eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

Almudena García-Ortiz, Noa B Martín-Cofreces, Sales Ibiza, Ángel Ortega, Alicia Izquierdo-Álvarez, Antonio Trullo, Víctor M Victor, Enrique Calvo, Begoña Sot, Antonio Martínez-Ruiz, Jesús Vázquez, Francisco Sánchez-Madrid, Juan M Serrador

Journal: PLoS biology 2017;15(4):e2000653

PMID: 28394935

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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.

Address: Dpt. Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa" (CBMSO), CSIC-UAM, Madrid, Spain.; 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.; Immunobiology Unit, Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisbon, Portugal.; Dpt. de Fisiología, Universitat de Valencia, Burjassot, Spain.; Servicio de Inmunología. Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain.; Unidad de Microscopía, CNIC, Madrid, Spain.; Center of Experimental Imaging, Ospedale San Raffaele, Milan, Italy.; 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.; Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.; Laboratorio de Proteómica Cardiovascular, CNIC, Madrid, Spain.; Fundación IMDEA-Nanociencia, Madrid, Spain.; Centro Nacional de Biotecnología (CNB-CSIC)-IMDEA Nanociencia Associated Unit, Madrid, Spain.; 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.

DOI: 10.1371/journal.pbio.2000653

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MeSH Terms: Actins, Amino Acid Substitution, Cell Line, Cells, Cultured, Cysteine, Enzyme Activation, Golgi Apparatus, Humans, Immunological Synapses, Isoenzymes, Luminescent Proteins, Mutation, Nitric Oxide, Nitric Oxide Synthase Type III, Profilins, Protein Kinase C, Protein Kinase C-theta, Protein Processing, Post-Translational, Protein Transport, Pseudopodia, RNA Interference, Recombinant Fusion Proteins, Recombinant Proteins, T-Lymphocytes