Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Authors

  • Carlos-Alberto Gonçalves,

    1. Neuroscience Group, Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, New South Wales; and
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    • The present address of Dr. C.-A. Gonçalves is Departamento De Bioquimica, UFRGS, Porto Alegre, Brazil.

  • Amanda Hall,

    1. Neuroscience Group, Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, New South Wales; and
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  • Alistair T. R. Sim,

    1. Neuroscience Group, Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, New South Wales; and
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  • Stephen J. Bunn,

    1. Neuroscience Group, Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, New South Wales; and
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  • Philip D. Marley,

    1. Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia
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  • Tat B. Cheah,

    1. Neuroscience Group, Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, New South Wales; and
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  • Peter R. Dunkley

    Corresponding author
    1. Neuroscience Group, Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, New South Wales; and
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Address correspondence and reprint requests to Prof. P. Dunkley at Discipline of Medical Biochemistry, University of Newcastle, Newcastle NSW 2308, Australia.

Abstract

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 [γ-32P]ATP, in the presence or absence of 10 µM Ca2+, 1 µM cyclic AMP, 1 µM 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.

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