title: Activation of tyrosine hydroxylase by intermittent hypoxia: involvement of serine phosphorylation.
abstract: Regulation of tyrosine hydroxylase (TH) by intermittent hypoxia (IH) was investigated in rat pheochromocytoma 12 (PC-12) cells by exposing them to alternating cycles of hypoxia (1% O2, 15 s) and normoxia (21% O2, 3 min) for up to 60 cycles; controls were exposed to normoxia for a similar duration. IH exposure increased dopamine content and TH activity by approximately 42 and approximately 56%, respectively. Immunoblot analysis revealed that comparable levels of TH protein were expressed in normoxic and IH cells. Removal of TH-bound catecholamines and in vitro phosphorylation of TH in cell-free extracts by the catalytic subunit of protein kinase A (PKA) increased TH activity in normoxic but not in IH cells, suggesting possible induction of TH phosphorylation and removal of endogenous inhibition of TH by IH. To assess the role of serine phosphorylation in IH-induced TH activation, TH immunoprecipitates and extracts derived from normoxic and IH cells were probed with anti-phosphoserine and anti-phospho-TH (Ser-40) antibody, respectively. Compared with normoxic cells, total serine and Ser-40-specific phosphorylation of TH were increased in IH cells. IH-induced activation of TH and the increase in total serine and Ser-40-specific phosphorylation of TH were inhibited by Ca2+/calmodulin-dependent protein kinase (CaMK) and PKA-specific inhibitors but not by inhibitors of the extracellular signal-regulated protein kinase pathway, suggesting that IH activates TH in PC-12 cells via phosphorylation of serine residues including Ser-40, in part, by CaMK and PKA. Our results also suggest that IH-induced phosphorylation of TH facilitates the removal of endogenous inhibition of TH, leading to increased synthesis of dopamine.
authors.affiliation: Department of Biochemistry,Western Reserve University, Cleveland, OH 44106-4935, USA. kgk@po.cwru.edu
authors.forename: Ganesh K
authors.initials: GK
authors.lastname: Kumar
authors.forename: Dong-Kyu
authors.initials: DK
authors.lastname: Kim
authors.forename: Myeong-Seon
authors.initials: MS
authors.lastname: Lee
authors.forename: Remya
authors.initials: R
authors.lastname: Ramachandran
authors.forename: Nanduri R
authors.initials: NR
authors.lastname: Prabhakar
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002395:Catecholamines / Q000378:metabolism; D002470:Cell Survival; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D004789:Enzyme Activation; D000860:Hypoxia / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D012008:Recurrence; D012694:Serine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
chemical_list: D002395:Catecholamines; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004298:Dopamine