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破坏它们， 转型 这里提出的工作涉及信号通路 用于通过多瘤中T抗原转化。 它强调 磷脂酰肌醇-3 '-激酶 了解PI-3 '-激酶 这不仅是因为它对中间T 转化，而且还因为这种酶与 其他转化基因和生长因子的生长调节 受体。 在这种酶中，P110亚基具有催化活性， 而p85亚基提供调节。 p85亚基含有SH 2， SH 3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。 对PI-3 '- 因此激酶具有普遍重要性。 我们的具体目标是： A. PI-3 '-激酶的调节及其在生长发育中的作用 转型 1.丝氨酸磷酸化位点的研究 2.阻断PI-3 '-激酶活化的效果分析 3. PI-3 '-激酶与其他生长因子之间关系的建立 调节途径 B。SH 2功能相关研究 1. SH 2的随机诱变以确定结构/功能 关系 2.确定中间T是否同时结合多于 一个SH 2 3.检验SH 2特异性的MXM假设，以确定设计的 中T突变体可以结合p85， 的特异性 4.确定是否可能存在额外的SH 2相互作用 重要的是确定肿瘤的仓鼠中T。 C. SH 3功能相关研究 1. PI-3 '-激酶SH 3的突变 2.结合p85 SH 3的蛋白质的鉴定 3. dl 1015区域中T的突变 4.筛选中间T和其他SH 3之间的相互作用 D.小G蛋白、PI-3 '-激酶和细胞凋亡的相互作用研究 中间T 1. PI-3 '-激酶rac/rho/cdc 42相互作用的测定 2.应用流式细胞术分析rac/rho在多瘤转化中的作用 负显性。

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.