The ability to exit from the cell cycle in its G1-phase is crucial for the well-being of the organism and is perhaps the most important feature that distinguishes a healthy cell from a cancer cell. This ability depends on intact pathways that converge on the retinoblastoma protein, RB, whose major function is to prevent cells from committing to the next cell division. To allow for proliferation, RB activity must be abrogated by multiple phosphorylations catalyzed by cyclin-dependent kinases (Cdks) in late G1 through M-phase. RB is activated, wholly or in part, by protein phosphatase 1 (PP1). This finding, reported by several laboratories including this one, prompted us to investigate the role of PP1alpha in the cell cycle. Thus, we found that, unless it is itself inhibited by Cdks, PP1alpha induces G1 arrest in cells that express RB. In RB-negative cells, PP1alpha appeared to cause cell death without apparent arrest. Consistent with this result, PP1alpha was induced by drugs causing experimental apoptosis. Therefore, we hypothesize that PP1alpha, is able to cause exit from the cell cycle, resulting in G1 arrest or cell death, by two distinct pathways. To test this hypothesis, we will 1. characterize the control of RB function by the PP1alpha catalytic subunit. 2. determine the conditions under which PP1alpha induces cell cycle arrest or cell death. 3. assess the relationship between oncogenic transformation and PP1alpha deregulation. These studies will help to fill important gaps in our understanding of cell cycle control, which, so far, has been focused on Cdks. Ultimately, these experiments are designed to evaluate whether PP1alpha may serve as a target for novel or additional anti-cancer strategies.
In Aim 1, we will determine whether the sites specifically dephosphorylated by PP1alpha in vitro and in vivo activate the known tumor suppressor functions of RB.
Aim 2 is an extension of our previous studies and will define the phases of the cell cycle in which PP1alpha may be required, induce cell cycle arrest and/or cell death. Further, we will ask whether these responses depend on the expression status of p53, RB, and Cdk inhibitors. Finally, we will examine whether PP1alpha is required for drug induced apoptosis.
In Aim 3, the previous results will be tested by asking the question whether oncogene mediated immortalization and transformation is associated with changes affecting PP1's ability to maintain active RB.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA054167-07
Application #
6362564
Study Section
Metabolic Pathology Study Section (MEP)
Program Officer
Yang, Shen K
Project Start
1993-09-01
Project End
2005-02-28
Budget Start
2001-03-01
Budget End
2002-02-28
Support Year
7
Fiscal Year
2001
Total Cost
$218,661
Indirect Cost
Name
Children's Hospital of Los Angeles
Department
Type
DUNS #
094878337
City
Los Angeles
State
CA
Country
United States
Zip Code
90027
Flores-Delgado, Guillermo; Liu, Cathy W Y; Sposto, Richard et al. (2007) A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis. J Proteome Res 6:1165-75
Liu, Cathy W Y; Wang, Rui-Hong; Berndt, Norbert (2006) Protein phosphatase 1alpha activity prevents oncogenic transformation. Mol Carcinog 45:648-56
Berndt, Norbert; Ludlow, John W (2004) Interaction between the retinoblastoma protein and protein phosphatase 1 during the cell cycle. Methods Mol Biol 281:17-32
Berndt, N (1999) Protein dephosphorylation and the intracellular control of the cell number. Front Biosci 4:D22-42
Dohadwala, M; Berndt, N (1998) Expression of functional protein phosphatase 1 catalytic subunit in E. coli. Methods Mol Biol 93:191-9
Berndt, N (1998) Control of PP1 activity through phosphorylation by cyclin-dependent kinases. Methods Mol Biol 93:67-77
Berndt, N; Dohadwala, M; Liu, C W (1997) Constitutively active protein phosphatase 1alpha causes Rb-dependent G1 arrest in human cancer cells. Curr Biol 7:375-86