SIGNAL TRANSDUCTION PATHWAYS AND POLYOMA MIDDLE T

总体目标是了解细胞生长调节的途径。 并了解多瘤病毒如何颠覆它们以产生 转型。这里提出的工作涉及信号通路。 用于多瘤中T抗原的转化。它强调 磷脂酰肌醇-3‘-激酶。对PI-3‘-激酶的认识 是至关重要的，不仅因为它似乎对中期T 转化，但也因为这种酶被牵连到 其他转化基因和生长因子对生长的调节 感受器。在这种酶中，p110亚基具有催化活性， 而P85亚单位则提供调节。P85亚基含有SH2， SH3和BCR结构域。这些元素存在于各种各样的 参与信号反应的蛋白质。得到了PI-3‘-的结果。 因此，激酶将具有普遍的重要性。我们的具体目标是： A.PI-3‘-激酶的调节及其在生长和发育中的作用 变换 1.丝氨酸磷酸化位点的研究 2.阻断PI-3‘-激酶活化的作用分析 3.PI-3‘-激酶与其他生长发育关系的建立 调控途径 B.与SH2功能相关的研究 1.通过随机诱变确定SH2的结构/功能 关系 2.确定中间T是否同时绑定到超过 一个SH2 3.检验SH2特异性的MXM假设，以查看设计的 中间T突变体可以将p85与适当改变的 专一性。 4.确定是否可能存在额外的SH2相互作用 对确定仓鼠中T的肿瘤形态有重要意义。 C.与SH3功能相关的研究 1.PI-3‘-激酶SH3的诱变 2.P85SH3结合蛋白的鉴定 3.DL1015区域中T基因的诱变 4.筛选中T与其他SH3的相互作用 D.小G蛋白、PI-3‘-蛋白和PI-3’-K之间相互作用的研究 中T 1.PI-3‘-激酶Rac/Rho/CDC 42相互作用的测定 2.分析Rac/Rho在多发性肿瘤转化中的作用 显性否定。

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.