Nitric oxide homeostasis is mediated by endothelial argininosuccinate synthase phosphorylation

Argininosuccinate synthase (AS) is part of the nitric oxide metabolic complex. It provides a dedicated source of arginine for endothelial nitric oxide synthase (eNOS) catalyzed nitric oxide (NO) production, protects endothelial cells from apoptosis and mediates the shear stress response. AS is regulated at the level of transcription, but due to the need to quickly alter NO levels in response to cellular cues, we hypothesized that AS activity could be mediated via post‐translational modifications (PTM). Bioinformatic searches identified several putative PTM. Due to the important role of serine/threonine phosphorylation for NO production, we focused our studies on these modifications. We found 9 serine and 6 threonine residues with a high probability of being phosphorylated and several putative kinases for these sites that are associated with regulating NO production. We utilized four approaches to validate our in‐silico findings: immunoprecipitation, overexpression, affinity chromatography and His‐tag purification. All methods indicate that AS is phosphorylated at serine/threonine residues. We also have evidence indicating that AS phosphorylation is physiologically relevant as both insulin and bradykinin impact the level of phosphorylation. Future studies are designed to identify the sites of phosphorylation and their functional relevance.