Control of cell death is vital for normal physiology, and its altered regulation underlies many diseases, including cancer. This project will explore the molecular mechanisms by which members of the Bcl-2 family promote either cell survival or apoptosis, and influence the cell cycle. Over the next three years, its aims are: 1) To test the hypothesis that the pro-survival members function by directly sequestering mammalian homologs of the nematode Ced-4. To extend the finding that Bcl-XL binds to Ced-4 and to characterize this central regulatory complex, multiple Bcl-2 family members will be tested for binding to the first mammalian homolog, Apaf-1, and additional components of the complex and novel Ced-4 homologs will be sought. 2) To explore whether Bcl-2 docks on mitochondria, which may be critical for its function, by binding to components of the mitochondrial pores that gate small molecules. Our preliminary evidence of interaction will be extended to multiple Bcl-2 relatives, and its physiological importance assessed by comparing the binding of Bcl-2 mutants and by determining how mutated pore components affect Bcl-2 survival function. 3) To clarify how Bcl-2 impedes progression from quiescence into cell cycle. Clarifying the signal pathway to nuclear regulators of the cell cycle, such as the pRB pocket proteins and E2F transcription factors, will be aided by a Bcl-2 point mutant (Y28F) lacking this function. The possibility that this tyrosine is phosphorylated and the potential regulatory role of proteins (eg calcineurin, Raf) that bind nearby in Bcl-2 will be explored 4) To investigate the functions of the novel pro-apoptotic protein Bim, which binds to Bcl-2 via a small conserved domain. Since Bim may be a 'death ligand' for certain pro-survival family members, its affinity for them and its regulation during apoptosis will be investigated. Its physiological roles will be explored by determining its expression pattern and characterizing mice now generated with the gene disrupted. Justification: Altered regulation of apoptosis is central to oncogenesis and all cytotoxic therapies. As the proposal addresses central issues about the regulation, builds on productive research, and exploits novel findings and special resources, it should significantly advance the molecular dissection of apoptosis.
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