Intracellular calcium serves as a second messenger to mediate the effects of many hormones, growth, factors, neurotransmitters and other extracellular signals. Although the actions of calcium do not appear to be mediated by a universal biochemical mechanism, this proposal aims to test the possibility that certain of the biochemical and physiological effects of calcium may be mediated by phosphorylation of a diverse array of substrate proteins catalyzed by the multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase). The proposal will examine its regulation by calcium can by autophosphorylation, will determine which type of receptor is subserved by the kinase and will expand on the putative role of the kinase in the regulation of carbohydrate and amino acid metabolism and in nuclear membrane breakdown. Site directed mutagenesis will be used to either block or mimic autophosphorylation sites that are involved in activating or inhibiting the CaM kinase. The mechanism for regulation by autophosphorylation will be investigated. The inhibitory """"""""pseudosubstrate"""""""" hypothesis of kinase regulation will be tested with truncation mutants lacking the inhibitory domain. These experiments will generate a calcium-independent form of the kinase to be used in examining two of its many cellular functions. One such function, the calcium-dependent regulation of carbohydrate and amino acid metabolism will be examined in Reuber H35 hepatoma cells. Inhibitory antibodies to the kinase or calcium-independent kinase preparations will be microinjected into the hepatoma cells and the ability of hormonal stimulation to regulate enzymes such as pyruvate kinase and phenylalanine hydroxylase examined. Similar studies are proposed to examine the role of the kinase in the calcium-triggered breakdown of nuclear membrane in sea urchin eggs, another of its postulated functions. Finally, involvement of the CaM kinase in mediating the effect of receptors that evaluate calcium via the phosphatidylinositol and other signalling systems will be examined. To achieve this, a model substrate of the kinase, tyrosine hydroxylase, will be transfected into Swiss 3T3 cells which have numerous receptors with diverse mechanisms for elevating calcium. Cells will be stimulated by signals such as vasopressin, bombesin and platelet-derived growth factor and the activation of the kinase monitored by its phosphorylation of tyrosine hydroxylase.
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