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.

Il-1β and Reactive Oxygen Species Differentially Regulate Neutrophil Directional Migration and Basal Random Motility in a Zebrafish Injury–Induced Inflammation Model

“…We further demonstrated that the il-1b-myd88 axis and NADPH oxidase-mediated ROS pathway are differentially required for basal random movement and injury-induced directional migration of neutrophils. Although MO knockdown is a transient and incomplete inactivation of the targeted genes and has a nonspecific toxic effect (55), the MOs used in this study are highly specific and have minimal toxic effect, as well documented by others (23,(28)(29)(30) and ourselves. We believe that the phenotypes we have observed in our study will likely represent the loss-of-function of the targeted genes.…”

“…We speculated that a similar mechanism might be used in zebrafish. To test this hypothesis, two MOs, myd88atg and myd88e2i2, which interfere with myd88 mRNA translation and splicing, respectively (28,29), were coinjected to effectively knock down myd88 expression in zebrafish embryos followed by tail fin amputation and bacterial injection (Supplemental Fig. 2G).…”

The Evolving Impact of G Protein-Coupled Receptor Kinases in Cardiac Health and Disease

“…Genome-wide RNA interference in Drosophila identified G protein-coupled receptor kinase (Gprk)2, which in other organisms is homologous to GRK5, to be a conserved regulator of NF〉 (255). Further studies have also confirmed GRK5 as a mediator of NF〉 signaling in mammals, with potential implications in cardiac hypertrophy and other cardiovascular pathologies, although the mechanism of action is still not fully understood (114,170,232).…”

The Drosophila Imd Signaling Pathway

“…For example, the ubiquitination state of various pathway components is delicately regulated. The E3-ligase Iap2 has been identified in many large-scale screens for Imd pathway regulators (60,66,69,70), and it also was shown to be essential for Imd pathway-mediated AMP expression and bacterial resistance in vivo (60,65,71). In addition, a number of ubiquitinating and deubiquitinating enzymes have been implicated in the negative regulation of the signaling pathway.…”