Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade

Abstract

We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3

“…Whereas other studies have demonstrated that release of cyt c from mitochondria induces a cas-3-mediated apoptotic cascade (Li et al, 1997;Liu et al, 1996;Zou et al, 1997), we asked if heat-shock treatment inhibits cyt cmediated activation of cas-3. As demonstrated previously for other cell types, expression of Hsp27 was induced in 293T cells at 6 ± 8 h after mild heat shock (Figure 1a).…”

“…Recent studies have shown that Apaf-1 promotes both activation of cas-9 and cas-9-mediated activation of cas-3 in mammalian cells (Li et al, 1997). These studies have further suggested that Apaf-1 functions as a docking protein for cas-9 and cyt c in mediating cleavage of cas-3 (Li et al, 1997).…”

“…Previous studies have demonstrated that release of cyt c from mitochondria to cytosol is an important step in inducing cas-3-mediated apoptosis (Li et al, 1997;Liu et al, 1996;Zou et al, 1997). To assess whether Hsp27 functions upstream or downstream to cyt c release from mitochondria, L929/pRc and L929/Hsp27 cells were treated with various agents, such as methylmethane sulphonate (MMS), 1-b-D-arabinofuranosyl-cytosine (ara-C) or staurosporine (STSP), that are known to cause the release of mitochondrial cyt c (Kharbanda et al, 1997a), and cytosols were analysed by immunoblotting (IB) with anti-cyt c. The results demonstrate that MMS treatment of both L929/pRc and L929/Hsp27 cells is associated with the release of cyt c ( Figure 3a, upper panel).…”

“…The ®nding that activated cas-9 in turn cleaves and activates cas-3 (executioner caspase) has supported a model in which the Apaf-1/cas-9 complex functions upstream to cas-3 in the apoptotic protease cascade (Li et al, 1997;Xue et al, 1996;Zou et al, 1997). Activation of the Apaf-1/cas-9 complex appears to be a common pathway employed by diverse stimuli that induce apoptosis in mammalian cells (Li et al, 1997). Cas-3 usually exists in the cytoplasm as an inactive precursor that becomes proteolytically activated in cells undergoing apoptosis (Schlegel et al, 1996;Wang et al, 1996).…”

“…Recent studies have shown that Apaf-1 binds to cas-9 (initiator caspase) in the presence of cyt c and that Apaf-1-mediated oligomerization leads to cas-9 activation (Li et al, 1997). The ®nding that activated cas-9 in turn cleaves and activates cas-3 (executioner caspase) has supported a model in which the Apaf-1/cas-9 complex functions upstream to cas-3 in the apoptotic protease cascade (Li et al, 1997;Xue et al, 1996;Zou et al, 1997). Activation of the Apaf-1/cas-9 complex appears to be a common pathway employed by diverse stimuli that induce apoptosis in mammalian cells (Li et al, 1997).…”