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Keywords:

  • Adrenal chromaffin cells;
  • Permeabilization;
  • Tyrosine hydroxylase;
  • Protein phosphorylation;
  • Calcium;
  • Protein kinases;
  • Protein phosphatases

Abstract

  1. Top of page
  2. 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.

Abbreviations used: BACC, bovine adrenal chromaffin cell; CaM-PKII, calcium/calmodulin-stimulated protein kinase II; CaM-PKII 273–302, CaM-PKII peptide inhibitor; CaM-PKII 291–317, peptide derived from CaM-PKII; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; KN92,2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; KN93, 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; MAPK, mitogen-activated protein kinase; MAPKAP kinases 1 and 2, mitogen-activated protein kinase-activated protein kinases 1 and 2; MARCKS, myristoylated alanine-rich C kinase substrate; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 12,13-dibutyrate; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PKA, protein kinase A; PKAi 5–22 amide, PKA peptide inhibitor; PKC, protein kinase C; PKCi 19–31, PKC peptide inhibitor; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; Ro 31-8220, 3-[1-[3-(amidinothio)propyl]-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimidemethane sulfonate; SDS, sodium dodecyl sulfate; TFP, trifluoperazine; TH, tyrosine hydroxylase; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride.