ROLE OF E1A IN INDUCING CELLULAR DNA SYNTHESIS

本提案的主要目的是更准确地界定 蛋白质的活性，通过使用 腺病毒E1 A蛋白。 E1 A是一种多功能磷蛋白。 其 多功能性反映在诱导细胞DNA合成的能力， 克服TGF-β在上皮细胞中的生长抑制作用，和 超越细胞ras活性的要求， S期 E1 A的这些活动超越了RAS的要求， 活动 已经提出了实验来确定，如果有的话， E1 A相关蛋白是E1 A诱导DNA合成所必需的， 失去ras活性的细胞。 我们假设 这些蛋白质将是重要的，因为它将建立， 时间，生长抑制因子如pRb、p107和/或p130是否 RAS的下游靶点。 本建议的第二个目的是确定 E1 A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E1 A可以恢复激酶 通过使TGF-β 1中p27 Kipl的抑制作用失效， β-处理的细胞，并提出了两个实验模型，其中这可能 发生. 这项提议的另一个目的是鉴定细胞蛋白质 E1 A在静止期诱导细胞DNA合成所需的 细胞 突变E1 A蛋白不能与一组特定的 细胞蛋白质将通过显微注射产生和测试， 启动DNA合成的能力。 此外，抗体或反义 可以中和细胞周期蛋白依赖性的活性的寡核苷酸， 将使用激酶和其他对G1到S转变重要的蛋白质 作为工具，以确定E1 A是否需要这些蛋白质中的任何一种， 刺激DNA合成。 最后，由于E1 A是磷蛋白， 假设磷酸化可能在某些 E1 A的活动仍然是一种可能。 因此，E1 A突变体在 磷酸化也将用于一些拟议的研究中。

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.