S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells.
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.
Clavreul
Nicolas
N
Vascular Biology, Evans Department of Medicine, Boston University Medical Center, Boston, Massachusetts, USA.
Adachi
Takeshi
T
Pimental
David R
DR
Ido
Yasuo
Y
Schöneich
Christian
C
Cohen
Richard A
RA
D000098:Acetophenones / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D002070:Butadienes / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009570:Nitriles / Q000494:pharmacology; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D000077191:Wortmannin
D000098:Acetophenones; D000730:Androstadienes; D002070:Butadienes; D008077:Lipoproteins, LDL; D009570:Nitriles; D011993:Recombinant Fusion Proteins; C113580:U 0126; C099252:oxidized low density lipoprotein; D006153:Guanosine Diphosphate; D030421:Peroxynitrous Acid; C056165:acetovanillone; D019001:Nitric Oxide Synthase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D016283:Proto-Oncogene Proteins p21(ras); D005978:Glutathione; D003545:Cysteine; D019331:NG-Nitroarginine Methyl Ester; D000077191:Wortmannin