EFFECTORS AND REGULATORS OF NORMAL AND ONCOGENIC RAS

RAS基因对真核生物的细胞生长控制是必不可少的。 此外，激活的ras基因是最常见的癌基因之一。 在人类肿瘤中。在酿酒酵母中，一种激活的ras 等位基因(类似于哺乳动物ras的致癌突变)导致 腺酰环化酶过度刺激。循环酶活性升高的结果 在不受控制的细胞增殖中，无法阻止对 营养缺乏和对热休克的敏感度。尽管很棒 了解酵母中ras的生物化学方面的进展 哺乳动物细胞中的信号转导途径各不相同，目前尚不清楚。这项建议 通过选择抑制热量的能力来描述隔离 携带激活的ras等位基因的酵母细胞的冲击敏感性 哺乳动物编码ras相互作用蛋白的cDNA。 我们最初的cdna分离株包括磷酸二酯酶，这种酶被 降低细胞内cAMP水平。来自我们的另外三个克隆 哺乳动物的cDNA表达文库显然编码的蛋白质 通过与RAS(两者)直接相互作用干扰RAS功能 野生型和致癌性)。这些可能会抑制ras功能。 或者通过降低单元中的活动RAS的比例(向下 监管)，或通过与RAS形成非生产性复合体并降低 其有效浓度(显性负性干扰)。后者 类应包括绑定到RAS但被 无法传输生长信号。所有这些分离株都是部分 CDNA。为了确定这些基因产物的正常功能，完整的 长度的cDNA将被分离并在我们的模型系统中表达。这个 干扰RAS功能所需的最小部分也将是 由删除映射确定。 这项工作的一个中心重点是细胞培养实验，以 确定正常基因产物在哺乳动物细胞中的作用。如果 他们通常充当ras的抑制者或下行调节者，然后 过度生产应该会干扰哺乳动物细胞中的ras功能，因为 根据它们阻止或逆转RAS变换的能力来判断。 如果我们的全长克隆编码效应蛋白，那么它们的 在哺乳动物细胞中的过度表达可能会提高而不是拮抗 RAS功能。对ras诱导的分化的影响也将是 检查过了。 这些研究的结果应该会极大地扩展我们对 Ras在哺乳动物细胞中的功能。它们也应该带来更好的 对肿瘤发生的一般理解。

ras genes are essential for cellular growth control in eucaryotes. Moreover, activated ras genes are among the most commonly found oncogenes in human tumors. In the yeast Saccharomyces cerevisiae, an activated ras allele (analogous to oncogenic mutations in mammalian ras) causes the overstimulation of adenylyl cyclase. Elevated cyclase activity results in uncontrolled cell proliferation, an inability to arrest in response to nutrient starvation and acute sensitivity to heat shock. Despite great strides in understanding the biochemistry of ras in yeast, the ras pathway in mammalian cells differs and remains unclear. This proposal describes the isolation, by selecting for the ability to suppress heat shock sensitivity in yeast cells harboring an activated ras allele, of mammalian cDNAs that encode ras-interacting proteins. Our initial cDNA isolates include phosphodiesterases that 'suppress' by lowering intracellular cAMP levels. Three additional clones from our mammalian cDNA expression library apparently encode proteins that interfere with ras function through a direct interaction with ras (both wild type and oncogenic). These are likely to suppress ras function either by decreasing the proportion of active ras in the cell (down regulation), or by forming a nonproductive complex with ras and lowering its effective concentration (dominant negative interference). The latter class should include truncated ras effectors that bind to ras but are unable to transmit growth signals. All of these isolates were partial cDNAs. To ascertain the normal functions of these gene products, full length cDNAs will be isolated and expressed in our model system. The minimum portion required for interference with ras function will also be determined by deletion mapping. A central focus of this work involves cell culture experiments to determine the role of the normal gene products in mammalian cells. If they normally act as ras suppressors or down regulators, then overproduction should interfere with ras function in mammalian cells, as judged by their ability to either block or reverse ras transformation. If our full length clones encode effector proteins, then their overexpression in mammalian cells may elevate, rather than antagonize, ras function. Effects on ras-induced differentiation will also be examined. The results of these studies should greatly expand our knowledge of the function of ras in mammalian cells. They should also lead to a better understanding of oncogenesis in general.