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 I kappa B alpha, a negative regulator of NF-kappa B signaling, as a substrate for GRK6. GRK6 directly phosphorylated I kappa B alpha at Ser(32)/Ser(36), and the kinase activity of GRK6 was required for the promotion of NF-kappa B signaling after TNF-alpha stimulation. Knockout of GRK6 in peritoneal macrophages remarkably attenuated the transcription of inflammatory genes after TNF-alpha 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-alpha. In summary, our study demonstrates that TNF-alpha induces GRK6 activation, and GRK6 promotes inflammatory responses through the phosphorylation of I kappa B alpha. (C) 2015 Elsevier Inc. All rights reserved.