Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medicine

Major Professor

Duane Eichler, Ph.D.

Committee Member

Robert Deschenes, Ph.D.

Committee Member

Gloria Ferreira, Ph.D.

Committee Member

Andreas Seyfang, Ph.D.

Committee Member

Craig Doupnik, Ph.D.

Keywords

endothelial nitric oxide synthase, protein kinase C, caveolin, phosphorylation, localization, calcium signaling

Abstract

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays a role, and how phosphorylation affects AS function in endothelial cells. We developed a phospho-specific antibody directed against pS328 AS and assayed for increases or decreases in phosphorylation relative to physiological factors. We found that AS phosphorylation at S328 occurred when endothelial cells were stimulated with physiological factors that stimulate nitric oxide production through calcium-dependent stimulation of eNOS. Furthermore, by utilizing kinase inhibitors and kinase knockdown experiments, we showed that phosphorylation at S328 significantly decreased when PKCá was knocked down, suggesting that S328 phosphorylation of AS is involved in PKCá signaling. In addition, by confocal microscopy, immunoprecipitation, and membrane fractionation, we showed that phosphorylation at S328 of AS promotes its co-localization with eNOS in the perinuclear region. These findings describe a novel pathway involving AS regulation of nitric oxide production, and may serve as a novel drug target in the restoration of vascular nitric oxide homeostasis.

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