TY - JOUR
T1 - Depolarization-stimulated catecholamine biosynthesis
T2 - Involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ
AU - Salvatore, Michael F.
AU - Waymire, Jack C.
AU - Haycock, John W.
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
KW - Blot immunolabeling
KW - Ca/calmodulin-dependent protein kinase II
KW - Extracellular signal-regulated protein kinase
KW - PC12
KW - PD98059
KW - cAMP-dependent protein kinase
UR - http://www.scopus.com/inward/record.url?scp=0034770233&partnerID=8YFLogxK
U2 - 10.1046/j.1471-4159.2001.00593.x
DO - 10.1046/j.1471-4159.2001.00593.x
M3 - Article
C2 - 11677263
AN - SCOPUS:0034770233
SN - 0022-3042
VL - 79
SP - 349
EP - 360
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
IS - 2
ER -