Synergistic Activation of Endothelial Nitric-oxide Synthase (eNOS) by HSP90 and Akt
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m304471200
ISSN1083-351X
AutoresSatoru Takahashi, Michael E. Mendelsohn,
Tópico(s)Physiological and biochemical adaptations
ResumoEndothelial nitric-oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. We recently used purified proteins to characterize the mechanisms by which heat shock protein 90 (HSP90) increases eNOS activity at low and high Ca2+ levels (Takahashi, S. and Mendelsohn, M. E. (2003) J. Biol. Chem. 278, 9339–9344). Here we extend these studies to explore interactions between HSP90, Akt, and eNOS. In studies with purified proteins, HSP90 increased the initial rate and maximal extent of Akt-mediated eNOS phosphorylation and activation at low Ca2+ levels. Akt was not observed in the eNOS complex in the absence of HSP90, but both active and inactive Akt associated with eNOS in the presence of HSP90. Direct binding of Akt to HSP90 was observed even in the absence of eNOS. HSP90 also facilitated CaM binding to eNOS irrespective of Akt presence. Geldanamycin (GA) disrupted HSP90-eNOS binding, reduced HSP90-stimulated CaM binding, and blocked both recruitment of Akt to the eNOS complex and phosphorylation of eNOS at Ser-1179. Akt phosphorylated only CaM-bound eNOS, in an HSP90-independent manner. HSP90 and active Akt together increased eNOS activity synergistically, which was reversed by GA. In bovine aortic endothelial cells (BAECs), the effects of vascular endothelial growth factor (VEGF) and insulin on eNOS-HSP90-Akt complex formation and eNOS activation were compared. BAPTA-AM inhibited VEGF- but not insulin-induced eNOS-HSP90-Akt complex formation and eNOS phosphorylation. Insulin caused rapid, transient increase in eNOS activity correlated temporally with the formation of eNOS-HSP90-Akt complex. GA prevented insulin-induced association of HSP90, Akt and CaM with eNOS and inhibited eNOS activation in BAECs. Both platelet-derived growth factor (PDGF) and insulin induced activation of Akt in BAECs, but only insulin caused HSP90-Akt-eNOS association and eNOS phosphorylation. These results demonstrate that HSP90 and Akt synergistically activate eNOS and suggest that this synergy contributes to Ca2+-independent eNOS activation in response to insulin. Endothelial nitric-oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. We recently used purified proteins to characterize the mechanisms by which heat shock protein 90 (HSP90) increases eNOS activity at low and high Ca2+ levels (Takahashi, S. and Mendelsohn, M. E. (2003) J. Biol. Chem. 278, 9339–9344). Here we extend these studies to explore interactions between HSP90, Akt, and eNOS. In studies with purified proteins, HSP90 increased the initial rate and maximal extent of Akt-mediated eNOS phosphorylation and activation at low Ca2+ levels. Akt was not observed in the eNOS complex in the absence of HSP90, but both active and inactive Akt associated with eNOS in the presence of HSP90. Direct binding of Akt to HSP90 was observed even in the absence of eNOS. HSP90 also facilitated CaM binding to eNOS irrespective of Akt presence. Geldanamycin (GA) disrupted HSP90-eNOS binding, reduced HSP90-stimulated CaM binding, and blocked both recruitment of Akt to the eNOS complex and phosphorylation of eNOS at Ser-1179. Akt phosphorylated only CaM-bound eNOS, in an HSP90-independent manner. HSP90 and active Akt together increased eNOS activity synergistically, which was reversed by GA. In bovine aortic endothelial cells (BAECs), the effects of vascular endothelial growth factor (VEGF) and insulin on eNOS-HSP90-Akt complex formation and eNOS activation were compared. BAPTA-AM inhibited VEGF- but not insulin-induced eNOS-HSP90-Akt complex formation and eNOS phosphorylation. Insulin caused rapid, transient increase in eNOS activity correlated temporally with the formation of eNOS-HSP90-Akt complex. GA prevented insulin-induced association of HSP90, Akt and CaM with eNOS and inhibited eNOS activation in BAECs. Both platelet-derived growth factor (PDGF) and insulin induced activation of Akt in BAECs, but only insulin caused HSP90-Akt-eNOS association and eNOS phosphorylation. These results demonstrate that HSP90 and Akt synergistically activate eNOS and suggest that this synergy contributes to Ca2+-independent eNOS activation in response to insulin. Endothelial nitric-oxide synthase (eNOS) 1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; peNOS, phospho(Ser-1179)-eNOS; pAkt, phospho(Ser-473)-Akt; HSP90, heat shock protein 90; CaM, calmodulin; l-NAME, N G-nitro-l-arginine methyl ester; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; BAEC, bovine aortic endothelial cells; BAPTA-AM, 1, 2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid; GA, geldanamycin; CaM, calmodulin; EC, endothelial cells.1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; peNOS, phospho(Ser-1179)-eNOS; pAkt, phospho(Ser-473)-Akt; HSP90, heat shock protein 90; CaM, calmodulin; l-NAME, N G-nitro-l-arginine methyl ester; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; BAEC, bovine aortic endothelial cells; BAPTA-AM, 1, 2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid; GA, geldanamycin; CaM, calmodulin; EC, endothelial cells. is a highly regulated, Ca2+/calmodulin (CaM)-dependent enzyme responsible for the physiological production of nitric oxide (NO) in the vasculature (1Ignarro L.J. Cirino G. Casini A. Napoli C. J. Cardiovasc. Pharmacol. 1999; 34: 879-886Crossref PubMed Scopus (646) Google Scholar). eNOS is also regulated by subcellular localization, post-translational modification such as phosphorylation by Akt/protein kinase B (2Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papopetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 3Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 4Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. Ortiz de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1991; 9: 845-848Abstract Full Text Full Text PDF Scopus (409) Google Scholar), and interactions with several regulatory proteins, such as heat shock protein 90 (HSP90) (5Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (855) Google Scholar, 6Gratton J.-P. Fontana J. O'Connor D.S. Garcia-Cardena G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: 22268-22272Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 7Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 9339-9344Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).Akt/protein kinase B increases eNOS activity by phosphorylation at Ser-1177 and Ser-1179 for human and bovine eNOS, respectively (2Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papopetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 3Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 4Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. Ortiz de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1991; 9: 845-848Abstract Full Text Full Text PDF Scopus (409) Google Scholar), while HSP90 promotes eNOS activity by direct interaction with the enzyme (5Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (855) Google Scholar, 6Gratton J.-P. Fontana J. O'Connor D.S. Garcia-Cardena G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: 22268-22272Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 7Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 9339-9344Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Exposure of endothelial cells (ECs) to vascular endothelial growth factor (VEGF), estrogen or fluid shear stress induces both an increased association of HSP90 with eNOS and eNOS phosphorylation by Akt, leading to elevation of NO production (2Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papopetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 3Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 4Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. Ortiz de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1991; 9: 845-848Abstract Full Text Full Text PDF Scopus (409) Google Scholar, 5Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (855) Google Scholar, 8Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 9Simoncini T. Hafezi-Moghadam A. Brazil D.P. Ley K. Chin W.W. Liao J.K. Nature. 2000; 407: 538-541Crossref PubMed Scopus (1217) Google Scholar, 10Russell 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 (165) Google Scholar, 11Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 12Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 13Brouet A. Sonveaux P. Dessy C. Balligand J.-L. Feron O. J. Biol. Chem. 2001; 276: 32663-32669Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). It has been shown further that in endothelial cells activated by the above stimuli, inhibition of either HSP90 or Akt results in a marked reduction of NO production. Though the activation of eNOS is well characterized for HSP90 and Akt individually, only a few studies have addressed the potential interplay of these two proteins in eNOS activation. Brouet et al. (13Brouet A. Sonveaux P. Dessy C. Balligand J.-L. Feron O. J. Biol. Chem. 2001; 276: 32663-32669Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) reported cooperative stimulation of eNOS by HSP90 and Akt in Ca2+-dependent VEGF-stimulated ECs. Their data supported that HSP90 association is a prerequisite for subsequent Akt-mediated stimulation of eNOS. In addition, in heterologous COS cell expression studies, it was suggested that synergistic enhancement of eNOS activation is induced by HSP90 and Akt. Recently, Fontana et al. (14Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Circ. Res. 2002; 90: 866-873Crossref PubMed Scopus (295) Google Scholar) also suggested that HSP90 might function as a scaffold protein for eNOS and Akt, facilitating eNOS phosphorylation and activation in VEGF-stimulated ECs. However, the synergy between HSP90 and Akt in eNOS activation cannot be explained by only the scaffolding effect of HSP90. The present study examines potential interactions between eNOS, HSP90, and Akt in vitro using purified proteins. Our results provide evidence that HSP90 and Akt synergistically activate eNOS at physiological calcium levels, and show that this occurs for Ca2+-independent activation of eNOS by insulin in endothelial cells.EXPERIMENTAL PROCEDURESMaterials—The enzymes, antibodies and reagents used in this study and their sources are as follows: active Akt1/PKBα, inactive Akt1/PKBα, crosstide, anti-CaM antibody, protein G-agarose beads, and LY294002 (Upstate Biotechnology, Lake Placid, NY), bovine brain HSP90, Triton X-100, Dowex AG50WX8, tetrahydrobiopterin, NADPH, FMN, FAD, l-NAME, l-arginine, sodium orthovanadate, sodium fluoride, β-glycerophosphate, and polyethylene glycol 400 (Sigma), CHAPS, recombinant chicken CaM, staurosporin, vascular endothelial growth factor (VEGF), insulin, platelet-derived growth factor-BB (PDGF), geldanamycin (GA), protease inhibitor mixture set III, and BAPTA-AM (Calbiochem, La Jolla, CA), anti-eNOS antibody and anti-HSP90 antibody (BD Transduction Laboratories, Lexington, KY), anti-peNOS antibody (phospho-Ser-1177) and anti-Akt antibody, anti-pAkt antibody (phospho-Ser-473) and Phototope-HRP Western blot detection system (Cell Signaling, Beverly, MA), 2′,5′-ADP Sepharose 4B, CaM Sepharose 4B, HiTrap Q, and PD10 columns (Amersham Biosciences), Bradford protein assay kit (Bio-Rad), polyvinylidene difluoride transfer membrane Immobilon-P (Millipore, Bedford, MA), l-[2,3,4-3H]arginine (45–70 Ci/mmol) and [γ-32P]ATP (3000 Ci/mmol) (PerkinElmer Life Sciences, Boston, MA). All other chemicals were of reagent grade.eNOS Purification—Recombinant bovine wild-type eNOS, expressed in Sf9 cells, was purified from the lysate by sequential chromatographies on 2′,5′-ADP Sepharose and CaM Sepharose columns according to the method of List et al. (15List B.M. Klosch B. Volker C. Gorren A.C. Sessa W.C. Werner E.R. Kukovetz W.R. Schmidt K. Mayer B. Biochem. J. 1997; 323: 159-165Crossref PubMed Scopus (138) Google Scholar). The eNOS was further purified by HiTrap Q chromatography with a linear gradient of 0.1–1 m NaCl (7Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 9339-9344Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The eNOS protein was stored at –80 °C in 50 mm Tris-HCl (pH 7.5) buffer containing 1% CHAPS, 1 mm dithiothreitol, 100 mm NaCl, and 5 mm EGTA. Protein concentration was determined with bovine serum albumin as a standard. CaM-bound eNOS was prepared as we previously reported (7Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 9339-9344Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).Phosphorylation of eNOS by Akt—eNOS (9.8 nm) and Akt (7.1 nm) were mixed with vehicle or HSP90 (45 nm) in the presence of CaM (300 nm) with 100 nm Ca2+ or 10 mm EGTA, and then left at room temperature for 10 min. In some experiments, HSP90 was pretreated with 1 μm GA. Phosphorylation of eNOS was initiated by addition of 50 μm ATP and 5 mm MgCl2 in kinase reaction buffer consisting of 50 mm HEPES-NaOH (pH 7.5), 1 mm dithiothreitol, 0.05% Triton X-100, and 5% glycerol. After incubation at 37 °C for the indicated time, the reaction was terminated by 10 μm staurosporine, which had no effect on eNOS activity, but completely inhibited Akt-mediated eNOS phosphorylation.In addition to eNOS phosphorylation, Akt activity was also evaluated by using the synthetic peptide substrate, crosstide (GRPRTSSFAEG). Crosstide (50 μm) was preincubated with HSP90 in the same manner as above, and then was incubated with active Akt at 37 °C for 5 min in the kinase reaction buffer containing 50 μm [γ-32P]ATP. Akt-dependent incorporation of radioactivity to crosstide was measured with a liquid scintillation analyzer.Immunoprecipitation and Immunoblotting of eNOS, HSP90, Akt, and CaM—eNOS complex was incubated with anti-eNOS antibody in the corresponding NOS reaction buffer at 4 °C for 2 h, and successively with protein G-agarose beads at 4 °C overnight, unless otherwise stated. The immunoprecipitates were subjected to SDS-PAGE and then blotted onto polyvinylidene difluoride membranes. The blots were incubated with the primary antibody at 4 °C overnight and then probed with the secondary antibody linked to peroxidase. Immunoreactive proteins were visualized on x-ray film by an enhanced chemiluminescent method. Intensity of each band was measured by densitometry and then normalized to the corresponding control. Thereafter, relative intensity to the defined group as described in each figure legend was calculated.eNOS Activity Assay—NOS activity was measured as the conversion of l-[3H]arginine to l-[3H]citrulline, as described by Bredt and Snyder (16Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3114) Google Scholar). Free calcium concentrations were calculated at pH 7.5 and 100 mm NaCl with K D (Ca2+-EGTA) of 27.9 nm at 37 °C, and the desired concentrations were achieved by mixing EGTA and Ca2+-EGTA (17Tsien R. Pozzan T. Methods Enzymol. 1989; 172: 230-262Crossref PubMed Scopus (394) Google Scholar). eNOS was mixed with the reaction buffer consisting of 25 mm Tris-HCl (pH 7.5), 5 mm CHAPS, 100 mm NaCl, 1 mm dithiothreitol, 1 mm NADPH, 20 μm FAD, 20 μm FMN, 20 μm tetrahydrobiopterin, and 300 nm CaM in the presence of 10 mm EGTA or 100 nm Ca2+. NOS reaction was initiated by addition of 100 μml-[3H]arginine, and incubation was done at 37 °C for 10 min, unless otherwise stated. The reaction was terminated by addition of 50 mm HEPES-NaOH (pH 5.5) buffer containing 2 mm EGTA, 2 mm EDTA, and 2 mml-NAME. l-[3H]citrulline was separated through a Dowex AG50WX8 (Na+-form) column and then counted on a liquid scintillation analyzer. l-NAME-inhibitable activity was determined as specific eNOS activity.eNOS Activity and Complex Formation in Endothelial Cells—Bovine aortic endothelial cells (BAECs) were allowed to grow to confluence in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 1.25 μg/ml amphotericin B and 10% fetal bovine serum. BAECs were starved in serum-free medium over night prior to experiments. The cells were pretreated with vehicle, 100 μm BAPTA-AM for 15 min or 10 μm GA for 1 h, and then stimulated with 1 nm VEGF, 200 nm insulin, or 200 nm PDGF for 10 min, unless otherwise stated. The cells were lysed in 20 mm Tris-HCl (pH 7.5), 1 mm EDTA, 100 mm NaCl, 10 mm sodium orthovanadate, 10 mm sodium fluoride, 10 mm β-glycerophosphate, 1% Triton X-100, 0.5% CHAPS, protease inhibitor mixture, and 5% PEG 400. The lysates were left on ice for 10 min and then were centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatants were passed through a PD10 column to remove endogenous arginine. The void fractions were collected as cell extract and subjected to eNOS activity and immunoprecipitation studies.Statistical Analysis—Data are presented as means ± S.E. Statistical difference was evaluated by Student's t test. p value of < 0.05 was regarded as significant.RESULTSSynergistic Effect of HSP90 and Akt on eNOS Activation in Vitro—Recombinant bovine eNOS was purified to homogeneity by sequential chromatography as recently described (7Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 9339-9344Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) to a specific activity of 12–15 nmol/min/nmol protein in the presence of 10 μm Ca2+ and 300 nm CaM. The eNOS preparation did not contain detectable HSP90, Akt, or CaM by immunoblotting.The time course of eNOS phosphorylation by Akt was examined for eNOS preincubated with active Akt, low Ca2+ (100 nm) and CaM in the presence or absence of HSP90 (Fig. 1). Phosphorylation of eNOS at Ser-1179 was detectable at 2.5 min in the absence of HSP90 and proceeded linearly for 10 min, when it plateaued out. In contrast, in the presence of HSP90, eNOS phosphorylation was detectable by 1 min and increased dramatically in the first 5–10 min, after which a plateau level was reached. Both the initial rates and the final amount of Akt-mediated eNOS phosphorylation were significantly higher in the presence of HSP90 than in the absence of HSP90. Pretreatment of HSP90 with 10 μm GA inhibited the enhancement of Akt phosphorylation of eNOS by HSP90 almost completely (data not shown).The degree of Akt-mediated eNOS phosphorylation at Ser-1179 and the presence of potential eNOS-associated proteins were analyzed at physiological calcium levels in the absence or presence of HSP90 (Fig. 2A). When HSP90 was present in the reaction mixture, it was found associated with immunoprecipitated eNOS, and this association was not affected by the presence of inactive or active Akt. HSP90 increased CaM detected with eNOS whether or not Akt was present. As expected, eNOS at Ser-1179 was phosphorylated by active Akt, but not by inactive Akt. Active Akt was not detected with eNOS in the absence of HSP90, but both active and inactive Akt associated with eNOS in the presence of HSP90. The extent of eNOS phosphorylation by Akt was greater in the presence of HSP90 than in its absence (see Fig. 1). GA disrupted HSP90 binding to eNOS, and reduced the amount of CaM recovered with eNOS. GA also blocked the recruitment of Akt to the eNOS complex and eNOS Ser-1179 phosphorylation. As shown in Fig. 2B, when eNOS, HSP90, and Akt were incubated, and HSP90 was then immunoprecipitated, both active and inactive Akt were present in the complex with HSP90 and eNOS. In addition, Akt was found associated with HSP90 in the absence of eNOS. Further studies were done to examine proteins remaining in the postimmunoprecipitation supernatant (data not shown). In these studies, eNOS (9.8 nm), HSP90 (45 nm), and Akt (7.1 nm) were mixed as in the studies shown in Fig. 2 and immunoprecipitated with either anti-eNOS or anti-HSP90 antibody using antibody concentrations chosen so that all detectable eNOS or HSP90 were precipitated, respectively. After immunoprecipitation of eNOS, HSP90 was easily detected in the residual supernatant, but only trace Akt was detected in the residual supernatant. After immunoprecipitation of HSP90, ∼10–15% of eNOS remained in the supernatant and Akt was not detected in the supernatant. These results indicate that virtually all Akt was bound tightly to HSP90 and incorporated into a ternary complex with eNOS. These data and those shown in Fig. 2 support that Akt is recovered in the eNOS complex only in the presence of HSP90, and that HSP90 is required for formation of the eNOS-HSP90-Akt ternary complex.Fig. 2Immunoprecipitation of eNOS and associated proteins. A, eNOS and active or inactive Akt were incubated with vehicle, HSP90, or GA-treated HSP90 in the presence of 100 nm Ca2+ and 300 nm CaM. eNOS was then immunoprecipitated and the immunoprecipitates were immunoblotted for eNOS, peNOS, HSP90, Akt, and CaM. B, HSP90 was incubated with eNOS and/or Akt, followed by immunoprecipitation of HSP90 and the immunoprecipitates were immunoblotted for eNOS, HSP90, and Akt. Data are representative of four experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next examined whether the presence of HSP90 bound to eNOS alters the degree of eNOS phosphorylation by Akt (Fig. 3). Control experiments for these studies were performed first using the synthetic Akt substrate, crosstide, to assess the level of Akt activity in the presence of Ca2+ or EGTA. Active Akt phosphorylated the crosstide peptide similarly in the presence of Ca2+ or EGTA in both the absence and presence of HSP90 (EGTA without HSP90, 102.2 ± 3.7%; EGTA with HSP90, 103.4 ± 3.6%; Ca2+ without HSP90, 100 ± 5.0%; Ca2+ with HSP90, 99.3 ± 1%, n = 4). To exclude HSP90-mediated increases of CaM binding to eNOS, CaM was pre-bound to eNOS and then separated from residual free (unbound) CaM (7Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 9339-9344Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). CaM-bound and -free eNOS were then mixed with active Akt in the presence or absence of HSP90. Akt phosphorylated only the CaM-bound form of eNOS, and CaM-bound eNOS was phosphorylated to a similar degree in the absence and presence of HSP90. Akt was unable to phosphorylate CaM-bound eNOS in the presence of EGTA or staurosporine (data not shown). Akt did not phosphorylate CaM-free eNOS even with HSP90 present, indicating that HSP90 binding to eNOS is not sufficient to support eNOS phosphorylation by Akt.Fig. 3Akt phosphorylation of CaM-free and CaM-bound eNOS in the absence or presence of HSP90. CaM-free or CaM-bound eNOS was prepared and preincubated with active Akt in the presence of vehicle (open bars) or HSP90 (filled bars), followed by incubation under kinase reaction conditions (37 °C, 10 min) in the presence of 10 mm EGTA or 1 mm Ca2+, respectively. eNOS was then immunoprecipitated and the immunoprecipitates were immunoblotted for eNOS, peNOS, HSP90, Akt, and CaM. eNOS phosphorylation is expressed as a percentage of basal phosphorylation for CaM-bound eNOS in the absence of HSP90. Data are mean ± S.E. for four determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using the same conditions as the previous experiments, eNOS activity next was assayed in the presence of HSP90, active Akt, or both and in the absence or presence of EGTA (Fig. 4). In all cases, eNOS activity was negligible in the presence of EGTA. eNOS activity was easily detectable in the presence of 100 nm Ca2+ and HSP90 increased the activity of eNOS in the presence of inactive Akt by 3.5-fold, similar to our recent study (7Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 9339-9344Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). GA abolished the increase in eNOS activity observed with HSP90 and inactive Akt. Basal and HSP90-stimulated eNOS activity in the absence of any form of Akt were nearly identical to basal and HSP90-stimulated eNOS activity in the presence of inactive Akt, indicating that inactive Akt itself has virtually no effect on eNOS activity. Active Akt alone increased eNOS activity by 2-fold. The presence of both HSP90 and active Akt produced a synergistic (9-fold) increase in eNOS activity. GA also reversed the increase in eNOS activity observed in the presence of HSP90 and active Akt to the level of active Akt alone.Fig. 4Effects of HSP90 and Akt on eNOS activity. eNOS was incubated with vehicle or HSP90 and inactive or active Akt in the presence of 10 mm EGTA (open bars) or 100 nm Ca2+ (filled bars) for 10 min. In some cases, GA was included in the reaction mixture. eNOS activity is expressed as a percentage of basal activity, defined as the activity of eNOS in the presence of inactive Akt at 100 nm Ca2+. Data are mean ± S.E. for five determinations. * and #, p < 0.05 from eNOS with inactive Akt at 100 nm Ca2+, and the corresponding group without GA, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Role of HSP90 in Insulin- and VEGF-induced eNOS Activation in Endothelial Cells—The in vitro studies above with purified proteins support that HSP90 facilitates Akt-mediated phosphorylation of eNOS, and that HSP90 and Akt coordinately promote eNOS activity at physiologic calcium concentrations. HSP90 has been shown to serve as a scaffold for Akt recruitment and to be required for activation of eNOS in VEGF-stimulated cells (14Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Circ. Res. 2002; 90: 866-873Crossref PubMed Scopus (295) Google Scholar). VEGF activates eNOS in Ca2+-dependent manner (13Brouet A. Sonveaux P. Dessy C. Balligand J.-L. Feron O. J. Biol. Chem. 2001; 276: 32663-32669Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In contrast, insulin activation of eNOS, which is also mediated by Akt, is Ca2+-independent (18Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). We therefore compared the effects of VEGF and insulin on eNOS activation and formation of an eNOS-HSP90-Akt ternary complex in intact BAECs. VEGF (1 nm) and insulin (200 nm) each caused a significant increase in eNOS activity (Fig. 5A). Consistent with previous reports (13Brouet A. Sonveaux P. Dessy C. Balligand J.-L. Feron O. J. Biol. Chem. 2001; 276: 32663-32669Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 18Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar), pretreatment of the cells with BAPTA-AM completely inhibited VEGF-induced eNOS activation, but had no effect on insulin-induced eNOS activation. In response to both VEGF and insulin, HSP90, and Akt both were co-precipitated with phospho(Ser-1179)-eNOS (Fig. 5B). BAPTA-AM inhibited most eNOS-HSP90-Akt complex formation and eNOS phosphorylation induced by VEGF. However, BAPTA-AM did not decrease eNOS-HSP90-Akt complex formation and Ser-1179 phosphorylation by the Ca2+-independent agonist insulin. With both agonists, eNOS activation is therefore correlated directly with eNOS-HSP90-Akt complex formation and eNOS Ser-1179 phosphorylation. We next examined whether inhibition of HSP90 by GA abrogates the insulin-induced increase in eNOS activity in BAECs (Fig. 6A). Insulin caused eNOS Ser-1179 phosphorylation and an increase in both HSP90 and Akt associated with eNOS (Fig. 6B; see Fig. 5B). Akt in this complex was phosphorylated at Ser-473, its active form, as expected (pAkt, Fig. 6B). Interestingly, though insulin stimulation of eNOS was Ca2+-independent in these experiments, insulin also increased the bindin
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