ONCOGENESIS & CONTROL OF PHOSPHOINOSITIDE CYCLE/KINASE C

本提案的目标是了解p2 l 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。 差距SR 3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。 差距SH 3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p21 Ras与GAP结合时，它诱导构象变化， 暴露GAP的SH 2/3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，允许进入差距SH 2/3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， 研究差距SH 3结构域的作用。 分离的SH 3结构域的表达 抑制NIH 3 T3细胞的毒蕈碱受体依赖性转化。 将使用来自其他基因的SH 3结构域确定特异性， 定点诱变。 将研究抑制机制。 与差距SH 3结构域特异性相互作用的蛋白质将是 使用重组SH 3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SH 3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

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.