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S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells

Nicolas Clavreul^*, Takeshi Adachi, David R. Pimental, Yasuo Ido, Christian Schöneich^|| and Richard A. Cohen^*,1

^* Vascular Biology,
Myocardial Biology and
Diabetes and Metabolism Units Evans Department of Medicine Boston University Medical Center, Boston, Massachusetts, USA;
Department of Biochemistry and Integrative Medical Biology, Keio University, School of Medicine, Tokyo, Japan; and
^|| Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, USA

¹Correspondence: Vascular Biology, 650 Albany St., X704, Boston, MA 02118, USA. E-mail: racohen{at}bu.edu

SPECIFIC AIMS

The endogenous production of oxidant species, such as peroxynitrite, triggers a broad range of signaling events regulating vascular function. This study was aimed to determine the role of direct oxidant-mediated S-glutathiolation of p21ras in regulating its activity and whether endogenously formed peroxynitrite can do so sufficiently to mediate signaling in endothelial cells in response to NO donors or oxidant stimuli such as oxidized LDL.

PRINCIPAL FINDINGS

1. Peroxynitrite promotes p21ras activation and its downstream signaling in endothelial cells via direct S-glutathiolation of cysteine-118
Bolus addition of peroxynitrite (100 µM) to BAEC increased p21ras activity as evaluated with a Raf-1 pull-down assay. Activity peaked at 5 min (2.48-±0.4-fold, P<0.01, n=5) and returned to a level significantly below baseline at 30 min. Phosphorylation of Erk (11-±1.8-fold) and Akt (20-±1.1-fold, Fig. 1 A) was noted as early as 1 min and peaked at 15 min. Phosphorylation of both Erk and Akt following peroxynitrite were prevented by adenoviral-mediated transfection of the cells with an N17 dominant-negative form of p21ras, indicating that their phosphorylation depended on p21ras activation.

View larger version (54K): [in this window] [in a new window]   Figure 1. Peroxynitrite S-glutathiolates p21ras on cysteine-118. A) BAEC were transfected with an adenoviral vector to express a p21ras C118S mutant or Lac Z and treated 48 h later with peroxynitrite (100 µM). Proteins were separated by SDS-PAGE and analyzed by immunoblot for phosphorylated Erk and Akt. B) BAEC transfected with the p21ras C118S mutant were loaded with BGSH and treated with peroxynitrite (100 µM). Cells were scraped into Tris-sucrose buffer and the membrane fraction separated by ultracentrifugation. S-glutathiolated proteins in the membrane fraction were purified on streptavidin beads, separated by SDS-PAGE, and analyzed by immunoblot for p21ras.

Several cysteines on p21ras have been described as redox sensitive. To detect S-glutathiolation of p21ras, BAEC were loaded with biotin-labeled GSH ethyl ester, treated with peroxynitrite (100 µM), and S-glutathiolation (GSS-) of p21ras was detected by immunoblot analysis for p21ras among the biotin-labeled proteins precipitated with streptavidin. GSS-p21ras was present 5 min after the addition of peroxynitrite (1.7-fold±0.3) and peaked at 10 min (3.2-fold±0.7, P<0.05, n=3), similar to the time course of p21ras activity. Biotinylated GSS-p21ras was not detected in the membrane fraction under control conditions, but increased dramatically 10–15 min after peroxynitrite (Fig. 1B ). S-glutathiolated p21ras was present in the cytosol at baseline. Because protein from the same amount of cells was blotted, it is apparent that most of the newly S-glutathiolated p21ras translocated to the membrane where p21ras is known to exert its activity. The precise role of p21ras S-glutathiolation in its activation was further investigated. In cells treated with peroxynitrite separation of the GSS-p21ras from the nonglutathiolated p21ras using streptavidin beads confirmed that the increase in p21ras activity only occurs in the S-glutathiolated fraction of p21ras. This finding was also supported by the fact that treatment with dithiothreitol (1 mM) reversed the redox activation of p21ras following peroxynitrite; ascorbate (1 mM) did not show any significant effect, indicating the involvement of a disulfide. The guanine nucleotide exchange rate of a recombinant p21ras protein, measured by the binding of a fluorescent GDP adduct, was increased by peroxynitrite only in the presence of free GSH.

2. Cysteine 118 is the main target of S-glutathiolation and is required for oxidative activation of p21ras
Cysteine-118, located in the nucleotide binding domain, is an accessible, redox-sensitive cysteine that is crucial for redox activation of p21ras. A p21ras mutant of cysteine 118 has the same basal activity as well as the same basal nucleotide exchange rate in vitro. Transfection of BAEC with an adenoviral vector to express a C118S mutant p21ras efficiently inhibited peroxynitrite-induced signaling to Erk (3.8-±1.2-fold vs. 13±2.2-fold, P<0.05, n=3) and Akt (2.9-±0.7-fold vs. 15-±1.9-fold, P<0.05, n=3, Fig. 1A ), indicating a central role for this cysteine in p21ras activation and downstream signaling induced by peroxynitrite. In cells loaded with biotinylated GSH ethyl ester, GSS-p21ras did not increase in the membrane fraction of cells transfected with the C118S mutant and treated with peroxynitrite (1.1±0.2) unlike cells transfected with an adenoviral vector expressing ß-galactosidase (4.0±0.9, P<0.05, n=3, Fig. 1B ). To estimate the amount of p21ras that is S-glutathiolated in cells treated with peroxynitrite (100 µM), the total amount of nonglutathiolated p21ras remaining in the supernatant after pull-down of the S-glutathiolated proteins was estimated. The GSS-p21ras formed by peroxynitrite represented 40 ± 13% of the total p21ras in control cells but was only 16 ± 9% in cells transfected with the p21ras C118S mutant. These results indicate that although other p21ras cysteines may be involved, the principal site of p21ras S-glutathiolation is cysteine-118, and glutathiolation of this cysteine is primarily responsible for p21ras-mediated downstream signaling induced by peroxynitrite.

3. OxLDL and NO donors generate a sufficient concentration of endogenous peroxynitrite in BAEC to promote oxidative-dependent activation of p21ras
To determine the relevance of physiologic generation of peroxynitrite in BAEC, we exposed cells to oxidized LDL (oxLDL). We observed a strong increase in Erk and Akt phosphorylation similar to that obtained with peroxynitrite (Fig. 2 A). The effect of oxLDL was attenuated by treating the cells with L-NAME, a nitric oxide (NO) synthase inhibitor, or MnTBAP, a superoxide anion and peroxynitrite scavenger. As NO and superoxide anion react together to make ONOO^–, these results suggest that the effect of oxLDL is promoted through peroxynitrite generation. We also found that oxLDL phosphorylates Erk and Akt via oxidant-dependent p21ras activation. Indeed, oxLDL in 15 min triggered p21ras S-glutathiolation formation in the membrane (Fig. 2) and adenoviral transfection of the p21ras C118S mutant blocked the signaling to Erk and Akt (Fig. 2A ).

View larger version (34K): [in this window] [in a new window]   Figure 2. Oxidized LDL promotes peroxynitrite-mediated activation of p21ras. A) BAEC were exposed to oxLDL (25 or 100 µg/mL, 15 min). The cells were transfected 48 h before with Lac Z or a p21ras C118S mutant. Cell lysate proteins were separated by SDS PAGE and analyzed by immunoblot to detect Erk and Akt phosphorylation. B) BAEC were loaded with biotin-labeled glutathione (250 µM, 1 h), then exposed to oxLDL (100 µg/mL). Membrane fraction was separated from the cytosol by ultracentrifugation. Glutathiolated proteins were purified with streptavidin beads in a nonreducing buffer, separated by SDS-PAGE, and blotted for p21ras. (n=4,*P<0.05).

We also tested the ability of a nitric oxide donor (DEANO) to mimic the activation of p21ras by peroxynitrite. In cells loaded with biotin-labeled GSH ethyl ester, DEANO (1 mM, 15 min) markedly increased S-glutathiolation of p21ras, which was prevented by MnTBAP, suggesting the formation of peroxynitrite. Moreover, DEANO triggered Erk and Akt phosphorylation, and this could be blocked by adenoviral overexpression of MnSOD. Because MnSOD transforms superoxide anion to H₂O₂, this result indicates that, in endothelial cells, the oxidant activation of p21ras is mediated primarily by generation of peroxynitrite rather than by any potential secondary production of H₂O₂.

CONCLUSIONS AND SIGNIFICANCE

After transient exposure of endothelial cells to peroxynitrite, formation of GSS-p21ras is associated with increased p21ras activity. Not only is the time course of thiol modification coincident with the increase in Raf-1 binding activity, but GSS-p21ras enters the active membrane fraction. Expression of a p21ras C118S mutant prevents formation of GSS-p21ras as well as downstream signaling to Erk and Akt, suggesting an essential role for this posttranslational modification of p21ras in its activation by oxidants. The direct activation of recombinant p21ras by peroxynitrite and GSH or by GSSG confirms that formation of GSS-p21ras increases its activity. Endogenous generation of peroxynitrite stimulated by oxidized LDL or formed from an NO donor and endogenous superoxide anion leads to S-glutathiolation and activation of p21ras. Several reactions may account for p21ras S-glutathiolation. In solution, however, peroxynitrite can be protonated to peroxynitrous acid (ONOOH; pK_a=6.8, of which up to 30% may undergo homolytic dissociation yielding hydroxyl radical (HO^•) and ^•NO. Alternatively, peroxynitrite efficiently reacts with CO₂ (k=3x10⁴ M^–1 s^–1) to form the intermediate complex, ONO₂CO₂^–, which spontaneously dissociates into carbonate anion radical (CO₃^•–) and ^•NO₂. All these radicals can react with low molecular weight or protein thiols to yield thiyl radicals. Based on the large excess of GSH over p21ras in cells, it is likely that peroxynitrite and the peroxynitrite-derived radicals react predominantly with GSH. The resulting glutathione thiyl radicals (GS^•) or glutathione sulfenic acid (GSOH) will either react with excess glutathione to yield the symmetric disulfide GSSG, or with protein thiols to yield mixed disulfides. GSSG has the potential to S-glutathiolate p21ras at Cys-118 and activate it. The relatively slow kinetics of p21ras S-glutathiolation and activation is consistent with GSSG being the major mediator.

The most direct evidence that thiol modification of p21ras initiated downstream signaling came from studies demonstrating that a C118S p21ras mutant attenuated downstream signaling to Erk and Akt. This p21ras mutant was demonstrated to have nearly normal basal activity, but to abrogate signaling initiated by what was thought to represent S-nitrosation by NO donors. Although it is possible that NO, peroxynitrite, or its products can S-nitrosate p21ras, perhaps transiently, it is unlikely that this thiol modification accounts for the increase in p21ras activity caused by peroxynitrite or NO in BAEC. The fact that MnSOD prevented S-glutathiolation and signaling via p21ras induced by the NO donor suggests that S-glutathiolation following the intracellular formation of peroxynitrite explains the redox regulation. S-glutathiolation is also supported by the fact that ascorbate, which should have reduced any S-nitrosated cysteines that may have formed, failed to reverse the S-glutathiolation or the increase in p21ras activity caused by peroxynitrite. Expression of the C118S mutant not only prevented the downstream signaling but also dramatically decreased the binding of biotinylated glutathione to p21ras. This strongly suggests that Cys-118 is not only the major site of S-glutathiolation of p21ras caused by peroxynitrite, but also that S-glutathiolation of this site is associated with increased activity and membrane translocation that leads to downstream signaling. The S-glutathiolation of p21ras by peroxynitrite was stoichiometrically large, accounting for 40% of the total, and this occurred largely at Cys-118 in intact BAEC.

Our studies therefore implicate direct effects of peroxynitrite on p21ras in BAEC that cause S-glutathiolation of Cys-118 and increase p21ras activity, Raf-1 binding, and signaling via Mek/Erk and PI₃ kinase/Akt. Precisely how S-glutathiolation of p21ras increases activity is unknown, but the data presented here clearly indicate that GSS-p21ras is activated p21ras. It is logical to suggest that steric effects or those of the negative charge on the glutamate residue of the tripeptide may be responsible. Because Cys-118 borders the guanine nucleotide binding domain, it is not unlikely that S-glutathiolation could affect the interaction with GTP, increasing p21ras activity. Finally, S-glutathiolation of cell proteins would logically be affected by the concentration of GSH that is known to be affected by pathological conditions such as during exposure to oxLDL when, despite an increase in total GSH, the fraction of reduced to oxidized GSH may decrease. It is notable that most of the past determinations of GSH/GSSG ratio under pathological conditions have been performed after acid precipitation of cell proteins, and therefore may not have included a large pool of protein-bound GSH.

View larger version (66K): [in this window] [in a new window]   Figure 3. Mechanism of p21ras redox-dependent activation by oxidized LDL. OxLDL promotes peroxynitrite generation via mitochondria, activation of NADPH oxidase, and eNOS, and is responsible for the S-glutathiolation of cysteine 118 on p21ras. This modification increases the rate of GDP release allowing GTP binding. The p21ras-GTP complex is active and triggers downstream signaling by interacting with the two main p21ras targets, Raf-1 and PI3K.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4875fje;

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