ROLE OF E1A IN INDUCING CELLULAR DNA SYNTHESIS

E1A 在诱导细胞 DNA 合成中的作用

• 批准号: 2415350
• 负责人: MLNikki L Harter
• 金额: $ 17.98万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415350
• 关键词: 3T3 cells; Adenoviridae; DNA replication; antisense nucleic acid; biological signal transduction; cell cycle; cell cycle proteins; cell growth regulation; cyclins; growth inhibitors; guanine nucleotide binding protein; host organism interaction; immunoprecipitation; microinjections; monoclonal antibody; mutant; phosphoproteins; phosphorylation; protein structure function; site directed mutagenesis; tissue /cell culture; transfection; transforming growth factors; virus protein; western blottings

The primary goal of this proposal is to define more precisely the activities of proteins that mediate cell cycle control through the use of the adenovirus E1A protein. E1A is a multifunctional phosphoprotein. Its versatility is reflected in the ability to induce cellular DNA synthesis, overcome the growth inhibitory effect of TGF-beta in epithelial cells, and override the requirement for cellular ras activity in promoting cells into S phase. These activities of E1A in overriding the requirement for ras activity. Experiments have been proposed to determine which, if any, of the E1A-associated proteins are required by E1A to induce DNA synthesis in cells that have lost ras activity. We hypothesize that the identities of these proteins will be important since it will establish, for the first time, whether growth suppressors such as pRb, p107 and/or p130 are downstream targets of ras. The second aim of this proposal is to determine the mechanisms by which E1A can overcome the growth-inhibitory effects of TGF-beta in epithelial cells. We hypothesize that E1A can restore kinase activity to cdks by disabling the inhibitory effects of p27Kipl in TGF- beta-treated cells, and present two experimental models in which this may occur. Another aim of this proposal is to identify the cellular proteins that are required by E1A to induce cellular DNA synthesis in quiescent cells. Mutant E1A proteins that fail to interact with a specific set of cellular proteins will be created and tested by microinjection for their ability to initiate DNA synthesis. Also, antibodies or anti-sense oligonucleotides that can neutralize the activities of cyclindependent kinases and other proteins important to the G1 to S transition will be used as tools, to determine whether any of these proteins are needed by E1A to stimulate DNA synthesis. Finally, since E1A is a phosphoprotein, the hypothesis that phosphorylation may have regulatory importance in some of E1A's activities remains a possibility. Thus, E1A mutants impaired in phosphorylation will also be used in some of the proposed studies.

这项提案的主要目标是更准确地定义 调节细胞周期控制的蛋白质的活性通过使用 腺病毒E1a蛋白。E1a是一种多功能的磷酸蛋白。它的 多功能性反映在诱导细胞DNA合成的能力上， 克服转化生长因子-β对上皮细胞的生长抑制作用，以及 在促进细胞转化为 S阶段。E1a的这些活动推翻了RAS的要求 活动。已经有人提议进行实验，以确定哪些(如果有的话) E1a相关蛋白是E1a诱导DNA合成所必需的。 失去ras活性的细胞。我们假设这些人的身份 这些蛋白质将是重要的，因为它将建立第一个 时间，无论pRb、p107和/或p130等生长抑制因子是否 RAS的下游目标。这项提案的第二个目标是确定 E1a克服细菌生长抑制作用的机制 上皮细胞中的转化生长因子-β。我们假设E1a可以恢复激酶 阻断p27Kipl在转化生长因子中的抑制作用对CDK的活性 贝塔处理的细胞，并提出了两个实验模型，其中可能 发生。这项提议的另一个目的是鉴定细胞蛋白质 E1a在静止状态下诱导细胞DNA合成所需的 细胞。突变的E1a蛋白不能与一组特定的 细胞蛋白质将通过显微注射产生并测试其 启动DNA合成的能力。此外，抗体或反义 能中和细胞周期蛋白依赖性的寡核苷酸 将使用激酶和其他对G1向S过渡至关重要的蛋白质 作为工具，来确定E1a是否需要这些蛋白质中的任何一种来 刺激DNA合成。最后，由于E1a是一种磷蛋白， 假设磷酸化可能在某些情况下具有调节作用 E1a的活动仍有可能。因此，E1a突变体在 磷酸化也将在一些拟议的研究中使用。