ROLE OF TYROSINE KINASES IN MIDDLE T AG TRANSFORMATION

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Middle t antigen (MTAg) is the transforming protein of polyoma virus. A critical unanswered question in polyoma virus biology is how MTAg expression elicits the dramatic changes associated with oncogenic transformation. MTAg has no known catalytic activities but has been shown to associate with two cellular proteins; pp60c- src and pp62c-yes, both of which are tyrosine kinases. MTAg is thought to function by activating the tyrosine kinase activity of these and possibly other cellular tyrosine kinases. The experiments proposed here utilize recently developed techniques and unique reagents to analyze, in greater detail than has previously been possible, how MTAg associates with and alters the biochemical and biological properties of the src family of tyrosine kinases. MTAg and pp60c-src will be overproduced in insect cells using a baculovirus expression system. pp60c-src and MTAg associate quantitatively when co-produced in insect cells and large quantities of complex are easily isolated. Because pp60c-src and MTAg fail to interact when co-produced in bacteria or yeast and only small quantities of complex are produced in mammalian cells, the baculovirus expression system is the system of choice for studying complex formation. Therefore, this expression system will be used to identify how MTAg specifically enhances the tyrosine kinase activity of pp60c-src interactions are regulated in mammalian cells. Genetic studies will be performed to precisely define the MTAg binding site on pp60c-src and to predict associations with other src family members. Finally, genetic studies will be performed to precisely define the contributions of other src family members to MTAg mediated transformation.