Apoptosis is fundamental to oncogenesis, and members of the BCL2 family of apoptotic molecules are central to the regulation of apoptosis. Reversible phosphorylation, catalyzed by kinases and phosphatases, is an important mechanism modulating diverse cellular pathways and has been shown to regulate the activity of the BCL2 molecules. BAD is a potent pro-apoptotic member of the BCL2 family whose function is tightly regulated by serine phosphorylation. In survival conditions, phosphorylation inactivates BAD, which binds 14-3-3 in the cytosol and dissociates from BCL-xL or BCL2. The pro-apoptotic function of BAD is activated by dephosphorylation, dissociation from 14-3-3, and binding to BCL-xL or BCL2. While the roles of kinases in apoptosis are well studied, far less is known about phosphatases. Using an IL-3-dependent cell line, we have identified a physiologically significant PP2A phosphatase activity that dephosphorylates and activates BAD. Preliminary data indicate that the susceptibility of BAD to this phosphatase activity in response to an apoptotic stimulus requires prior dissociation from 14-3-3, suggesting that BAD dephosphorylation is regulated by competitive interactions between 14-3-3. BAD, and PP2A. Reversible phosphorylation also regulates apoptosis signaling by the forkhead transcription factors FKHR and FKHRL1, which are involved in rhabdomyosarcoma and leukemia translocations. Like BAD, FKHR and FKHRL1 phosphorylated by Akt are bound by 14-3-3 in the cytosol, and dephosphorylated FKHR and FKHRL1 are nuclearly localized, transactivating cell death genes. We hypothesize that the serine/threonine phosphatase PP2A activates the pro-apoptotic function of BAD, and that the activity of PP2A on BAD is regulated by complex interactions between BAD, PP2A, and 14-3-3. To test this hypothesis, we will elucidate the PP2A BAD phosphatase structure and regulation, and investigate the interactions between 14-3-3 and BAD that regulate BAD phosphatase activity. In addition, we postulate that activation of the pro-apoptotic function of FKHR and FKHRL1 is regulated by a similar mechanism. We will use parallel experimental approaches to extend the model of activation of pro-apoptotic molecules by phosphatase to FKHR and FKHRL1. These experiments will provide a mechanistic understanding of phosphatase regulation in apoptosis.
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