STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RAS P21 PROTEINS

RAS P21 蛋白的结构和功能表征

• 批准号: 3916837
• 负责人: J C LACAL
• 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/3916837
• 关键词: Escherichia coli; Retroviridae disease; Xenopus; acid base balance; chemical structure function; diacylglycerols; egg /ovum; gene expression; genetic manipulation; guanine nucleotide binding protein; guanosine triphosphate; guanosinetriphosphatases; human tissue; inositol phosphates; laboratory mouse; lipid metabolism; microinjections; molecular biology; monoclonal antibody; mouse sarcoma virus; neoplastic transformation; oncogenes; oncoproteins; phorbols; phosphatidylcholines; phosphatidylethanolamines; phospholipids; platelet derived growth factor; protein engineering; protein kinase C; protein sequence; protein structure function; virus protein

We have found that microinjection of the transforming but not the normal p21 protein into Xenopus laevis oocytes induced the production of 1,2-diacylglycerol (DAG) and inositol triphosphate (IP-3). While the transforming H-ras p21 was an effective mitogen for normal 3T3 cells, its mitogenic function was substantially reduced (about 80%) in protein kinase C (PKC)-depleted cells. The activity was almost completely recovered by co-microinjection of the ras p21 protein and PKC. These results provide evidence for a functional requirement of PKC for the mitogenic activity of the H.ras protein. In contrast with the results obtained in Xenopus oocytes, 3T3 cells transformed by a variety of ras oncogenes do not show any increase of basal levels of IP-3. However, DAG levels are increased about 40-50% over control, normal cells, suggesting a different source for the production of DAG than the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP-2). We have observed elevated levels of the catabolites resulting from the hydrolysis of other major phospholipids, like phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE). Transformation by the sis oncogene, as well as treatment with serum or PDGF, induced production of DAG and IP-3 but not the hydrolysis of PC or PE. These results provide evidence for at least two independent mechanisms for the production of DAG, and that both mechanisms can be activated by individual oncogene products. In a different set of experiments, we have been able to express in E. coli and purify to homogeneity the product of the rho gene from Aplysia californica, a ras-related gene. We have demonstrated that, indeed, the gene product of the rho gene (p21 rho) is a G. protein with a similar GTPase activity to that of the normal ras protein. Finally, we have been able to restore membrane localization and transforming activities of ras p21 mutants devoid of both activities by insertion of a membrane signaling peptide of the p60src product from the amino terminal.

我们已经发现，显微注射转化细胞，而不是 正常p21蛋白进入非洲爪蟾卵母细胞诱导 生产1，2-二酰基甘油（DAG）和三磷酸肌醇 （IP-3）。 而转化型H-ras p21是一种有效的丝裂原， 对于正常的3 T3细胞，其促有丝分裂功能基本上是 在蛋白激酶C（PKC）耗尽的细胞中减少（约80%）。 的 活性几乎完全恢复共微量注射 rasp 21蛋白和PKC。 这些结果提供了证据， 蛋白激酶C的促有丝分裂活性的功能要求， H.ras蛋白。 与在非洲爪蟾中获得的结果相反， 卵母细胞，3 T3细胞转化的各种ras癌基因不 显示IP-3基础水平的任何增加。 然而，DAG水平是 比对照正常细胞增加约40-50%，表明 不同的来源，用于生产DAG比水解 磷脂酰肌醇4，5-二磷酸（PIP-2）。 我们观察到 水解产生的催化剂水平升高 其他主要磷脂，如磷脂酰胆碱（PC）和 磷脂酰乙醇胺（PE）。 SIS的改造 癌基因以及血清或血小板衍生生长因子治疗，诱导 产生DAG和IP-3，但不水解PC或PE。 这些结果提供了证据，至少有两个独立的 生产DAG的机制，并且这两种机制都可以 被个别致癌基因产物激活。 在不同的布景里 的实验，我们已经能够表达在E.大肠杆菌并纯化 为了使来自Astrasia的rho基因的产物同质化， californica，一个ras相关基因。 我们已经证明， 实际上，rho基因的基因产物（p21 rho）是G.蛋白 具有与正常ras蛋白相似的GT3活性。 最后，我们已经能够恢复膜定位， 缺乏这两种基因的ras p21突变体的转化活性 活性通过插入膜信号肽的 p60 src产物的氨基末端。