Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a nonacylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGFregulated to produce NO. Endothelial nitric-oxide synthase (eNOS)1 is the NOS isoform responsible for cardiovascular homeostasis including regulation of blood pressure, vessel remodeling, and angiogenesis. In addition to the profound physiological role of eNOS-derived NO, eNOS is unique among NOS family members since it is a peripheral membrane protein that is modified by co-translational N-myristoylation and post-translational cysteine palmitoylation (1, 2). Both N-myristoylation and cysteine palmitoylation are necessary for the subcellular targeting of eNOS onto peripheral aspects of the Golgi complex and to cholesterol-rich microdomains of the plasma membrane including caveolae/ lipid rafts (3, 4). Moreover, mislocalization of the enzyme to either domain impairs agonist-stimulated NO release from cells, implying that the proper subcellular localization of eNOS is critical for stimulus-dependent coupling to the enzyme (5, 6). Recently many investigators have shown that protein phosphorylation of eNOS by several serine/threonine kinases is a critical control step for NO production by endothelial cells. Phosphorylation by AMP kinase (7), Akt (or protein kinase B)

The relative importance of lipid rafts vs. specialized rafts termed caveolae to influence signal transduction is not known. Here we show that in cells lacking caveolae, the dually acylated protein, endothelial nitric oxide synthase (eNOS), localizes to cholesterolrich lipid raft domains of the plasma membrane. In these cells, expression of caveolin-1 (cav-1) stimulates caveolae biogenesis, promotes the interaction of cav-1 with eNOS, and the inhibition of NO release from cells. Interestingly, in cells where cav-1 does not drive caveolae assembly, despite equal levels of cav-1 and eNOS and localization of both proteins to raft domains of the plasmalemma, the physical interaction of eNOS with cav-1 is dramatically less resulting in less inhibition of NO release. Thus, cav-1 concentrated in caveolae, not in rafts, is in closer proximity to eNOS and is necessary for negative regulation of eNOS function, thereby providing the first clear example of spatial regulation of signaling in this organelle that is distinct from raft domains.T he lipid raft hypothesis formulated more than 10 years ago (1), postulated the existence of lipid rafts as dynamic assemblies of cholesterol and sphingolipids in the plasma membrane. Caveolae are specialized lipid rafts because of the ability of caveolins to initiate caveolae biogenesis from raft-derived components. The proposed functions of rafts͞caveolae are diverse and somewhat controversial, including cholesterol transport (2, 3), endocytosis (4), potocytosis (5), and signal transduction (6-9). In addition, there is much confusion in the literature where the distinction among lipid rafts, flattened caveolae, or caveolae proper is less than clear, thus making it difficult to ascertain which cellular functions might be attributable to rafts, caveolae, or caveolins, per se.For example, because cholesterol-modifying drugs such as cyclodextrins remove cholesterol from plasmalemmal rafts and caveolae, these reagents cannot distinguish between signaling events occurring in these compartments. Moreover, from a biophysical perspective, it is unknown whether the curvature and structure of plasmalemma-attached caveolae with caveolin-1 (cav-1) as the coat protein or whether the presence of cav-1 protein in the plasma membrane, per se, is necessary for the effects of cav-1 on any target protein or function. This distinction is important because although cav-1 is the main structural protein of caveolae, it may also exist in lipid rafts without the formation of plasmalemma-attached caveolae (10), be found in the trans-Golgi network as originally described (9), or found in other organelles (11).Endothelial nitric-oxide synthase (eNOS) is a dually acylated signaling protein responsible for the production of nitric oxide (NO) in the cardiovascular system. N-myristoylated and cysteine palmitoylation of eNOS targets eNOS to the cytoplasmic face of the Golgi and to plasmalemmal caveolae. eNOS in these domains can interface with several aspects of signal transduction systems through direct interactions wi...

Caveolae are cholesterol and glycosphingolipid-rich flask-shaped invaginations of the plasma membrane which are particularly abundant in vascular endothelium and present in all other cell types of the cardiovascular system, including vascular smooth-muscle cells, macrophages, cardiac myocytes, and fibroblasts. Caveolins and the more recently discovered cavins are the major protein components of caveolae. When caveolae were discovered, their functional role was believed to be limited to transport across the endothelial cell barrier. Since then, however, a large body of evidence has accumulated, suggesting that these microdomains are very important in regulating many other important endothelial cell functions, mostly due to their ability to concentrate and compartmentalize various signaling molecules. Over the course of several years, multiple studies involving knockout mouse and small interfering RNA approaches have considerably enhanced our understanding of the role of caveolae and caveolin-1 in regulating many cardiovascular functions. New findings have been reported implicating other caveolar protein components in endothelial cell signaling and function, such as the understudied caveolin-2 and newly discovered cavin proteins. The aim of this review is to focus primarily on molecular and cellular aspects of the role of caveolae, caveolins, and cavins in endothelial cell signaling and function. In addition, where appropriate, the possible implications for the cardiovascular and pulmonary physiology and pathophysiology will be discussed.

Caveolin-1 and -2 are the two major coat proteins found in plasma membrane caveolae of most of cell types. Here, by using adenoviral transduction of either caveolin-1 or caveolin-2 or both isoforms into cells lacking both caveolins, we demonstrate that caveolin-2 positively regulates caveolin-1-dependent caveolae formation. More importantly, we show that caveolin-2 is phosphorylated in vivo at two serine residues and that the phosphorylation of caveolin-2 is necessary for its actions as a positive regulator of caveolin-1 during organelle biogenesis in prostate cancer cells. Mutation of the primary phosphorylation sites on caveolin-2, serine 23 and 36, reduces the number of plasmalemma-attached caveolae and increases the accumulation of noncoated vesicles, but does not affect trafficking of caveolin-2, interaction with caveolin-1 or its biophysical properties. Thus, the phosphorylation of caveolin-2 is a novel mechanism to regulate the dynamics of caveolae assembly.