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Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-kappa B Signaling
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2010 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 184, no 11, 6188-6198 p.Article in journal (Refereed) Published
Abstract [en]

Because NF-kappa B 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-kappa B pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappa B 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 I kappa B homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappa B 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-kappa B signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappa B 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-kappa B signaling. The Journal of Immunology, 2010, 184: 6188-6198.

Place, publisher, year, edition, pages
2010. Vol. 184, no 11, 6188-6198 p.
National Category
Biological Sciences Microbiology in the medical area
URN: urn:nbn:se:su:diva-49704DOI: 10.4049/jimmunol.1000261ISI: 000278439600032OAI: diva2:379302

authorCount :16

Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2016-08-25Bibliographically approved
In thesis
1. Signaling pathways in Drosophila immunity
Open this publication in new window or tab >>Signaling pathways in Drosophila immunity
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drosophila relies on innate immunity to protect itself from its hostile environment throughout its life cycle. Despite the remarkable progress in understanding many aspects of Drosophila immunity, there are still big gaps in our knowledge. The general aim of this thesis was to gain a better understanding about the regulatory mechanisms controlling gene expression in Drosophila, with a focus on immunity. 

To enable isolation of Drosophila genes involved in immunity, we developed a method that allows visualization of immune gene expression in large number of embryos.  Reporter gene expression in wild type and mutant embryos was used to validate this approach, which should be a valuable complement to existing genetic and RNAi screens. 

Cactus, the Drosophila IκB protein, is known as a cytoplasmic inhibitor of Dif and Dorsal. We discovered that Cactus is also present in the cell nucleus. In response to Toll pathways signaling, cytoplasmic Cactus degrades rapidly in a proteasome-dependent manner, while a nuclear form of Cactus is stable and persists throughout signaling. This suggests that Cactus also has a function in the nucleus.

A genome-wide RNAi-based screen was performed in cultured S2 cells. Several novel components of NF-κB pathways were isolated as putative regulators of Drosophila immunity. One of them, the G protein-coupled receptor kinase-2 (Gprk2), was shown to be required for Drosomycin expression and for resistance to infection. Gprk2 interacts with Cactus, but is not required for Cactus degradation upon Toll pathway activation.  

The dpld/wech gene was previously found to affect periferal nervous system development. Here, we show that wech belongs to the LIN-41 subclade of the TRIM protein superfamily, and contains target sites for microRNAs. Genetic and cell transfection assays confirmed that wech expression is regulated by the microRNA let-7. This seems to be a conserved regulatory mechanism throughout the LIN-41 subclade. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2011. 67 p.
Drosophila immunity, signaling pathways, Cactus, NFκB
National Category
Natural Sciences
Research subject
Molecular Biology
urn:nbn:se:su:diva-64256 (URN)978-91-7447-398-8 (ISBN)
Public defence
2011-12-15, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2011-11-23 Created: 2011-11-14 Last updated: 2013-12-06Bibliographically approved

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Esfahani, Shiva SeyedoleslamiEngström, Ylva
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