ONCOGENESIS & CONTROL OF PHOSPHOINOSITIDE CYCLE/KINASE C

癌发生

• 批准号: 2007480
• 负责人: IAN G MACARA
• 金额: $ 26.45万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2007480
• 关键词: 3T3 cells; biological signal transduction; cell transformation; conformation; fusion gene; genetic mapping; guanine nucleotide binding protein; guanosinetriphosphatase activating protein; 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.

本提案的目标是了解p2l Ras的调节 通过Ras-GRF（鸟嘌呤核苷酸释放因子）和 p120 Ras-GAP（GTP酶激活蛋白），并阐明p120 Ras-GAP的功能。 GAP SR3结构域。 主要有三个目的：（1）绘制序列 在Ras中，指定对GRF敏感性;（2）NEV的分支 Ras与GAP相互作用的模型;（3）Ras与GAP相互作用的机制。 GAP SH3结构域与通过毒蕈碱的信号转导的相互作用 受体，使用一种新的生物测定这种相互作用。 第一个目标是完成正在进行的绘制拉斯地区地图的工作 蛋白质，赋予敏感性Ras特异性GRF，丙氨酸- 扫描诱变消除对GRF的敏感性的突变体将是 通过引入显性阴性形式的Ras进行生物学测试 （S17N）。 第二个目标的目的是了解 Ras与GAP的相互作用。 将测试一个模型，该模型预测， 当p21Ras与GAP结合时，它诱导构象变化， 暴露GAP的SH2/3结构域，使其能够攻击目标， 特异性酪氨酸磷酸化蛋白。 GAP-靶标复合物是 具有下游效应子功能。 GAP的亲和力 对于Tyr-磷酸化的蛋白质，预期在不存在 比在p21 Ras：GTP存在的情况下更明显。 这一预测将在以下两个方面得到检验： 在完整细胞中，使用显性负性N17 Ras抑制Ras：GTP 在体外，使用GAP片段的GST融合物，磷酸化， 肽和p21 Ha-c-Ras。 GAP与特定脂质的相互作用 也将进行研究，以确定脂质是否干扰 假定的构象变化，允许进入GAP SH2/3结构域。 第三个目的是利用一种新的GAP功能的焦点抑制测定， 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。 将使用来自其他基因的SH3结构域确定特异性， 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作为探针鉴定，并使用酵母克隆 双混合动力系统 这些研究将提供重要的新信息 对SH3结构域功能的影响，以及对信号转导机制的影响。 毒蕈碱受体