CYTOCHROME-C AND DATP-DEPENDENT FORMATION OF APAF-1 CASPASE-9 COMPLEXINITIATES AN APOPTOTIC PROTEASE CASCADE/

We report here the purification of the third protein factor, Apaf-3, t hat participates in caspase-3 activation in vitro. Apaf-3 was identifi ed 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 activ ates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenua ted the activation of caspase-3 and cellular apoptotic response in viv o, indicating that caspase-9 is the most upstream member of the apopto tic protease cascade that is triggered by cytochrome c and dATP.