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The Role of Vimentin in the Tumor Marker Nup88-Dependent Multinucleated Phenotype Masaki Makise* , Hideaki Nakamura and Akihiko Kuniyasu

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The Role of Vimentin in the Tumor Marker Nup88-Dependent Multinucleated Phenotype Masaki Makise* , Hideaki Nakamura and Akihiko Kuniyasu Makise et al. BMC Cancer (2018) 18:519 https://doi.org/10.1186/s12885-018-4454-y RESEARCH ARTICLE Open Access The role of vimentin in the tumor marker Nup88-dependent multinucleated phenotype Masaki Makise* , Hideaki Nakamura and Akihiko Kuniyasu Abstract Background: Nucleoporin Nup88, a component of nuclear pore complexes, is known to be overexpressed in several types of tumor tissue. The overexpression of Nup88 has been reported to promote the early step of tumorigenesis by inducing multinuclei in both HeLa cells and a mouse model. However, the molecular basis of how Nup88 leads to a multinucleated phenotype remains unclear because of a lack of information concerning its binding partners. In this study, we characterize a novel interaction between Nup88 and vimentin. We also examine the involvement of vimentin in the Nup88-dependent multinucleated phenotype. Methods: Cells overexpressing tagged versions of Nup88, vimentin and their truncations were used in this study. Coprecipitation and GST-pulldown assays were carried out to analyze protein-protein interactions. Vimentin knockdown by siRNA was performed to examine the functional role of the Nup88-vimentin interaction in cells. The phosphorylation status of vimentin was analyzed by immunoblotting using an antibody specific for its phosphorylation site. Results: Vimentin was identified as a Nup88 interacting partner, although it did not bind to other nucleoporins, such as Nup50, Nup214, and Nup358, in HeLa cell lysates. The N-terminal 541 amino acid residues of Nup88 was found to be responsible for its interaction with vimentin. Recombinant GST-tagged Nup88 bound to recombinant vimentin in a GST-pulldown assay. Although overexpression of Nup88 in HeLa cells was observed mainly at the nuclear rim and in the cytoplasm, colocalization with vimentin was only partially detected at or around the nuclear rim. Disruption of the Nup88-vimentin interaction by vimentin specific siRNA transfection suppressed the Nup88-dependent multinucleated phenotype. An excess amount of Nup88 in cell lysates inhibited the dephosphorylation of a serine residue (Ser83) within the vimentin N-terminal region even in the absence and presence of an exogenous phosphatase. The N-terminal 96 amino acid residues of vimentin interacted with both full-length and the N-terminal 541 residues of Nup88. Conclusions: Nup88 can affect the phosphorylation status of vimentin, which may contribute to the Nup88-dependent multinucleated phenotype through changing the organization of vimentin. Keywords: Nup88, Nucleoporin, Vimentin, Cancer, Multinucleated phenotype, Kinases, Interaction * Correspondence: [email protected] Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Makise et al. BMC Cancer (2018) 18:519 Page 2 of 12 Background Proteomic approaches indicate that the N-terminal Nuclear pore complexes (NPCs) are channels that medi- region of vimentin contains many amino acid (aa) residues ate bidirectional trafficking between the cytoplasm and that are phosphorylated by a variety of kinases, such as the nucleus in eukaryotic cells. In vertebrates, multiple mitotic kinases [20, 25, 28, 29]. For instance, phosphoryl- copies of approximately 30 polypeptides, termed nucleo- ation by cyclin-dependent kinase 1 (Cdk1) promotes porins (Nups), assemble to form NPCs [1, 2]. A single vimentin IF disassembly into non-filamentous oligomers. NPC exhibits an eight-fold symmetric cylinder-like In addition, Cdk1 promotes Plk1 specific vimentin structure that can be divided into three distinct regions phosphorylation during early mitosis [30–32]. Both Rho- [2, 3]. One such region is the central channel, which associated kinase and Aurora B kinase promote the segre- spans the inner and outer nuclear membrane. The other gation of vimentin IF cooperatively at the cleaved furrow two regions are the nuclear basket and the cytoplasmic from late mitosis to cytokinesis [30, 33, 34]. Although filaments, both of which are joined by the central chan- vimentin overexpression in cancer cells has been reported nel. Approximately one-third of nucleoporins have a var- to be closely correlated with accelerated tumor growth, iety of phenylalanine-glycine (FG) repeats [3, 4]. FG migration, and invasion, specific regulation of the phos- repeat-containing Nups form unstructured barriers in phorylation status of vimentin during the development of the central channel, which are crucial for the selective cancer remains poorly understood [20]. trafficking of macromolecules [4, 5]. In the present study, to gain insights into the molecu- Nup88 is a non-FG Nup located on the cytoplasmic lar basis of how overexpressed Nup88 induces multinu- face of the NPC that participates in nuclear export by clei in HeLa cells, we screened Nup88 interacting interacting with other FG Nups, such as Nup358 and proteins using a proteomic approach. Here, we demon- Nup214 [3, 6–8]. Intriguingly, abnormally high levels strate that a novel interaction of Nup88 with vimentin of Nup88 has been detected in the cytoplasm of vari- can affect the phosphorylation status of vimentin. We ous tumor cells [3, 9, 10]. Furthermore, it has been also propose that the interaction between Nup88 and reported that elevated Nup88 expression in tumor vimentin is involved in the Nup88-dependent multinu- cells is correlated with tumor grade such as in colo- cleated phenotype. rectal cancer, breast cancer and hepatocellular carcin- oma [11–15]. Consequently, it has been proposed that Methods Nup88 might be used as a cancer prognostic and Plasmid constructs diagnostic marker [3]. Plasmids expressing GFP-tagged proteins under the con- Recent reports indicate that Nup88 overexpression is trol of a CMV promoter were generated by cloning their responsible, at least in part, for the early steps of tumori- PCR products into pEGFP-N2 (Clontech, Mountain genesis. For instance, overexpression of Nup88 in HeLa View, CA). The expression plasmids for FLAG-tagged cells induces multipolar spindles and the appearance of proteins were constructed by replacing the EGFP genes multinucleated cells [16]. In addition, elevated Nup88 in pEGFP-N2 with synthetic oligonucleotides encoding 3 expression in a mouse model induces multinuclei by x FLAG. A plasmid that expresses GST-fused Nup88 promoting the premitotic destruction of Polo-like kinase was constructed by inserting the Nup88 gene down- 1 (Plk1), a kinase required for bipolar spindle formation stream of the GST gene in pGEX4T-3 (GE Healthcare and chromosome segregation [17, 18]. However, the mo- Life Sciences, Piscataway, NJ). A plasmid that expresses lecular basis of how Nup88 leads to a multinucleated Halo tagged Nup214 or Nup358 was purchased from phenotype is poorly understood because of a lack of in- Kazusa Genome Technologies Inc. [35]. formation concerning its interacting partners [19]. Proteomic analysis reveals a variety of proteins that as- Cell culture and stable cell lines sociate with NPCs [1]. One such protein is vimentin, Cells were cultured in Dulbecco’s modified Eagle’s which is classified as a type III intermediate filament (IF) medium (DMEM) supplemented with 10% fetal bovine protein abundant in motile mesenchymal cells, various serum and penicillin/streptomycin at 37 °C in a humidi- types of tumors, and cancer cell lines [20]. Vimentin fied incubator supplied with 5% CO2. forms insoluble filamentous networks that distribute T-REx HeLa cells containing a single Flp recombin- widely in the cytoplasm from the perinuclear region to ation site in the genome were established from HeLa the cell periphery. Phosphorylation of vimentin turns its R19 cells [36]. The T-REx HeLa stable cell lines were insoluble filaments into a soluble or insoluble non- established according to the manufacturer’s instructions filamentous form [21–23]. This organizational change of (Life Technologies, Carlsbad, CA). Briefly, the T-REx vimentin endows the cells with mechanical resistance, HeLa cells were cotransfected with pcDNA5/FRT/TO allowing them to undergo morphological change and encoding an EGFP-tagged version of the genes of inter- motility [20, 24–27]. est and pOG44 encoding the Flp recombinase. Following Makise et al. BMC Cancer (2018) 18:519 Page 3 of 12 transfection, the cells were transferred into medium con- Antibodies taining 100 μg/ml hygromycin B and 3 μg/ml blasticidin Mouse monoclonal anti-human Nup88 (#611896) was pur- S to select for drug-resistant clones. The clones were chased from BD Biosciences (Franklin Lakes, NJ). Rabbit collected by trypsinization and stored frozen in liquid polyclonal anti-GFP (#598), anti-αtubulin (#PM054), and N2 before use. Protein expression in T-REx HeLa cells anti-DDDDK (#PM020) were purchased from Medical & was induced using medium containing 1 μg/ml doxycyc- Biological Laboratories (Tokyo, Japan). Rabbit polyclonal line (DOX) for 24-48 h. anti-GFP (#ab290), mouse monoclonal anti-vimentin
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