STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RAS P21 PROTEINS

RAS P21 蛋白的结构和功能表征

• 批准号: 3963547
• 负责人: S A AARONSON
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3963547
• 关键词: Escherichia coli; gene expression; genetic mapping; molecular biology; monoclonal antibody; mouse sarcoma virus

A series of ras p21 proteins carrying activating point mutations at position 12, 59 or 61, as well as the normal protein, have been expressed in E. coli. The purified proteins were analyzed for in vitro and in vivo activities. We have found that there are at least two different potential mechanisms of activation of transforming properties of ras p21 proteins. One is by means of a reduction of the GTPase activity of the protein, as found in some of the activating point mutations at position 12 or 61. Another mechanism involves acceleration of the interchange of bound nucleotides, as found in mutants with substitutions of Ala to Thr at position 59. Both mechanisms are consistent with the hypothesis that ras p21s are G-binding proteins which act through their association with GTP. After hydrolysis of GTP to GDP + Pi, the p21 molecule loses its ability to interact with other component(s) in the pathway which regulates cell growth. In parallel studies, we have expressed in E. coli a series of deletion mutants of the Harvey-ras p21 protein and utilized them to map epitopes recognized by a series of newly generated or previously reported monoclonal antibodies. By means of this strategy, we have been able to map different regions of the ras p21 molecule related to its biochemical activities. Two regions, from 5-69 and 107-164 are related to GTP-binding activity. A region from 70 to 106 was found to be related to the biological activity of the protein. Finally, microinjection experiments have been performed in which the mitogenic activity of ras p21 proteins has been linked to very early pH alterations induced in microinjected cells. Attempts are being made to correlate pH alterations induced by ras p21 proteins and other protein kinase activities.

一系列携带激活点突变的ras p21蛋白， 位置12、59或61以及正常蛋白质，已经表达 在大肠杆菌 对纯化的蛋白进行了体外和体内分析 活动 我们发现至少有两种不同的势能 ras p21蛋白转化特性的激活机制。 一种是通过降低蛋白质的GTP酶活性， 在12或61位的一些激活点突变中发现。 另一种机制涉及加速结合的交换， 核苷酸，如在具有Ala至Thr的取代的突变体中发现的。 位置59. 这两种机制都与ras p21是G结合蛋白，其通过与GTP结合而起作用。 在GTP水解为GDP + Pi后，p21分子失去了其在细胞中表达的能力。 与调节细胞的途径中的其他组分相互作用 增长 在平行研究中，我们在E.大肠杆菌一系列 Harvey-ras p21蛋白的缺失突变体，并利用它们来定位 由一系列新产生的或先前报道的表位识别 克隆抗体 通过这种策略，我们已经能够绘制出 ras p21分子的不同区域与其生物化学性质有关， 活动 5-69和107-164两个区域与GTP结合相关 活动 70到106的区域被发现与 蛋白质的生物活性。 最后，显微注射实验 其中ras p21蛋白的促有丝分裂活性 与显微注射细胞中诱导的非常早期的pH值改变有关。 人们试图将rasp 21诱导的pH改变与 蛋白质和其他蛋白激酶活性。