Reconstitution of an Endothelial Nitric-oxide Synthase (eNOS), hsp90, and Caveolin-1 Complex in Vitro

Abstract

The activity of endothelial nitric-oxide synthase (eNOS) is regulated by its subcellular localization, phosphorylation and through its interaction with different proteins. The association of eNOS with caveolin-1 (Cav) is believed 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 as opposing regulatory proteins on eNOS function. Immunoprecipitation of Cav-1 from bovine lung microvascular endothelial cells shows that eNOS and hsp90 are present in the Cav-1 complex. eNOS and hsp90 from the lysate also interact with exogenous glutathione S-transferase-linked caveolin-1 (GST-Cav), and the addition of calcium-activated calmodulin (CaM) to the GST-Cav complex partially inhibited the association of eNOS and hsp90. Purified eNOS associates with GST-Cav specifically through the caveolin-scaffolding domain (residues 82–101); however, the addition of CaM slightly, but nonstatistically, reduces eNOS binding to GST-Cav. When hsp90 is present in the binding reaction, the addition of increasing concentrations of CaM significantly displaces eNOS and hsp90 from GST-Cav. eNOS enzymatic activity is also less sensitive to inhibition by the caveolin scaffolding peptide (residues 82–101) when eNOS is prebound to hsp90. Collectively, our results show that the actions of CaM on eNOS dissociation from caveolin are facilitated in the presence of hsp90. The activity of endothelial nitric-oxide synthase (eNOS) is regulated by its subcellular localization, phosphorylation and through its interaction with different proteins. The association of eNOS with caveolin-1 (Cav) is believed 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 as opposing regulatory proteins on eNOS function. Immunoprecipitation of Cav-1 from bovine lung microvascular endothelial cells shows that eNOS and hsp90 are present in the Cav-1 complex. eNOS and hsp90 from the lysate also interact with exogenous glutathione S-transferase-linked caveolin-1 (GST-Cav), and the addition of calcium-activated calmodulin (CaM) to the GST-Cav complex partially inhibited the association of eNOS and hsp90. Purified eNOS associates with GST-Cav specifically through the caveolin-scaffolding domain (residues 82–101); however, the addition of CaM slightly, but nonstatistically, reduces eNOS binding to GST-Cav. When hsp90 is present in the binding reaction, the addition of increasing concentrations of CaM significantly displaces eNOS and hsp90 from GST-Cav. eNOS enzymatic activity is also less sensitive to inhibition by the caveolin scaffolding peptide (residues 82–101) when eNOS is prebound to hsp90. Collectively, our results show that the actions of CaM on eNOS dissociation from caveolin are facilitated in the presence of hsp90. endothelial nitric-oxide synthase nitric-oxide synthase nitric oxide calmodulin 90-kDa heat shock protein bovine lung microvascular endothelial cell(s) caveolin antibody Endothelial nitric-oxide synthase (eNOS)1 is the NOS isoform responsible for the physiological production of nitric oxide (NO) in the cardiovascular system. Endothelium-derived NO is an important regulator of systemic blood pressure, angiogenesis, and vascular architecture (1.Huang P.L. Huang Z. Mashimo H. Bloch K.D. Moskowitz M.A. Bevan J.A. Fishman M.C. Nature. 1995; 377: 239-242Crossref PubMed Scopus (1788) Google Scholar, 2.Murohara T. Asahara T. Silver M. Bauters C. Masuda H. Kalka C. Kearney M. Chen D. Symes J.F. Fishman M.C. Huang P.L. Isner J.M. J. Clin. Invest. 1998; 101: 2567-2578Crossref PubMed Scopus (1092) Google Scholar, 3.Rudic R.D. Shesely E.G. Maeda N. Smithies O. Segal S.S. Sessa W.C. J. Clin. Invest. 1998; 101: 731-736Crossref PubMed Scopus (706) Google Scholar). eNOS was originally identified as a membrane-associated, NADPH- and tetrahydrobiopterin-requiring enzyme activated by calcium/calmodulin (CaM) (4.Forstermann U. Pollock J.S. Schmidt H.H. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (551) Google Scholar). However, recent biochemical and structural studies have indicated additional regulatory controlsin situ, by subcellular targeting, protein phosphorylation, and protein-protein interactions (5.Papapetropoulos A. Rudic R.D. Sessa W.C. Cardiovasc. Res. 1999; 43: 509-520Crossref PubMed Scopus (167) Google Scholar). eNOS can directly interact with at least four proteins in vitro and in cellular extracts: caveolins 1 and 3 (6.Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 7.Garcia-Cardena G. Fan R. Stern D.F. Liu J. Sessa W.C. J. Biol. Chem. 1996; 271: 27237-27240Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), the intracellular domains of certain G-protein coupled receptors (8.Marrero M.B. Venema V.J. Ju H. He H. Liang H. Caldwell R.B. Venema R.C. Biochem. J. 1999; 343: 335-340Crossref PubMed Scopus (96) Google Scholar), CaM (4.Forstermann U. Pollock J.S. Schmidt H.H. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (551) Google Scholar), and heat shock protein 90 (hsp90) (9.Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (865) Google Scholar). The first two proteins inhibit NOS activity, whereas the latter two are stimulatory. In the case of caveolins, in vitro data support the concept that when eNOS is localized in the caveolae, it has the capacity to interact with caveolin. This interaction may tonically down-regulate NO production and is most likely associated with inactivation of the enzyme at rest. According to this model, agonists that promote an elevation of intracellular calcium can promote the dissociation of caveolin from eNOS via a proposed exclusive binding of either caveolin or calmodulin to eNOS, thus removing the negative regulation (10.Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 11.Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). However, it is not clear if the time course of dissociation of caveolin from eNOS is consistent with eNOS activation and NO release and whether this occurs in a calcium/CaM-dependent manner with all forms of stimulation leading to NO production (12.Kuchan M.J. Frangos J.A. Am. J. Physiol. 1994; 266: C628-C636Crossref PubMed Google Scholar, 13.Rizzo V. McIntosh D.P. Oh P. Schnitzer J.E. J. Biol. Chem. 1998; 273: 34724-34729Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Recently, we have shown that agonists that promote NO release through distinct signaling mechanisms (histamine, estradiol, vascular endothelial growth factor, and fluid shear stress) stimulate the recruitment of hsp90 to eNOS in a time frame consistent with activation of eNOS and NO production (9.Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (865) Google Scholar, 14.Russell K.S. Haynes M.P. Caulin-Glaser T. Rosneck J. Sessa W.C. Bender J.R. J. Biol. Chem. 2000; 275: 5026-5030Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Moreover, the ansamycin antibiotic geldanamycin, which specifically binds in the unique ATP binding pocket of hsp90, inhibits agonist-stimulated NO production from cultured endothelial cells and endothelium-dependent relaxations of isolated blood vessels (15.Shah V. Wiest R. Garcia-Cardena G. Cadelina G. Groszmann R.J. Sessa W.C. Am. J. Physiol. 1999; 277: G463-G468PubMed Google Scholar). Collectively, these results are consistent with the idea that hsp90 directly or indirectly modulates eNOS. This concept of hsp90 facilitating NOS activation has been extended to another NOS isoform, neuronal NOS. Neuronal NOS also interacts with hsp90 in cell lysates, and geldanamycin blocks ionomycin-stimulated NO release from cells stably expressing neuronal NOS (16.Bender A.T. Silverstein A.M. Demady D.R. Kanelakis K.C. Noguchi S. Pratt W.B. Osawa Y. J. Biol. Chem. 1999; 274: 1472-1478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The relationship between caveolin and hsp90 as opposing regulatory proteins that control the production of NO has not been explored. Therefore, the purposes of this study are to 1) examine the interactions of eNOS with caveolin-1 and hsp90 in cells and in vitro; 2) determine the role of hsp90 in the calmodulin-induced dissociation of eNOS from caveolin; and 3) explore the interaction of these regulatory proteins on eNOS function. Bovine lung microvascular endothelial cells (BLMVEC) were cultured in high glucose Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum, penicillin, streptomycin, and l-glutamine as described previously (17.Garcia-Cardena G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (578) Google Scholar). 2′-5′ ADP-Sepharose 4B was obtained from Amersham Pharmacia Biotech. Peptides, corresponding to the amino acid 82–101 scaffolding domain of caveolin-1 (Cav-(82–101) (18.Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar), were synthesized, purified, and analyzed by reversed phase high pressure liquid chromatography and mass spectrometry by the W. M. Keck biotechnology resource center at Yale University School of Medicine. A scrambled version of the caveolin-1 peptide (Cav-X) was also used as a negative control (10.Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Purified hsp90 from bovine brain was obtained from Stressgen, and purified calmodulin was from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant bovine eNOS was purified fromEscherichia coli as described previously (19.Martasek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar). eNOS stock solutions (1 mg/ml) for all of the studies were in a buffer composed of 50 mm Tris-HCl (pH 7.5), 10% glycerol, 200 mmNaCl, 0.5 mml-arginine, and 40 μm BH4. eNOS and caveolin-1 were immunoprecipitated from cultured BLMVEC as described previously (7.Garcia-Cardena G. Fan R. Stern D.F. Liu J. Sessa W.C. J. Biol. Chem. 1996; 271: 27237-27240Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). Immunocomplexes were isolated, electrophoresed, and either Western blotted or exposed to autoradiography in the case of [35S]methionine-labeled BLMVEC (20.Liu J. Garcia-Cardena G. Sessa W.C. Biochemistry. 1995; 34: 12333-12340Crossref PubMed Scopus (108) Google Scholar). Western blots were sequentially probed with anti-caveolin-1 monoclonal Ab (Transduction Laboratories), with anti-eNOS monoclonal Ab (9D10; Zymed Laboratories Inc. Laboratories), and with anti-hsp90 monoclonal Ab (SPA 830; Stressgen). The caveolin fusion protein expression was performed as described previously (21.Song S.K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). The interaction of GST-caveolin fusion protein with eNOS and hsp90 from BLMVEC cells lysates was performed as follows. Cells were lysed in a modified RIPA buffer (50 mmTris-Cl, pH 7.4, 1% Nonidet P-40, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% SDS, 0.1% deoxycholic acid, 1 mm Pefabloc, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 2 μg/ml pepstatin) for 1 h at 4 °C. The samples were Dounce-homogenized (20 strokes) and centrifuged for 10 min at 16,000 × g at 4 °C. Lysates (500 μg of proteins) were incubated overnight at 4 °C with beads containing ∼100 pmol of either GST alone or GST-Cav (∼20 μl of packed volume) in a total volume of 400 μl. After binding, the beads were washed (three times) with wash buffer containing 50 mm Tris-Cl (pH 7.4), 125 mm NaCl, 1 mm EDTA, and 1 mm EGTA. In some experiments, the immobilized proteins were washed with 50 mm Tris-Cl (pH 7.4), 125 mm NaCl containing 1 mm CaCl2. The beads were then resuspended in the same buffer, and CaM (0.1 and 1 μm) was then added for 1 h at 4 °C. In vitro interactions of GST-Cav with purified or recombinant proteins (hsp90, eNOS, or CaM) were performed for 2 h at 4 °C in binding buffer (50 mmTris-Cl, pH 7.4, in 20% glycerol). After binding, the beads were extensively washed (five times) with a high salt wash buffer (50 mm Tris-Cl, pH 7.7, 400 mm NaCl, and 1 mm EDTA). Beads were eluted by boiling in SDS-sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. Interacting proteins were identified by colloidal brilliant blue (Sigma) staining of the gels or by Western blotting with anti-eNOS monoclonal, anti-hsp90 monoclonal Ab or anti-calmodulin monoclonal Ab (Upstate Biotechnology, Inc., Lake Placid, NY). The binding of eNOS, hsp90, and calmodulin to ADP-Sepharose beads was performed in the above binding buffer for 2 h at 4 °C. Following incubation, the beads were washed three times, and elution from the beads was done by boiling (10 min) in SDS-sample buffer, electrophoresis, and Western blotting (see above). The conversion of3H-l-arginine to 3H-labeledl-citrulline was used to determine NOS activity (22.Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3128) Google Scholar, 23.Garcia-Cardena G. Martasek P. Masters B.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). To examine the effects of either caveolin peptides or hsp90 on NOS activity, Me2SO vehicle, Cav peptide (residues 82–101; 10 μm), or hsp90 (1 μg) was preincubated with recombinant eNOS (0.5 μg) in a 100-μl reaction for 15 min at room temperature. The reaction was initiated by the addition of l-arginine and NOS co-factors for 10 min at 37 °C. The coat protein for caveolae, caveolin-1 (6.Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 7.Garcia-Cardena G. Fan R. Stern D.F. Liu J. Sessa W.C. J. Biol. Chem. 1996; 271: 27237-27240Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), and hsp90 (9.Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (865) Google Scholar) have been shown independently to co-precipitate with eNOS from endothelial cell Therefore, we if these three proteins in a in endothelial Immunoprecipitation of eNOS from [35S]methionine-labeled BLMVEC lysates the presence of four proteins in the eNOS and A 90-kDa and a protein were previously identified as hsp90 (9.Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (865) Google Scholar, V.J. M.B. Venema R.C. Biochem. Biophys. Res. Commun. 1996; PubMed Scopus Google Scholar) and caveolin-1 (6.Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google G. Fan R. Stern D.F. Liu J. Sessa W.C. J. Biol. Chem. 1996; 271: 27237-27240Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), however, the of the and proteins are The of the the proteins and eNOS that a of hsp90 or caveolin-1 interacts with eNOS in endothelial cells these the and that the of and the of proteins may the associated proteins are binding The interaction of caveolin-1 and hsp90 with eNOS is not a of the total eNOS is in in most endothelial cells (7.Garcia-Cardena G. Fan R. Stern D.F. Liu J. Sessa W.C. J. Biol. Chem. 1996; 271: 27237-27240Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, V.J. R. Ju H. M.B. Venema R.C. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar, V. H. A. C. Res. 1999; PubMed Scopus Google Scholar), and the of hsp90 is associated with or in the M.J. M.J. Pratt W.B. J. Biol. 1996; Google Scholar). the interaction of eNOS with its associated regulatory proteins to signaling the binding is and Immunoprecipitation of caveolin-1 from BLMVEC lysates in the of eNOS and hsp90 by Western the idea that these proteins as a complex in 1 we if eNOS and hsp90 isolated from BLMVEC lysates caveolin-1 as a to the complex. in 1 eNOS and hsp90 with GST-Cav but not with GST To examine if an important of eNOS, the interaction of with exogenous CaM was added to the GST-Cav complex. The addition of calcium-activated CaM (0.1 and 1 μm) to the GST-Cav complex partially inhibited the association of eNOS and hsp90 to the complex. results that in endothelial eNOS hsp90 are of with thus that the three proteins are in a complex in endothelial cells and that exogenous CaM can the binding of eNOS and hsp90 to caveolin. we if we the complex in vitro purified proteins. the scaffolding domain of caveolin-1 82–101) is responsible for the interaction of caveolin with proteins as and eNOS M.A. Sessa W.C. J.A. Okamoto T. Lisanti M.P. Biol. 1999; PubMed Scopus Google Scholar). of peptides from the scaffolding domain of caveolin-1 the association of eNOS with GST-Cav However, (10.Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) not the binding of eNOS to the of the in vitro interactions between the two proteins. shows that purified eNOS and hsp90 not interact with GST alone and that hsp90 not interact with GST-Cav. However, in the presence of eNOS, hsp90 is in the complex with GST-Cav. This shows that the interaction of hsp90 with the caveolin-1 is through its association with eNOS, not via binding to as described previously A. Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The association of eNOS with caveolin-1 in endothelial cells is to following activation of eNOS by that promote CaM binding to eNOS J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). The binding of CaM to eNOS is believed to the binding of caveolin-1 to the a exclusive interaction with either an (CaM) or of eNOS. This has been described cell lysates for the of eNOS and caveolin-1 by the addition of purified CaM to the proteins or by the of the scaffolding domain peptide as a caveolin that eNOS binding to J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, H. R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the scaffolding domain peptide is an of eNOS activity that to with to CaM in NOS activity (10.Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, S. R. C. C. Lisanti M.P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of caveolin-1 calmodulin binding to eNOS as a regulatory has not been directly with caveolin-1 Therefore, we eNOS hsp90 with GST alone or GST-Cav in the presence of CaM (1 μm in 1 mm and analyzed the complex by staining or Western in eNOS or hsp90 not to GST 10% of the eNOS specifically to GST-Cav The addition of CaM not eNOS binding to GST-Cav of eNOS with hsp90 results in of the complex with with 10% of eNOS and hsp90 4 and of the complex with CaM results in less eNOS and hsp90 we performed Western blotting to proteins to GST-Cav. eNOS interacts with but not In presence of concentrations of CaM to 1 CaM slightly, but nonstatistically, reduces eNOS binding to GST-Cav in 3 in 3 When hsp90 is present in the binding reaction, the of eNOS to GST-Cav not 2 with however, the addition of increasing concentrations of CaM significantly displaces eNOS from GST-Cav hsp90 is also from the caveolin complex in to CaM the that hsp90 directly binds to eNOS and eNOS as a for the hsp90 association with caveolin-1 2 results that the in eNOS following the addition of CaM are not to its association with caveolin-1 and that the presence of hsp90 the actions of CaM in eNOS from The latter may the of eNOS observed in endothelial cell lysates (see 1 J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). eNOS to caveolin-1 is believed to in an inactive we the of an eNOS complex by of the complex ADP-Sepharose eNOS binds to this via its binding Purified eNOS (0.5 μg or in the of CaM binds to ADP-Sepharose whereas hsp90 (1 μg or and CaM (1 μm) not directly to the However, in presence of eNOS, hsp90 is to the via its interaction with eNOS the addition of CaM μm) the binding of eNOS to the that CaM the of eNOS for ADP-Sepharose through increased for binding of eNOS for increased of hsp90 and CaM in the complex. data that CaM activation of eNOS hsp90 the of in the complex. To examine the between hsp90 and caveolin as of eNOS we NOS activity in In the presence of of CaM (0.1 μm), the caveolin scaffolding domain (10 μm), inhibits and hsp90 NOS of eNOS with hsp90 reduces the effects of To examine this we increasing concentrations of the on eNOS activity in the or in the presence of hsp90 preincubated with eNOS. in inhibits NOS activity with an of μm However, in the presence of hsp90, an of μm is that the of caveolin-1 are when eNOS is to hsp90. Collectively, these data that hsp90 the actions of caveolin-1 on eNOS. In support of this idea are data that 1) hsp90, eNOS, and caveolin-1 can as a complex in endothelial 2) The complex can lysates or purified with eNOS the between hsp90 and caveolin; 3) CaM displaces eNOS from whereas if hsp90 is in the reaction CaM a on eNOS from caveolin; and hsp90 binds to eNOS in a manner and reduces the actions of the caveolin-1 scaffolding peptide on eNOS In a cellular the presence of hsp90 and caveolin-1 associated to eNOS may activation of eNOS and release of nitric an in calcium-activated CaM may in the recruitment of hsp90 to the complex and the release of the caveolin This was previously to on whereas we that hsp90, and regulatory D. J. Y. K. Papapetropoulos A. Sessa W.C. Nature. 1999; PubMed Scopus Google Scholar), as in the release of eNOS from The presence of a calmodulin binding on hsp90 that the interaction between eNOS and the protein can regulated by the binding CaM Y. H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), as by CaM facilitating the effects of hsp90 on the eNOS dissociation from GST-Cav 3) and association of hsp90 and eNOS with CaM in a complex to ADP-Sepharose The dissociation of eNOS from caveolin in to agonists has been however, our results that hsp90 a role in this the cellular mechanisms of eNOS with CaM and hsp90 as of eNOS activation and this the interaction our of endothelial cells