Evaluation and Characterization of Novel Signal Transduction Pathways in Striatum
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In the mammalian central nervous system, protein kinases and protein phosphatases control the function of myriad target proteins in the pre- and postsynaptic compartments, including other protein kinases and phosphatases, neurotransmitter receptors, ion channels, transporters, metabolic enzymes, transcription factors, cytoskeletal elements, and vesicle-docking proteins. Using biochemical and pharmacological approaches, a number of novel striatal signal transduction pathways were evaluated and characterized in the following studies, with emphasis on protein kinase C-mediated signaling. 1) A known and novel form of mouse Adk encoding splice variants of adenosine kinase, the principal enzyme of adenosine metabolism, were cloned from a mouse brain cDNA library and expressed and purified as recombinant proteins with high enzymatic activity. The tissue distribution of adenosine kinase isoform expression was defined. A polyclonal anti adenosine kinase antibody was generated for further characterization of the enzyme. In vitro protein phosphorylation studies using purified protein kinases and in vivo radioimmunoprecipitation assays using the novel antibody for adenosine kinase indicated, however, that this metabolic enzyme is unlikely to be regulated by phosphorylation. 2) Further studies using a candidate approach demonstrated the regulation of several postsynaptic phosphoproteins by striatal adenosine A2A receptor signaling, including ionotropic glutamate receptor subunits, mitogen-activated protein kinase isoforms, a striatal inhibitor of protein phosphatase 1, a protein phosphatase 1- and actin-binding protein, and the cAMP-response element-binding protein. 3) In parallel studies, inhibitor-1, a protein phosphatase 1 inhibitor activated by cAMPdependent protein kinase, was characterized as a novel protein kinase C substrate in vitro and in vivo. Phosphorylation state-specific antibodies raised against this novel phosphorylation site showed that it is dephosphorylated by protein phosphatase 1 and positively regulated by group I metabotropic glutamate receptors in the striatum. Furthermore, protein kinase C-dependent phosphorylation was shown to reduce the efficiency with which inhibitor-1 serves as a substrate for cAMP-dependent protein kinase in vitro and in vivo. 4) Finally, protein kinase C activation was shown to decrease the level of phosphorylation of cyclin-dependent kinase 5 substrates in the striatum, suggesting a possible role for protein kinase C in regulating cyclin-dependent kinase 5 activity.