PROTEIN PHOSPHORYLATION AND SECRETION AND ETHANOL ACTIONS

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The phosphorylation of protein has been proposed to be one of the molecular mechanisms involved in cellular regulation. The protein kinases catalyze these phosphorylation reactions. In this investigation, properties of protein kinase activity associated with cell cytoskeletal proteins have been studied in both neuronal and non-neuronal tissues. Extensive studies were carried out to characterize the squid axon neurofilament kinase. These investigations led to conclude that this kinase is a cyclic nucleotide- and calcium-independent protein kinase which is different from casein type I and type II kinases. The axoplasm and neurofilament preparation had no detectable protein kinase inhibitor activity, but strong inhibitor activity, which was not dialyzable but was heat inactivitable, was found in ganglion cells. This inhibitor activity may account for the low phosphorylation activity found in the stellite ganglion cells and may indicate inhibitory regulation of squid axon neurofilament kinase activity in the ganglion cell bodies. In rat brain, the kinase activity is associated with microtubule-associated proteins and phosphorylates them. Effects of ethanol on phosphorylation of microtubule associated protein (MAP 2) were investigated. Ethanol (4-24 mM) increased phosphorylation of MAP 2. In the presence of cAMP or mM ethanol, increased phosphorylation of MAP 2 was observed over control. Much higher phosphorylation of MAP 2 was observed in the presence of cAMP and ethanol than the sum of phosphorylation of MAP 2 by cAMP and ethanol separately. Kinetic studies of the influence of ethanol on MAP 2 phosphorylation reveal an increased rate of phosphorylation of MAP 2 and a decreased Km in the presence of ethanol. These studies suggest that protein kinase(s) other than cAMP dependent protein kinase are influenced by ethanol and the enzyme(s) phosphorylate at distinct sites on MAP 2. The observation that ethanol affects MAP 2 provides an experimental basis for investigating the effects of ethanol on the structure and function of these cytoskeletal proteins and enzymes.