PROTEIN INTERACTIONS AND REPRESSION FUNCTION OF THE E1A

E1A 的蛋白质相互作用和抑制功能

• 批准号: 3198310
• 负责人: Elizabeth Moran
• 金额: $ 25.15万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-09 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198310
• 关键词: Adenoviridae; DNA binding protein; gene expression; genetic mapping; genetic transcription; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; molecular cloning; neoplastic transformation; oncogenes; phosphoproteins; posttranslational modifications; protein structure function; retinoblastoma; transcription factor; transforming virus; virus protein

The long term objective of this proposal is to understand the biochemical steps which transform a normal resting cell to one with malignant characteristics. This proposal focuses on the mechanism by which the Adenovirus E1A oncogene induces proliferation of quiescent primary cells. Recent evidence indicates that this process is related to E1A binding of an unidentified 300kDa cellular product. The specific aims of this project are to characterize the 300kDa product by (1) purifying and raising antibodies to it; (2) cloning and overexpressing it; (3) characterizing its posttranslational modifications, and (4) determining its intrinsic biological activities. The identification and characterization of p300 will focus largely on obtaining specific antibodies that recognize this product. When antibodies or some sequence are obtained, cDNA libraries will be screened to identify a p300 clone. The isolation and expression of a p300 clone will permit more extensive characterization including in vitro mutagenesis to study its biological activity. The biochemical and biological properties of the 300kDa protein will be characterized with particular emphasis on properties related to cell growth control and regulation of gene expression. The 300kDa product is a phosphoprotein which appears to undergo specific hyperphosphorylation during mitosis (preliminary results). The phosphorylation patterns of interphase and mitotic p300 will be mapped using techniques such as phosphopeptide analysis. Ultimately, phosphorylation events will be correlated with biological activity. The overall goal of the present proposal is to determine what biological activity of p300 makes it an apparent target for E1A cell growth regulating activity. Obtaining purified p300 will allow us to test its intrinsic biological activity. We will determine, for example, whether it is a DNA binding protein. We will determine the subcellular localization of p300, and the level of protein expression in various normal and aberrantly regulated cells. Possible interactions between p300 and other cellular products will be probed to identify cellular factors that may influence p300 function. Recent discoveries (reviewed in Green, 1989) have emphasized the relationship between E1A transforming functions and human cancer. The transforming mechanisms of adenoviruses are closely related to those of the human papillomaviruses, implicated in human malignancies. E1A association with the retinoblastoma protein provides further evidence that study of the E1A-associated 300-kilodalton product will promote our understanding of human tumorigenesis.

该提议的长期目标是了解生化 将正常休息单元转换为具有恶性肿瘤的步骤 特征。 该提议着重于 腺病毒E1a癌基因诱导静态原代细胞的增殖。 最近的证据表明，此过程与E1a的结合有关 身份不明的300KDA细胞产品。 该项目的具体目的 通过（1）净化和升高来表征300KDA产品 它的抗体； （2）克隆和过表达； （3）表征其 翻译后修饰，（4）确定其内在的 生物活动。 p300的识别和表征将主要集中在 获得识别该产品的特定抗体。 当抗体时 或获得某些序列，将筛选cDNA库以识别 P300克隆。 p300克隆的隔离和表达将允许 更广泛的表征包括体外诱变，以研究其 生物活性。 生化和生物学特性 300KDA蛋白将被特征在于特性 与细胞生长控制和基因表达的调节有关。 这 300KDA产品是一种磷蛋白，似乎经历了特定的 有丝分裂期间的高磷酸化（初步结果）。 这 将映射相间和有丝分裂p300的磷酸化模式 使用诸如磷酸肽分析之类的技术。 最终， 磷酸化事件将与生物活性相关。 这 本提案的总体目标是确定什么生物学 P300的活性使其成为E1A细胞生长调节的明显靶标 活动。 获得纯化的P300将使我们能够测试其内在的 生物活性。 例如，我们将确定它是否是DNA 结合蛋白。 我们将确定p300的亚细胞定位 以及各种正常和异常的蛋白质表达水平 调节细胞。 P300与其他细胞之间的可能相互作用 将探测产品以识别可能影响的细胞因素 P300功能。 最近的发现（绿色审查，1989年）强调了 E1A转化功能与人类癌症之间的关系。 这 转化腺病毒的机制与 人类乳头瘤病毒，涉及人类恶性肿瘤。 E1A协会 与视网膜细胞瘤蛋白有关，提供了进一步的证据表明 E1A相关的300千洛达尔顿产品将促进我们对 人类肿瘤发生。