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• W2014175763 abstract "The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function. The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function. Endothelial cells rely on the dynamic release of nitric oxide to maintain cardiovascular homeostasis (1Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). To cope with the continuously changing environment, endothelial cells need to control their nitric oxide production by various mechanisms. Hence, the function of endothelial nitric-oxide synthase (eNOS) 1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; PDK1, 3-phosphoinositide-dependent kinase 1; Hsp90, 90-kDa heat shock protein; PP2A, protein phosphatase 2A; siRNA, small interfering RNA; BAEC, bovine aortic endothelial cell. 1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; PDK1, 3-phosphoinositide-dependent kinase 1; Hsp90, 90-kDa heat shock protein; PP2A, protein phosphatase 2A; siRNA, small interfering RNA; BAEC, bovine aortic endothelial cell. is regulated in an exceedingly complex fashion (2Papapetropoulos A. Rudic R.D. Sessa W.C. Cardiovasc. Res. 1999; 43: 509-520Crossref PubMed Scopus (164) Google Scholar, 3Fulton D. Gratton J.P. Sessa W.C. J. Pharmacol. Exp. Ther. 2001; 299: 818-824PubMed Google Scholar). In the past, elevations of intracellular free Ca2+ concentrations were thought to play the principal role in eNOS activation and regulation (4Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). We now know that serine 1179-phosphorylated eNOS can fully function without the rise of cytosolic Ca2+ concentrations (5Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3016) Google Scholar, 7McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Besides protein phosphorylation, eNOS function is critically modulated by a number of protein-protein interactions (2Papapetropoulos A. Rudic R.D. Sessa W.C. Cardiovasc. Res. 1999; 43: 509-520Crossref PubMed Scopus (164) Google Scholar, 3Fulton D. Gratton J.P. Sessa W.C. J. Pharmacol. Exp. Ther. 2001; 299: 818-824PubMed Google Scholar, 8Fleming I. Busse R. Am. J. Physiol. 2003; 284: R1-R12PubMed Google Scholar). For example, by interacting with caveolin-1, eNOS is targeted in the caveolae of endothelial cells and kept in an idle state (9Feron 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 (593) Google Scholar, 10García-Cardeña 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). eNOS was also reported to directly bind with the intracellular domain of certain G-protein coupled receptors such as angiotensin II AT1, bradykinin B2, and endothelin-1 ETB (3Fulton D. Gratton J.P. Sessa W.C. J. Pharmacol. Exp. Ther. 2001; 299: 818-824PubMed Google Scholar, 11Ju H. Venema V.J. Marrero M.B. Venema R.C. J. Biol. Chem. 1998; 273: 24025-240259Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Binding with these receptors was shown to cause reversible inhibition on eNOS activity. Recent yeast two-hybrid screen identified more eNOS-associated proteins (NOSIP, NOSTRIN, dynamin, etc.) (3Fulton D. Gratton J.P. Sessa W.C. J. Pharmacol. Exp. Ther. 2001; 299: 818-824PubMed Google Scholar, 8Fleming I. Busse R. Am. J. Physiol. 2003; 284: R1-R12PubMed Google Scholar, 12Dedio J. Konig P. Wohlfart P. Schroeder C. Kummer W. Muller-Esterl W. FASEB J. 2001; 15: 79-89Crossref PubMed Scopus (144) Google Scholar, 13Zimmermann K. Opitz N. Dedio J. Renne C. Muller-Esterl W. Oess S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 17167-17172Crossref PubMed Scopus (142) Google Scholar). Thus, it has become increasingly clear that either by influencing eNOS subcellular localization or by directly acting on its catalytic process, protein-protein interactions provide a particularly versatile way to modulate eNOS function.Another intermediate protein that plays crucial roles in eNOS regulation is the 90-kDa heat shock protein (Hsp90) (14García-Cardeña G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (856) Google Scholar, 15Balligand J.L. Cir. Res. 2002; 90: 838-841Crossref PubMed Scopus (43) Google Scholar). Hsp90 belongs to a group of highly conserved stress proteins expressed in all eukaryotic cells. As one of the most abundant cytosolic proteins (1–2% total cellular protein), Hsp90 functions as a molecular chaperone participating in protein folding and signal transduction (16Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4369) Google Scholar, 17Pratt W.B. Proc. Soc. Exp. Biol. Med. 1998; 217: 420-434Crossref PubMed Scopus (412) Google Scholar). Hsp90 was found to associate with eNOS in resting endothelial cells (14García-Cardeña G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (856) Google Scholar). The interaction between Hsp90 and eNOS can be further enhanced by a variety of stimuli such as histamine, bradykinin, vascular endothelial growth factor, shear stress, and estrogen (14García-Cardeña G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (856) Google Scholar, 18Russell 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). Further structure analysis revealed that Hsp90 binds with the N-terminal oxygenase domain of eNOS (residues 300–400) (19Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Cir. Res. 2002; 90: 866-873Crossref PubMed Scopus (295) Google Scholar). Binding with Hsp90 significantly increases eNOS activity. Inhibition of Hsp90 function attenuates endothelium-dependent vascular relaxation, suggesting that Hsp90 is coupled with eNOS in vascular tissues and that this coupling augments nitric oxide production (14García-Cardeña G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (856) Google Scholar). The effect of Hsp90 on eNOS is mediated, at least in part, by the enhancement of calmodulin binding affinity to eNOS (20Song Y. Zweier J.L. Xia Y. Biochem. J. 2001; 355: 357-360Crossref PubMed Scopus (79) Google Scholar, 21Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 30821-30827Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 22Song Y. Zweier J.L. Xia Y. Am. J. Physiol. 2001; 281: C1819-C1824Crossref PubMed Google Scholar). Hsp90 has also been shown to facilitate the ability of Ca2+/calmodulin to dissociate the interaction between caveolin-1 and eNOS, thereby reversing the inhibitory action of caveolin-1 on eNOS (23Gratton J.P. Fontana J. O'Connor D.S. García-Cardeña G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: 22268-22272Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar).In addition to its allosteric action, Hsp90 was found to be crucial in eNOS serine 1179/1177 (bovine/human) phosphorylation (2Papapetropoulos A. Rudic R.D. Sessa W.C. Cardiovasc. Res. 1999; 43: 509-520Crossref PubMed Scopus (164) Google Scholar, 8Fleming I. Busse R. Am. J. Physiol. 2003; 284: R1-R12PubMed Google Scholar). Hsp90 was shown previously to associate Akt (24Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar). Further studies demonstrated that Hsp90 serves as a module to recruit Akt to phosphorylate the eNOS serine 1179 residue (19Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Cir. Res. 2002; 90: 866-873Crossref PubMed Scopus (295) Google Scholar). Indeed, Hsp90 inhibition resulted in decrease of eNOS serine 1179 phosphorylation (25Brouet A. Sonveaus 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). However, the details on how Hsp90 inhibition decreases eNOS serine 1179 phosphorylation are not fully understood. For example, whether the reduction of eNOS serine 1179 phosphorylation in Hsp90-inhibited cells was caused by the loss of Akt binding to eNOS or by other mechanisms remains unknown. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1) (26Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 27Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1387) Google Scholar). Whether the upstream kinase PDK1 involves in the actions of Hsp90 on eNOS phosphorylation and function is also unknown. In the present study, we demonstrate that the Hsp90 inhibition-induced decrease of eNOS serine 1179 phosphorylation is caused by Akt deactivation rather than loss of Akt binding to eNOS. We further reveal that depletion of PDK1 in Hsp90-inhibited cells is primarily responsible for Akt deactivation. We also provide evidence demonstrating that increase of PDK1 expression can prevent Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and accordingly preserve enzyme function.EXPERIMENTAL PROCEDURESMaterials—Cell culture materials were obtained from Invitrogen. Bovine aortic endothelial cells (BAECs) were from Cell Systems (Kirkland, WA). Geldanamycin and radicicol were purchased from Sigma. MG132 was obtained from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone was obtained from Calbiochem. 2′,5′-ADP-Sepharose 4B was obtained from Amersham Biosciences. Antibody against eNOS was purchased from BD Transduction Laboratories. Antibodies against phospho-eNOS (serine 1179), Akt, and phospho-Akt (serine 473) were products of Cell Signaling Technology (Beverly, MA). Antibody against phospho-eNOS (threonine 497) was purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies against PDK1, protein phosphatase 2A (PP2A)-Aα/β, PP2A-B56-α, PP2A-C, and β-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against β-actin was purchased from Sigma. l-[14C]Arginine was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). The protease inhibitor tablet was obtained from Roche Applied Science. Calmodulin, NADPH, l-arginine, tetrahydrobiopterin, N-nitro-l-arginine methyl ester, and other reagents were purchased from Sigma unless otherwise indicated.Cell Culture and Transfection—Human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco' modified Eagle' medium with 10% fetal bovine serum (Invitrogen). HEK 293 cells neither contain eNOS mRNA nor express eNOS protein (28Liu J. Garcia-Cardena G. Sessa W.C. Biochemistry. 1995; 34: 12333-12340Crossref PubMed Scopus (107) Google Scholar). Wild-type bovine eNOS cDNA in mammalian expression vector pcDNA3 was transfected into HEK 293 cells using Lipofectamine and PLUS reagents (Invitrogen) according to the manufacturer' instruction. Transfected cells were cultured in selective media (complete Dulbecco' modified Eagle' medium containing 500 μg/ml G-418). G-418-resistant colonies were isolated with cloning cylinders, trypsinized, and proliferated in selective media containing 500 μg/ml G418. After two cycles of the colony selection process, eight G418-resistent cell lines were obtained, and further characterization showed that they stably expressed eNOS. One of the lines (eNOS.C4) was designated as the eNOS-HEK 293 cell and used in this study.For some studies, eNOS-HEK 293 cells were transiently transfected with pCMV5-PDK1 or pCMV5-Akt vector (kindly provided by Dr. Brian A. Hemmings from Friedrich Miescher Institute) using FuGENE6 (Roche Molecular Biochemicals) according to the manufacturer' instructions. The expression of appropriate proteins was confirmed after 24 h of transfection.siRNA—The siRNA oligonucleotides corresponding to human PDK1 (5′-GUCCGCCUGUAAGAGUUCATT-3′) were purchased from Dharmacon, Inc. A nonspecific oligonucleotide (5′-AUUGUAUGCGAUCGCAGACUU-3′) (Dharmacon) was used as a control. In 12-well plates, cells were plated the day before transfection and were grown to 30–50% confluence. siRNA oligonucleotides (100 nm) were transfected into cells with Oligofectamine reagent (Invitrogen). After 48 h of transfection, Western blottings were carried out to examine the knockdown of targeted proteins.Pull-down Assay—Cells were harvested and lysed on ice for 30 min in lysis buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF, 1 mm Na3VO4, 5 mm sodium pyrophosphate, and protease inhibitor tablet). The cell lysates were centrifuged at 14,000 × g for 15 min, and the supernatants were recovered. Supernatants containing equal amounts of proteins were incubated with 2′,5′-ADP-Sepharose 4B resins (50 μl in 50% slurry) for 2 h at 4 °C.The resins were washed once with regular washing buffer (50 mm Tris-HCl, 100 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40), twice with high-salt washing buffer (50 mm Tris-HCl, 500 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40), and another time with regular washing buffer. Pulled-down proteins were then eluted by 5-min boiling of the beads in SDS/PAGE buffer and analyzed by Western blotting.Western Blotting—Cells were lysed on ice for 30 min in modified radioimmunoprecipitation assay buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mm NaF, 1 mm Na3VO4, 5 mm sodium pyrophosphate, and protease inhibitor tablet. Cell lysates were centrifuged at 14,000 × g for 15 min, and the supernatant was recovered. The total protein concentrations were determined by using the detergent compatible protein assay reagent (Bio-Rad). The lysates were denatured by boiling in SDS sample buffer. The proteins were separated by SDS/PAGE on 4–20% gradient gels (Invitrogen) and then transferred to nitrocellulose membranes by using a semidry transfer cell (Bio-Rad). After blocking, the membranes were probed with the appropriate primary antibodies. Membrane-bound primary antibodies were detected using secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique (SuperSignal West Pico; Pierce). Densitometry was performed by using an AlphaImager 3300 gel documentation and image analysis system.Nitric-oxide Synthase Activity Assay—eNOS activity was measured by the l-[14C]arginine to l-[14C]citrulline conversion assay (29Xia Y. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12705-12710Crossref PubMed Scopus (132) Google Scholar). To measure the activity of phospho-eNOS, the assay was performed in the presence of 10 nm Ca2+ as reported previously (6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3016) Google Scholar, 7McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). In brief, cells were harvested in homogenate buffer (50 mm Tris-HCl, pH7.4, 2 mm DTT, 50 mm NaF, 1 mm Na3VO4, and protease inhibitor mixture) and homogenated by pulse sonication. After centrifugation (14,000 g for 15 min at 4 °C), the pellets were recovered, washed, and resuspened in homogenate buffer. Cell lysates (45 μg of protein) was added to the reaction mixture containing 50 mm Tris-HCl, pH 7.4, 0.5 mm NADPH, 10 nm CaCl2, 10 μg/ml calmodulin, 10 μm tetrahydrobiopterin, 5 μm l-[14C]arginine, and 45 μm l-arginine. After 45 min incubation at 37 °C, the reactions were terminated by ice-cold stop buffer. l-[14C]Citrulline was separated by passing the reaction mixture through Dowex AG 50W-X8 (Na+ form; Sigma) cation exchange columns and quantitated by liquid scintillation counting.Statistics—Data were expressed as mean ± S.E. Comparisons were made using a two-tailed Student' paired or unpaired t test. Differences were considered statistically significant at p < 0.05.RESULTSTo determine the roles of Hsp90 in maintaining eNOS phosphorylation, we treated the bovine eNOS stably transfected HEK 293 cells with Hsp90 inhibitor geldanamycin (1 μm). As shown in Fig. 1A, geldanamycin time-dependently decreased serine 1179-phophorylated eNOS. This was a highly specific effect because the phosphorylation status of another residue in eNOS, threonine 497, was unchanged. The total eNOS or Hsp90 level in cells was also not affected by geldanamycin. Corresponding to the decrease of serine 1179 phosphorylation, eNOS activity was markedly attenuated in Hsp90-inhibited cells (Fig. 1B). The specificity of the eNOS assay was evidenced by the fact that the classic nitric-oxide synthase inhibitor N-nitro-l-arginine methyl ester (1 mm) completely abolished the nitric-oxide synthase activity measured. Although geldanamycin had been used as an Hsp90 inhibitor to probe the effects of Hsp90 on eNOS, there was a concern that geldanamycin might redox cycle with eNOS (30Billecke S.S. Bender A.T. Kanelakis K.C. Murphy P.J.M. Lowe E.R. Kamada Y. Pratt W.B. Osawa Y. J. Biol. Chem. 2002; 277: 20504-20509Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 31Dikalov S. Landmesser U. Harrison D.G. J. Biol. Chem. 2002; 277: 25480-25485Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). To rule out this concern, we examined the effects of radicicol, a non-quinone Hsp90 inhibitor known to be redox insensitive (30Billecke S.S. Bender A.T. Kanelakis K.C. Murphy P.J.M. Lowe E.R. Kamada Y. Pratt W.B. Osawa Y. J. Biol. Chem. 2002; 277: 20504-20509Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Similar to the effects of geldanamycin, radicicol (10 μm) also selectively reduced eNOS phosphorylation of serine 1179 but not that of threonine 497 (Fig. 1C). These two lines of evidence demonstrated the crucial roles of Hsp90 in maintaining eNOS serine 1179 phosphorylation and function.To ascertain that our findings from eNOS-HEK 293 cells occurred in native endothelial cells, we determined that the effect of Hsp90 inhibition on eNOS phosphorylation in BAECs. In accordance with the results from eNOS-HEK 293 cells, geldanamycin also selectively decreased serine 1179-phosphorylated eNOS in a time-dependent manner (Fig. 1D). These data confirmed that the eNOS-HEK 293 cell was a valid model to study the regulation of eNOS phosphorylation by Hsp90 in endothelial cells.Because Hsp90 recruits Akt to phosphorylate eNOS serine 1179, we pulled down eNOS and determined whether alterations of eNOS-associated Akt was responsible for the decrease of serine 1119-phosphorylated eNOS in Hsp90-inhibited cells. As shown in Fig. 2A, eNOS-associated phospho-Akt, the active form of Akt, was markedly attenuated by Hsp90 inhibition. It is interesting that the total levels of Akt associated with eNOS remained unchanged. eNOS-associated Hsp90 was not altered by geldanamycin treatment either. These data indicated that Hsp90-induced decrease of eNOS serine 1179 phosphorylation was caused by Akt deactivation rather than the loss of Akt association with eNOS. We also measured the alterations of phospho-Akt and total Akt levels in the whole lysates of Hsp90-inhibited cells. Similar to the changes in eNOS-associated Akt, the levels of phospho-Akt were reduced, whereas the total Akt levels remained the same (Fig. 2B). In line with the decrease in phospho-Akt, the phosphorylation of cytosolic glycogen synthase kinase 3β was also attenuated (Fig. 2C). In addition to declined kinase activity, elevations of phosphatase can also give rise to the decrease of phosphoproteins. eNOS serine 1179 was reported to be selectively dephosphorylated by PP2A (32Greif D.M. Kou R. Michel T. Biochemistry. 2002; 41: 15845-15853Crossref PubMed Scopus (91) Google Scholar). We therefore monitored PP2A expressions in the absence and presence of the Hsp90 inhibitor. PP2A consists of a structure subunit A, a regulatory subunit B, and a catalytic subunit C (33Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar). As shown in Fig. 2D, the expression levels of the three PP2A subunits were all not significantly altered by Hsp90 inhibition. Thus, loss of eNOS serine 1179 phosphorylation in Hsp90-inhibited cells was not caused by the elevated PP2A expression.Fig. 2Deactivation of eNOS-associated Akt in Hsp90-inhibited cells. A, Hsp90 inhibition caused dephosphorylation of eNOS-associated Akt. After geldanamycin (GA; 1 μm) incubation for 24 h, eNOS in cells was pulled down and the eNOS-associated phospho-Akt, Akt, and Hsp90 were detected by Western blotting. As shown, with equal amounts of eNOS input, Hsp90 inhibition decreased eNOS-associated phospho-Akt without affecting the total Akt and Hsp90 bound to eNOS. B, alterations of phospho-Akt and total Akt in the whole lysates of control and geldanamycin-treated cells. Tubulin blottings were employed as loading control. C, effect of geldanamycin on the phosphorylation of glycogen synthase kinase 3 (GSK-3). D, effects of Hsp90 inhibition on PP2A expression in cells. Geldanamycin treatment did not alter the expression levels of all three subunits of PP2A. These data are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Akt is activated by the phosphorylation of PDK1 (26Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 27Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1387) Google Scholar). Because neither total Akt levels in cells nor the amounts of Akt bound with eNOS were changed by Hsp90 inhibition, we then sought to determine whether alterations of PDK1 resulted in Akt deactivation in Hsp90-inhibited cells. As shown in Fig. 3A, both geldanamycin and radicicol time-dependently depleted cytosolic PDK1 concentrations. PDK1 reduction synchronized the decrease of eNOS serine 1179 phosphorylation. Similar effects were also seen in BAECs (Fig. 3B). We then determined by what mechanism PDK1 was depleted. Because the ubiquitin-proteasome system is responsible for the majority of protein degradation in cells, we reasoned that the PDK1 in Hsp90-inhibited cells was degraded by proteasome. Indeed, proteasome blockade by MG132 (10 μm) prevented the loss of PDK1 in Hsp90-inhibited cells. In contrast, treating the cells with the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had no effect on Hsp90 inhibition-PDK1 depletion (Fig. 3C). Together, these data strongly suggested that Hsp90 inhibition promoted PDK1 degradation in proteasome and lack of PDK1 resulted in Akt deactivation and subsequent loss of eNOS serine 1179 phosphorylation.Fig. 3Alterations of PDK1 in Hsp90-inhibited cells. A, both geldanamycin and radicicol time-dependently depleted intracellular PDK1 levels. B, geldanamycin decreased PDK1 in BAECs. Representative data from three independent experiments are shown. The signal intensity of the Western blots was quantitated by densitometer. IDV, integrated density value. **, p < 0.01; ***, p < 0.001, versus control untreated groups, n = 3. C, proteasome inhibitor MG132 (10 μm), but not the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) (20 μm), prevented the loss of PDK1 caused by Hsp90 inhibition. Data were shown as mean ± S.E. (**, p < 0.01; versus geldanamycin (GA)- or radicicol-treated group, n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further prove that loss of PDK1 is responsible for Akt deactivation as well as decrease of eNOS serine 1179 phosphorylation, we investigated whether down-regulation of PDK1 can recapitulate the effects of Hsp90 inhibition on Akt and eNOS serine 1179 phosphorylation. siRNA was used to knock down PDK1. As shown in Fig. 4, transfection of PDK1 siRNA dramatically reduced the PDK1 content in cells. As a result, phospho-Akt and serine 1179-phosphorylated eNOS were decreased. Consistent with the effects of Hsp90 inhibition on eNOS, PDK1 knockdown did not change the phosphorylation of eNOS threonine 497. Transfection of the nonspecific control siRNA had no effect on PDK1, phospho-Akt, or eNOS phosphorylation in cells. These results demonstrated that PDK1 knock-down was sufficient to induce Akt deactivation as well as reduction of eNOS serine 1179 phosphorylation.Fig. 4PDK1 gene silencing induced Akt deactivation and eNOS serine 1179 dephosphorylation. PDK1 siRNA and a control siRNA were transfected to cells. After 48-h transfection, PDK1 was dramatically reduced by PDK1 siRNA but not the control siRNA (A and B). PDK1 knockdown deactivated Akt, leading to decrease of eNOS serine 1179 phosphorylation (A and C). This was a selective action, because the phosphorylation of eNOS threonine 497 was not affected by PDK1 knockdown. The total eNOS levels in control or siRNA-transfected cells remained unchanged. **, p < 0.01, versus control, n = 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, we investigated whether overexpression of PDK1 can reverse Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation. As shown in Fig. 5A, transfections of the control empty vectors n" @default.
• W2014175763 created "2016-06-24" @default.
• W2014175763 creator A5021170475 @default.
• W2014175763 creator A5034513446 @default.
• W2014175763 date "2005-05-01" @default.
• W2014175763 modified "2023-09-28" @default.
• W2014175763 title "Roles of 3-Phosphoinositide-dependent Kinase 1 in the Regulation of Endothelial Nitric-oxide Synthase Phosphorylation and Function by Heat Shock Protein 90" @default.
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