Jason Fontana

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

Domain Mapping Studies Reveal That the M Domain of hsp90 Serves as a Molecular Scaffold to Regulate Akt-Dependent Phosphorylation of Endothelial Nitric Oxide Synthase and NO Release

Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.

Reconstitution of an Endothelial Nitric-oxide Synthase (eNOS), hsp90, and Caveolin-1 Complex in Vitro EVIDENCE THAT hsp90 FACILITATES CALMODULIN STIMULATED DISPLACEMENT OF eNOS FROM CAVEOLIN-1

The activity of endothelial nitric-oxide synthase (eNOS) is regulated by 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.

Heat Shock Protein 90 Mediates the Balance of Nitric Oxide and Superoxide Anion from Endothelial Nitric-oxide Synthase

The balance of nitric oxide (·NO) and superoxide anion (O⨪2) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ·NO. As eNOS is capable of generating both ·NO and O⨪2, we hypothesized that Hsp90 might also mediate eNOS-dependent O⨪2 production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 μg/ml; 17.8 μm) and then stimulated with the calcium ionophore,A23187 (5 μm). GA significantly decreasedA23187-stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased A23187-stimulated eNOS-dependent O⨪2production (p < 0.001, n = 8).A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ·NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O⨪2production.

Regulation of survivin function by Hsp90

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis. This interaction involves the ATPase domain of Hsp90 and the survivin baculovirus inhibitor of apoptosis repeat. Global suppression of the Hsp90 chaperone function or targeted Abmediated disruption of the survivin–Hsp90 complex results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis, and cell cycle arrest with mitotic defects. These data link the cellular stress response to an antiapoptotic and mitotic checkpoint maintained by survivin. Targeting the survivin–Hsp90 complex may provide a rational approach for cancer therapy.

Erratum: Regulation of endothelium-derived nitric oxide production by the protein kinase Akt (Nature (1999) 399 (597-601))

This corrects the article DOI: 21218

Native low-density lipoprotein induces endothelial nitric oxide synthase dysfunction: role of heat shock protein 90 and caveolin-1.

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (*NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O(2)(*-)), which inactivates *NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for *NO, O(2)(*-), phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O(2)(*-) by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by approximately 34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (approximately 85%) and cytosol (approximately 15%). Only 3-6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O(2)(*-), which scavenges *NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O(2)(*-).

Dominant-Negative Hsp90 Reduces VEGF Stimulated Nitric Oxide Release and Migration in Endothelial Cells

Objectives—Heat-shock protein 90 (Hsp90) coordinates the regulation of diverse signaling proteins. We try to develop a new tool to explore the regulatory functions of Hsp90 in endothelial cells (ECs) instead of the existing chemical approaches. Methods and Results—We designed a dominant-negative Hsp90 construct by site-direct mutagenesis of residue Asp-88 to Asn (D88N-Hsp90) based on the structure of the ATP/ADP-binding site. Recombinant wild-type Hsp90 protein binds ATP-Sepharose beads in manner inhibited by ATP or 17-AAG, a specific inhibitor for Hsp90, however the binding activity of D88N-Hsp90 was markedly reduced and the inhibitory effects of ATP or 17-AAG were negligible. The dimerization between endogenous Hsp90&agr; and exogenous HA-Hsp90&bgr; was confirmed by immunoprecipitation, however the association between eNOS and D88N-Hsp90 was less than WT-Hsp90. Furthermore, adenoviral transduction of bovine aortic ECs with D88N-Hsp90 suppressed VEGF-induced phosphorylation of Akt, eNOS, and NO release and the inhibitory effect was blocked by okadaic acid. Moreover, D88N-Hsp90 abolished VEGF-stimulated Rac activation and suppressed VEGF-induced stress fiber formation. Transduction with D88N-Hsp90 decreased growth medium mediated migration of wild-type ECs, but not Akt1(−/−) ECs suggesting that Akt is key target of Hsp90. Conclusions—Our data demonstrate that dominant-negative Hsp90 modulates endothelial cell mobility mainly through PP2A-mediated dephosphorylation of Akt and Rac activation.

Molecular Control of Endothelial Derived Nitric Oxide: A New Paradigm for Endothelial NOS Regulation by Posttranslational Modifications

Publisher Summary This chapter highlights recent developments in the field of endothelial nitric oxide synthase (NOS) regulation by posttranslational control mechanisms as they pertain to both basal and stimulated production of nitric oxide (NO). The physiological production of NO by vascular endothelium is important for cardiovascular homeostasis. NO synthesized by NOS family member, endothelial NOS (eNOS), is the enzyme that produces endothelium-derived relaxing factor, identified as NO, as originally demonstrated by the classic studies of Furchgott, Ignarro, and Moncada. Endothelium-derived NO is important for blood-pressure control, angiogenesis, and vascular remodeling as demonstrated by the profound perturbations in these processes in mice deficient in this NOS isoform. Regulation of NO production is an important area of research relevant to various aspects of vascular biology including vasomotor control, thrombosis, inflammation, vascular remodeling, and angiogenesis.