GRK6 phosphorylates IκBα at Ser³²/Ser³⁶ and enhances TNF-α-induced inflammation

G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine kinases that phosphorylate agonist-activated G protein-coupled receptors (GPCRs). It has recently been reported that GRKs regulate GPCR-independent signaling through the phosphorylation of intracellular proteins. To date, several intracellular substrates for GRK2 and GRK5 have been reported. However, those for GRK6 are poorly understood. Here we identified IκBα, a negative regulator of NF-κB signaling, as a substrate for GRK6. GRK6 directly phosphorylated IκBα at Ser³²/Ser³⁶, and the kinase activity of GRK6 was required for the promotion of NF-κB signaling after TNF-α stimulation. Knockout of GRK6 in peritoneal macrophages remarkably attenuated the transcription of inflammatory genes after TNF-α stimulation. In addition, we developed a bioluminescence resonance energy transfer (BRET) probe to monitor GRK6 activity. Using this probe, we revealed that the conformational change of GRK6 was induced by TNF-α. In summary, our study demonstrates that TNF-α induces GRK6 activation, and GRK6 promotes inflammatory responses through the phosphorylation of IκBα.