Structure and Function of Protein Kinases: Molecular Mechanism of Neuronal Signal Transduction

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The importance of protein kinases as target molecules of second messengers in the cellular signal transduction mechanisms activated by hormones and neurotransmitters are well established. Protein kinase C and cAMP-dependent protein kinase are thought to be major protein kinases in these mechanisms. In this study, we tried to elucidate the molecular mechanisms of neuronal signal transduction related to these protein kinases.1) Localization and function of protein kinase C isozymes: Light- and electron-microscopic localization of alpha, beta-I, beta-II, and gamma subtypes of protein kinase C in rat brain were elucidated using specific antibodies. Immunohistochemical analysis of developing rat brain showed that each subtype expresses at different stage. Isozymes related to neurorite outgrowth and synaptic formation were identified. Specific expression of these isozymes was also shown by the in situ hybridization technique.2) The role of protein kinase C in the synaptic transmission: Invol … More vement of protein kinase C in calcium-dependent vesicular neurotransmitter release was shown. Gamma subtype of protein kinase C which is activated by arachidonic acid was shown to be involved in the GABA release from Purkinje cell in cerebellum.3) Molecular cloning and expression of regulatory subunit of cAMP-dependent protein kinase: A cDNA clone containing an entire coding region of the regulatory subunit was cloned from a rat brain cDNA library. Wild or mutant regulatory subunits were expressed in E. coli. After incubation with catalytic subunit purified from bovine heart, holo-enzymes were activated by CANW. Although these subunits bound cAMP with high affinity, holo-enzymes contains mutant subunits could not be activated by high concentrations of cAMP, indicating the importance of the presence of intrachain two cAMP-binding sites. These studies suggest that subtypes of protein kinase C have specific roles in neurorite outgrowth, synaptic formation, neurotransmitter release and long term potentiation, and showed the importance of protein phosphorylation in the neuronal signal transduction. Less