Protein phosphatase 2A (PP2A) is a serine/threonine specific phosphatase composed of catalytic subunit C and two regulatory subunits, A and b. There are several distinct forms of the B subunit that generate enzyme diversity. SV40 and polyoma tumor antigens bind to overlapping regions in the N terminus of the A subunit and compete with the B subunit for binding sites. The long-term goal of this proposal is to understand the role of T antigen- PP2A interactions in transformation by SV40 and polyoma virus. This will be achieved by analyzing the molecular structure of the A subunit and of A subunit-T antigen complexes and by investigating the normal function of PP2A in growth control.
The specific aims are: 1. To determine the crystal structure of the A subunit and of complexes of the A subunit with SV40 small t antigen and polyoma middle T. This work will elucidate how the structure of tumor antigens has evolved to associate with a highly conserved cellular protein. 2. To inhibit the interaction of middle T antigen with the A subunit by specific peptides that mimic binding sites. The investigator proposes to investigate whether inhibition of middle T binding to the A subunit results in dissociation of the c-Src protein from middle T, and in reversion of the transformed phenotype of middle T-transformed cells. Attempts will be made to develop a system for introducing peptides into cells by uptake from the medium. These studies could have important implications for the development of anticancer drugs. 3. To transiently express an A subunit mutant in cells that does not bind T antigens or B subunit but binds the C subunits and causes formation of A-C dimer not controlled by the B subunit. A subunit antisense constructs will also be expressed in attempts to reduce A subunit levels and an increase in free B and C subunits. The effects of mutant and antisense RNA expression on general protein phosphorylation and phosphorylation of specific proteins involved in growth control will be investigated. 4. To determine structural and functional changes of PP2A during the cells cycle. The synthesis, turnover, and possible phosphorylation of all PP2A subunits will be analyzed, and a search will be conducted for proteins that are associated with the PP2A holoenzyme. This will be carried out by cell fractionation and by using poly- and monoclonal antibodies that recognize and immunoprecipitate native A subunit bound to the other subunits and/or to unknown proteins.
Showing the most recent 10 out of 24 publications
