; C484481:Cradd protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D019014:fas Receptor; D053143:Caspase 2; D020169:Caspases