Protein Phosphatase 2A and Polyomavirus-induced Oncogenesis

Polyomavirus, a small DNA tumour virus, causes a variety of neoplasms in its natural host, the mouse and is therefore used as a model system for the study of carcinogenesis. The transforming protein of polyomavirus is the middle tumor antigen (MT). MT has no intrinsic enzymatic activity, but rather transforms by associating with and modulating the activities of cellular proteins involved in control of the cell cycle. The association of polyomavirus MT with protein phosphatase 2A (PP2A), a cellular serinehreonine phosphatase, appears to be necessary for MT- mediated transformation. Mutational analysis of MT provided evidence that binding of PP2A is required for the subsequent assembly of a MT transformation complex containing cellular proteins like the tyrosine kinase pp60c- src , phosphatidylinositol 3-kinase (PI 3-kinase) and Shc. However, it was not known if PP2A activity plays a role in assembly of the MT transformation complex or if PP2A binding has only a structural, non-enzymatic function in this process. Therefore the major goal of the proposed project was to elucidate the role of PP2A activity in polyomavirus MT-mediated transformation. We were able to show that PP2A activity is not essential in cis for the assembly of a MT transformation complex. In addition our mutational analysis not only provided first experimental data on PP2A catalytic site residues but also supports the model of a common catalytic mechanism among serinehreonine phosphatases. The second major goal of the project consisted in the identification, isolation and characterization of proteins, which are specifically associated with catalytically inactive C subunit mutants and which potentially could be substrates or regulators of PP2A. Several inactive C subunit mutants were able to form stable complexes between PP2A and cellular proteins. One of these cellular proteins was identified as a protein phosphatase methylesterase-1 (PME-1), purified, microsequenced, and its cDNA was cloned. Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. PME-1 not only represents the first mammalian protein methylesterase to be cloned but also an enzyme, which regulates PP2A`s modification status and activity. Our work does not only provide new insights into the interactions between MT and PP2A but also contributes to the understanding of PP2A regulation in general. Although our work is at the moment "only" a contribution to basic science, it might at the same time, serve as a starting point for the development of therapeutic concepts for cancer treatment. During the course of the project we generated poly- and monoclonal antibodies against PP2A subunits, which were essential for the realization of the project. For the commercial sale of antibodies I concluded material license agreements with the American company Upstate Biotechnology (UBI). The project was scientifically as well as commercially successful.