The retinoblastoma protein (RB), a tumor suppressor gene product, is regulated by phosphorylation and inhibits cell cycle progression in its dephosphorylated form. Our own work has identified a type-1 protein phosphatase (PP1) as being responsible for RB dephosphorylation, suggesting an anti-proliferative role for PP1. We will use our experience in the field of protein phosphorylation to test this hypothesis by investigating the role of PP1 in oncogene-mediated transformation of mammalian cells.
The Specific Aims of this project are 1. to identify the specific mammalian isoform(s) of PP1 catalyzing RB dephosphorylation 2. to examine whether stimulation of RB phosphatase a) affects neoplastic transformation in vitro, and b) can suppress the phenotype of transformed cells 3. to examine whether inactivation of RB phosphatase a) affects neoplastic transformation of normal cells, and b) the transformed phenotype of transformed cells. (l) The PP1 isozyme acting on RB will be identified by characterizing the effect of PP1 isozyme-specific antibodies on the RB phosphorylation in cell extracts. Secondly, RB phosphatase activity will be purified utilizing recombinant RB as a substrate. (2) To induce high levels of PP1 we will transfect murine C3H 10T 1/2 cells with expression vectors carrying PP1-cDNA and isolate cell lines stably over-expressing PP1. Transformation efficiency by mos will be assessed by focus formation. To characterize the transformed phenotype we will quantitate anchorage dependence, glucose uptake, and growth factor requirements. (3) To block PP1 expression or activity we intend to transfect cells with vectors encoding i) PP1 antisense mRNA or ii) an inhibitor-1 fragment. Ultimately these studies will help to define the role of protein phosphatase 1 in cellular transformation at the molecular level. It is envisaged that they will eventually lead to new strategies regarding the prevention or treatment of malignant cell growth.
