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.
Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, 1500 East Medical Center Drive, 4219 CCGC, Ann Arbor, MI 48109, USA.
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Benedict
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Ding
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D017398:Alternative Splicing; D019943:Amino Acid Substitution; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D004792:Enzyme Precursors; D011506:Proteins; D000255:Adenosine Triphosphate; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate