Characterization of cytoplasmic caspase-2 activation by induced proximity.
Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore, we identify Hsp90alpha as a key negative regulator of heat shock-induced caspase-2 activation.
Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Lisa
L
Bouchier-Hayes
Andrew
A
Oberst
Gavin P
GP
McStay
Samuel
S
Connell
Stephen W G
SW
Tait
Christopher P
CP
Dillon
Jonathan M
JM
Flanagan
Helen M
HM
Beere
Douglas R
DR
Green
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D015374:Biosensing Techniques; D053420:CRADD Signaling Adaptor Protein / Q000378:metabolism; D053143:Caspase 2 / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D000076249:Heat Shock Transcription Factors; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013312:Stress, Physiological; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D050257:Tubulin Modulators / Q000494:pharmacology
D053477:Apoptotic Protease-Activating Factor 1; D001426:Bacterial Proteins; D053420:CRADD Signaling Adaptor Protein; C484481:Cradd protein, mouse; D004268:DNA-Binding Proteins; D053200:Fas-Associated Death Domain Protein; D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; D000076249:Heat Shock Transcription Factors; C490017:Hsf1 protein, mouse; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050257:Tubulin Modulators; C054562:yellow fluorescent protein, Bacteria; D053143:Caspase 2