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Simian Virus 40 Large T Antigen Targets the Microtubule-Stabilizing Protein TACC2

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Simian Virus 40 Large T Antigen Targets the Microtubule-Stabilizing Protein TACC2 3190 Research Article Simian virus 40 large T antigen targets the microtubule-stabilizing protein TACC2 Shuchin Tei1,2, Noriko Saitoh1, Tetsushi Funahara1, Shin-ichi Iida3, Yuko Nakatsu1, Kayo Kinoshita1, Yoshikazu Kinoshita2, Hideyuki Saya3 and Mitsuyoshi Nakao1,* 1Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan 2Department of Gastroenterology and Hepatology, Shimane University School of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan 3Division of Gene Regulation, Institute for Advanced Medical Research, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan *Author for correspondence ([email protected]) Accepted 15 June 2009 Journal of Cell Science 122, 3190-3198 Published by The Company of Biologists 2009 doi:10.1242/jcs.049627 Summary The large T antigens of polyomaviruses target cellular proteins missegregation. These mitotic defects are caused by N-terminal- that control fundamental processes, including p53 and the RB deleted T antigen, which minimally interacts with TACC2, family of tumor suppressors. Mechanisms that underlie T- whereas T-antigen-induced microtubule destabilization is antigen-induced cell transformation need to be fully addressed, suppressed by overexpressing TACC2. Thus, TACC2 might be because as-yet unidentified target proteins might be involved a key target of T antigen to disrupt microtubule regulation and in the process. In addition, recently identified polyomaviruses chromosomal inheritance in the initiation of cell transformation. are associated with particular human diseases such as aggressive skin cancers. Here, we report that simian virus 40 (SV40) large T antigen interacts with the transforming acidic coiled-coil- Supplementary material available online at containing protein TACC2, which is involved in stabilizing http://jcs.biologists.org/cgi/content/full/122/17/3190/DC1 microtubules in mitosis. T antigen directly binds TACC2 and induces microtubule dysfunction, leading to disorganized Key words: Large T antigen, TACC2, Chromosomes, Nucleus, mitotic spindles, slow progression of mitosis and chromosome Mitosis Introduction members, WU polyomavirus and KI polyomavirus, were found from Journal of Cell Science Cellular transformation is essential for progression toward tumor respiratory tract specimens (Allander et al., 2007; Gaynor et al., development. The proteins involved in this process have been 2007). More recently, Feng et al. described a new virus, Merkel identified as oncogene products and tumor suppressors. Simian virus cell polyomavirus, the genome of which is integrated into cellular 40 (SV40), a polyomavirus of rhesus macaque origin, is a DNA DNA in the aggressive human skin cancers (Feng et al., 2008). These tumor virus that can induce tumors in rodents and transform many lines of evidence raise the essential question of how their types of cultured mammalian cells, including those of human origin oncoproteins function in the cells. In addition to the known targets (Ahuja et al., 2005; Fanning and Knippers, 1992; Poulin and of T antigen, this protein was recently reported to interact with Bub1, DeCaprio, 2006). The SV40-encoded replication protein, designated which has dual roles in spindle assembly checkpoint and large T antigen and referred to as T antigen, is a viral oncoprotein chromosome congression (Cotsiki et al., 2004; Hein et al., 2009). that modulates diverse cellular activities in genome integrity and Thus, SV40 T antigen might target as-yet unidentified cellular cell-cycle regulation, thereby promoting the early steps of oncogenic proteins to cause cell transformation and oncogenesis (Chang et al., transformation. Although many studies have reported that T antigen 1997; Moens et al., 2007; Sachsenmeier and Pipas, 2001; Woods inactivates p53 and the RB family of tumor suppressors, it is et al., 1994). suggested that other regulatory proteins are required for SV40- During our investigations, we found that T antigen interacts with induced cellular transformation (Chang et al., 1997; Moens et al., transforming acidic coiled-coil (TACC) protein 2, designated 2007; Sachsenmeier and Pipas, 2001). TACC2. There are at least three TACC family proteins (TACC1, Polyomaviruses are highly specific to their host species but TACC2 and TACC3), all of which contain the conserved TACC encode a similar T antigen (Ahuja et al., 2005; Chang et al., 1997; domain and have been possibly implicated in tumorigenesis Fanning and Knippers, 1992; Moens et al., 2007; Poulin and (Gergely, 2002; Raff, 2002). TACC1 and TACC3 were found to be DeCaprio, 2006; Sachsenmeier and Pipas, 2001). In humans, JC overexpressed in human cancers, and overexpression of TACC1 polyomavirus and BK polyomavirus were discovered to be induced the transformation of primary mouse cells in culture (Still responsible for progressive multifocal encephalopathy and renal et al., 1999a; Still et al., 1999b). By contrast, TACC2 was nephropathy, respectively (Imperiale, 2000; Moens et al., 2007; downregulated as breast tumors became more malignant (Chen et Poulin and DeCaprio, 2006). Analogous to SV40, both viruses al., 2000). Overexpression of TACC2 in these cells reverted encode T antigens that transform the cells in culture and promote malignant phenotypes to benign phenotypes both in vivo and in tumor formation in animals (Allander et al., 2007; Moens et al., vitro, suggesting that TACC2 might function as a potential tumor 2007; Poulin and DeCaprio, 2006). Additionally, two human suppressor. T antigen interacts with TACC2 3191 Here, we report that human TACC2 has a crucial role in Results microtubule stabilization in cultured cells, and that T antigen binds T antigen interacts with TACC2 TACC2 for inducing mitotic defects and chromosomal instability. To test the effect of T antigen on cellular function independently In addition to changes in chromosomes, T antigen, or the loss of of p53 and RB, we introduced GFP-fused T antigen in HeLa cells TACC2 function, causes abnormalities in nuclear structural after that lacked these functional proteins but stably transmitted cell division. Our findings suggest that TACC2 is a key target of chromosomes during cell division (Fig. 1A). Abnormally nucleated T antigen for promoting mitotic defects leading to abnormal cells, which had multiple nuclei and/or micronuclei, were increased chromosomal and nuclear inheritance. These findings provide the by about fourfold at 72-96 hours after transfection, compared with first evidence that a viral oncoprotein can directly disrupt control cells. This result suggests that T antigen might rapidly target microtubule regulation, and shed light on the molecular basis of other cellular factors rather than p53 and RB. In addition, we found the initiation of cellular transformation. the similar effects of T antigen in rodent CHO cells (supplementary Journal of Cell Science Fig. 1. T antigen interacts with TACC2. (A) T antigen induces abnormally nucleated cells. HeLa cells were examined 96 hours after transfection with GFP-fused T antigen. Cells expressing GFP alone (control) or GFP-fused T antigen (GFP-TAg) were stained with DAPI. Micronuclei and multiple nuclei are indicated by the arrow and arrowhead, respectively (lower left). Abnormally nucleated cells were counted (right). Values are the means and standard deviations from five independent experiments (n=200). ***Statistical significant difference: P<0.001. (B) Schematic representation of SV40 large T antigen showing the LxCxE motif and bipartite region that bind RB family proteins and p53 for inactivation, respectively (Ahuja et al., 2005; Fanning and Knippers, 1992; Moens et al., 2007). The mitotic checkpoint protein Bub1 associates with the region containing amino acids 89-97 (Cotsiki et al., 2004). Yeast two-hybrid screening using the T antigen region containing amino acids 250-708 as bait identifies the TACC domain of TACC2 (GenBank accession no. NP996742; amino acids 788-996). (C) Human TACC family proteins. C-terminal TACC domains are conserved among the TACC family proteins. Deletion mutants of TACC2 are shown. (D) T antigen binds TACC2 in vitro. GST-fused T antigen(250-708) on glutathione-agarose beads was incubated with His-tagged TACC domains of TACC proteins and p53. Proteins bound on the beads were detected by western blot analysis with anti-His antibodies. The input shows 10% of each protein. (E) Partial colocalization of T antigen with TACC2 at the mitotic spindles. HeLa cells expressing GFP-fused T antigen (green) were stained with anti-TACC2 antibodies (red). 3192 Journal of Cell Science 122 (17) material Fig. S1A, left). Abnormally nucleated CHO cells promptly TACC protein family (TACC1, TACC2 and TACC3) have been augmented at 48 hours after transfection of T antigen, suggesting identified in human, and are differently implicated in naturally that these alterations induced by T antigen are unlikely to depend occurring cancers (Gergely, 2002; Raff, 2002). To determine a on cell type. direct interaction between T antigen and TACC2, we prepared To identify novel factors that interact with T antigen, we glutathione S-transferase (GST)-fused T antigen(250-708) and performed yeast two-hybrid screening using the region containing His-tagged TACC domains of human TACC proteins, and amino acids 250 to 708 of T antigen as bait (Fig. 1B). From a subjected them to an in vitro pull-down analysis (Fig. 1D). The screening of approximately 7ϫ106 independent transformants of TACC
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