CELL GROWTH CONTROL FUNCTIONS OF THE E1A ONCOGENE

我计划研究基因调节活动， 腺病毒E1A的12S产物的转化功能 癌基因 这一分析明显不同于以往的研究， 许多实验室的转录反式激活功能， 与13S E1A产品相关。 尽管最近取得了进展， 鉴定E1A功能结构域和E1A相关蛋白，包括 视网膜母细胞瘤（Rb）的产品，迄今为止，很少有人了解 E1A产物控制以下表达的具体步骤： 细胞产物以这样的方式激活休眠细胞。 已知 E1A 12S蛋白具有两种不同的功能， 增长 两者都需要指导细胞通过即使是一轮 细胞分裂，但表达的N-末端功能（不参与 Rb结合）足以驱动细胞进入S期并在此停滞 细胞周期的一个关键点。 在这个项目中，我计划专注于一个特定的 已知响应E1A细胞周期而调节的细胞产物 功能，并且已知在细胞周期控制中发挥核心作用，我将 研究哺乳动物cdc2产物，测定其E1A介导的 调控，分离并详细分析其启动子， 将特异性启动子元件的活性与 E1A产品中的特定功能域。 的关系 cdc2活性和E1A刺激的细胞生长将进一步研究， 测定CDC2磷酸化状态、激酶活性和亚基 与野生型和突变型E1A产物表达相关的结构。 特异性灭活单个E1A的突变体的可用性 转化域使其成为特别有吸引力的系统。 在项目的第二部分，我将重点关注cdc2的影响。 活动 cdc2激酶似乎是磷酸化剂， Rb基因产物。 目前Rb功能的模型提出， 磷酸化使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， 使细胞脱离静止状态，进入细胞周期。 在 与此模型相反，很明显Rb与E1A的结合完全不是 对于E1A介导的S期诱导至关重要，尽管这些细胞不能 进行有丝分裂。 我认为诱导cdc2蛋白表达 并且一定水平的CDC2激酶活性的激活可能是替代的 E1A可以诱导S期而不与Rb物理结合的机制。 初步结果表明，N-末端E1A功能的表达 可能足以用于E1A介导的cdc2活化。 探索这个 可能性，并阐明生物学的潜在机制， 由E1A指导的事件，我将描述翻译后 原代细胞中Rb基因产物上发生的修饰 由野生型E1A和选择的E1A突变体刺激， 细胞周期进程 E1A的表达也能抑制细胞的生长 抑制TGF β的作用。 初步结果表明，E1A CDC2活性诱导可能是该机制的一部分。 我会 表征TGF β处理的细胞中cdc2的性质， E1A野生型和突变体表达的存在和不存在，以详细说明 E1A抵消TGF β作用的机制。 我觉得我的计划 重点关注具有良好初步迹象的有限系统， 重要性将使我能够富有成效地进行。 该项目还具有 能够利用两个重要工具的优势， 在哺乳动物细胞研究中始终可行：正常原代细胞， 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.