Preganglionic stimulation increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion by both cAMP-dependent and Ca2+-dependent protein kinases.
Electrical stimulation of the preganglionic cervical sympathetic trunk increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat by a nicotinic mechanism and by a noncholinergic mechanism. We have measured the incorporation of [32P]Pi into specific tryptic phosphopeptides in tyrosine hydroxylase in order to identify the protein kinases that phosphorylate this enzyme in electrically stimulated ganglia. 32P-labeled tyrosine hydroxylase was isolated from the ganglion by immunoprecipitation and polyacrylamide gel electrophoresis and was subjected to tryptic hydrolysis. Seven tryptic peptides were resolved from these hydrolysates by two-dimensional thin-layer electrophoresis and chromatography. Preganglionic stimulation (20 Hz, 5 min) increased the incorporation of 32P into four of these peptides. In the presence of cholinergic antagonists, however, electrical stimulation increased the labeling of only one phosphopeptide. From a comparison of the effects of preganglionic stimulation with the effects of agonists that activate specific protein kinases, we conclude that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a cAMP-dependent protein kinase and a Ca2+/calmodulin-dependent protein kinase. The nicotinic component of preganglionic stimulation appears to be mediated by a Ca2+/calmodulin-dependent protein kinase, while the noncholinergic component appears to be mediated by cAMP-dependent protein kinase. Although protein kinase C can phosphorylate tyrosine hydroxylase, this kinase does not appear to participate in the stimulation-induced phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.
D000818:Animals; D002460:Cell Line; D004558:Electric Stimulation; D005728:Ganglia, Sympathetic / Q000201:enzymology / Q000502:physiology*; D007700:Kinetics; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D014357:Trypsin; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D010748:Phosphopeptides; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D014357:Trypsin