ONCOGENESIS & CONTROL OF PHOSPHOINOSITIDE CYCLE/KINASE C

这项提案的目标是了解p2l RAS的监管 原癌基因蛋白的Ras-GRF(鸟核苷酸释放因子)和 PL20RAS-GAP(GTP酶激活蛋白)，并阐明其功能。 缺口SR3结构域。主要有三个目标：(L)绘制序列图 在RAS中，对GRF指定敏感性；(2)新的分支 RAS与GAP相互作用的模型；(3)RAS与GAP的相互作用机理 GAP SH3结构域与M胆碱信号转导的相互作用 受体，使用一种新的生物测试来进行这种相互作用。 第一个目标是完成正在进行的绘制RAS区域(S)的努力 对RAS特异性GRF敏感的蛋白质，通过丙氨酸- 扫描诱变。消除对GRF敏感性的突变体将是 通过引入显性-阴性RAS进行生物学测试 (S17N)。 第二个目标的目的是为了了解 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，它诱导了构象变化， 暴露GAP的SH2/3域，使他们能够与以下目标交战 特定的酪氨酸磷酸化蛋白。缺口-目标复合体是 建议具备下游效应器功能。GAP的亲和力 在没有酪氨酸的情况下，酪氨酸磷酸化蛋白的含量预计会更低 而不是在p2l RAS：GTP存在下。这一预测将在两个方面得到验证 在完整细胞中，使用显性负N17Ras抑制RAS：GTP 在体外，使用GST-GST融合的GAP片段，磷酸化- 多肽和p21 Ha-c-Ras。GAP与特定脂类的相互作用 还将进行调查，以确定血脂是否会干扰 假定的构象变化，允许进入GAP SH2/3结构域。 第三个目标是利用一种新的、针对间隙函数的焦点抑制分析来 研究GAP SH3结构域的作用。分离的SH3结构域的表达 抑制NIH3T3细胞的M受体依赖性转化。 特异性将使用其他基因的SH3结构域和 定点突变。并对其抑制机理进行了探讨。 与GAP SH3结构域特异相互作用的蛋白质将是 以重组SH3为探针进行鉴定，酵母克隆 双杂交系统。这些研究将提供重要的新信息 关于SH3结构域的功能，以及关于SH3的信号转导机制 毒鼠碱受体。

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.