title: WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation.
abstract: The casp9 protein plays a critical role in apoptosis induced by a variety of death stimuli. A regulator of apoptosis, Apaf-1, binds to and activates pro-casp9 in the presence of cytochrome c and dATP, a requirement that is bypassed by deletion of the WD-40 repeats located in the C-terminal half of Apaf-1. In this report, we used constitutively active Apaf-1 mutant lacking the WD-40 repeat region to study the mechanism and regulation of pro-casp9 activation. Mutational analysis revealed that only a small portion of the CED-4 homologous region (residues 456-559) could be deleted without destroying the ability of Apaf-1-(1-559) to activate pro-casp9. Apaf-1 can self-associate to form oligomers. Disruption of Apaf-1 self-association by deletion (Delta109-559) or mutation of the P-loop region (K149R) abrogated Apaf-1-mediated pro-casp9 activation. Forced oligomerization of the caspase recruitment domain of Apaf-1 was sufficient for pro-casp9 activation. Dimerization of chimeric Fpk-pro-casp9 protein with the dimerizer drug FK1012 induced pro-casp9 processing and apoptosis in cells. Significantly, the C-terminal region containing WD-40 repeats interacted with its N-terminal CED-4 homologous region, as determined by immunoprecipitation experiments. Importantly, expression of the WD-40 repeat region inhibited Apaf-1 self-association and proteolytic activation of pro-casp9. These studies provide a mechanism by which Apaf-1 promotes autoactivation of pro-casp9 through Apaf-1 self-association, a process that is negatively regulated by the WD-40 repeats.
authors.affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.forename: L
authors.initials: L
authors.lastname: Ding
authors.forename: D M
authors.initials: DM
authors.lastname: Spencer
authors.forename: G
authors.initials: G
authors.lastname: Núñez
mesh_terms: D000818:Animals; D053477:Apoptotic Protease-Activating Factor 1; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C506592:APAF1 protein, human; C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D004792:Enzyme Precursors; D010446:Peptide Fragments; D011506:Proteins; C506433:CASP9 protein, human; C506434:Casp9 protein, mouse; D053453:Caspase 9; D020169:Caspases