Abstract-Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation. Key Words: nitric oxide Ⅲ signaling Ⅲ scaffold Ⅲ hsp90 Ⅲ Akt E ndothelial nitric oxide synthase (eNOS) continually produces low levels of nitric oxide (NO) to regulate several aspects of cardiovascular homeostasis. In endothelial cells and cells transfected with the eNOS cDNA, eNOS behaves as a peripheral membrane protein that is regulated by the allosteric activator, calmodulin (CaM). In vitro, the addition of CaM to recombinant eNOS markedly accelerates NOS catalytic function and NO synthesis. 1 However, in vivo, additional regulatory mechanisms other than CaM participate in eNOS activation/inactivation. This concept is supported by studies demonstrating that mislocalization of eNOS secondary to mutations that block its membrane association do not influence its catalytic function or calcium dependency in vitro; however, agonist-stimulated NO release from cells is markedly diminished. 2-4 These studies imply that spatial and temporal regulation of membrane associated eNOS function must involve other protein-protein or protein-lipid interactions that impact on its activation state.In the past several years, many protein partners that interact with eNOS have been described, including caveolins-1 and -3, 5,6 heat shock protein 90 (hsp90), 7 dynamin-2, 8 G protein-coupled receptors, 9 and certain kinases including Akt and mitogen-activated protein kinase family members. 10,11 All these proteins have been show to interact with eNOS in conventional in vitro assays, including coprecipitations, affinity chromatography, and yeast 2-hybrid analysis. In vivo, there is compelling evidence supporting the importance of caveolin-1, hsp90, and Akt in regulating NO release because overe...

Tumor vasculature is hyperpermeable to macromolecules compared to normal vasculature; however, the relationship between tumor hyperpermeability and tumor progression is poorly understood. Here we show that a cell-permeable peptide derived from caveolin-1, termed cavtratin, reduces microvascular hyperpermeability and delays tumor progression in mice. These antipermeability and antitumor actions of cavtratin occur in the absence of direct cytostatic or antiangiogenic effects. Cavtratin blocks microvascular permeability by inhibiting endothelial nitric oxide synthase (eNOS), as the antipermeability and antitumor actions of cavtratin are markedly diminished in eNOS knockout mice. Our results support the concepts that hyperpermeability of tumor blood vessels contributes to tumor progression and that blockade of eNOS may be exploited as a novel target for antitumor therapy.

). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of AspPro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WT versus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases.

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This study, although only introductory, indicates the potential for extrapedicular vertebral body fixation in the thoracic spine. Biomechanical evaluations are presently being conducted to evaluate the use of extrapedicular thoracic screw fixation.