Phosphorylation of argininosuccinate synthase at T131 correlates with changes in NO production

Abstract

The citrulline‐nitric oxide cycle is a tightly regulated system for the production of nitric oxide (NO) in vascular endothelial cells. Argininosuccinate synthase (AS) acts as the rate‐limiting step in the cycle which converts citrulline back to arginine, providing the essential substrate for endothelial nitric oxide synthase. Previous work has demonstrated that AS is a phospho‐protein, a post‐translational modification that allows AS to support acute changes in NO production. Bioinformatic analysis identified a putative AS phosphorylation site at threonine 131, which was confirmed by LC‐MS analysis showing that phosphorylation at T131 increased upon treatment of endothelial cells with the serine/threonine phosphatase inhibitor okadaic acid. To investigate the role of phosphorylation of AS at T131, analysis was carried out comparing the overexpression of phospho‐null (T to A) and phospho‐mimetic (T to D) mutations to wild type AS. After knockdown of endogenous AS by siRNA, rescue of AS function was measured by the recovery of NO production in endothelial cells under conditions of limited arginine. Expression of the phospho‐null mutation showed similar levels of NO production to wild type AS. However, expression of the T131 phospho‐mimetic AS construct demonstrated reduced NO upon stimulation with VEGF confirming the physiological significance of T131 phosphorylation. (NIH RO1 HL083153‐01A2).