Dephosphorylation of argininosuccinate synthase at serine 328 corresponds with stimulated vascular endothelial nitric oxide production

Deciphering the molecular mechanisms involved in the constitutive and stimulated production of nitric oxide (NO) by endothelial cells is critical to understanding how physiologic stimuli mediate their cardiovascular protective effects, or how risk factors mediate their deleterious effects on the vascular wall. Considering endothelial function and viability is highly dependent on argininosuccinate synthase (AS) function, very little is known about the regulation of AS function as it relates to endothelial NO production. The bulk of the literature that discusses the regulation of AS suggests that it occurs primarily at the level of transcription. In this report, we demonstrate that AS is phosphorylated at serine‐328 allowing for more immediate regulation of arginine availability to accommodate changes in vascular endothelial NO production. Using insulin and other stimulants, we show by western blot analysis that the dephosphorylation AS‐Ser328 correlates with dephosphorylation of eNOS‐Thr495, and phosphorylation of eNOS‐Ser1179, the activated state of eNOS in vascular endothelial cells. Significantly, these results represent the first report identifying a biologically significant phosphorylation site involved in AS regulation. As importantly, these results also demonstrate another level of control that provides for the coordinate regulation of eNOS and AS in the citrulline‐nitric oxide cycle.