NOSIP, a novel modulator of endothelial nitric oxide synthase activity

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector pro teins such as Ca2+-calmodulin, by posttranslational modifications such as p hosphorylation via protein kinase B, and by translocation of the enzyme fro m the plasma membrane caveolae to intracellular compartments. Reversible ac ylation of eNOS is thought to contribute to the intracellular trafficking o f the enzyme; however, protein factor(s) that govern the translocation of t he enzyme are still unknown, Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting prote in), which avidly binds to the carboxyl-terminal region of the eNOS oxygena se domain. Coimmunoprecipitation studies demonstrated the specific interact ion of eNOS and NOSIP in vitro and in vivo, and complex formation was inhib ited by a synthetic peptide of the caveolin-1 scaffolding domain. NO produc tion was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP, Stimulation with the calcium ionophore A2 3187 induced the reversible translocation of eNOS from the detergent-insolu ble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS, Im munofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that aas abolished in the presence of NOSIP. Subcellular f ractionation studies identified eNOS in the caveolin-rich membrane fraction s of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracel lular compartments, We conclude that NOSIP is a novel type of modulator tha t promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.