Role of cytochrome c and dATP/ATP hydrolysis in Apaf‐1‐mediated caspase‐9 activation and apoptosis

Apaf‐1 plays a critical role in apoptosis by binding to and activating procaspase‐9. We have identified a novel Apaf‐1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD‐40 repeat. The product of this Apaf‐1 cDNA activated procaspase‐9 in a cytochrome c and dATP/ATP‐dependent manner. We used this Apaf‐1 to show that Apaf‐1 requires dATP/ATP hydrolysis to interact with cytochrome c , self‐associate and bind to procaspase‐9. A P‐loop mutant (Apaf‐1K160R) was unable to associate with Apaf‐1 or bind to procaspase‐9. Mutation of Met368 to Leu enabled Apaf‐1 to self‐associate and bind procaspase‐9 independent of cytochrome c , though still requiring dATP/ATP for these activities. The Apaf‐1M368L mutant exhibited greater ability to induce apoptosis compared with the wild‐type Apaf‐1. We also show that procaspase‐9 can recruit procaspase‐3 to the Apaf‐1–procaspase‐9 complex. Apaf‐1(1–570), a mutant lacking the WD‐40 repeats, associated with and activated procaspase‐9, but failed to recruit procaspase‐3 and induce apoptosis. These results suggest that the WD‐40 repeats may be involved in procaspase‐9‐mediated procaspase‐3 recruitment. These studies elucidate biochemical steps required for Apaf‐1 to activate procaspase‐9 and induce apoptosis.