Inhibition of Cytochrome c-induced Caspase Activation

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DESCRIPTION (provided by applicant): Apoptosis is a program of cellular suicide wherein individual cells are removed from the midst of a living tissue without destroying overall tissue architecture. In response to apoptotic signals, cells activate a family of intracellular proteases known as caspases, which participate in the orderly dismantling and packaging of the dying cell. Under many circumstances (e.g. following treatment of cells with chemotherapeutic agents), caspase activation proceeds through a mitochondrial-dependent pathway, leading to translocation of the respiratory chain component, cytochrome c, from the mitochondria to the cytoplasm. Once cytoplasmic, cytochrome c interacts with an apoptotic regulator, Apaf-1 to promote ATP-dependent activation of a specific caspase, caspase 9. This complex of caspase 9/Apaf-1/cytochrome c is referred to as the apoptosome. It has been demonstrated that many signals promoting cell survival impinge upon the mitochondrial pathway, preventing the efflux of mitochondrial cytochrome c to the cytoplasm. This can effectively prevent caspase activation and forestall cell death. In this proposal, however, we focus on an alternative mode of cell survival, acting through post-cytochrome c inhibition of the apoptosome. Specifically, we have found that both activated Erk kinases and the oncoprotein Bcr-Abl can prevent activation of caspase 9, even in the presence of cytosolic cytochrome c. It is the goal of this proposal to elucidate the mechanism(s) underlying this inhibition. Towards this end we propose a careful characterization of the apoptosome (composition, post-translational modifications, binding partners) in the presence and absence of these activated kinases. Moreover, we propose to use a new methodology to identify and characterize direct substrates of these kinases in a cell-free apoptotic reconstitution system. Finally, we propose two screens to identify additional novel apoptosomal regulators. Collectively, these experiments should provide insight into cellular survival mechanisms acting after mitochondrial release of cytochrome c.