Cif (cytochrome c efflux-inducing factor) activity is regulated by Bcl- 2 and caspases and correlates with the activation of bid

Almost all cultured cells can be induced to undergo apoptosis by a plethora of stimuli, including but not limited to DNA damage, exposure to glucocorticords, and withdrawal of growth factors (reviewed in reference 56). Although these stimuli elicit varied initial cellular responses, the final stages of apoptosis are remarkably similar irrespective of the lethal stimulus. In particular, activation of a novel family of cytosolic proteases, termed caspases (1; reviewed in references 8 and 9), is a key common hallmark of apoptotic cells. Caspases are relatively widely and constitutively expressed as inactive zymogens (procaspases), which are themselves proteolytically cleaved to yield an active enzyme (reviewed in references 37 and 48). Caspases can be divided into “initiator” caspases (e.g., caspases 8 and 10), which activate downstream “effector” caspases (e.g., caspases 3, 6, and 7). Effector caspases then cleave a limited subset of cellular proteins (reviewed in reference 44), which appear ultimately to be responsible for the biochemical and morphological changes associated with programmed cell death. The remarkable similarity in the terminal events of apoptosis regardless of the initial apoptotic stimulus strongly implies that there is a common signal transduction pathway leading to activation of caspases in the cytosol. One of the most extensively studied processes of apoptotic signal transduction is initiated by activated Fas (CD95/Apo-1) (reviewed in reference 36). Fas is a cell membrane receptor which belongs to the tumor necrosis factor receptor superfamily (60). The binding of the Fas ligand (54) or agonistic antibodies against Fas (38) causes the activation of Fas and rapid apoptosis of many cell types (49). Fas contains a motif referred to as the death domain, which is required for apoptosis (21, 55). Biochemically, activated Fas interacts, through its death domain, with the death domain of the FADD/MORT-1 protein (reviewed in reference 7), which in turn recruits procaspase 8, thus forming a death-inducing signaling complex (DISC) (25, 43). Engagement of the Fas receptor results in the oligomerization and activation of caspase 8 (35, 64), which ultimately results in the activation of many downstream effector caspases (4, 33). Caspase 8 may activate other caspases by directly cleaving them (51), or it may indirectly activate them through the use of the cytochrome c:Apaf-1 signaling pathway (49). Thus, through an unidentified mechanism, caspase 8 activation results in the efflux of cytochrome c, normally an exclusively mitochondrial protein, into the cytosol (reviewed in reference 45). Once in the cytosol, cytochrome c, in the presence of dATP, binds to apoptotic protease-activating factor 1 (Apaf-1) (30), to which is tethered procaspase 9 (29, 42). The binding of cytochrome c to Apaf-1 then facilitates the activation of caspase 9, potentially through an autoactivation process facilitated by Apaf-1-mediated oligomerization (52). Active caspase 9, in turn, activates procaspases 3 and 7 by direct cleavage (29, 52). The activation of caspase 3 in particular is extremely important, since it is the most biologically relevant effector caspase identified to date, being responsible for the cleavage of a large number of target proteins (reviewed in references 9 and 44). The activation of caspase 3 generally also implies that the cell is irretrievably committed to dying. Thus, apoptotic signaling involves a caspase cascade that ultimately results in intracellular proteolysis and death (reviewed in references 37 and 48). Mitochondrial cytochrome c efflux, with the resultant activation of caspases 9, 3, and 7, appears to be a pivotal control point not only for Fas-mediated apoptosis in certain cell types (49) but also for the chemical induction of apoptosis. This hypothesis is supported by studies which demonstrated that overexpression of the apoptosis suppressor, Bcl-2, in mitochondria of various cells blocked cytochrome c efflux and apoptosis induced by a variety of stimuli (26, 63). Furthermore, the process of cytochrome c efflux during Fas-induced apoptosis in Jurkat cells was blocked by overexpression in mitochondria of the related apoptotic suppressor protein, Bcl-XL (58). Therefore, transduction not only of chemically induced but also of Fas-induced apoptotic signals to mitochondria can be inhibited by overexpression of apoptosis suppressors. Molecular and biochemical studies have demonstrated that apoptotic effectors can physically interact with and inhibit the function(s) of various apoptotic suppressors. In particular, Bad (62) and Bax (40) can inhibit Bcl-2, and Bad (62), Bak (6, 12, 24), and Bax (3) can inhibit Bcl-XL. Since Bax (19, 61) and Bad (65; reviewed in reference 13), which are normally localized in the cytosol, can be actively translocated to mitochondria during apoptosis induction, it is plausible that apoptotic effector proteins such as Bax and/or Bad could serve at the end of the common central apoptotic signal transduction pathway to induce cytochrome c efflux. This hypothesis was supported by a recent study which reported that recombinant Bax proteins directly induced cytochrome c efflux from mitochondria (11, 22). While apoptotic suppressors may have additional functions downstream of cytochrome c efflux (42, 47, 66), it is possible that Bcl-2 and Bcl-XL proteins block the effect of Bax, Bad or other apoptosis effectors on mitochondria and consequently block cytochrome c efflux. We recently developed a cell-free assay for studying regulation of mitochondrial cytochrome c efflux (17). Using this assay, we observed that treatment of HL-60 cells with the potent apoptosis inducer staurosporine (STS) induced a cytosolic activity, termed Cif (cytochrome c efflux-inducing factor), which rapidly facilitated cytochrome c efflux from purified mitochondria (17). Similar factors and activities have also been described by others (27, 53). Here we demonstrate that the activation of Cif, as well as its action in mitochondria, could be inhibited by Bcl-2 overexpression. This inhibition could be overcome in vitro by the addition of activated caspases. Furthermore, the addition of activated caspases to S-100 extracts prepared from normal cells was sufficient to induce Cif. Thus, Cif activation required an active caspase(s), and its effect could be blocked by apoptotic suppressors. Finally, Cif activity was selectively neutralized by anti-Bid antibodies and the activation of Cif correlated with the cleavage of Bid by caspases. These results suggest that Cif and Bid are one and the same and that it is the biologically relevant regulator of cytochrome c efflux and is thus an important factor in the process of apoptotic signal transduction.