Genome-wide RNA interference in Drosophila cells identifies G protein-coupled receptor kinase 2 as a conserved regulator of NF-kappaB signaling.
Because NF-kappaB 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-kappaB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappaB 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 IkappaB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappaB 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-kappaB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappaB reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll(10b)-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-kappaB signaling.
Valanne
Susanna
S
Institute of Medical Technology, University of Tampere, Tampere, Finland.
Myllymäki
Henna
H
Kallio
Jenni
J
Schmid
Martin Rudolf
MR
Kleino
Anni
A
Murumägi
Astrid
A
Airaksinen
Laura
L
Kotipelto
Tapio
T
Kaustio
Meri
M
Ulvila
Johanna
J
Esfahani
Shiva Seyedoleslami
SS
Engström
Ylva
Y
Silvennoinen
Olli
O
Hultmark
Dan
D
Parikka
Mataleena
M
Rämet
Mika
M
D000818:Animals; D015153:Blotting, Western; D004330:Drosophila; D029721:Drosophila Proteins / Q000276:immunology* / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000276:immunology* / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000276:immunology / Q000378:metabolism*; D006091:Gram-Negative Chemolithotrophic Bacteria / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D016328:NF-kappa B / Q000276:immunology* / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D015027:Zebrafish
D029721:Drosophila Proteins; D016328:NF-kappa B; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; C496574:Gprk2 protein, Drosophila