title: Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.
abstract: Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.
authors.affiliation: Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, E-28029 Madrid, Spain.
authors.forename: Sales
authors.initials: S
authors.lastname: Ibiza
authors.forename: Andrea
authors.initials: A
authors.lastname: Pérez-Rodríguez
authors.forename: Angel
authors.initials: A
authors.lastname: Ortega
authors.forename: Antonio
authors.initials: A
authors.lastname: Martínez-Ruiz
authors.forename: Olga
authors.initials: O
authors.lastname: Barreiro
authors.forename: Carlota A
authors.initials: CA
authors.lastname: García-Domínguez
authors.forename: Víctor M
authors.initials: VM
authors.lastname: Víctor
authors.forename: Juan V
authors.initials: JV
authors.lastname: Esplugues
authors.forename: José M
authors.initials: JM
authors.lastname: Rojas
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Madrid
authors.forename: Juan M
authors.initials: JM
authors.lastname: Serrador
mesh_terms: D000941:Antigens / Q000737:chemistry*; D017209:Apoptosis; D018106:CD28 Antigens / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D000941:Antigens; D018106:CD28 Antigens; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins; D003545:Cysteine