Rat pheochromocytoma tyrosine hydroxylase is phosphorylated on serine 40 by an associated protein kinase.
Tyrosine hydroxylase, a key enzyme in the biosynthesis of catecholamines, was previously shown to be phosphorylated on four distinct serine residues in PC12 cell cultures, each one being specific for the kinase system involved (McTigue, M., Cremins, J., and Halegoua, S. (1985) J. Biol. Chem. 260, 9047-9056). A cAMP- and Ca2+-independent protein kinase was found to be associated with tyrosine hydroxylase purified from rat pheochromocytoma tumor. The use of this activity and the availability of a large amount of purified tyrosine hydroxylase allowed identification of the site phosphorylated by this kinase activity. A peptide of 1.5 kDa (about 12 residues long), carrying the phosphorylation site, was released from 32P-labeled tyrosine hydroxylase by limited proteolysis with trypsin. This peptide was isolated from trypsinized tyrosine hydroxylase by sequential gel filtration and ion exchange chromatographies. Analysis by thin layer chromatography of an acid hydrolysate of the peptide revealed that it contained phosphoserine. The sequence determination of the peptide showed that it corresponded to the residues 38-45 in the tyrosine hydroxylase primary structure (Arg-Gln-Ser(P)-Leu-Ile-Glu-Asp-Ala). Thus, the associated kinase phosphorylated Ser-40, one of the phosphorylation sites for the cAMP-dependent protein kinase also found in rat pheochromocytoma tumors. These results are compared to those recently appearing in a report by Campbell et al. (Campbell, D. G., Hardie, D. G., and Vulliet, P. R. (1986) J. Biol. Chem. 261, 10489-10492).
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D010446:Peptide Fragments / Q000032:analysis; D010673:Pheochromocytoma / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D010446:Peptide Fragments; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D014357:Trypsin