IKKγ/NEMO Localization into Multivesicular Bodies

Abstract

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3β (GSK-3β) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3β leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3β and V-ATPase in NF-κB signaling activation.

Regulation of IkappaB Protein Expression by Early Gestation in the Thymus of Ewes

“…NEMO regulates IκB kinase activation to phosphorylate NF-κB inhibitors IκBs that modulate NF-κB signaling to participate in immune and inflammatory responses [ 44 ]. The downregulation of NEMO suppresses NF-κB signal transduction to modulate inflammatory and immune responses [ 45 ]. NEMO (also known as IKKγ) deficiency results in an increase in the ISG15 expression, which is a type I interferon signature [ 46 ].…”

Normoxic HIF-1α Stabilization Caused by Local Inflammatory Factors and Its Consequences in Human Coronary Artery Endothelial Cells

“…After the preparation of the donor vector (see Supplementary Methods ), HEK-293 cells were cotransfected with the donor and the two TALE-Nuclease vectors by the calcium phosphate method as described previously [ 11 ]. Knock-in HEK-293 cells were then selected using DMEM containing hygromycin B (AppliChem, Darmstadt, Germany).…”