Tyrosine hydroxylase phosphorylation in digitonin-permeabilized bovine adrenal chromaffin cells: the effect of protein kinase and phosphatase inhibitors on Ser19 and Ser40 phosphorylation.
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.
Gonçalves
C A
CA
Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, New South Wales, Australia.
Hall
A
A
Sim
A T
AT
Bunn
S J
SJ
Marley
P D
PD
Cheah
T B
TB
Dunkley
P R
PR
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*
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D010744:Phosphoric Monoester Hydrolases; D004072:Digitonin; D002118:Calcium