Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro - Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1

The activity of endothelial nitric-oxide synthase (eNOS) is regulated by it s subcellular localization, phosphorylation and through its interaction wit h different proteins. The association of eNOS with caveolin-1 (Cav) is beli eved to maintain eNOS in an inactive state; however, increased association of eNOS to heat shock protein 90 (hsp90) is observed following activation. In this study, we investigate the relationship between caveolin and hsp90 a s opposing regulatory proteins on eNOS function. Immunoprecipitation of Cav -1 from bovine lung microvascular endothelial cells shows that eNOS and hsp 90 are present in the Cav-1 complex. eNOS and hsp90 from the lysate also in teract with exogenous glutathione S-transferase-linked caveolin-1 (GST-Cav) , and the addition of calcium-activated calmodulin (CaM) to the GST-Cav com plex partially inhibited the association of eNOS and hsp90, Purified eNOS a ssociates with GST-Cav specifically through the caveolin-scaffolding do. ma in (residues 82-101); however, the addition of CaM slightly, but nonstatist ically, reduces eNOS binding to GST-Cav, When hsp90 is present in the bindi ng reaction, the addition of increasing concentrations of CaM significantly displaces eNOS and hsp90 from GST-Cav. eNOS enzymatic activity is also les s sensitive to inhibition by the caveolin scaffolding peptide (residues 82- 101) when eNOS is prebound to hsp90, Collectively, our results show that th e actions of CaM on eNOS dissociation from caveolin are facilitated in the presence of hsp90.