ONCOGENESIS & CONTROL OF PHOSPHOINOSITIDE CYCLE/KINASE C

癌发生

• 批准号: 2089661
• 负责人: IAN G MACARA
• 金额: $ 24.57万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2089661
• 关键词: 3T3 cells; biological signal transduction; cell transformation; conformation; fusion gene; genetic mapping; guanine nucleotide binding protein; guanosinetriphosphatases; intermolecular interaction; lipids; molecular cloning; molecular oncology; muscarinic receptor; mutant; oncoproteins; phosphatidylinositols; protein kinase C; protein structure function; site directed mutagenesis; tissue /cell culture; transcription factor

The goals of this proposal are to understand the regulation of the p2l Ras protoncogene protein by Ras-GRF (Guanine Nucleotide Release Factor) and pl20 Ras-GAP (GTPase Activating Protein), and to elucidate the function of the GAP SR3 domain. There are three major aims: (l) mapping the sequences in Ras that specify sensitivity to GRF; (2) the ramifications of a nev model for the interaction of Ras with GAP; and (3) the mechanism of interaction of the GAP SH3 domain with signal transduction via muscarinic receptors, using a novel biological assay for this interaction. The first aim will complete ongoing efforts to map region(s) of the Ras protein that confer sensitivity to the Ras-specific GRF, by alanine- scanning mutagenesis. Mutants that abolish sensitivity to GRF will be tested biologically by introduction into dominant-negative form of Ras (S17N). The purpose of the second aim is to understand the mechanism of interaction of Ras with GAP. A model will be tested which predicts that when p2l Ras associates with GAP, it induces a conformational change that exposes the SH2/3 domains of GAP, enabling them to engage targets such as specific Tyrosine-phosphorylated proteins. The GAP-target complex is proposed to possess an downstream effector function. The affinity of GAP for Tyr-phosphorylated proteins is expected to be lower in the absence than in the presence of p2l Ras:GTP. This prediction will be tested both in intact cells, using the dominant negative N17Ras to suppress Ras:GTP formation; and in vitro, using GST-fusions of GAP fragments, phospho- peptides and p2l Ha-c-Ras. The interaction of GAP with specific lipids will also be investigated to determine whether lipids interfere with the putative conformational change that allows access to the GAP SH2/3 domain. The third aim utilizes a new, focus suppression assay for GAP function to study the role of the GAP SH3 domain. Expression of isolated SH3 domain inhibits muscarinic receptor-dependent transformation of NIH 3T3 cells. Specificity will be determined using SH3 domains from other genes and site-directed mutagenesis. The inhibitory mechanism will be investigated. Proteins that interact specifically with the GAP SH3 domains will be identified using recombinant SH3 as a probe, and cloned using the yeast two-hybrid system. These studies will provide important new information on SH3 domain function, and on the mechanism of signal transduction by muscarinic receptors.

该提案的目标是了解 p2l Ras 的监管 Ras-GRF（鸟嘌呤核苷酸释放因子）的原癌基因蛋白和 pl20 Ras-GAP（GTP酶激活蛋白），并阐明其功能 GAP SR3 域。 有三个主要目标： (l) 绘制序列 在 Ras 中指定对 GRF 的敏感性； （2）新规的影响 Ras 与 GAP 相互作用的模型； (3) 机制 GAP SH3 结构域与毒蕈碱信号转导的相互作用 受体，使用一种新的生物测定法来检测这种相互作用。 第一个目标是完成绘制拉斯区域地图的持续努力 丙氨酸赋予 Ras 特异性 GRF 敏感性的蛋白质 扫描诱变。消除对 GRF 敏感性的突变体将是 通过引入 Ras 显性失活形式进行生物学测试 （S17N）。 第二个目标的目的是了解其机制 Ras 与 GAP 的相互作用。 将测试一个模型，该模型预测 当 p2l Ras 与 GAP 结合时，它会引起构象变化， 暴露 GAP 的 SH2/3 域，使它们能够参与诸如 特定的酪氨酸磷酸化蛋白。 GAP-目标复合体是 提议拥有下游效应器功能。 GAP亲和力 对于酪氨酸磷酸化蛋白，预计在不存在的情况下会较低 与 p2l Ras:GTP 存在时相比。 这一预测将得到检验 在完整细胞中，使用显性失活 N17Ras 抑制 Ras:GTP 形成；在体外，使用 GAP 片段的 GST 融合，磷酸化 肽和 p2l Ha-c-Ras。 GAP 与特定脂质的相互作用 还将进行研究以确定脂质是否会干扰 允许访问 GAP SH2/3 结构域的假定构象变化。 第三个目标利用一种新的 GAP 功能焦点抑制测定法来 研究 GAP SH3 结构域的作用。 分离的 SH3 结构域的表达 抑制 NIH 3T3 细胞的毒蕈碱受体依赖性转化。 特异性将使用其他基因的 SH3 结构域来确定 定点诱变。 将研究抑制机制。 与 GAP SH3 结构域特异性相互作用的蛋白质将是 使用重组SH3作为探针进行鉴定，并使用酵母进行克隆 双混合系统。 这些研究将提供重要的新信息 SH3结构域功能及信号转导机制 毒蕈碱受体。