Phosphorylation-dephosphorylation of proteins is one of the most important mechanisms for the regulation of cellular functions. Protein kinase C (PKC) has emerged as a pivotal regulatory element for the regulation of many cellular functions. Multiple PKC subspecies have been identified by molecular cloning, however, the functional role of each of these enzymes is largely unknown. These enzymes were found to have distinct tissue, cellular, and subcellular distributions and were differentially expressed during development. Since the activation of PKC results in the phosphorylation of target proteins, we have isolated several PKC substrates from rat brain for analysis of their physiological functions. Among them, a 78-amino acid CNS-specific calmodulin (CaM)-binding protein, neurogranin, has been characterized as a specific PKC substrate. This proteins binds CaM in the absence of Ca2+ at a region adjacent to the phosphorylation site and thus becomes a poor substrate for PKC. Phosphorylation of neurogranin by Pkc promotes its dissociation from CaM. The formation of neurogranin/CaM complexes in the absence of Ca2+ was also observed in rat brain homogenate, suggesting that neurogranin is a specific Ca2+ -independent CaM-binding protein. A synthetic peptide corresponding to the site of phosphorylation and CaM- binding domain of neurogranin was found to be phosphorylated by PKC but not by other protein kinases. A substitution of tryptophan for phenylalanine enables this peptide to increase fluorescence upon phosphorylation, a characteristic useful for spectrofluorimetric assay of PKC. The CA2+-independent group B PKC delta and epsilon have been isolated from rat brain particulate and soluble fractions, respectively. These enzymes are free from contamination of the Ca2+-independent group A PKC alpha, beta, and gama. Two forms of PKC epsilon, a PS/DAG- dependent epsilon1 and independent epsilon2, have been isolated by chromatography on Mono Q column. Conversion of PKC epsilon1 and epsilon2 was achieved by incubation of the former with Ca2+ and PS/DAG, although the kinase activity inherent in PKC epsilon is independent of Ca2+. Thus, the receptor-mediated hydrolysis of phosphoinositides could trigger the activation of all PKCs and promote the conversion of PKC epsilon into the effector-independent enzyme for sustained activation when the stimulatory signal subsides. The genomic structures of CNS- specific PKC gama and neurogranin were analyzed for the purpose of defining tissue-specific and development-regulated expression of these two proteins. Multiple nuclear protein binding elements in PKC gamma gene have been identified by footprint and gel mobility shift assays. A potential negative regulatory element-binding protein, its expression in inversely related to the expression of PKC gamma, was identified in fetal rat brain nuclear extract. The structural feature of the neurogranin gene is currently under investigation.