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的调节 RAS-GRF（鸟嘌呤核苷酸释放因子）和 PL20 RAS-GAP（GTPase激活蛋白），并阐明 GAP SR3域。 有三个主要目的：（l）映射序列 在指定对GRF敏感性的RA中； （2）NEV的后果 RAS与间隙相互作用的模型； （3）机制 GAP SH3结构域与通过毒桃体的信号转导的相互作用 受体，使用一种新型的生物学测定法进行这种相互作用。 第一个目标将完成持续绘制RAS区域的努力 通过丙氨酸 - 扫描诱变。废除对GRF敏感的突变体将是 通过引入主要阴性形式在生物学上测试的RAS （S17N）。 第二个目的的目的是了解 RAS与间隙的相互作用。 将测试模型，以预测 当P2L RA与间隙相关时，它会引起构象变化 揭示差距的SH2/3域，使它们能够参与等目标 特定的酪氨酸磷酸化蛋白。 缝隙目标综合体是 提议具有下游效应子函数。 差距的亲和力 因为在不存在的情况下，预计磷酸化的蛋白质将较低 与P2L RAS的存在相比：GTP。 该预测将两者都测试 在完整的细胞中，使用主要的负N17RA抑制RAS：GTP 形成；在体外，使用隙片段的GST融合，磷酸 肽和P2L HA-C-RAS。 间隙与特定脂质的相互作用 还将研究以确定脂质是否干扰 推定的构象变化，允许访问GAP SH2/3域。 第三个目标利用新的，焦点抑制差距功能 研究GAP SH3域的作用。 孤立的SH3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域确定，并且 定向诱变。 将研究抑制机制。 专门与GAP SH3结构域相互作用的蛋白质将是 使用重组SH3鉴定为探针，并使用酵母克隆 两个杂交系统。 这些研究将提供重要的新信息 在SH3域功能以及信号转导的机理上 毒蕈碱受体。