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Cytoskeleton Remodeling Factor Binding Protein 1 Is a Novel Actin Gamma Interferon-Induced Guanylate

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Cytoskeleton Remodeling Factor Binding Protein 1 Is a Novel Actin Gamma Interferon-Induced Guanylate Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor Nicole Ostler, Nathalie Britzen-Laurent, Andrea Liebl, Elisabeth Naschberger, Günter Lochnit, Markus Ostler, Downloaded from Florian Forster, Peter Kunzelmann, Semra Ince, Verena Supper, Gerrit J. K. Praefcke, Dirk W. Schubert, Hannes Stockinger, Christian Herrmann and Michael Stürzl Mol. Cell. Biol. 2014, 34(2):196. DOI: 10.1128/MCB.00664-13. Published Ahead of Print 4 November 2013. http://mcb.asm.org/ Updated information and services can be found at: http://mcb.asm.org/content/34/2/196 These include: REFERENCES This article cites 79 articles, 22 of which can be accessed free at: http://mcb.asm.org/content/34/2/196#ref-list-1 on December 20, 2013 by UNIVERSITAETSBIBLIOTHEK CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor Nicole Ostler,a Nathalie Britzen-Laurent,a Andrea Liebl,a Elisabeth Naschberger,a Günter Lochnit,b Markus Ostler,c Florian Forster,d Peter Kunzelmann,e Semra Ince,f Verena Supper,d Gerrit J. K. Praefcke,g Dirk W. Schubert,e Hannes Stockinger,d Christian Herrmann,f Michael Stürzla Downloaded from ‹Division of Molecular and Experimental Surgery, University Medical Center Erlangen, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germanya; Faculty of Medicine, Institute of Biochemistry, Justus Liebig University, Giessen, Germanyb; Institute of Physics, Chemnitz University of Technology, Chemnitz, Germanyc; Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austriad; Institute of Polymer Materials, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germanye; Physical Chemistry I, Ruhr University Bochum, Bochum, Germanyf; Institute for Genetics, University of Cologne, Cologne, Germanyg Gamma interferon (IFN-␥) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is http://mcb.asm.org/ among the cellular proteins that is the most abundantly induced by IFN-␥ and mediates its cell biologic effects. As yet, the mo- lecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-␥-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin re- on December 20, 2013 by UNIVERSITAETSBIBLIOTHEK modeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-␥-dependent defense strategies. amma interferon (IFN-␥) exerts regulatory functions in a Network confirmed that GBP-1 expression in CRC is associated Gvariety of physiological and pathophysiological processes (1, with lower tumor aggressiveness (40). 2). It is best known for its potent immunomodulatory activity Despite the significant clinical relevance of GBP-1 expression (3–5). Additionally, IFN-␥ plays an important role in host de- and its well-documented functions, the molecular mechanisms of fenses against infection with viral and microbial pathogens (6–8). action of the protein have not been resolved. It has been proposed For example, IFN-␥ can inhibit pathogen replication and intracel- that GBP-1 might inhibit cell proliferation through the suppres- lular trafficking and pathogen-induced vesicle formation (7, 9). In sion of ␤-catenin/T cell factor (TCF) signaling (19, 34). In addi- addition, IFN-␥ is an important trigger of antitumoral immune tion, the GTPase activity of GBP-1 is required for the upregulation responses (2, 10–14). The direct antitumorigenic activity of IFN-␥ of integrin ␣4 and the inhibition of MMP-1 expression, leading to on tumor cells includes the induction of apoptosis (15, 16) and the the inhibition of cell migration and invasion, respectively (35, 41). However, no direct molecular target of GBP-1 has been identified inhibition of cell proliferation, migration, and invasion (3, as yet. Accordingly, the goal of this study was to identify cellular 17–20). binding factors of GBP-1 and to determine the function of these Guanylate binding protein 1 (GBP-1) is among the most sig- molecules in the regulation of the cellular response to IFN-␥. nificantly induced proteins in cells exposed to IFN-␥ (21, 22). (Parts of this work were conducted by N. Ostler in partial ful- GBP-1 belongs to the dynamin superfamily of large GTPases (23, fillment of the requirements for a doctoral thesis.) 24), which is characterized by an oligomerization-dependent GTPase activity (25–28). GBP-1 has been shown to mediate the MATERIALS AND METHODS antibacterial and antiviral activities of IFN-␥ (29, 30). Similar Ϫ Ϫ antipathogen effects have been observed with mouse GBPs Plasmids. The plasmids pMCV1.4( ) and pMCV2.2( ), the latter of which contains a gentamicin resistance cassette, were obtained from (mGBPs), indicating that GBPs exert conserved functions in hu- mans and rodents (9, 31–33). In addition, GBP-1 is both necessary and sufficient for the inhibitory effects of IFN-␥ on cell prolifera- Received 3 June 2013 Returned for modification 22 July 2013 tion, migration, and invasion, as shown in endothelial cells and Accepted 28 October 2013 epithelial tumor cells (17, 19, 34–37). Of note, the putative murine Published ahead of print 4 November 2013 homologue of human GBP-1, mGBP-2, has also been shown to Address correspondence to Michael Stürzl, [email protected]. inhibit cell motility (38). In accordance with its cell biological N.O. and N.B.-L. contributed equally to this article. activities, the expression of GBP-1 in colorectal carcinoma (CRC) Copyright © 2014, American Society for Microbiology. All Rights Reserved. has been associated with a significantly improved prognosis (39). doi:10.1128/MCB.00664-13 A recent comprehensive study from the Cancer Genome Atlas 196 mcb.asm.org Molecular and Cellular Biology p. 196–209 January 2014 Volume 34 Number 2 GBP-1 Controls Actin Remodeling Mologen (Berlin, Germany). The Flag (F) tag sequence was cloned into seeded on uncoated surfaces and incubated overnight in DMEM–0.5% both vectors using the EcoRV/EcoRI restriction sites. Green fluorescent FCS. The cells were subsequently treated with IFN-␥ (100 U/ml or 50 protein (GFP), GBP-1 (NCBI accession number NM_002053), and vari- U/ml for cells transfected with small interfering RNA [siRNA]; Roche, ous GBP-1 mutants were inserted in frame into the pMCV1.4-Flag con- Grenzach-Wyhlen, Germany) in the same medium for 24 h. struct as previously described (42). The following expression vectors were Transfection. The transfection of HeLa cells with expression vectors created: F–GBP-1, F–GBP-1(R227E/K228) (28), F–GBP-1(R240A) (43), was performed using the calcium phosphate method (45). For the RNA F–GBP-1(⌬CaaX), and F–GBP-1(K51A) (26, 44). Furthermore, an ex- interference (RNAi) experiments, the transfection was performed with pression vector for the GFP–GBP-1 fusion protein (F–GFP–GBP-1) was RNAiMax in Opti-MEM medium (both obtained from Life Technolo- generated with the pMCV1.4 construct as previously described (42). Ad- gies/Invitrogen, Darmstadt, Germany) according to the manufacturer’s Downloaded from ditionally, the sequence of GBP-1 was inserted in frame into the instructions. The transfection was performed in either 4-well Lab-Tek pMCV2.2-Flag vector using the EcoRI restriction site (17). chamber slides or 6-well cell culture plates (both purchased from Nunc, The human ␤-actin gene (NCBI accession number NM_001101.3) Thermo Fisher Scientific, Bonn, Germany) at a cell density of 2.8 ϫ 104 was amplified from cDNA derived from mRNA isolated from HeLa cells. and 1.3 ϫ 105 cells, respectively. GBP-1 Stealth Select RNAi siRNA The forward primer harbors a restriction site for BamHI (underlined) (HSS104020) oligonucleotides specific for human GBP-1 and stealth non- (5 -CCGGGATCCAGGATGGATGATGATATCGCC-3 ), and the re- targeting negative-control RNAi duplexes (medium GC content) were verse= primer contains a restriction site for EcoRI (underlined)= (5 -GCGG used at a final concentration of 16.6 nM (both were purchased from Mo- AATTCTTGAAGCATTTGCGGTGG-3 ). The PCR product was= sub- lecular Probes/Invitrogen). HUVECs were transfected with the Promo- = jected to restriction digestion with BamHI and EcoRI and then inserted in Fectin transfection reagent (PromoCell) as described by the manufac- http://mcb.asm.org/
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