Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.
Li
P
P
Howard Hughes Medical Institute, and Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 75235, USA.
Nijhawan
D
D
Budihardjo
I
I
Srinivasula
S M
SM
Ahmad
M
M
Alnemri
E S
ES
Wang
X
X
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D001665:Binding Sites; D001943:Breast Neoplasms; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D004789:Enzyme Activation; D004847:Epithelial Cells; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D009097:Multienzyme Complexes; D011506:Proteins; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D003546:Cysteine Endopeptidases; C026600:2'-deoxyadenosine triphosphate