Role of Serine-19 Phosphorylation in Regulating Tyrosine Hydroxylase Studied with Site- and Phosphospecific Antibodies and Site-Directed Mutagenesis

Abstract: The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or Leu¹⁹]-recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser¹⁹ and Ser⁴⁰ in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in l -DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser¹⁹ (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser⁴⁰ phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser⁴⁰, as opposed to Ser¹⁹, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser⁴⁰ but not Ser¹⁹. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser¹⁹ phosphorylation had been eliminated (by substitution of Leu for Ser¹⁹). Thus, depolarization-induced increases in the stoichiometry of Ser¹⁹ phosphorylation do not appear to influence directly the activity of TH in situ.