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

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. © 2017 garcía-ortiz et al.