STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RAS P21 PROTEINS

我们发现微量注射转化，而不是 正常p21蛋白导入非洲爪哇卵母细胞诱导 1，2-二酰甘油(DAG)和三磷酸肌醇的生产 (IP-3)。而转化的H-ras p21是一种有效的有丝分裂原 对于正常的3T3细胞，它的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%)。这个 联合微量注射可使活动几乎完全恢复。 Ras p21蛋白和PKC。这些结果为 PKC对细胞有丝分裂活性的功能要求 H.ras蛋白。与在非洲爪哇获得的结果形成对比 卵母细胞，由多种ras癌基因转化的3t3细胞不 显示IP-3的基础水平有任何升高。但是，DAG级别为 比正常对照细胞增加约40%-50%，这表明 生产DAG的来源不同于水解液 磷脂酰肌醇4，5-二磷酸(PIP-2)。我们观察到 由水解物引起的分解代谢水平升高 其他主要磷脂，如磷脂酰胆碱(PC)和 磷脂酰乙醇胺(PE)。SIS的转型 癌基因，以及用血清或PDGF治疗 生产DAG和IP-3，但不能水解PC或PE。 这些结果为至少两个独立的 产生DAG的机制，这两种机制都可以 被个别癌基因产品激活。在不同的一组中 在实验中，我们已经能够在大肠杆菌中表达并纯化 海兔Rho基因产物的同源性研究 Calfornica，ras相关基因。我们证明了这一点， 事实上，Rho基因(P21Rho)的基因产物是一种G蛋白。 具有与正常ras蛋白相似的GTP酶活性。 最后，我们已经能够恢复膜的局部化和 两者缺失的ras p21突变体的转化活性 通过插入一种膜信号肽的活性 氨基末端的p60src产物。

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.