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Tyrosine Hydroxylase Phosphorylation in Digitonin‐Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser 19 and Ser 40 Phosphorylation
Author(s) -
Gonçalves CarlosAlberto,
Hall Amanda,
Sim Alistair T. R.,
Bunn Stephen J.,
Marley Philip D.,
Cheah Tat B.,
Dunkley Peter R.
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.69062387.x
Subject(s) - phosphorylation , okadaic acid , protein kinase c , protein kinase a , phosphatase , kinase , biology , protein phosphorylation , digitonin , calmodulin , mitogen activated protein kinase kinase , biochemistry , microbiology and biotechnology , protein tyrosine phosphatase , enzyme
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 [γ‐ 32 P]ATP, in the presence or absence of 10 µ M Ca 2+ , 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca 2+ increased the phosphorylation of Ser 19 and Ser 40 . Cyclic AMP, and phorbol dibutyrate in the presence of Ca 2+ , increased the phosphorylation of only Ser 40 . Ser 31 and Ser 8 were not phosphorylated. The Ca 2+ ‐stimulated phosphorylation of Ser 19 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 Ca 2+ ‐stimulated phosphorylation of Ser 40 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 Ser 19 and Ser 40 were dephosphorylated by PP2A.