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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / metabolism*
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Amino Acid Substitution
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Cell Line
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Cells, Cultured
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Cysteine / metabolism
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Enzyme Activation
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Golgi Apparatus / enzymology
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Golgi Apparatus / immunology
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Golgi Apparatus / metabolism
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Humans
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Immunological Synapses / enzymology*
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Immunological Synapses / immunology
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Immunological Synapses / metabolism
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Isoenzymes / chemistry
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Isoenzymes / genetics
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Isoenzymes / metabolism*
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Luminescent Proteins / antagonists & inhibitors
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Mutation
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Nitric Oxide / metabolism
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Nitric Oxide Synthase Type III / antagonists & inhibitors
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Nitric Oxide Synthase Type III / genetics
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Nitric Oxide Synthase Type III / metabolism*
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Profilins / genetics
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Profilins / metabolism*
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Protein Kinase C / chemistry
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Protein Kinase C / genetics
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Protein Kinase C / metabolism*
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Protein Kinase C-theta
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Protein Processing, Post-Translational*
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Protein Transport
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Pseudopodia
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RNA Interference
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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T-Lymphocytes / cytology
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T-Lymphocytes / immunology
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T-Lymphocytes / metabolism*
Substances
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Actins
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Isoenzymes
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Luminescent Proteins
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PFN1 protein, human
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Profilins
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Recombinant Fusion Proteins
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Recombinant Proteins
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Nitric Oxide
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NOS3 protein, human
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Nitric Oxide Synthase Type III
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PRKCQ protein, human
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Protein Kinase C
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Protein Kinase C-theta
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Cysteine