The study of those cellular proteins which are specifically bound by the tumor (T) antigens of papova viruses is proving increasingly important to our understanding of human cancer. These studies have given important insights into the workings of proto-oncogenes such as c-src and anti- oncogenes such as the retinoblastoma gene. This proposal attempts to fill in gaps in our knowledge concerning the most recently identified cellular enzyme found in complex with T antigens, protein phosphatase 2A (PP2A). This proposal has two major goals: 1) to determine what role (or roles) PP2A/T antigen complex formation plays in various small T antigen (ST) and middle T antigen (MT) functions and 2) to investigate both normal and T antigen-perturbed regulation of PP2A function. The approach will be to analyze in parallel the functioning of the normal 55 kDa PP2A regulatory subunit and the polyomavirus substitutes, ST and MT, in modulating the 36/63 kDa PP2A heterodimer. First, all three subunits of PP2A plus polyomavirus ST will be overproduced. The overproduced subunits will be used for biochemical studies in vitro as well as for production of immunoblotting and immunoprecipitating antisera needed to study the interactions of T antigens and PP2A in vitro and in vivo. In parallel we will perform a complete genetic analysis of the interaction of the 36/63 kDa PP2A heterodimer with ST and with the 55 kDa protein. In addition to determining the regions of each protein necessary for complex formation and activity, this approach will allow us to determine whether association of ST and of the 55 kDa protein with the 36/63 kDa heterodimer are affected by similar structural changes in the 36 kDa and 63 kDa proteins. A select group of the resulting ST mutants will be analyzed in functional assays to determine the importance of PP2A/ST association for individual function of polyomavirus ST. Parallel middle T versions of certain ST mutants will be constructed and analyzed to investigate more thoroughly the importance of middle T binding to PP2A for middle T-mediated transformation, and the interdependent binding of various middle T-associated proteins. The interdependency of binding of the components of middle T complexes will also be studied by attempting to reassemble the middle T complex from purified baculoviral proteins in vitro and in mixed infections of baculoviruses expressing each component. We will use our antisera to analyze of the role of PP2 for middle T-mediated transformation, and the interdependent binding of various middle T-associated proteins. The interdependency of binding of the components of middle T complexes will also be studied by attempting to reassemble the middle T complex from purified baculoviral proteins in vitro and in mixed infections of baculoviruses expressing each component. We will use our antisera to analyze of the role of PP2A in cell cycle regulation by probing biochemical modifications of PP2A subunits and the nature of the complexes they form over the course of the cell cycle using mammalian, frog egg, and yeast systems. We will test the abilities of our various proteins mutated in the interaction sites to modulate cellular function and phosphorylation events.es
Showing the most recent 10 out of 28 publications
