ROLE OF E1A IN INDUCING CELLULAR DNA SYNTHESIS

E1A 在诱导细胞 DNA 合成中的作用

• 批准号: 2701723
• 负责人: MLNikki L Harter
• 金额: $ 18.69万
• 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/2701723
• 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.

该提议的主要目标是更精确地定义 通过使用介导细胞周期控制的蛋白质的活性 腺病毒E1A蛋白。 E1a是一种多功能磷蛋白。 它是 多功能性反映在诱导细胞DNA合成的能力中， 克服TGF-β在上皮细胞中的生长抑制作用，并 超越了将细胞促进细胞中的细胞RAS活性的要求 s阶段。 E1A的这些活动覆盖了RAS的要求 活动。 已经提出了实验来确定哪个（如果有） E1A要求与E1A相关的蛋白诱导DNA合成 失去RAS活性的细胞。 我们假设 这些蛋白质将很重要，因为它将建立，因为 时间，诸如PRB，P107和/或P130之类的增长抑制剂是否为 RAS的下游目标。 该提议的第二个目的是确定 E1A可以克服生长抑制作用的机制 上皮细胞中的TGF-β。 我们假设E1A可以恢复激酶 通过禁用P27KIPL在TGF-的抑制作用来对CDK的活性 经β处理的细胞，并介绍了两个实验模型，其中可能 发生。 该建议的另一个目的是识别细胞蛋白 E1A需要诱导静态的细胞DNA合成所要求的 细胞。 突变的E1a蛋白无法与一组特定集相互作用 细胞蛋白将通过显微注射来创建和测试 启动DNA合成的能力。 另外，抗体或抗敏感性 可以中和环依赖性活性的寡核苷酸 将使用对G1至S过渡重要的激酶和其他蛋白质 作为工具，确定E1A是否需要这些蛋白质 刺激DNA合成。 最后，由于E1a是一种磷蛋白，因此 假设磷酸化可能具有调节性的重要性 E1A的活动仍然是可能的。 因此，E1a突变体受损 磷酸化也将用于一些提出的研究。