Phosphorylation and subcellular translocation of endothelial nitric oxide synthase.
In the vascular endothelium, diverse cell surface receptors are coupled to the Ca2+/calmodulin-dependent activation of nitric oxide (NO) synthase. We now report that, in intact cultured endothelial cells, several drugs and agonists are associated with increased serine phosphorylation of the endothelial NO synthase. We biosynthetically labeled bovine aortic endothelial cells with [32P]orthophosphoric acid, exposed the cells to various drugs and hormones, and then immunoprecipitated the enzyme from cell extracts using a highly specific anti-peptide antibody. The marked endothelial NO synthase phosphorylation induced by bradykinin is maximal only after 5 min of agonist exposure and is stable for at least 20 min. Basal and agonist-induced phosphorylation of the NO synthase in endothelial cells is completely inhibited by the calmodulin antagonist compound W-7. We prepared subcellular fractions of endothelial cells that had been biosynthetically labeled with [35S]methionine or [32P]orthophosphoric acid and immunoprecipitated the endothelial NO synthase from untreated (basal) and bradykinin-treated cells. In the basal state, [35S]methionine-labeled endothelial NO synthase is associated primarily with the particulate cellular fraction, but the phosphorylated enzyme is primarily cytosolic. Following exposure to bradykinin, a substantial fraction of the [35S]methionine-labeled NO synthase is now found in the cytosolic fraction, associated with a marked increase in the level of cytosolic enzyme phosphorylation. We propose that agonist-induced phosphorylation of NO synthase is associated with translocation of the enzyme from membrane to cytosol and may thereby regulate the biological effects of endothelial NO synthesis in situ.
Michel
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Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
Li
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GK
Busconi
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D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors
D010748:Phosphopeptides; D047428:Protein Kinase Inhibitors; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases