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 (⌬109 -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 Fpkpro-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 procasp9 through Apaf-1 self-association, a process that is negatively regulated by the WD-40 repeats.Programmed cell death, or apoptosis, an evolutionarily conserved and genetically regulated biological process, plays a critical role in development and tissue homeostasis of multicellular organisms (1-4). Dysfunction of this process contributes to the pathogenesis of several diseases in humans and mice (4 -6). A family of cysteine proteases (designated caspases) related to Caenorhabditis elegans CED-3 appears to represent the executionary arm of the apoptotic program. Each caspase contains conserved residues important for specific proteolytic activity cleaving substrates after aspartate residues (7-9). Caspases are synthesized in cells as inactive zymogens and, upon stimulation with apoptotic signals, are processed into mature forms composed of a tetramer of two large and two small subunits (10, 11). The apoptotic process is characterized by sequential activation of multiple caspases. Depending on their order of activation in the proteolytic cascade, caspases can be divided into upstream and downstream caspases. The casp9 protein is an upstream caspase that contains a caspase recruit domain (CARD) in its N terminus, a region that is also present in the prodomain of several death proteases including CED-3, casp1, and casp2 (12). Activation of casp9 initiates a protease cascade with subsequent activation of downstream casp3 and casp7, leading to cleavage of target proteins and execution of the apoptotic program (13).Our understanding of how caspases are activated in mammalian cell...

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