title: eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.
abstract: The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.
authors.affiliation: Dpt. Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa" (CBMSO), CSIC-UAM, Madrid, Spain.
authors.forename: Almudena
authors.initials: A
authors.lastname: García-Ortiz
authors.affiliation: Servicio de Inmunología. Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain.|Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.
authors.forename: Noa B
authors.initials: NB
authors.lastname: Martín-Cofreces
authors.affiliation: Immunobiology Unit, Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisbon, Portugal.
authors.forename: Sales
authors.initials: S
authors.lastname: Ibiza
authors.affiliation: Dpt. de Fisiología, Universitat de Valencia, Burjassot, Spain.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Ortega
authors.affiliation: Servicio de Inmunología. Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Izquierdo-Álvarez
authors.affiliation: Unidad de Microscopía, CNIC, Madrid, Spain.|Center of Experimental Imaging, Ospedale San Raffaele, Milan, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Trullo
authors.affiliation: Dpt. de Fisiología, Universitat de Valencia, Burjassot, Spain.|Servicio de Endocrinología y Nutrición, Hospital Universitario Dr. Peset Aleixandre, Fundación para la Promoción de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Valencia, Spain.
authors.forename: Víctor M
authors.initials: VM
authors.lastname: Victor
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.|Laboratorio de Proteómica Cardiovascular, CNIC, Madrid, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Calvo
authors.affiliation: Fundación IMDEA-Nanociencia, Madrid, Spain.|Centro Nacional de Biotecnología (CNB-CSIC)-IMDEA Nanociencia Associated Unit, Madrid, Spain.
authors.forename: Begoña
authors.initials: B
authors.lastname: Sot
authors.affiliation: Servicio de Inmunología. Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Martínez-Ruiz
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.|Laboratorio de Proteómica Cardiovascular, CNIC, Madrid, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Vázquez
authors.affiliation: Servicio de Inmunología. Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain.|Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Madrid
authors.affiliation: Dpt. Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa" (CBMSO), CSIC-UAM, Madrid, Spain.
authors.forename: Juan M
authors.identifier: 0000-0003-4710-3077
authors.initials: JM
authors.lastname: Serrador
mesh_terms: D000199:Actins / Q000378:metabolism*; D019943:Amino Acid Substitution; D002460:Cell Line; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D006801:Humans; D054992:Immunological Synapses / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051304:Profilins / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011554:Pseudopodia; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*
chemical_list: D000199:Actins; D007527:Isoenzymes; D008164:Luminescent Proteins; C495738:PFN1 protein, human; D051304:Profilins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D003545:Cysteine