Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites

Biochemistry. 2002 Dec 31;41(52):15845-53. doi: 10.1021/bi026732g.

Abstract

The endothelial isoform of nitric oxide synthase (eNOS) is a calcium/calmodulin-dependent enzyme that catalyzes the synthesis of nitric oxide, a key mediator of vascular homeostasis. eNOS undergoes a variety of posttranslational modifications, including phosphorylation on at least three residues: serines 116 and 1179 and threonine 497. Although the agonist-modulated protein kinase pathways that lead to eNOS phosphorylation have been studied in detail, the signaling pathways governing eNOS dephosphorylation remain less well characterized. The present study identifies protein phosphatase 2A (PP2A) as a key determinant of eNOS dephosphorylation and enzyme activity. We transfected bovine aortic endothelial cells (BAEC) with epitope-tagged cDNAs encoding wild-type eNOS or a series of phosphorylation-deficient eNOS mutants, immunoprecipitated [(32)P(i)] biosynthetically labeled recombinant proteins using antibodies directed against the epitope tag and treated the [(32)P(i)]-phosphorylated eNOS with protein phosphatases. We found that PP2A dephosphorylates eNOS residues threonine 497 and serine 1179 but not serine 116 and that an eNOS mutant lacking these three established phosphorylation sites is robustly labeled when expressed in BAEC and is dephosphorylated by PP2A. An inhibitor of PP2A increases eNOS enzymatic activity and augments overall levels of eNOS phosphorylation, specifically increasing phosphorylation of serines 116 and 1179. When transfected into BAEC or COS-7 cells, a "phospho-mimetic" eNOS mutant in which threonine 497 is changed to aspartate shows attenuated phosphorylation at serine 1179 as well as reduced enzyme activity in COS-7 cells. Our results indicate that regulation of eNOS dephosphorylation may be a key point for control of nitric oxide-dependent signaling pathways in vascular endothelial cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • COS Cells
  • Cattle
  • Cell Line / enzymology
  • Cell Line / metabolism
  • Enzyme Activation / drug effects
  • Enzyme Activation / genetics
  • Enzyme Inhibitors / pharmacology
  • Marine Toxins
  • Mutagenesis, Site-Directed*
  • Nitric Oxide Synthase / genetics*
  • Nitric Oxide Synthase / metabolism*
  • Nitric Oxide Synthase Type III
  • Okadaic Acid / pharmacology
  • Oxazoles / pharmacology
  • Phosphates / metabolism
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / chemistry*
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphoprotein Phosphatases / physiology
  • Phosphorus Radioisotopes / metabolism
  • Phosphorylation / drug effects
  • Protein Phosphatase 2
  • Serine / genetics
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Threonine / genetics
  • Transfection

Substances

  • Enzyme Inhibitors
  • Marine Toxins
  • Oxazoles
  • Phosphates
  • Phosphorus Radioisotopes
  • Okadaic Acid
  • Threonine
  • Serine
  • calyculin A
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type III
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2