1. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.
- Author
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García-Ortiz A, Martín-Cofreces NB, Ibiza S, Ortega Á, Izquierdo-Álvarez A, Trullo A, Victor VM, Calvo E, Sot B, Martínez-Ruiz A, Vázquez J, Sánchez-Madrid F, and Serrador JM
- Subjects
- Amino Acid Substitution, Cell Line, Cells, Cultured, Cysteine metabolism, Enzyme Activation, Golgi Apparatus enzymology, Golgi Apparatus immunology, Golgi Apparatus metabolism, Humans, Immunological Synapses immunology, Immunological Synapses metabolism, Isoenzymes chemistry, Isoenzymes genetics, Luminescent Proteins antagonists & inhibitors, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mutation, Nitric Oxide metabolism, Nitric Oxide Synthase Type III antagonists & inhibitors, Nitric Oxide Synthase Type III genetics, Profilins genetics, Protein Kinase C chemistry, Protein Kinase C genetics, Protein Kinase C-theta, Protein Transport, Pseudopodia, RNA Interference, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, T-Lymphocytes cytology, T-Lymphocytes immunology, Actins metabolism, Immunological Synapses enzymology, Isoenzymes metabolism, Nitric Oxide Synthase Type III metabolism, Profilins metabolism, Protein Kinase C metabolism, Protein Processing, Post-Translational, T-Lymphocytes metabolism
- 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.
- Published
- 2017
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