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.
