1998 — 2002 |
Yang, Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Bcl2 Related Gene, Bad, and Apoptosis
DESCRIPTION: (adapted from the investigator's abstract) BCL2, the proto-oncogene that is deregulated in the most common translocation of follicular lymphomas, enhances oncogenesis by preventing programmed cell death. The BCL2 family of proteins are central to the regulation of apoptosis and include a number of interacting molecules. Both BCL2 and its close homolog BCL-XL have cell cycle activities separable from their anti-apoptotic function. The pro-apoptotic protein BAD heterodimerizes with BCL2 and BCL-XL, and promotes cell death. We have found that while BAD counters BCL2 and BCL-XL's anti-apoptotic activity, it does not affect their cell cycle functions. When control cells growth arrest, cells expressing BAD continue to cycle in the absence of serum and undergo accelerated apoptosis. These data suggest the hypothesis that BAD functions both in cell cycle and cell death. This grant proposes to determine the molecular mechanism of the cell cycle and cell death functions of BAD and BAD/BCL-XL heterodimers. First, the precise effect of BAD and BAD/BCL-XL in the cell cycle response to growth arrest signals will be determined. The structure-function relationship of the cell cycle and cell death activities will be defined to determine whether these function co-segregate. To further investigate the mechanism of BAD function, downstream protein targets of BAD and BAD/BCL-XL will be identified and cloned. These data will provide mechanistic insight into the normal function of the BCL2 family of cell death regulators and how their dysfunction contribute to oncogenesis.
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2002 — 2006 |
Yang, Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Serine/Threonine Phosphatases in Apoptosis
DESCRIPTION: (provided by applicant) 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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2003 — 2007 |
Yang, Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cell Cycle Function of the Bcl2 Family
DESCRIPTION (provided by applicant): The anti-apoptosis molecules BCL2 and BCL-xL delay cell cycle entry. The decision of a cell to be in G0 quiescence or to re-enter the cell cycle is important in normal homeostasis and in oncogenesis. The significance of both the anti-apoptosis and anti-proliferative functions of BCL2 has been demonstrated in animal models of tumorigenesis and in human cancers. BCL2 and BCL-xL expression delays the transition from G0 to S by a process requiring the presence of p27. We found that in cells expressing BCL2 and BCLxL, induction of the early G1 genes c-Myc and cyclin D1 were unaffected, but p27 was significantly elevated and activation of G1 cyclin-dependent kinases was inhibited. BCL2 or BCL-xL blocked c-Myc-induced cell cycle progression efficiently, but did not delay cell cycle entry in myc -/- cells. Cell size and RNA content analyses indicated that BCL2 and BCL-xL delayed cell growth that normally accompanies entry into cell cycle from G0, suggesting that cell growth and cell cycle progression were uncoupled. Our data indicate that BCL2 and BCL-xL inhibit G1 progression and delay G0-G1 transition by elevating p27 and inhibiting events downstream of Myc. We hypothesize that BCL2 and BCL-xL regulate cell growth during cell cycle entry by playing a role in mitochondria bioenergetics. We propose to test our hypothesis by 1) elucidating the mechanism of p27 upregulation, 2) identifying the specific cell cycle functions of Myc affected by BCL2 and BCL-xL, and 3) determining the role of BCL2 and BCL-xL in mitochondrial proliferation during emergence from quiescence. The goals of this grant are to elucidate the molecular mechanism of cell cycle delay by BCL2 and BCL-xL and to determine the physiologic significance of regulation of cell cycle entry by BCL2 or BCL-xL.
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