PTDINS-4-PHOSPHATE 5-KINASE IN P21RAC SIGNALING

描述：(改编自摘要)这项工作的主要目标 应用是确定磷脂酰肌醇激酶在 通过小的GTP结合蛋白Rac进行信号传递。RAC参与了 肌动蛋白细胞骨架在细胞膜褶皱、运动、细胞中的调控 细胞形态的黏附和维持。申请者提交 RAC与PI-4 5K和PI-3相互作用和可能调节的证据 激酶(等电点3K)和研究结果表明，RAC对 细胞骨架可以通过产生特定的磷脂来调节， 由于磷脂酰肌醇-4，5-二磷酸(PIP2)结合并调节 肌动蛋白调节蛋白的功能。建议进行实验，以 确定PI-4 5K在体内的作用及Rac对其的影响 这种酶的活性和在细胞中的定位。与RAC关联的 对PI-45K进行纯化，得到一个克隆。RAC的区域 而相互作用所需的激酶将是 经鉴定，并构建了具有特定能力丧失的突变体 参与这种相互作用，用作主要的阴性试剂 在研究RAC/PIP2/细胞骨架信号转导中。RAC、RAC之间的相互作用 PI-45K和其他调节蛋白(RhoGDI、交换蛋白等)。 将用纯重组蛋白进行研究以确定 PIP2合成的活化机制。此外，实验还包括 描述以确定细胞是否需要RAC/PI 3K复合体 粘附性以及PI 3K是否作用于RAC下游，使用PI 3K 显性否定结构。

DESCRIPTION: (Adapted from the abstract) The principal goal of this application is to define the role of phosphatidylinositol kinases in signaling by the small GTP-binding protein Rac. Rac is involved in regulating the actin cytoskeleton in membrane ruffling, motility, cell adherence and maintenance of cell shape. The applicant presents evidence that Rac interacts with and may regulate a PI-4 5K and PI-3 kinase (PI 3K) and findings suggesting that Rac's regulation of the cytoskeleton may be mediated by production of specific phospholipids, since phosphatidylinositol-4,5-bisphosphate (PIP2) binds to and regulates function of actin regulatory proteins. Experiments are proposed to determine the role of PI-4 5K in vivo and to determine effects of Rac on this enzyme's activity and localization in cells. The Rac-associated PI-4 5K will be purified and a cDNA clone obtained. The regions of Rac and the kinase necessary for interacting with one another will be identified, and mutants constructed with specific loss of ability to participate in this interaction, for use as dominant negative reagents in studying Rac/PIP2/cytoskeleton signaling. Interactions among Rac, PI-4 5K and other regulatory proteins (RhoGDI, exchange proteins, etc.) will be studied with pure recombinant proteins to determine the mechanism of activation of PIP2 synthesis. In addition, experiments are described to determine whether a Rac/PI 3K complex is necessary for cell adherence and whether PI 3K acts downstream of Rac, using a PI 3K dominant negative construct.