G-protein-coupled-receptor kinases mediate TNFα-induced NFκB signalling via direct interaction with and phosphorylation of IκBα.
Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in Raw264.7 macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibits TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene, macrophage inflammatory protein-1β. Consistent with these results, over-expression of GRK2 or 5 enhances TNFα-induced NFκB activity. In addition,we show that GRK2 and 5 interact with IκBα via the N-terminal domain of IκBα and that IκBα isa substrate for GRK2 and 5 in vitro. Furthermore, we also find that GRK5 but not GRK2 phosphorylates IκBα at the same amino acid residues (Ser32/36) as that of IKKβ. Interestingly,associated with these results, knockdown of IKKβ in Raw264.7 macrophages did not affect TNFα-induced IκBα phosphorylation. Taken together, these results demonstrate that both GRK2 and 5 are important and novel mediators of a non-traditional IκBα-NFκB signaling pathway.
Department of Physiology and Division of Pathology, Michigan State University, East Lansing, MI 48824, USA.
Sonika
S
Patial
Jiansong
J
Luo
Katie J
KJ
Porter
Jeffrey L
JL
Benovic
Narayanan
N
Parameswaran
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000235:genetics / Q000378:metabolism; D054768:G-Protein-Coupled Receptor Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C496568:GRK2 protein, mouse; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C517895:Grk5 protein, mouse