PROTEIN INTERACTIONS AND REPRESSION FUNCTION OF THE E1A

这项提案的长期目标是了解生物化学 将正常静止细胞转化为恶性细胞的步骤 特色 这项建议的重点是， 腺病毒E1A癌基因诱导静止原代细胞增殖。 最近的证据表明，这一过程与E1A结合有关， 未鉴定的300kDa细胞产物。 该项目的具体目标 通过（1）纯化和提高300kDa产物的纯度， （2）克隆和过表达它;（3）表征它的特性。 翻译后修饰，和（4）确定其内在的 生物活动。 p300的识别和表征将主要集中在 获得识别该产物的特异性抗体。 当抗体 或获得一些序列，将筛选cDNA文库以鉴定 p300克隆 p300克隆的分离和表达将允许 更广泛的表征，包括体外诱变，以研究其 生物活性 的生物化学和生物学特性， 300 kDa蛋白质将特别强调其特性 与细胞生长控制和基因表达调控有关。 的 300kDa产物是一种磷蛋白， 有丝分裂过程中的过度磷酸化（初步结果）。 的 将绘制间期和有丝分裂期p300的磷酸化模式 使用诸如磷酸肽分析的技术。 最后， 磷酸化事件将与生物活性相关。 的 本提案的总体目标是确定生物学 p300的活性使其成为E1A细胞生长调节的明显靶点 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一个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组织中的蛋白表达水平 调节细胞。 p300与其他细胞因子的相互作用 产品将被探测，以确定细胞因子，可能会影响 p300函数。 最近的发现（在绿色，1989年回顾）强调了 E1A转化功能与人类癌症的关系。 的 腺病毒的转化机制与 人乳头瘤病毒，与人类恶性肿瘤有关。 E1A缔合 与视网膜母细胞瘤蛋白的关系提供了进一步的证据， E1A相关的300千道尔顿产物将促进我们对 人类肿瘤发生

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.