Genome-Wide RNA Interference inDrosophilaCells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-κB Signaling

Abstract Because NF-κB signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-κB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-κB regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk)2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila IκB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-κB regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-κB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-κB reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll10b-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-κB signaling.