REGULATION OF CASEIN KINASE II BY EGF IN MAMMALIAN CELLS

哺乳动物细胞中 EGF 对酪蛋白激酶 II 的调节

• 批准号: 6236860
• 负责人: NEIL OSHEROFF
• 金额: $ 2.68万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-31 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6236860
• 关键词: DNA topoisomerases; active sites; casein kinase; cell growth regulation; clone cells; enzyme activity; epidermal growth factor; hormone regulation /control mechanism; immunologic assay /test; peptide hormone; phosphorylation; protein purification

Casein kinase II is a highly conserved serine/threonine kinase which is essential to eukaryotic cells. In vivo and in vitro, it phosphorylates a broad spectrum of proteins which are critically involved in the regulation of cellular growth, metabolism, transformation, and morphology. Casein kinase II is rapidly and transiently stimulated following treatment of cells with a number of polypeptide hormones such as epidermal growth factor (EGF), insulin, or insulin-like growth factor I. Despite the apparent involvement of casein kinase II in a number of growth-related processes, little is understood concerning the mechanism by which it is regulated in the eukaryotic cell. The ultimate goal of the proposed research is to describe the physiological regulation of casein kinase II results from a hyperphosphorylation of the kinase's beta subunit. Furthermore, this regulatory event is mediated by an EGF-activated stimulatory factor which is proteinaceous in nature. Thus, the specific aims of this proposal are: 1) to characterize the hyperphosphorylation event which activates casein kinase II; 2) to identify the EGF-regulated stimulatory factor; and 3) to determine the physiological effects of casein kinase II activation on nuclear substrates such as DNA topoisomerases I and II. Human A-431 carcinoma cells will serve as the research model for this project. This line is well established and contains an extreme abundance of EGF receptors per cell. The stimulatory phosphorylation of casein kinase II will be characterized by a variety of experimental approaches. Studies will determine whether it is mediated by a novel autophosphorylation reaction or by a separate kinase, identify the primary site(s) of hormone-induced modification on the kinase's beta subunit, characterize the relationship between EGF-induced hyperphosphorylation and hormone-independent autophosphorylation, and determine whether other polypeptide hormones regulate casein-kinase II by a common biochemical mechanism. The EGF-regulated stimulatory factor will be purified primarily by chromatographic methods and characterized by a variety of biochemical and immunological techniques. Finally, the effect of casein kinase II activation on the phosphorylation state and catalytic function of DNA topoisomerases (and potentially other nuclear substrates) will be monitored by biochemical and immunological assays.

酪蛋白激酶II是一种高度保守的丝氨酸/苏氨酸激酶 对真核细胞必不可少的。 体内和体外，它磷酸化A 广泛参与调节的蛋白质 细胞生长，新陈代谢，转化和形态。 酪蛋白 在处理后，激酶II迅速，瞬时刺激 具有多种多肽激素（例如表皮生长因子）的细胞 （EGF），胰岛素或类似胰岛素样生长因子I。 酪蛋白激酶II参与许多与生长相关的过程， 几乎没有理解有关其调节机制的知识 真核细胞。 拟议研究的最终目标是描述生理 酪蛋白激酶II的调节是由过度磷酸化的 激酶的β亚基。 此外，此监管事件是由 EGF激活的刺激因子本质上是蛋白质的。 因此，该提案的具体目的是：1）表征 激活酪蛋白激酶II的高磷酸化事件； 2）识别 EGF调节的刺激因子； 3）确定生理 酪蛋白激酶II激活对核底物（例如DNA）的影响 拓扑异构酶I和II。 人A-431癌细胞将作为 该项目的研究模型。 这条线已经确定， 每个细胞中包含大量的EGF受体。 刺激性 酪蛋白激酶II的磷酸化将以多种为特征 实验方法。 研究将确定它是否由 新型的自磷酸化反应或单独的激酶，确定 激素诱导的ββ诱导的修饰的主要位点 亚基，表征EGF诱导的关系 高磷酸化和非激素独立的自磷酸化，并且 确定其他多肽激素是否通过 一种常见的生化机制。 EGF调节的刺激因子将 主要通过色谱方法纯化，并以A的特征 多种生化和免疫学技术。 最后，效果 酪蛋白激酶II激活在磷酸化状态和催化 DNA拓扑异构酶的功能（以及潜在的其他核底物） 将通过生化和免疫学测定法监测。