CHARACTERIZATION OF FACTORS CRITICAL FOR RAS ACTIVITY

研究增殖的分子控制的一个中心焦点是 细胞ras基因。这种基因在人类进化过程中的重要作用 细胞增殖的控制是通过它的频率来指示的 在自然发生的肿瘤中的突变，以及注射 抑制性抗体或突变体可阻止细胞增殖。目标是 这项提议的目的是进一步调查 基因是受控制的。Ras蛋白被认为是生物学上的。 在与GTP结合时激活，直到RAS的天然GTP酶活性为止 将结合的核苷酸转换为GDP。其他细胞因素包括 显然参与了对这种转换的控制，包括一个 GTP酶激活蛋白(GAP)。这个实验室过去的研究 表明GAP活性在某些物质存在时被抑制 已知在有丝分裂时产生的脂类 刺激。在脂质研究的过程中，确定了一个因素 其具有抑制脂质中ras的GTP酶活性的能力- 依赖的态度。这是这里描述的工作的第一个目标 为了纯化这种GTP酶抑制蛋白，克隆该基因，并进一步 描述其活动的特征。朝着这一目标取得的重要进展是 这是本提案首次提交以来所作的决定。这些信息 在这些研究中获得的结果可能有助于确定这种蛋白质在 正常对照的增殖，以及正常对照的变化 在肿瘤发展过程中的作用机制。存在多个 细胞ras活性所需的细胞因子显然是 在显微注射研究中显示出两个独立的显性 抑制性ras突变蛋白。这项提议的第二个目标是 为了确定GAP、GTP酶抑制蛋白还是NF-1蛋白 是这两种抑制剂的细胞靶点。关键人物 这一努力的重要性与这样一个事实有关，即其中一个突变体 对突变的ras蛋白有特异性的抑制作用。智能交通系统的定义 靶标可能提供了一种选择性抑制酶活性的方法 肿瘤中突变的ras基因。最后，最近的证据表明，这种基因 突变型神经纤维瘤病1型(NF-1与GAP有关) 核苷酸序列和刺激GTP酶活性的能力 拉斯。此外，该实验室最近的研究表明， 脂类抑制了核因子-1蛋白。有相似之处， 能够抑制核因子-1的脂类的显著差异 与GAP相比。不幸的是，因为核因子-1蛋白非常 大的，只有核因子-1的催化片段可供研究。 现在必须对全长蛋白质进行分析。该基因的突变 是常见的，并具有重要的临床后果。获得的信息 在这项提案中将是至关重要的，以确定脂类在 核因子-1的控制，这种控制的变化可能 是由自然发生的突变造成的，如果这种改变发挥作用 在临床表现中的作用。总而言之，所获得的信息 在这里概述的研究预计将有助于理解 参与控制增殖的一般分子机制， 并提供如何控制这些过程的线索。

A central focus in studies of the molecular control of proliferation is the cellular ras gene. The critical importance of this gene in the control of cellular proliferation is indicated by the frequency of its mutation in naturally occurring tumors, and the fact that injection of inhibitory antibodies or mutant blocks cellular proliferation. The goal of this proposal is to further investigate the mechanism by which this gene is controlled. The ras protein is believed to be biologically active when bound to GTP, until the native GTPase activity of ras converts the bound nucleotide to GDP. Other cellular factors are apparently involved in the control of this conversion, including a GTPase activating protein (GAP). Past studies from this laboratory indicate that GAP activity is inhibited in the presence of certain lipids which are known to be produced at the time of mitogenic stimulation. In the course of lipid studies a factor was identified which had the ability to inhibit GTPase activity of ras in a lipid- dependent manner. It is the first objective of the work described here to purify this GTPase inhibitory protein, clone the gene, and further characterize its activity. Important progress toward this goal has been made since the initial submission of this proposal. The information gained in these studies might help define the role of this protein in the normal control proliferation, and alterations of this normal control mechanism during the development of tumors. The presence of multiple cellular factors required for the activity of cellular ras was clearly demonstrated in microinjection studies with two separate dominant inhibitory ras mutant proteins. A second objective of this proposal is to determine whether GAP, the GTPase inhibitory protein, or NF-1 protein is the cellular target of either of these inhibitors. The critical importance of this effort relates to the fact that one of these mutants is specifically inhibitory to mutant ras proteins. Definition of its target might provide a means to selectively inhibit the activity of mutant ras in tumors. Finally, recent evidence indicates that the gene mutated in neurofibromatosis type 1 (NF-1 is related to GAP both in nucleotide sequence and in the ability to stimulate the GTPase activity of ras. Furthermore, recent studies from this laboratory indicate that lipids inhibit the NF-1 protein. There were similarities and significant differences in the types of lipids able to inhibit NF-1 compared to GAP. Unfortunately, because the NF-1 protein is extremely large, only the catalytic fragment of NF-1 has been available for study. The full-length protein must now be analyzed. Mutations in this gene are common and have important clinical consequences. Information gained in this proposal will be essential to determine the role of lipids in the control of NF-1, alterations in this type of control which might result from naturally occurring mutations, and if such alterations play a role in clinical manifestations. In summary, the information gained in the studies outlined here are expected to aid in understanding the general molecular mechanisms involved in the control of proliferation, and provide clues as to how these processes might be controlled.