This research is designed to examine the role of phosphorylation in the regulation of metabolism. These studies will examine the role of four multipotential protein kinases, casein kinase I and II and protease activated kinase (PAK) I and II. Emphasis will be placed on the role of PAK II in the regulation of metabolism, since PAK II has been shown to be activated in serum-starved cells in response to insulin, epidermal growth factor (EGF), and phorbol esters. During the coming grant period, we propose to identify the mode of activation of PAK II in response to EGF and phorbol esters at a molecular level. Total enzyme and amounts of active and inactive PAK II will be examined and compared in normal and transformed cells with regard to hormonal status and growth phase. Levels of the enzyme will also be examined with regard to position in the cell cycle and with gene amplification upon acquired drug resistance. PAK II and PAK I will be purified to homogeneity, polyclonal and monoclonal antibodies produced, and the enzymes characterized physically and chemically. Purified proteins phosphorylated by PAK II in vitro will be examined as substrate in vivo; the effects of phosphorylation on enzymic/protein activity will be analyzed. Regulation of levels of casein kinase I, casein kinase II and PAK I will be examined with regard to cell type, position in the cell cycle, and hormonal status, and the interrelationship between these enzymes and PAK II will be examined. Purified proteins, identified as substrate for these three multipotential protein kinases, will be analyzed and the functional consequences of individual and multiple phosphorylation events will be determined.
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