S-nitrosoglutathione and endothelial nitric oxide synthase-derived nitric oxide regulate compartmentalized ras S-nitrosylation and stimulate cell proliferation.
OBJECTIVE
S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane.


RESULTS
Ras signaling pathway stimulation by 50 and/or 100 μM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 μM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras.


METHODS
The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways.


CONCLUSIONS
S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.
Department of Biological Sciences, Universidade Federal de São Paulo/Campus Diadema, São Paulo, Brazil.
Wagner L
WL
Batista
Fernando T
FT
Ogata
Marli F
MF
Curcio
Rodrigo B
RB
Miguel
Roberto J
RJ
Arai
Alisson L
AL
Matsuo
Miriam S
MS
Moraes
Arnold
A
Stern
Hugo P
HP
Monteiro
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D019556:COS Cells; D020013:Calcium Signaling; D002462:Cell Membrane / Q000201:enzymology; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000201:enzymology; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism* / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010084:Oxidation-Reduction; D051966:Phospholipase C gamma / Q000037:antagonists & inhibitors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000494:pharmacology*; D026403:S-Nitrosothiols / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D020030:Nitric Oxide Donors; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; C021315:S-nitrosocysteine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019061:src-Family Kinases; D051966:Phospholipase C gamma; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D003545:Cysteine; D001920:Bradykinin