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

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-theta (PKC-theta) 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 beta-actin and PKC-theta from the lamellipodium-like distal (d)-SMAC, promoting PKC-theta. activation. Furthermore, eNOS-derived NO S-nitrosylated beta-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-theta. was corroborated by overexpression of PFN1- and actin-binding defective mutants of beta-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-theta. 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. ; Institute de Salud Carlos III (ISCIII, Spanish Government) (grant number PI10/02136) to J.M.S. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ERC (grant number ERC-2011-AdG294340-GENTRIS) to F.S.M. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. MINECO/FEDER (grant number SAF2015-69396-R) to J.M.S. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. MINECO (grant number B102015-67580-P) to J.V. The funder had no role in study design, data collection and analysis, decision to publish, or ...