Receptor-regulated Dynamic S-Nitrosylation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells

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221

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-L-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.atherosclerosis ͉ nitrosation ͉ vascular wall ͉ chaperone R ecent years have witnessed an increasing interest in the roles of nitric oxide (NO) in signal transduction pathways other than its activation of the cGMP pathway. Many of these roles rely on NO's ability to alter protein function through posttranslational modifications. Among these modifications, S-nitrosylation has emerged as a potential and fundamental regulator of protein function. S-nitrosylation is a covalent modification of thiol groups by formation of a thionitrite (-S-NϭO) group, facilitated by the formation of higher nitrogen oxides (1, 2). To date, several dozens of proteins have been shown to become S-nitrosylated and, in many cases, this modification was accompanied by altered function (see table S1 of ref. 1 for review).Nitric oxide, synthesized in the endothelium by endothelial nitric oxide synthase (eNOS), plays crucial roles in the vascular wall, including the maintenance of vascular tone. The possibility that NO might modify eNOS, or elements of the complex system involved in its activation, is an attractive hypothesis, suggesting a potential autoregulatory feedback mechanism. The eNOS enzyme is regulated by several posttranslational modifications including myristoylation, palmitoylation, and phosphorylation (3). This enzyme is also tightly regulated by specific interactions with inhibitory proteins such as caveolin-1 and by positive modulation by the scaffolding protein Hsp90. These interactions have been described in detail, and a relatively complete picture is beginning to emerge (4).We have previously used a proteomic approach to identify several proteins that were S-nitrosylated after exposure of vascular endothelial cells to the physiological nitrosothiol, Snitroso-L-cysteine (CSNO) (5). Further work led to the identification of Hsp90 as a protein susceptible to S-nitrosylation. This chaperone protein, known for its functions in protein folding, degradation, and scaffolding, has attracted renewed ...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

Martínez‐Ruiz

Villanueva²,

Orduña³

et al. 2005

279

221

“…eNOS is activated in a Ca 2ϩ -dependent manner by the phosphoinositide 3-kinase (PI3K)/Akt pathway through the phosphorylation of Ser 1179 (5,6). eNOS activation also entails denitrosylation of eNOS, which is constitutively S-nitrosylated (7,8). Another level of regulation is provided by posttranslational NH 2 -terminal acylations, which determine eNOS subcellular localization: palmitoylation of Cys 15 and Cys 26 specifically targets eNOS to the plasma membrane, whereas myristoylation of Gly 2 provides the general membrane association required for S-nitrosylation and localization on the Golgi apparatus (7,9).…”

mentioning

confidence: 99%

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

Ibiza

Pérez-Rodríguez

Ortega

et al. 2008

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigenpresenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys 118 , suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys 118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. S-nitrosylation ͉ eNOS ͉ apoptosis

“…The enzyme is modified by N-myristoylation and palmitoylation, which targets it to the caveolae in the plasma membrane where it is kept in a basal state by binding to caveolin-1 through a consensus site (1,2). Agonists activate eNOS through multiple mechanisms: phosphorylation/ dephosphorylation of specific residues, interaction with different proteins, S-nitrosylation, and specific subcellular localization (1,(3)(4)(5)(6)(7). Agonists such as platelet-activating factor (PAF) and VEGF phosphorylate eNOS via Akt (8,9).…”

mentioning

confidence: 99%

Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability

Sánchez

Rana²,

Kim³

et al. 2009

The molecular mechanisms of endothelial nitric oxide synthase (eNOS) regulation of microvascular permeability remain unresolved. Agonist-induced internalization may have a role in this process. We demonstrate here that internalization of eNOS is required to deliver NO to subcellular locations to increase endothelial monolayer permeability to macromolecules. Using dominant-negative mutants of dynamin-2 (dyn2K44A) and caveolin-1 (cav1Y14F), we show that anchoring eNOS-containing caveolae to plasma membrane inhibits hyperpermeability induced by plateletactivating factor (PAF), VEGF in ECV-CD8eNOSGFP (ECV-304 transfected cells) and postcapillary venular endothelial cells (CVEC). We also observed that anchoring caveolar eNOS to the plasma membrane uncouples eNOS phosphorylation at Ser-1177 from NO production. This dissociation occurred in a mutant-and cell-dependent way. PAF induced Ser-1177-eNOS phosphorylation in ECVCD8eNOSGFP and CVEC transfected with dyn2K44A, but it dephosphorylated eNOS at Ser-1177 in CVEC transfected with cav1Y14F. Interestingly, dyn2K44A eliminated NO production, whereas cav1Y14F caused reduction in NO production in CVEC. NO production by cav1Y14F-transfected CVEC occurred in caveolae bound to the plasma membrane, and was ineffective in causing an increase in permeability. Our study demonstrates that eNOS internalization is required for agonist-induced hyperpermeability, and suggests that a mechanism by which eNOS is activated by phosphorylation at the plasma membrane and its endocytosis is required to deliver NO to subcellular targets to cause hyperpermeability.caveolae ͉ endothelial nitric oxide synthase ͉ endothelial permeability ͉ inflammation ͉ protein traffic N itric oxide (NO) is the signal for several outcomes in the control of circulation. Its main source in the cardiovascular system is endothelial (e)NOS. The enzyme is modified by N-myristoylation and palmitoylation, which targets it to the caveolae in the plasma membrane where it is kept in a basal state by binding to caveolin-1 through a consensus site (1, 2). Agonists activate eNOS through multiple mechanisms: phosphorylation/ dephosphorylation of specific residues, interaction with different proteins, S-nitrosylation, and specific subcellular localization (1, 3-7). Agonists such as platelet-activating factor (PAF) and VEGF phosphorylate eNOS via Akt (8, 9). Despite advances in our understanding of the biochemistry of eNOS, the mechanisms by which these molecular modifications determine the functional outcome of eNOS activation remain unexplored.NO derived from eNOS is a key mediator in the hyperpermeability response to PAF and VEGF (7,10,11). We previously showed that eNOS internalization (endocytosis) is a required step in the signaling cascade leading to PAF-induced hyperpermeability (7). In this study, we tested the hypothesis that caveolar internalization of eNOS is a requirement for localized NO production and NO delivery to a subcellular target to cause hyperpermeability. To address this hypothesis, we used ECV-304 cells (...