Bcl-2 regulates amplification of caspase activation by cytochrome c

Caspases, a family of specific proteases, have central roles in apoptosis [ 1], Caspase activation in response to diverse apoptotic stimuli involves th e relocalisation of cytochrome c from mitochondria to the cytoplasm where i t stimulates the proteolytic processing of caspase precursors. Cytochrome c release is controlled by members of the Bcl-2 family of apoptosis regulato rs [2,3]. The anti-apoptotic members Bcl-2 and Bcl-x(L) may also control ca spase activation independently of cytochrome c relocalisation or may inhibi t a positive feedback mechanism [4-7], Here, we investigate the role of Bcl -2 family proteins in the regulation of caspase activation using a model ce ll-free system. We found that Bcl-2 and Bcl-x(L) set a threshold in the amo unt of cytochrome c required to activate caspases, even in soluble extracts lacking mitochondria, Addition of dATP (which stimulates the procaspase-pr ocessing factor Apaf-1 [8,9]) overcame inhibition of caspase activation by Bcl-2, but did not prevent the control of cytochrome c release from mitocho ndria by Bcl-2, Cytochrome c release was accelerated by active caspase-3 an d this positive feedback was negatively regulated by Bcl-2, These results p rovide evidence for a mechanism to amplify caspase activation that is suppr essed at several distinct steps by Bcl-2, even after cytochrome c is releas ed from mitochondria.