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Characterization of a putative AS regulatory phosphorylation site implicated in vascular endothelial nitric oxide production

Argininosuccinate synthase (AS) catalyzes the rate‐limiting step of the nitric oxide (NO) cycle in vascular endothelial cells (EC); however, little is known about its regulation. Recently, we reported that AS is regulated by phosphorylation. Using in silico, as well as proteomic analysis, we have identified a putative phosphorylation site at serine 328 which is located adjacent to a caveolin binding motif located in the central region of AS tertiary structure. In this report, we examined the role of the S328 site relative to supporting NO production in ECs by mutation analysis, mutating S328 to either an alanine (S328A) or to the phospho‐mimetic aspartate (S328D). These constructs were overexpressed in ECs, and NO production was measured after stimulation with either the calcium ionophore plus ortho‐vanadate, or vascular endothelial growth factor (VEGF). In both cases, the S328D AS mutant produced significantly higher NO than wild type AS (n=3, p<0.05), while the S328A mutant had no effect over background. Notably, the S328A mutant also did not support cell viability when the BAECs were serum starved before VEGF incubation. These data suggest that the S328D mutant promotes stimulated NO production and maintains cell viability, and supports the proposal that S328 may be a biologically significant site for AS regulation.