| Apoptosis is an internally programmed form of cell death induced by a variety of physiological and pharmacological stimuli. Members of the Bcl-2 family of proteins regulate this highly ordered process. The results presented here demonstrate that in unstressed cells the pro-apoptotic protein Bax is located in the cytosol, and that following exposure to the protein kinase inhibitor, staurosporine, or ligation of the Fas receptor, Bax translocates specifically to the mitochondria. Relocalization of Bax occurred in close temporal association with mitochondrial release of cytochrome c and activation of DEVD-specific caspases. Addition of the broad range caspase inhibitor, z-VAD-fmk, prevented Bax translocation induced by anti-Fas, but not by staurosporine, indicating that different upstream signals may be responsible for Bax relocalization. Bax translocation was closely associated with cell death, either in the presence or absence of caspase activity, suggesting that Bax translocation is part of the common execution machinery and may represent an irreversible commitment to cell death. The data presented here also demonstrate that Fas ligation results in a conformational change in the N-terminus of Bax, which precedes its relocalization to the mitochondria. These results support a model in which, following the application of an apoptotic stress, Bax undergoes an N-terminal conformational change while residing in the cytosol, thereby unmasking epitopes in the C-terminal hydrophobic domain that are required for insertion into the mitochondrial membrane. Overexpression of the anti-apoptotic protein Bcl-2 inhibited the Bax conformational change and mitochondrial translocation, cytochrome c release, caspase processing and activation, and cell death induced by treatment with staurosporine or anti-Fas. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein, Bid, indicating that Bcl-2 functions downstream of these events. These results demonstrate that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not through direct interaction with, or by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change. Prevention of Bax conformational change and subsequent translocation to the mitochondria represents a novel mechanism by which Bcl-2 inhibits apoptosis. |
