Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells

J Biol Chem. 2012 Jul 27;287(31):26168-76. doi: 10.1074/jbc.M112.378794. Epub 2012 Jun 13.


Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetophenones / pharmacology
  • Amino Acid Substitution
  • Animals
  • Aorta / cytology*
  • Argininosuccinate Synthase / genetics
  • Argininosuccinate Synthase / metabolism*
  • Benzopyrans / pharmacology
  • Bradykinin / pharmacology
  • Calcium / metabolism
  • Calcium / physiology*
  • Calcium Signaling
  • Cattle
  • Cells, Cultured
  • Endothelial Cells / enzymology*
  • Enzyme Activation
  • Gene Knockdown Techniques
  • Indoles / pharmacology
  • Isoenzymes / metabolism
  • Maleimides / pharmacology
  • Mutagenesis, Site-Directed
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase Type III / metabolism*
  • Okadaic Acid / pharmacology
  • Phosphorylation
  • Protein Kinase C-alpha / antagonists & inhibitors
  • Protein Kinase C-alpha / genetics
  • Protein Kinase C-alpha / metabolism*
  • Protein Phosphatase 1 / antagonists & inhibitors
  • Protein Phosphatase 2 / antagonists & inhibitors
  • Protein Processing, Post-Translational
  • RNA Interference
  • Serine / metabolism*


  • Acetophenones
  • Benzopyrans
  • Indoles
  • Isoenzymes
  • Maleimides
  • Okadaic Acid
  • Nitric Oxide
  • Serine
  • rottlerin
  • Nitric Oxide Synthase Type III
  • Protein Kinase C-alpha
  • Protein Phosphatase 1
  • Protein Phosphatase 2
  • Argininosuccinate Synthase
  • bisindolylmaleimide I
  • Bradykinin
  • Calcium