Phosphorylation of purified rat striatal tyrosine hydroxylase by Ca2+/calmodulin-dependent protein kinase II: effect of an activator protein.
The phosphorylation of tyrosine hydroxylase, purified from rat striatum, was investigated using purified Ca2+/calmodulin (CaM)-dependent protein kinase II. This kinase catalyzed the Ca2+-dependent incorporation of up to 0.8 mol 32PO4/mol tyrosine hydroxylase subunit (62 kilodaltons). Reverse-phase high-performance liquid chromatography mapping of tryptic 32P-peptides established that the Ca2+/CaM-dependent protein kinase II phosphorylated a different serine residue than was phosphorylated by the cyclic AMP-dependent protein kinase. Limited proteolysis sequentially reduced the subunit Mr from 62 to 59 kilodaltons and finally to 57 kilodaltons, resulting in loss of the site phosphorylated by the Ca2+/CaM-dependent protein kinase II, but not the site phosphorylated by the cyclic AMP-dependent protein kinase. Phosphorylation by the Ca2+/CaM-dependent protein kinase II had little direct effect on the kinetic properties of tyrosine hydroxylase, but did convert it to a form that could be activated twofold by addition of an activator protein. This heat-labile activator protein increased the Vmax without affecting the Km for the pterin cofactor. This effect was specific in that the activator protein was without effect on nonphosphorylated tyrosine hydroxylase or on tyrosine hydroxylase phosphorylated by the cyclic AMP-dependent protein kinase. These results are consistent with the hypothesis that the "Vmax-type" activation of tyrosine hydroxylase observed upon depolarization of neural and adrenal tissues may be mediated by the Ca2+/CaM-dependent protein kinase II.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002851:Chromatography, High Pressure Liquid; D003342:Corpus Striatum / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D014357:Trypsin; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D010710:Phosphates; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin