biostudies-literatureWackernagel LMDeutsche Forschungsgemeinschaft6778https://www.ebi.ac.uk/biostudies/studies/S-EPMC9223704biostudies-literatureUnknown23(12)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. International journal of molecular sciencesIKKγ/NEMO Localization into Multivesicular Bodies.PMC922370497850925-SFB854361210922/GRK2408Medunjanin SFischer KDWackernagel LMWeinert SZuschratter WRichter KBraun-Dullaeus RCAbdi Sarabi MfalseIKKγ/NEMO Localization into Multivesicular Bodies.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. 2022-01-01T00:00:00Z2022 Jun2025-04-26T15:34:06.538Z2025-02-19T03:51:38.833ZS-EPMC92237043574322010.3390/ijms23126778