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Nuclear localization of the Drosophila IκB protein Cactus and its response to the Toll signaling pathway
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Ylva Engström)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Ylva Engström)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Ylva Engström)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In Drosophila, the Toll signaling pathway is known as a regulator of both dorso-ventral patterning during embryogenesis and regulation of immunity. Activation of of the Toll pathway results in nuclear accumulation of the NFκB/Rel transcription factors Dif and Dorsal, and the subsequent activation of downstream target genes. The current model is that Cactus is a strictly cytoplasmic protein, interacting with Dorsal and Dif to inhibit their nuclear translocation. However, immunostaining revealed that Cactus is present both in the nucleus and cytoplasm of fat body cells and S2 cells. Activation of Toll signaling in cell culture demonstrated that a nuclear form of Cactus is stable and persists during signaling, while cytoplasmic Cactus is degraded in a proteosome-dependent manner and then re-synthesized. Alternative splicing of Cactus pre-mRNA produces two Cactus isoforms, differing by 18 amino acids in the C-terminus. We show that both isoforms act as inhibotors of Dif- and Dorsal-mediated Drosomycin-luciferase expression, although the longer isoform of Cactus was a slightly better inhibitor. Both isoforms showed similar subcellular distribution, being present both in the cytoplasm and nucleus of larval fat body cells. Thus, the present finding suggest that Cactus does not act as a degradeable, cytoplasmic inhibitor of Dif and Dorsal, but also plays a role in the nucleus during immune challenge. 

National Category
Natural Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-64254OAI: oai:DiVA.org:su-64254DiVA: diva2:456477
Available from: 2011-11-14 Created: 2011-11-14 Last updated: 2011-11-15Bibliographically 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.
Keyword
Drosophila immunity, signaling pathways, Cactus, NFκB
National Category
Natural Sciences
Research subject
Molecular Biology
Identifiers
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)
Opponent
Supervisors
Note

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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