CONTROL OF PHOSPHOLIPASE C IN V-SRC TRANSFORMED CELLS

V-SRC 转化细胞中磷脂酶 C 的控制

• 批准号: 6311496
• 负责人: JAMES Carlton GARRISON
• 金额: $ 1.8万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6311496
• 关键词: G protein; Sf9 cell line; active sites; biological signal transduction; endothelin; intermolecular interaction; isozymes; membrane model; membrane proteins; mitogens; mutant; neoplastic transformation; oncogenes; phosphatidylinositols; phospholipase C; protein kinase C; protein tyrosine kinase; receptor expression; tissue /cell culture; transfection; western blottings

Both receptor tyrosine kinases as exemplified by growth factor receptors and expression of non-receptor kinases such as pp60v-src can stimulate inositol lipid breakdown and generate signals such as diacylglycerol (DAG) and Ca2+ within the cell. The long term goal of the project is to determine how tyrosine kinases interact with the mechanisms regulating inositol lipid signalling. Data in the Progress Report suggest that expression of the v-src tyrosine kinase in Rat-1 fibroblasts causes a dramatic enhancement of the ability of mitogens such as endothelin to stimulate the production of Ins(1,4,5)P3 and DAG from the membrane. Thus, one effect of these changes is to generate increased levels of signalling molecules in the cell. This finding suggests that v-src transformation results in a cell that is hyper-responsive to the hormones and mitogens in serum yielding constant activation of the pathways participating in transformation. The current data points to an effect of v-src transformation on the interaction of the G protein, Gq, with phospholipase C as a primary event causing the hyper-responsive state. Therefore, this proposal is focused on examining the nature of the receptor - Gq - PLC-beta system in Rat-1 cells, determining the site of action of src on these membrane proteins and exploring the possibility that v-src transformation removes feedback inhibition from the system. The project will be conducted via three Specific Aims: (Aim 1) To define the role of the functional domains of the v-src protein kinase in the dramatic amplification of endothelin-stimulated InsP3 production. This aim will be approached by using Rat-1 cells transfected with a variety of src constructs designed to allow correlation of src expression with appearance of endothelin hyper-responsiveness, to determine the role of the molecule's SH2 domain in the response and the need for attachment of the v-src kinase to the membrane. (Aim 2) To determine the site of action of v-src within the endotheline receptor - Gq - PLC-Beta signalling complex. This aim will be approached by purifying components of the signalling complex from normal and transformed cells prior to assaying Gq-stimulated PLC activity in lipid vesicles containing exogenous [3H]-PIP2 as the substrate. The interaction of the endothelin receptor and Gq will be examined in membranes from Sf9 cells expressing recombinant endothelin receptors. (Aim 3) To explore the hypothesis that the v-src effect is caused by a loss of feedback control on receptor- stimulated phosphatidylinositol breakdown. Since feedback inhibition of this system potentially occurs via protein kinases, these experiments will be performed by examining whether Gq and/or PLC-Beta can be phosphorylated with various purified protein kinases followed by assay of Gq-stimulated PLC activity in lipid vesicles. The ability of the proteins to be phosphorylated in intact Rat-1 cells will also be examined and compared with the results obtained with purified proteins.

以生长因子受体为例的两种受体酪氨酸激酶 而非受体激酶的表达，如pp60v-src，可以刺激 肌醇能分解脂质并产生二酰甘油等信号 (DAG)和细胞内的钙离子。该项目的长期目标是 确定酪氨酸激酶如何与调节机制相互作用 肌醇脂质信号。进度报告中的数据表明， V-src酪氨酸激酶在大鼠成纤维细胞中的表达引起 内皮素等有丝分裂原的能力显著增强 刺激膜产生Ins(1，4，5)P3和DAG。 因此，这些变化的一个影响是产生更多的 细胞中的信号分子。这一发现表明，v-src 转化后的细胞对激素高度敏感 和血清中的有丝分裂原产生持续激活的通路 参与转型。当前的数据指向了一个效果 V-src转化对G蛋白Gq与G蛋白相互作用的影响 磷脂酶C是导致高反应状态的主要事件。 因此，这项建议的重点是审查 受体-GQ-PLC-β系统在大鼠-1细胞中的定位 Src对这些膜蛋白的作用及其可能性探讨 该v-src变换从系统中消除了反馈抑制。 该项目将通过三个具体目标进行：(目标1)确定 V-src蛋白激酶功能结构域在血管紧张素转换酶中的作用 显著放大内皮素刺激的InsP3的产生。这 用不同种类的转基因大鼠-1细胞来接近目的 的src构造，以允许src表达与 内皮素高反应性的出现，以确定其作用 分子在反应中的SH2结构域和对连接的需要 V-src蛋白结合到细胞膜上。(目标2)确定地点 V-src在内皮素受体-Gq-PLC-Beta中的作用 信令复合体。这一目标将通过净化组件来实现 来自正常细胞和转化细胞的信号复合体 测定GQ刺激的脂泡中PLC活性 以外源[~3H]-PIP2为底物。内皮素的相互作用 受体和GQ将在Sf9细胞膜上进行检测，表达 重组内皮素受体。(目标3)探索假设 V-src效应是由于失去了对受体的反馈控制- 刺激磷脂酰肌醇分解。由于反馈抑制 这个系统可能通过蛋白激酶发生，这些实验 将通过检查GQ和/或PLC-Beta是否可以 用各种纯化的蛋白激酶磷酸化后的检测 对GQ刺激的脂泡PLC活性的影响。美国人的能力 还将检测在完整的大鼠1号细胞中被磷酸化的蛋白质 并与纯化蛋白的结果进行了比较。