REGULATION OF CASEIN KINASE II BY EGF IN MAMMALIAN CELLS

酪蛋白激酶II是一种高度保守的丝氨酸/苏氨酸激酶 真核细胞所必需的。在体内和体外，它使一种 在调控中起关键作用的广谱蛋白质 细胞的生长、新陈代谢、转化和形态。酪蛋白 激酶II在治疗后迅速和短暂地被刺激 含有多种多肽激素的细胞，如表皮生长因子 (EGF)、胰岛素或胰岛素样生长因子I，尽管表面上 酪蛋白激酶II参与了许多与生长相关的过程， 人们对它的调控机制知之甚少。 真核细胞。 所提议的研究的最终目标是描述生理学 酪蛋白激酶II的调节是由于酪蛋白激酶II过度磷酸化所致 激酶的β亚基。此外，这一监管事件是由 一种EGF激活的刺激因子，本质上是蛋白质类的。 因此，这项提案的具体目标是：1)描述 激活酪蛋白激酶II的过度磷酸化事件；2)识别 EGF调节的刺激因子；3)测定生理性的 酪蛋白激酶II激活对DNA等核底物的影响 拓扑异构酶I和II。人A-431癌细胞将作为 本项目的研究模型。这条线路很好，而且 每个细胞都含有极其丰富的EGF受体。刺激性 酪蛋白激酶II的磷酸化将通过各种不同的 实验方法。研究将确定它是否由 一种新的自动磷酸化反应或通过单独的激酶，鉴定 激素诱导的激酶β修饰的原发部位(S) 亚单位，表征EGF诱导的 过度磷酸化和激素非依赖性自磷酸化，以及 确定其他多肽激素是否通过以下方式调节酪蛋白激酶II 一种常见的生化机制。EGF调节的刺激因子将 主要通过层析方法进行纯化，其特征是 各种生化和免疫学技术。最后，效果 酪蛋白激酶II激活对磷酸化状态和催化作用的影响 DNA拓扑异构酶(以及潜在的其他核底物)的功能 将通过生化和免疫学分析进行监测。

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.