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

Abstract

The highly reactive species, peroxynitrite, is produced in endothelial cells in pathological states in which the production of superoxide anion and NO is increased. Here, we show that peroxynitrite added exogenously or generated endogenously in response to exposure to an NO donor or oxidized low-density lipoproteins (oxLDL) increases p21ras activity in bovine aortic endothelial cells. The activation is not dependent on upstream elements but rather is due to direct targeting of p21ras by reversible S-glutathiolation of cysteine thiols as demonstrated by biotin-labeling techniques. The time course of p21ras S-glutathiolation following peroxynitrite corresponds to the increase in its Raf-1 binding activity and translocation to the membrane. Moreover, p21ras S-glutathiolation and activation can be reversed by dithiothreitol, confirming the importance of a disulfide bond. S-glutathiolation also promoted guanine nucleotide exchange of recombinant p21ras. In addition, the oxidant-induced activation of Mek/Erk and PI3 kinase/Akt was abrogated by dominant-negative and Cys-118 p21ras mutants, and the latter also prevented S-glutathiolation of p21ras. These results indicate that peroxynitrite arising from NO donors or pathological stimuli such as oxLDL triggers direct S-glutathiolation of p21ras Cys-118, which increases p21ras activity and mediates downstream signaling.

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

“…Mallis et al [12] showed that recombinant p21ras can be S-glutathiolated on the C-terminal cysteines by hydrogen peroxide and glutathione or by GSSG, but the effect on activity was not assessed. In a recent study from our lab, we found that direct oxidant-mediated S-glutathiolation of recombinant p21ras increases its activity, and that peroxynitrite formed endogenously can mediate signaling in endothelial cells in response to nitric oxide donors, ONOO − , or stimuli which produce endogenous ONOO − , such as oxidized LDL [17]. In addition, angiotensin II in vascular smooth muscle cells [6], and alpha-adrenergic stimulation [8] or mechanical stretch [18] of cardiac myocytes increase endogenous hydrogen peroxide and initiate MAP kinase and Akt signaling via p21ras S-glutathiolation.…”

“…[17,24,25] Recombinant H-p21ras (2 μg) was loaded with a fluorescent nucleotide analogue N-methylanthraniloyl GDP (Mant-GDP), which exhibits fluorescence only when bound to the protein. The p21ras was incubated 1 h at 37°C with an excess of Mant-GDP in a binding buffer (potassium phosphate buffer (50 mM), NaCl (50 mM), MgCl 2 (5 mM) and EDTA (5 mM)) and the protein-Mant-GDP complex was purified on a PL-6 spin column.…”

“…The decrease in fluorescence was quantified with a fluorimeter with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The results were normalized by the fluorescence before addition of peroxynitrite or GSSG as described [17].…”

“…Although the Cterminal cysteines, Cys 181 , Cys 184 and Cys 186 , are normally adducted by lipid moieties in intact cells, S-nitroso-or S-glutathione thiol adducts of Cys 118 have been implicated as regulating normal and pathological cellular events [6,7,[12][13][14]. Redox modifications of Cys 118 , which is part of the guanine nucleotide binding site ( 117 KCDL 120 ), have been implicated in activation of p21ras and downstream signaling to Raf-1/Mek/Erk [15][16][17]. Although it is generally accepted that p21ras participates in redox reactions, the relative extent to which oxidative modification of its thiols directly regulates its functional activity remains poorly understood.…”

“…The modified cysteine residue is identified from the liquid chromatography (LC) mass spectrometry (MS and MS/ MS) analysis of the peptides obtained after proteolysis of the protein(s) [22,23]. For the present experiments we used ONOO − and GSSG which regulate the activity of p21ras by introducing various oxidative modifications [17,19].…”