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

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

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism*
  • Amino Acid Substitution
  • Cell Line
  • Cells, Cultured
  • Cysteine / metabolism
  • Enzyme Activation
  • Golgi Apparatus / enzymology
  • Golgi Apparatus / immunology
  • Golgi Apparatus / metabolism
  • Humans
  • Immunological Synapses / enzymology*
  • Immunological Synapses / immunology
  • Immunological Synapses / metabolism
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • 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
  • Nitric Oxide Synthase Type III / metabolism*
  • Profilins / genetics
  • Profilins / metabolism*
  • Protein Kinase C / chemistry
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein Kinase C-theta
  • Protein Processing, Post-Translational*
  • 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
  • T-Lymphocytes / metabolism*

Substances

  • Actins
  • Isoenzymes
  • Luminescent Proteins
  • PFN1 protein, human
  • Profilins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Nitric Oxide
  • NOS3 protein, human
  • Nitric Oxide Synthase Type III
  • PRKCQ protein, human
  • Protein Kinase C
  • Protein Kinase C-theta
  • Cysteine