S-Glutathiolation of Ras Mediates Redox-sensitive Signaling by Angiotensin II in Vascular Smooth Muscle Cells

Abstract

Angiotensin II (AII) increases production of reactive oxygen species from NAD(P)H oxidase, a response that contributes to vascular hypertrophy. Here we show in cultured vascular smooth muscle cells that S-glutathiolation of the redox-sensitive Cys 118 on the small GTPase, Ras, plays a critical role in AII-induced hypertrophic signaling. AII simultaneously increased the Ras activity and the S-glutathiolation of Ras (GSS-Ras) detected by biotin-labeled GSH or mass spectrometry. Both the increase in activity and GSS-Ras was labile under reducing conditions, suggesting the essential nature of this thiol modification to Ras activation. Overexpression of catalase, a dominant-negative p47 phox , or glutaredoxin-1 decreased GSS-Ras, Ras activation, p38, and Akt phosphorylation and the induction of protein synthesis by AII. Furthermore, expression of a Cys 118 mutant Ras decreased AII-mediated p38 and Akt phosphorylation as well as protein synthesis. These results show that H 2 O 2 from NAD(P)H oxidase forms GSS-Ras on Cys 118 and increases its activity leading to p38 and Akt phosphorylation, which contributes to the induction of protein synthesis. This study suggests that GSS-Ras is a redox-sensitive signaling switch that participates in the cellular response to AII.

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

“…8A), indicating that Grx1 is an important cellular mechanism of de-glutathionylation, as shown in several previous studies [21,22,66,[68][69][70][71]. For example, de-glutathionylation of actin in NIH3T3 fibroblast cells, and its polymerization and migration to the cell periphery, in response to EGF-stimulation was shown to be abolished in cells in which Grx1 was knocked down by interference RNA [71].…”

“…S-glutathionylation can inactivate the catalytic function of many proteins such as transcription factors NF-κB and NF-1 [59,60], protein tyrosine phosphatase 1B (PTP-1B) [61,62], protein kinase C-α [63], etc. However, the activities of a number of other proteins, such as HIV-1 protease, glutathione S-transferase, and Ras, are enhanced by Sglutathionylation [64][65][66]. In certain cases, the altered protein function due to Sglutathionylation has been shown to play a role in regulation of cell physiology.…”

“…For example, S-glutathionylation-mediated inactivation of PTP-1B in cells treated with epidermal growth factor may be critical for sustaining/enhancing the receptor tyrosine kinase-mediated signaling pathway [67]. On the other hand, activation of Ras due to S-glutathionylation in vascular smooth muscle cells in response to angiotensin II results in stimulation of protein synthesis, potentially leading to cell hypertrophy [66]. Therefore, S-glutathionylation of proteins may represent a novel mechanism by which redox modulates cell signaling and physiology [3].…”

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

“…[1] In addition to its well-characterized regulation by guanine nucleotide exchange factors which release GDP and allow GTP to bind [2,3], and the prenylation of terminal cysteines, which mediates membrane binding [4,5], p21ras activity may be regulated by oxidative post-translational modification of cysteines [6][7][8]. Of the six cysteine residues of p21ras ( Figure 1) four (118, 181, 184 and 186) are surfaceexposed, as shown by structural, chemical and mutational studies [9][10][11].…”

“…Of the six cysteine residues of p21ras ( Figure 1) four (118, 181, 184 and 186) are surfaceexposed, as shown by structural, chemical and mutational studies [9][10][11]. 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].…”