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.