Regulation of caspase-9 activity by differential binding to the apoptosome complex

Proteolytic processing of procaspase- 9 is required for its activation, but processing in itself appears to be insufficient for its activity. We studied caspase activation in a cell- free system and found that incubation of cytosol from rat kidney proximal tubule cells with Cytochrome c ( Cyt c) and dATP results in rapid autocatalytic processing of procaspase- 9 from similar to 50 kD to similar to 38 kD size fragment. Moreover, Cyt c concentration influences the production of alternatively processed forms of caspase-9. At lower Cyt c concentration ( 0.01-0.05 mg/ ml), two fragments of caspase- 9 of the size 38 and 40 kD are produced. In contrast, at higher concentrations of Cyt c (> 0.1 mg/ ml) only 38 kD fragment will prevail. However, our failure to capture processed caspase- 9 by affinity labeling or coelution with Apaf- 1 suggested that caspase- 9 undergoes a conformational change during its enzymatic action on effector caspases, resulting in its release from the apoptosome complex and inactivation. In support of this hypothesis, catalytic inhibitors of caspase- 9 prevented its release from the apoptosome complex without affecting its auto- processing and allowed successful capture of active caspase- 9 ( 38 kD) and its complex by affinity labeling. These observations suggest that complex allosteric interactions with the apoptosome complex influence caspase- 9 activity and function by controlling not only the induction of its enzymatic activity, but also its rapid termination.