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.

两种受体酪氨酸激酶，例如生长因子受体 并且非受体激酶如pp 60 v-src的表达可以刺激 肌醇脂质分解并产生信号如甘油二酯 (DAG)细胞内的Ca 2+。 该项目的长期目标是 确定酪氨酸激酶如何与调节 肌醇脂质信号传导。 进度报告中的数据表明， 大鼠-1成纤维细胞中v-src酪氨酸激酶的表达引起了 有丝分裂原如内皮素 刺激膜上Ins（1，4，5）P3和DAG的产生。 因此，这些变化的一个影响是产生更高水平的 细胞中的信号分子。 这一发现表明，v-src 转化导致细胞对激素高度反应 血清中的有丝分裂原产生持续激活的途径 参与转型。 目前的数据表明 v-src转化对G蛋白Gq与 磷脂酶C作为引起高反应状态的主要事件。 因此，本建议的重点是审查 Rat-1细胞中的受体-Gq-PLC-β系统，确定 src对这些膜蛋白的作用，并探讨其可能性 该V-SRC变换从系统中移除反馈抑制。 该项目将通过三个具体目标进行：（目标1）确定 v-src蛋白激酶的功能结构域在 内皮素刺激的InsP 3产生的显著放大。 这 将通过使用转染有多种细胞的Rat-1细胞来达到目的。 设计的src构建体，以允许src表达与 内皮素高反应性的出现，以确定其作用， 分子的SH 2结构域在反应和需要连接的 将v-src激酶转移到细胞膜上。 (Aim（2）确定场地 v-src在内皮素受体-Gq-PLC-β中的作用 信号复合体 这一目标将通过纯化组分来实现 正常细胞和转化细胞的信号复合物， 测定含有Gq的脂质囊泡中Gq刺激的PLC活性 外源性[3 H]-PIP 2作为底物。 内皮素的相互作用 受体和Gq将在来自表达 重组内皮素受体 (Aim（3）假设： V-SRC效应是由受体上反馈控制的丧失引起的， 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-β是否可以 用各种纯化的蛋白激酶磷酸化，然后测定 Gq刺激的PLC活性的脂质囊泡。 的能力 还将检查完整Rat-1细胞中待磷酸化的蛋白质 并与用纯化蛋白质获得的结果进行比较。