Studies in a variety of tissues have rekindled interest in the possible second messenger role of calcium in mediating the action of numerous hormones, neurotransmitters, growth factors and other regulatory agents. Stimulation of neural tissue by membrane depolarization as well as hormonal regulation of liver cell function may involve calcium-dependent phosphorylation. Although the actions of calcium do not appear to be mediated by a universal biochemical mechanism, I propose to study 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 a calcium-dependent kinase with broad substrate specificity. The major calcium/calmoldulin-dependent protein kinase phospharylating microtubule-associated protein-2 (MAP-2) in rat brain has been purified to homogeneity. The enzyme has a broad substrate specificity characteristic of 'general' protein kinase, such as the cAMP-dependent protein kinase. The possibility that this protein kinase is a mediator of calcium action in diverse tissues will be examined. The physical characterization of the kinase will be extended and the role of autophosphorylation tested. The tissue and subcellular localization of the kinase will be determined by biochemical and immunological assays. The ability of this kinase to phosphorylate numerous potential substrates will be investigated. Differences in the specificity of the calcium kinase and the cAMP kinase will be analyzed by sequencing the phosphorylation sites of selected substrates and by testing synthetic peptide substrates. Activation of the calcium/calmodulin kinase in vivo will be examined in PC12 cells which contain three putative substrates - tyrosine hydroxylase and two groups of microtubule-associated proteins, MAP-2 and tau. Calcium influx into PC12 will be stimulated by depolarization of the cell membrane or by activation of the acetylcholine receptor. 32P incorporation into the target proteins will be analyzed and sites of phosphorylation compared with in vitro phosphorylation by the calcium kinase. Functional changes in the three substrates will be correlated with phosphorylation. Numerous biochemical effects of calcium may be mediated in a variety of tissues via activation of this calcium/calmodulin-dependent protein kinase.
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