SIGNAL TRANSDUCTION PATHWAYS AND POLYOMA MIDDLE T

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The overall goals are to understand pathways of cell growth regulation and to understand how polyomavirus subverts them to produce transformation. The work proposed here concerns signaling pathways employed for transformation by polyoma middle T antigen. It emphasizes the enzyme phosphatidylinositol-3'-kinase. Understanding PI-3'-kinase is essential not only because it appears critical for middle T transformation, but also because this enzyme has been implicated in growth regulation by other transforming genes and growth factor receptors. In this enzyme, the P110 subunit has catalytic activity, while the p85 subunit provides regulation. The p85 subunit contains SH2, SH3 and bcr domains. These elements are found in a wide variety of proteins involved in signal response. The results obtained for PI-3'- kinase will therefore be of general importance. Our specific goals are: A. Studies on regulation of PI-3'-kinase and its role in growth and transformation 1. Studies on sites of serine phosphorylation 2. Analysis of the effect of blocking PI-3'-kinase activation 3. Establishment of connections between PI-3'-kinase and other growth regulatory pathways B. Studies related to SH2 function 1. Random mutagenesis of SH2s to determine structure/function relationships 2. Determination of whether middle T binds simultaneously to more than one SH2 3. Test of the MXM hypothesis of SH2 specificity to see if a designed middle T mutant can bind a p85 with an appropriately altered specificity. 4. Determination of whether an additional SH2 interaction might be important for determining tumor profiles of hamster middle T. C. Studies related to SH3 function 1. Mutagenesis of the SH3 of PI-3'-kinase 2. Identification of proteins that bind p85 SH3 3. Mutagenesis of middle T in the region of dl 1015 4. Screening for interactions between middle T and other SH3s D. Studies of the interactions among small G-proteins, PI-3'-kinase and middle T 1. Determination of PI-3'-kinase rac/rho/cdc 42 interactions 2. Analysis of the role of rac/rho in polyoma transformation using dominant negatives.