ROLE OF TYROSINE KINASES IN MIDDLE T AG TRANSFORMATION

中间t抗原（MTAg）是多瘤性肿瘤的转化蛋白 病毒 多瘤病毒生物学中一个关键的未解问题是 MTAg的表达是如何引起与 致癌转化 MTAg没有已知的催化活性 但已显示与两种细胞蛋白质相关; pp 60 c- src和pp 62 c-yes，两者都是酪氨酸激酶。 MTAg是 被认为是通过激活酪氨酸激酶活性来发挥作用的， 这些和可能的其他细胞酪氨酸激酶。 的 这里提出的实验利用最近开发的技术， 独特的试剂进行分析，比以前更详细 MTAg是如何与生物化学物质结合并改变生物化学物质的， 和酪氨酸激酶SRC家族的生物学特性。 使用一种方法，MTAg和pp 60 c-src将在昆虫细胞中过量产生。 杆状病毒表达系统 pp 60 c-src和MTAg关联 当在昆虫细胞和大细胞中共同产生时， 大量的复合物很容易分离。 因为pp 60 c-src和 当在细菌或酵母中共同产生时，MTAg不能相互作用， 在哺乳动物细胞中仅产生少量的复合物， 杆状病毒表达系统是选择用于 研究复杂的构造 因此，该表达系统将 用于鉴定MTAg如何特异性增强酪氨酸 pp 60 c-src相互作用的激酶活性在 哺乳动物细胞 基因研究将被精确地 确定pp 60 c-src上的MTAg结合位点，并预测 与其他SRC家庭成员的联系。 最后，基因 将进行研究，以精确地确定 其他src家族成员转化为MTAg介导的转化。

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.