title: Tyrosine hydroxylase phosphorylation in digitonin-permeabilized bovine adrenal chromaffin cells: the effect of protein kinase and phosphatase inhibitors on Ser19 and Ser40 phosphorylation.
abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [gamma-32P]ATP, in the presence or absence of 10 microM Ca2+, 1 microM cyclic AMP, 1 microM phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40. Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+, increased the phosphorylation of only Ser40. Ser31 and Ser8 were not phosphorylated. The Ca2+-stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273-302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+-stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5-22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19-31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A.
authors.affiliation: Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, New South Wales, Australia.
authors.forename: C A
authors.initials: CA
authors.lastname: Gonçalves
authors.forename: A
authors.initials: A
authors.lastname: Hall
authors.forename: A T
authors.initials: AT
authors.lastname: Sim
authors.forename: S J
authors.initials: SJ
authors.lastname: Bunn
authors.forename: P D
authors.initials: PD
authors.lastname: Marley
authors.forename: T B
authors.initials: TB
authors.lastname: Cheah
authors.forename: P R
authors.initials: PR
authors.lastname: Dunkley
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000201:enzymology*; D004072:Digitonin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011506:Proteins / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D010744:Phosphoric Monoester Hydrolases; D004072:Digitonin; D002118:Calcium