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PLoS Biol. 2017 Apr 10;15(4):e2000653. doi: 10.1371/journal.pbio.2000653. eCollection 2017 Apr.

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

Author information

1
Dpt. Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa" (CBMSO), CSIC-UAM, Madrid, Spain.
2
Servicio de Inmunología. Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain.
3
Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
4
Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.
5
Immunobiology Unit, Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisbon, Portugal.
6
Dpt. de Fisiología, Universitat de Valencia, Burjassot, Spain.
7
Unidad de Microscopía, CNIC, Madrid, Spain.
8
Center of Experimental Imaging, Ospedale San Raffaele, Milan, Italy.
9
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.
10
Laboratorio de Proteómica Cardiovascular, CNIC, Madrid, Spain.
11
Fundación IMDEA-Nanociencia, Madrid, Spain.
12
Centro Nacional de Biotecnología (CNB-CSIC)-IMDEA Nanociencia Associated Unit, Madrid, Spain.

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.

PMID:
28394935
PMCID:
PMC5386235
DOI:
10.1371/journal.pbio.2000653
[Indexed for MEDLINE]
Free PMC Article

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