Calcium/diacylglycerol-dependent protein phosphorylation in the nervous system

Katherine Ann Albert, Yale University.

This is an Open Access Thesis


Calcium/diacylglycerol-dependent protein kinase, most often referred to as protein kinase C, is of interest to research groups across many scientific disciplines for two major reasons. First, protein kinase C is of general importance in signal transduction because its second messenger activator, diacylglycerol, is generated by phosphoinositide metabolism in response to a host of biologically active molecules. Second, protein kinase C has been identified as the cellular receptor for tumor-promoting phorbol esters. Protein kinase C is of particular interest to neuroscientists because its highest activity is in the nervous system. The studies described in this dissertation were undertaken to analyze the role of protein kinase C in the nervous system. Three different approaches were employed. First, a major endogenous substrate for protein kinase C, the 87-kDa protein, was identified in brain, was purified, and was characterized with respect to its physicochemical properties and its distribution. Second, the effect of phosphorylation by protein kinase C was examined with a nervous system substrate of known function, namely tyrosine hydroxylase. Third, the regulation of protein kinase C by calcium-binding proteins was examined.The 87-kDa protein was identified in bovine brain and was purified from bovine forebrain supernatant with the use of conventional chromatographic methods. The physicochemical properties of the 87-kDa protein indicated that the molecular weight was 68 kDa, and that the protein was an extremely elongated monomer. The 87-kDa protein was phosphorylated by protein kinase C to a stoichiometry of 2.2 mol $\sp{32}$P per mol protein, exclusively on serine residues.The 87-kDa protein was characterized with respect to its species, tissue, and subcellular distribution. A similar protein was present in monkey, human, rat, mouse, and bovine brain, in Torpedo californica electric organ, and in a variety of non-neuronal rat and bovine tissues. The rat protein was of M$\sb{\rm r}$ 4,000-7,000 lower, and was slightly more acidic, than the bovine protein. The distribution of the 87-kDa protein was found to be similar to the distribution of protein kinase C.Protein kinase C was found to phosphorylate and activate tyrosine hydroxylase. The results suggest that protein kinase C and cAMP-dependent protein kinase phosphorylate the same site(s) on tyrosine hydroxylase, and activate tyrosine hydroxylase by the same mechanism.Calmodulin was found previously to inhibit the calcium/phospholipid-dependent phosyphorylation of the 87-kDa protein, in a crude extract prepared from rat brain synaptosomal cytosol. In the present work, rabbit brain calmodulin and some other calcium-binding proteins were found to inhibit the phosphorylation of the 87-kDa protein by protein kinase C, in the purified system. These results suggest that protein kinase C may be regulated by calcium-binding proteins.