Ordering the cytochrome c-initiated caspase cascade: Hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to t he CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation o f caspase-9. Caspase-9 is thought to propagate the death signal by triggeri ng other caspase activation events, the details of which remain obscure. He re, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 sele ctively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 di d not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-ind ucible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that c aspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 an d reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pat hway and also participates in a feedback amplification loop involving caspa se-9.