Endothelial Nitric Oxide Synthase Is Regulated by Tyrosine Phosphorylation and Interacts with Caveolin-1

Abstract

The regulation of endothelial nitric oxide synthase (eNOS) by phosphorylation is poorly understood. Here, we demonstrate that eNOS is tyrosine-phosphorylated in bovine aortic endothelial cells (BAEC) using 32 P metabolic labeling followed by phosphoamino acid analysis and by phosphotyrosine specific Western blotting. Treatment of BAEC with hydrogen peroxide and the protein tyrosine phosphatase inhibitor, sodium orthovanadate, increases eNOS tyrosine phosphorylation. Utilizing a novel immunoNOS assay, the increase in tyrosine phosphorylation is associated with a 50% decrease in the specific activity of the enzyme. Because eNOS is localized in plasmalemma caveolae, we examined if tyrosine phosphorylated eNOS interacts with caveolin-1, the coat protein of caveolae. Immunoprecipitation of eNOS from bovine lung microvascular endothelial cells resulted in the co-precipitation of caveolin-1. Conversely, immunoprecipitation of caveolin-1 resulted in the co-precipitation of tyrosine-phosphorylated eNOS. Thus, tyrosine phosphorylation is a novel regulatory mechanism for eNOS and caveolin-1 is the first eNOSassociated protein. Collectively, these observations provide a novel regulatory mechanism for eNOS and suggest that tyrosine phosphorylation may influence its activity, subcellular trafficking, and interaction with other caveolin-interacting proteins in caveolae.Post-translational modification of proteins is a widely used mechanism for transmitting biological signals and regulating the activities, biosynthesis, and degradation of enzymes. One of these modifications, protein phosphorylation, plays a central role in signal transduction pathways regulating many biological processes, including cellular proliferation, migration, and differentiation. More specifically, phosphorylation of tyrosine residues is an essential step in cellular activation by many external signals, including growth factors, cytokines, and cellular stress (1, 2).In the cardiovascular and nervous systems, activation of cell surface receptors triggers the immediate synthesis of nitric oxide (NO).1 In the cardiovascular system, NO is derived from one of the chemically equivalent guanidino nitrogens of L-arginine, in a reaction catalyzed by endothelial NO synthase (eNOS or NOS 3). In intact blood vessels and in cultured endothelial cells (EC), eNOS is rapidly activated by agonists that mobilize intracellular calcium. Increases in intracellular calcium facilitate interactions with calmodulin and the activated calciumcalmodulin complex can stimulate NADPH-dependent electron flux through eNOS to produce NO (3). Recent evidence suggests that eNOS can also be activated in a calcium-independent manner by fluid shear stress (4 -6) and insulin-like growth factor (7), presumably mediated through a tyrosine kinase cascade, since inhibitors of tyrosine kinases, but not chelation of intracellular calcium, inhibits NO release. Whether eNOS is directly tyrosine-phosphorylated or indirectly linked to a tyrosine kinase cascade is not known. Once NO is produced by either ...

Prostate-specific membrane antigen expression in regeneration and repair

“…1 Therefore, our findings of PSMA expression in endothelial cells associated with neovasculature may indicate that it is functioning to increase folic acid levels at the site of neovasculature formation, so that folic acid can exert its proangiogenic effects (Figure 4). In support of this, PSMA has been reported to coimmunoprecipitate with the caveolae marker, caveolin 1, 37 which also coimmunoprecipitates with eNOS, 38,39 suggesting the in vivo proximity of PSMA and eNOS.…”

Analysis of the CAVEOLIN-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines

“…Puri®ed caveolae membranes are enriched with speci®c lipids (cholesterol and glycosphingolipids (Murata et al, 1995;Fra et al, 1995)) and a plethora of lipidmodi®ed signalling molecules including H-ras, c-src, other src-like kinases, eNOS, plasminogen activator and heterotrimeric G-proteins Li et al, 1995Garcia-Cardena, et al, 1996;Stahl and Mueller, 1995). In addition, a number of membrane associated receptors, including several that are coupled to G-proteins or possess intrinsic tyrosine kinase activity, localize to or are internalized by caveolae (Lisanti et al, 1994).…”

Recombinant Expression of Caveolin-1 in Oncogenically Transformed Cells Abrogates Anchorage-independent Growth

“…In addition, evidence has been presented suggesting that caveolin-1 interacts directly with many of these proteins: G-protein ␣ subunits (8,17,22,25,33), Src-family tyrosine kinases (27), H-Ras (26), eNOS (71,72), and Shc (73). Binding to caveolin-1 appears to be mediated by a cytoplasmic membrane-proximal region of caveolin-1, termed the caveolin scaffolding domain (27,33).…”