CHARACTERIZATION OF FACTORS CRITICAL FOR RAS ACTIVITY

RAS 活性关键因素的表征

• 批准号: 3198208
• 负责人: DENNIS W STACEY
• 金额: $ 25.56万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198208
• 关键词: cell growth regulation; chromatography; cow; enzyme inhibitors; gene mutation; guanine nucleotide binding protein; guanosinetriphosphatases; laboratory mouse; laboratory rabbit; microinjections; molecular cloning; neoplastic transformation; nucleic acid sequence; oncogenes; oncoproteins; phospholipids; prostaglandins; protein sequence; tissue /cell culture

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.

增殖的分子控制研究的中心焦点是 细胞ras基因。 这个基因在人类基因组中的重要性 细胞增殖的控制由其频率指示。 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阻断细胞增殖。 目标 这项建议的目的是进一步研究这种机制， 基因被控制。 ras蛋白被认为是生物学上 当与GTP结合时有活性，直到ras的天然GTP酶活性 将结合的核苷酸转化为GDP。 其他细胞因子包括 显然参与控制这一转换，包括一个 GTP酶激活蛋白（GAP）。该实验室过去的研究 表明GAP活性在某些 已知在促有丝分裂时产生的脂质 刺激. 在脂质研究过程中， 其具有抑制脂质中ras的GT3活性的能力， 依赖的方式。 这是这里所描述的工作的第一个目标 为了纯化该GT3抑制蛋白，克隆该基因，并进一步 描述其活动。 在实现这一目标方面取得了重要进展， 自首次提交该提案以来， 的信息 在这些研究中获得的结果可能有助于确定这种蛋白质在 正常对照增殖，以及该正常对照的改变 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， 在显微注射研究中证实， 抑制性Ras突变蛋白。 本建议的第二个目标是 以确定GAP、GT3抑制蛋白或NF-1蛋白 是这些抑制剂的细胞靶点。 临界 这一努力的重要性与这些突变体中的一种 特异性抑制突变ras蛋白。 界定其 靶点可能提供一种选择性抑制 肿瘤中的突变ras。 最后，最近的证据表明， 在神经纤维瘤病1型中突变（NF-1与GAP相关， 核苷酸序列和刺激GT3活性的能力 的RAS。 此外，该实验室最近的研究表明， 脂质抑制NF-1蛋白。 有相似之处， 能够抑制NF-1的脂质类型的显著差异 与GAP相比。 不幸的是，由于NF-1蛋白质是非常 由于NF-1的分子量很大，只有NF-1的催化片段可用于研究。 现在必须分析全长蛋白质。 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 在这项建议将是至关重要的，以确定脂质的作用， NF-1的控制，这种控制类型的改变， 自然发生的突变，如果这种变化发挥作用， 在临床表现中的作用。 总之，所获得的信息 在这里概述的研究，预计将有助于了解 参与增殖控制的一般分子机制， 并提供如何控制这些过程的线索。