Synergistic Activation of eNOS by HSP90 and Akt: Calcium-independent Endothelial Nitric Oxide Synthase Activation Involves For

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 Ca levels (Takahashi, S. and Mendelsohn, M. E., J. Biol. Chem. 278, 9339-9344, 2003). 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 Ca 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 Ser1179. 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 VEGFbut not insulin-induced eNOS-HSP90-Akt complex formation and eNOS phosphorylation. Insulin caused a rapid, transient increase in eNOS activity correlated temporally with the formation of eNOSHSP90-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 HSP90Akt-eNOS association and eNOS phosphorylation. These results demonstrate that HSP90 and Akt synergistically activate eNOS and support the importance of this mechanism for Ca-independent eNOS agonists such as insulin. 3 by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from INTRODUCTION Endothelial nitric oxide synthase (eNOS) is a highly regulated, Ca/calmodulin (CaM)-dependent enzyme responsible for the physiological production of nitric oxide (NO) in the vasculature (1). eNOS is also regulated by subcellular localization, post-translational modification such as phosphorylation by Akt/protein kinase B (2-4), and interactions with several regulatory proteins, such as heat shock protein 90 (HSP90) (5-7). Akt/protein kinase B increases eNOS activity by phosphorylation at Ser1177 and Ser1179 for human and bovine eNOS, respectively (2-4), while HSP90 promotes eNOS activity by direct interaction with the enzyme (5-7). 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 (2-5, 8-13). 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. (13) reported cooperative stimulation of eNOS by HSP90 and Akt in Ca-dependent VEGF-stimulated ECs. Their data supported that HSP90 4 by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from 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. (14) 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 Ca-independent activation of eNOS by insulin in endothelial cells. 5 by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from EXPERIMENTAL PROCEDURES Materials. 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 Gagarose beads and LY294002 (Upstate Biotechnology, Lake Placid, NY), bovine brain HSP90, Triton X-100, Dowex AG50WX8, tetrahydrobiopterin, NADPH, FMN, FAD, LNAME, L-arginine, sodium orthovanadate, sodium fluoride, β-glycerophosphate and polyethyleneglycol 400 (Sigma, St. Louis, MO), CHAPS, recombinant chicken CaM, staurosporin, vascular endothelial growth factor (VEGF), insulin, platelet-derived growth factor-BB (PDGF), geldanamycin (GA), protease inhibitor cocktail 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-Ser1177) and anti-Akt antibody, anti-pAkt antibody (phospho-Ser473) 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 Pharmacia Biotech, Piscataway, NJ), Bradford protein assay kit (Bio-Rad, Hercules, CA), PVDF transfer membrane Immobilon-P (Millipore, Bedford, MA), L-[2,3,4-H]arginine (45-70 Ci/mmol) and [γ-P]ATP (3000 Ci/mmol) (New England Nuclear, 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 6 by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from according to the method of List et al. (15). The eNOS was further purified by HiTrap Q chromatography with a linear gradient of 0.1-1 M NaCl (7). The eNOS protein was stored at –80C 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 (7). 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 Ca 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 37C 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 37C for 5 min in the kinase reaction buffer containing 50 μM [γ-P]ATP. Aktdependent incorporation of radioactivity to crosstide was measured with a liquid scintillation analyzer. Immunoprecipitation and Immunoblotting of eNOS, HSP90, Akt and CaM. 7 by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from eNOS complex was incubated with anti-eNOS antibody in the corresponding NOS reaction buffer at 4C for 2 h, and successively with protein G-agarose beads at 4C over night, unless otherwise stated. The immunoprecipitates were subjected to SDS-PAGE and then blotted onto PVDF membranes. The blots were incubated with the primary antibody at 4C over night 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-[H]arginine to L-[H]citrulline, as described by Bredt and Snyder (16). Free calcium concentrations were calculated at pH 7.5 and 100 mM NaCl with KD (Ca-EGTA) of 27.9 nM at 37C, and the desired concentrations were achieved by mixing EGTA and Ca-EGTA (17). 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 Ca. NOS reaction was initiated by addition of 100 μM L-[H]arginine, and incubation was done at 37C 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 mM L-NAME. L-[H]citrulline was separated through a Dowex AG50WX8 (Na-form) column and then counted on a liquid 8 by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from 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 cocktail and 5% PEG400. The lysates were left on ice for 10 min and then were centrifuged at 10,000 rpm for 10 min at 4C. 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. 9 by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from