Ibiza, SalesPérez-Rodríguez, AndreaOrtega, ÁngelMartínez Ruiz, AntonioBarreiro, OlgaCarlota A. García-DomínguezVíctor M. VíctorJuan V. EspluguesJosé M. RojasFrancisco Sánchez-MadridJuan M. Serrador2024-05-222024-05-222008-07-290027-84241091-649010.1073/pnas.0711062105https://hdl.handle.net/20.500.14352/104316Ras/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 antigenpresenting 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.engAttribution-NonCommercial-NoDerivatives 4.0 Internationaljournal articlehttps://doi.org/10.1073/pnas.071106210open access577.1Bioquímica (Farmacia)2302 Bioquímica