ERK1 and ERK2, two microtubule-associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ.
Tyrosine hydroxylase (TH) is phosphorylated at four sites in situ and in vivo, and the protein kinases that phosphorylate three of these sites (Ser8,Ser19,Ser40) have been identified. In intact cells, the phosphorylation of the fourth site (Ser31) is increased in response to phorbol esters or nerve growth factor (NGF). Here, we show that Ser31 is phosphorylated by ERK1 and ERK2, two myelin basic protein and microtubule-associated protein kinases. Extracts of NGF- or bradykinin-treated PC12 rat pheochromocytoma cells were fractionated on Mono Q columns. Protein kinase activity toward Ser31 in TH was present in two peaks corresponding to myelin basic protein kinase activities previously identified as ERK1 and ERK2. Phosphorylation of purified TH in vitro by both kinases was selective for Ser31 up to at least 0.6 mol of phosphate per mol of TH subunit. Treatment of intact PC12 cells with bradykinin or NGF increased both the phosphorylation of TH-Ser31 in situ and the catalytic activity of ERKs (measured subsequently in vitro with myelin basic protein as substrate). Pretreatment of the cells with genistein (a protein-tyrosine kinase inhibitor) decreased the bradykinin- but not the NGF-induced changes in both TH-Ser31 phosphorylation and ERK activity. Genistein also inhibited the increases in Ser31 phosphorylation produced by phorbol dibutyrate, muscarine, and Ba2+. The data indicate that ERK activity is responsible for phosphorylating TH at Ser31 in intact cells and suggest that TH-Ser31 phosphorylation may be regulated by multiple signaling pathways that converge at or prior to the activation of the ERKs.
Department of Biochemistry, Louisiana State University Medical Center, New Orleans 70119.
J W
JW
Haycock
N G
NG
Ahn
M H
MH
Cobb
E G
EG
Krebs
D000595:Amino Acid Sequence; D000818:Animals; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D007529:Isoflavones; D046911:Macromolecular Substances; D010455:Peptides; D012694:Serine; D019833:Genistein; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases