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 Ca 21 -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 in¯uence 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 1 -stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-de®cient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using siteand phosphorylation state-speci®c antibodies. Treatment with elevated K 1 (158 mM) for 5 min increased TH PS at each site in a Ca 21 -dependent manner. Pretreatment with PD98059 prevented elevated K 1 -stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not signi®cantly increased by forskolin, and elevated K 1 -stimulated Ser40 PS was three-to ®ve-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K 1 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. Keywords: blot immunolabeling, Ca 21 /calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, extracellular signal-regulated protein kinase, PC12, PD98059.

Targeting Tyrosine Hydroxylase to Improve Bradykinesia

“…This supported an earlier observation, before ser31 was identified, that the peptide fragment associated with TH activation had ser31 in the sequence (Tachikawa et al, 1987). Indeed, increased ser31 phosphorylation, alone from NGF treatment or in conjunction with depolarization-stimulated ser19 phosphorylation, enhances L-DOPA accumulation and is independent of any affect on ser40 phosphorylation (Mitchell et al, 1990;Harada et al, 1996;Salvatore et al, 2001). There certainly is substantial evidence that ser40 phosphorylation can modulate TH activity from in vitro and in situ work.…”

“…These observations are highly relevant when applied to interpreting the impact of changes in ser31 and ser40 phosphorylation observed in vivo. Indeed, the results obtained in PC12 cells may have direct relevance to the in vivo situation because the basal phosphorylation of TH at ser31 and ser40 in the PC12 cell line versus that in the CNS are very similar, ranging from a phosphorylation stoichiometry of 0.02-0.05 for ser40 compared to 0.05 to 0.35 for ser31 (Salvatore et al, 2001;2009a;2009b) (Table 1). Therefore, in order to definitively answer whether any change in ser40 phosphorylation observed in vivo is of any consequence to L-DOPA biosynthesis capabilities, we must first define how much phosphorylation is necessary at ser40 and ser31 to affect L-DOPA biosynthesis capabilities in vivo.…”

“…Furthermore, treatment with NGF increased TH activity for out to 1 hour (Greene et al, 1984). Subsequent studies indicated that the effect of NGF on TH phosphorylation is solely due to enhanced ser31 phosphorylation (Haycock 1990;Salvatore et al, 2001). There is also evidence that NGF-signaling can increase TH promoter activity (Suzuki et al, 2004).…”

“…Therefore, in order to definitively answer whether any change in ser40 phosphorylation observed in vivo is of any consequence to L-DOPA biosynthesis capabilities, we must first define how much phosphorylation is necessary at ser40 and ser31 to affect L-DOPA biosynthesis capabilities in vivo. (Salvatore et al, 2001) versus the ranges reported in the nigrostriatal pathway in vivo (Salvatore et al, 2000;2009a;2009b). The in vivo TH phosphorylation ranges in striatum and substantia nigra represents results from mice (C57B1/6) and rats (Sprague-Dawley, Fischer 344, and Brown-Norway/Fischer 344 F 1 hybrid).…”

Protein Kinase Cδ Negatively Regulates Tyrosine Hydroxylase Activity and Dopamine Synthesis by Enhancing Protein Phosphatase-2A Activity in Dopaminergic Neurons

“…Of the different phosphorylation sites, Ser40 is the major regulatory site contributing to increased TH activity and dopamine synthesis in vivo (Meligeni et al, 1982;Waymire et al, 1991;Daubner et al, 1992;Wu et al, 1992;McCulloch et al, 2001). Studies using PC12 cells either responsive or nonresponsive to cAMP stimuli demonstrated that PKA is an important kinase in THser40 phosphorylation (Wilson et al, 1996;Salvatore et al, 2001). Recently, Kobori et al (2004) reported that glial cell line-derived neurotrophic factor increases TH-ser31 and TH-ser40 phosphorylation, which contributes to enhanced dopamine synthesis in mesencephalic cultures.…”