Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 2008-07, Vol.105 (30), p.10507-10512
• Main Authors: Ibiza, Sales, Pérez-Rodríguez, Andrea, Ortega, Ángel, Martínez-Ruiz, Antonio, Barreiro, Olga, García-Domínguez, Carlota A, Víctor, Víctor M, Esplugues, Juan V, Rojas, José M, Sánchez-Madrid, Francisco, Serrador, Juan M
• Format: Article
• Language: English
• Subjects: Antigens; Antigens - chemistry; Apoptosis; Biological Sciences; CD28 Antigens - chemistry; Cell membranes; Cysteine - chemistry; Extracellular Signal-Regulated MAP Kinases - metabolism; Fluorescence; Gene Expression Regulation, Enzymologic; Golgi apparatus; Golgi Apparatus - metabolism; Humans; Models, Biological; Mutation; Neurons; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - metabolism; Oxides; Physiological regulation; Protein isoforms; Protein Structure, Tertiary; Proto-Oncogene Proteins c-raf - metabolism; ras Proteins - metabolism; Receptors; T cell antigen receptors; T cell receptors; T lymphocytes; T-Lymphocytes - immunology; T-Lymphocytes - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0711062105

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 Cys¹¹⁸, 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 Cys¹¹⁸ 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.