CELL GROWTH CONTROL FUNCTIONS OF THE E1A ONCOGENE

我计划研究基因调控活动，特别是与 腺病毒E1a 12S产物的转化功能 致癌基因。这一分析与以前的研究在以下方面有明显的不同 转录反式激活功能的许多实验室 与13S E1A产品相关联。尽管最近在以下方面取得了进展 确定E1a功能结构域和E1a相关蛋白，包括 视网膜母细胞瘤(RB)产品，到目前为止，人们对 E1a产品控制表达的具体步骤 细胞产物以激活静息细胞的方式。这是众所周知的 E1A12S蛋白具有调节细胞的两种不同功能 成长。这两种方法都需要引导细胞通过甚至一轮 细胞分裂，但N-末端功能的表达(未参与 Rb结合)足以驱动细胞进入S期并在此停滞 细胞周期中的一个点。在这个项目中，我计划将重点放在一个特定的 已知细胞产物受E1a细胞周期调控 功能，并已知在细胞周期控制中发挥核心作用，我将 研究哺乳动物的cdc2产物，确定其E1a介导的水平 调控，分离并详细分析其启动子，以及 特定启动子元件的活性与酶活性之间的关系 E1a产品中的特定功能域。两国之间的关系 Cdc2活性和E1a刺激的细胞生长将通过 Cdc2磷酸化状态、激酶活性和亚基的测定 与野生型和突变型E1a产物表达相关的结构。 特异性灭活个体E1a的突变体的可用性 域转换使其成为一个特别吸引人的系统。 在项目的第二部分，我将重点介绍cdc2的影响。 活动。Cdc2激酶似乎是一种磷酸化试剂 Rb基因产物。目前的RB函数模型提出 磷酸化使Rb和Rb的细胞生长抑制作用失活 E1a蛋白对活性Rb的滴定模拟Rb失活和 允许细胞离开静息状态，进入细胞周期。在……里面 与这个模型相比，显然E1a与Rb的结合根本不是 E1a介导的S期诱导是必不可少的，尽管这些细胞失败了 才能进行有丝分裂。我认为诱导cdc2蛋白表达 而激活一定水平的cdc2激酶活性可能是一种替代方案。 E1a诱导S相而不与Rb物理结合的机制。 初步结果表明，N-末端E1a功能的表达 对于E1a介导的CDC2的激活可能是足够的。要探索这一点 并阐明其潜在的生物学机制 由E1A执导的事件，我将描述翻译后的 原代细胞中Rb基因产物的修饰 用野生型E1a和精选的E1a突变体刺激导致流产 细胞周期进程。E1a的表达也可以阻断细胞的生长 抑制转化生长因子β的作用。初步结果表明，E1a Cdc2活性的诱导可能是这种机制的一部分。这就做 在转化生长因子β处理的细胞中表征cdc2的特性，在 E1a野生型和突变型表达的存在和缺失，以详细说明 E1a中和转化生长因子β效应的机制。我觉得我的计划 专注于具有良好初步迹象的有限系统 意义将使我能够富有成效地继续前进。这个项目还有 能够利用两个重要工具的优势是 在哺乳动物细胞研究中总是可行的：正常的原代细胞，以及 良好的遗传系统。

I plan to study the gene-regulating activities specifically associated with the transforming functions of the 12S product of the adenovirus E1A oncogene. This analysis is distinctly different from previous studies in many laboratories of the transcriptional transactivation function associated with the 13S E1A product. In spite of recent progress in identifying E1A functional domains and E1A associated proteins, including the retinoblastoma (Rb) product, there is as yet, very little insight into the specific steps by which the E1A products control the expression of cellular products in such a way as to activate resting cells. It is known that the E1A 12S protein carries two separate functions which regulate cell growth. Both are required to direct cells through even a single round of cell division, but expression of the N-terminal function (not involved in Rb binding) is sufficient to drive cells into S-phase and to stall at this point in the cell cycle. In this project, I plan to focus on a specific cellular product known to be regulated in response to E1A cell-cycle functions, and known to play a central role in cell cycle control, I will study the mammalian cdc2 product, determine the level of its E1A-mediated regulation, isolate and make a detailed analysis of its promoter, and correlate the activity of specific promoter elements with the activity of specific functional domains in the E1A products. The relationship between cdc2 activity and E1A-stimulated cell growth will be studied further by determining the cdc2 phosphorylation state, kinase activity and subunit structure in relation to expression of wild-type and mutant E1A products. The availability of mutants specifically inactivating individual E1A transforming domains makes this a particularly attractive system. In the second part of the project, I will focus on the effects of cdc2 activity. The cdc2 kinase appears to be the phosphorylating agent for the Rb gene product. Current models of Rb function propose that phosphorylation inactivates the cell growth-suppressing effect of Rb and that titration of active Rb by the E1A proteins mimics Rb inactivation and allows cell to leave the resting state and enter the cell cycle. In contrast to this model, it is clear that Rb binding by E1A is not at all essential for E1A-mediated induction of S-phase, although these cells fail to proceed to mitosis. I propose that induction of cdc2 protein expression and activation of some level of cdc2 kinase activity may be an alternate mechanism by which E1A can induce S-phase without physically binding Rb. Preliminary results suggest that expression of the N-terminal E1A function may be sufficient for E1A-mediated activation of cdc2. To explore this possibility and to elucidate the mechanisms underlying the biological events directed by E1A, I will characterize the post-translational modifications that occur on the product of the Rb gene in primary cells stimulated by wild-type E1A and selected E1A mutants which induce abortive cell cycle progression. E1A expression can also block the cell growth suppressing effects of TGFbeta. Preliminary results suggest that E1A induction of cdc2 activity may be part of this mechanism. I will characterize the properties of cdc2 in TGFbeta treated cells, in the presence and absence of E1A wild-type and mutant expression, to detail the mechanism by which E1A counteracts the effects of TGFbeta. I feel my plans to focus on limited systems with good preliminary indications of significance will enable me to proceed productively. This project also has the advantage of being able to utilize two important tools that are not always feasible in mammalian cell studies: normal primary cells, and a good genetic system.