This proposal concerns the gene products of polyoma virus required for virus growth and transformation. We are particularly concerned with how post-synthetic modifications regulate protein function. We therefore concentrate on large T and middle T, which are both phosphoproteins. Our goal is to learn how these proteins function and how phosphorylation contributes to that function. Our approach is both biochemical and genetic. Middle T associates with pp60c-src, the cell homologue of the transforming protein of Rous sarcoma virus. It is likely that the action of middle T in transformation results from this interaction. We will test this directly by creating mutants of pp60c-src designed to block middle T mediated transformation. We will probe the interaction between these two proteins with hybrid molecules containing pp60c-src and pp60v-src sequences and with pp60v-src mutants. Biochemical evidence suggests middle T serine phosphorylation is required for association with pp60c-src. This will be tested directly. The serine phosphorylation sites will be identified. Oligonucleotide mutagenesis will then be used to create mutants lacking the phosphorylation sites. The biology and biochemistry of these mutants will be compared to wild type. The middle T/pp-60c-src complex possesses tyrosine kinase activity; tyrosine phosphorylation of cell proteins has been demonstrated in RSV-transformed cells. However, the complexes also possess phosphatidylinositol kinase activity. Experiments involving calcium release and protein kinase C activation will be carried out to decide whether in vivo alterations in phosphatidylinositol metabolism are important for transformation. Large T antigen, which is important for DNA replication and RNA transcription, is a valuable model for non-histone chromosomal proteins. Using ts-a and hr-t mutants, we have obtained evidence that some of the phosphorylations may be required for function. Mapping of the sites and mutagenesis will be used to test this hypothesis. Phosphorylation patterns will be examined in different cells and different subcellular locations to to try to assess the importance of the other phosphorylations of large T. In these analyses, we will use both wild type virus and the large number of large T mutants now available.
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