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: 6217364
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6217364
• 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.

两种受体酪氨酸激酶，以生长因子受体为例 非受体激酶如 pp60v-src 的表达可以刺激 肌醇脂质分解并产生信号，例如二酰甘油 (DAG) 和细胞内的 Ca2+。 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 肌醇脂质信号传导。 进度报告中的数据表明 Rat-1 成纤维细胞中 v-src 酪氨酸激酶的表达导致 显着增强有丝分裂原（例如内皮素）的能力 刺激膜产生 Ins(1,4,5)P3 和 DAG。 因此，这些变化的影响之一是产生更高水平的 细胞内的信号分子。 这一发现表明 v-src 转化导致细胞对激素过度反应 和血清中的丝裂原产生持续激活的途径 参与转型。 目前的数据表明了效果 v-src 转化对 G 蛋白 Gq 相互作用的影响 磷脂酶 C 作为导致高反应状态的主要事件。 因此，本提案的重点是审查 Rat-1 细胞中的受体 - Gq - PLC-beta 系统，确定 src对这些膜蛋白的作用并探索可能性 v-src 变换消除了系统的反馈抑制。 该项目将通过三个具体目标进行：（目标 1）定义 v-src 蛋白激酶功能域在 内皮素刺激的 InsP3 产生显着放大。 这 将通过使用转染多种物质的 Rat-1 细胞来实现这一目标 src 构造的设计允许 src 表达式与 内皮素高反应性的出现，以确定其作用 响应中分子的 SH2 结构域以及连接的需要 v-src 激酶连接到膜上。 （目标 2）确定 内皮素受体内 v-src 的作用 - Gq - PLC-Beta 信号复合体。 这个目标将通过纯化成分来实现 之前正常细胞和转化细胞的信号复合物 测定含有 Gq 刺激的脂质囊泡中的 PLC 活性 外源[3H]-PIP2作为底物。 内皮素的相互作用 受体和 Gq 将在表达 Sf9 细胞的膜中进行检查 重组内皮素受体。 （目标 3）探索以下假设： v-src 效应是由受体反馈控制丧失引起的 刺激磷脂酰肌醇分解。 由于反馈抑制 该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 是否可以执行 用各种纯化的蛋白激酶磷酸化，然后进行测定 脂质囊泡中 Gq 刺激的 PLC 活性。 的能力 还将检查完整 Rat-1 细胞中磷酸化的蛋白质 并与纯化蛋白质获得的结果进行比较。