STRUCTURE AND REGULATION OF THE E4F TRANSCRIPTION FACTOR

描述(改编自申请者摘要)：本研究的目标 的结构和功能组织的特征 细胞转录因子E4F的性质和测定方法 主要受腺病毒E1a癌基因的调控。E1a 正常作用于反式激活腺病毒基因表达 在早期的溶血性感染期间。然而，异常的E1a表达可能具有 对包括细胞在内的细胞表型的深远影响 永生化和肿瘤性转化。一个鲜为人知的函数 对细胞转移特性的抑制是由 其他癌基因(如erb-B2/neu)。所有这些功能都被认为是 由于E1a有能力影响活动和 细胞转录因子的功能。细胞因子E4F 是这样的一个靶点，其DNA结合活性随着 E1a表达。有证据表明，这种激活是由 E4F的磷酸化以及E1a区域参与了 抑制肿瘤转移是激活E4F- 依赖转录。作为理解E4F作用的前奏 在正常细胞和转化细胞中，其结构的分子细节 监管必须确定下来。 通过对E4F基因的表达筛选，获得了一个编码E4F的基因克隆 Lambda噬菌体文库并经序列识别证实， 免疫学指标及其对E4启动子的激活能力 依赖E1a的方式。这项建议的第一个具体目标是 确定E4F中关键的结构域和特定氨基酸残基 DNA结合，可能的二聚化和转录激活。这个 第二个特殊目的是阐明支配E1a的分子事件 激活E4F。这将需要将E1a的地点本地化- 诱导磷酸化，鉴定E4F和In中的其他残基 激活和分析激酶所需的E1a(S)和细胞 涉及的组件。第三个具体目标是确定 激酶(S)和/或参与E4F激活的其他细胞成分和 克隆它们以备将来调查之用。综上所述，这方面的研究 该提案可能阐明E4F如何发挥作用和 是规范的，从而为更好地理解 E4F在E1a的正常和调节后果中所起的作用 表情。

DESCRIPTION (Adapted from Applicant's Abstract): The goals of this study are to characterize the structure and functional organization of the cellular transcription factor E4F and to determine the nature and means of its regulation, primarily by the E1A oncogene of adenovirus. E1A normally functions to the trans-activate adenoviral gene expression during early lytic infection. However, aberrant E1A expression can have profound effects upon cellular phenotype including cellular immortalization and neoplastic transformation. A lesser known function is the suppression of metastatic properties of cells transformed by other oncogene (e.g., erb-B2/neu). All of these functions are thought to be due to the ability of E1A to influence the activities and functions of cellular transcription factors. The cellular factor E4F is one such target whose DNA binding activity is markedly increased with E1A expression. Evidence suggests this activation is controlled by phosphorylation of E4F and that the region of E1A involved in the suppression of metastasis is required for the activation of E4F- dependent transcription. As a prelude to understanding the role of E4F in normal and transformed cells, the molecular details of its structure and regulation must be determined. A cDNA clone encoding E4F has been isolated by expression screening of lambda phage libraries and confirmed by sequence recognition, immunological criteria and its ability to stimulate the E4 promoter in an E1A-dependent manner. The first specific aim of this proposal is to identify domains and specific amino acid residues in E4F critical for DNA binding, possible dimerization and transcriptional activation. The second specific aim is to elucidate the molecular events governing E1A activation of E4F. This will entail localization of the site of E1A- induced phosphorylation, identification of other residues in E4F and in E1A required for activation and analysis of the kinase(s) and cellular components involved. The third specific aim is to identify the kinase(s) and/or other cellular components involved in E4F activation and clone them for future investigation. Taken together, the studies in this proposal may elucidate the precise mechanisms of how E4F functions and is regulated, thereby laying the ground work for a better understanding of the role E4F plays normally and in mediating consequences of E1A expression.