Co-regulation of AS and eNOS phosphorylation by calcium 1 Protein Kinase C α Phosphorylates a Novel Argininosuccinate Synthase Site at Serine 328 During Calcium-Dependent Stimulation of Endothelial Nitric Oxide Synthase in Vascular Endothelial Cells*

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 (AL), providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS and AL, 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 report, we showed that AS Ser328 phosphorylation was coordinately regulated with eNOS Ser1179 phosphorylation when bovine aortic endothelial cells (BAECs) 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 C alpha (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser328 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. _______________________________________