Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity.
nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.
Kimberly W
KW
Raines
Guan-Liang
GL
Cao
Eun Kyoung
EK
Lee
Gerald M
GM
Rosen
Paul
P
Shapiro
D017209:Apoptosis; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D015398:Signal Transduction
D007476:Ionophores; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium