Depolarization-stimulated catecholamine biosynthesis

Involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ

Michael Francis Salvatore, Jack C. Waymire, John W. Haycock

Research output: Contribution to journalArticleResearchpeer-review

82 Citations (Scopus)

Abstract

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca2+-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K+-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K+ (+ 58 mM) for 5 min increased TH PS at each site in a Ca2+-dependent manner. Pretreatment with PD98059 prevented elevated K+-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K+-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K+ and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.

Original languageEnglish
Pages (from-to)349-360
Number of pages12
JournalJournal of Neurochemistry
Volume79
Issue number2
DOIs
StatePublished - 1 Nov 2001

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Phosphorylation
Biosynthesis
Depolarization
Tyrosine 3-Monooxygenase
Protein Kinases
Catecholamines
Proteins
Stoichiometry
PC12 Cells
Phospho-Specific Antibodies
MAP Kinase Signaling System
Extracellular Signal-Regulated MAP Kinases
Colforsin
Protein Kinase Inhibitors
Cyclic AMP-Dependent Protein Kinases
Cells

Keywords

  • Blot immunolabeling
  • Ca/calmodulin-dependent protein kinase II
  • Extracellular signal-regulated protein kinase
  • PC12
  • PD98059
  • cAMP-dependent protein kinase

Cite this

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title = "Depolarization-stimulated catecholamine biosynthesis: Involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ",
abstract = "Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca2+-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K+-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K+ (+ 58 mM) for 5 min increased TH PS at each site in a Ca2+-dependent manner. Pretreatment with PD98059 prevented elevated K+-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K+-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K+ and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.",
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Depolarization-stimulated catecholamine biosynthesis : Involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ. / Salvatore, Michael Francis; Waymire, Jack C.; Haycock, John W.

In: Journal of Neurochemistry, Vol. 79, No. 2, 01.11.2001, p. 349-360.

Research output: Contribution to journalArticleResearchpeer-review

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T2 - Involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ

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