SV40 encodes two structurally related tumor (T) antigens, large T and small T; polyoma virus encodes three related T antigens, large, medium and small T. Large T of either virus is a multifunctional protein involved in viral DNA replication and in controlling transcription. All tumor antigens are implicated to some extent in malignant transformation, but SV40 large T and polyoma virus medium T are most intimately involved in this process. The main goals of this proposal are: (1) to investigate the possible role of phosphorylation in the function(s) of SV40 large T. We have previously determined eight phosphorylation sites clustered in two separate regions of large T. It seems likely that the introduction of one or several negative charges into the large T molecule will alter its function(s). By studying various mutants, defective in viral DNA replication, transcription control, or transformation, we expect to establish correlations between mutant defects and alterations in phosphorylation at individual sites. Sites that seem to be potentially important for functions will be removed by site-directed mutagenesis, and the effect of the mutation of function will be studied. We also intend to investigate the kinetics of phosphorylation, phosphate turnover at individual sites, and subcellular location of protein kinases involved in phosphorylation of large T. Furthermore, the effect of phosphorylation on in vitro measurable activities or properties of large T, such as DNA binding, ATPase activity, oligomerization, DNA synthesis, and transcription will be investigated. (2) to determine the phosporylation sites in polyoma virus large T and to study their role in function. We suspect that certain phosphorylation sites have been conserved in evolution because they are important for function. (3) to investigate two proteins (50.000 and 34.000 daltons) from SV40-transformed rat cells, precipitated with antiserum against a synthetic peptide, corresponding to the carboxy terminus of an alternate reading frame at the 3 feet end of the SV40 early region. We plan to examine whether these proteins are truly SV40-coded and to study their properties. (4) to purify polyoma virus medium T further and to characterize its associated tyrosine-specific protein kinase activity. The kinase-active fraction of medium T sediments is 200K complex. The goal is to find out whether this complex is a tetramer of medium T, or medium T associated with a cellular enzyme, and to understand its role in transformation by polyoma virus.
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