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.
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