Oncogene (2008) 27, 6334–6346 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen S Kotadia1,LRKao1, SA Comerford2, RT Jones1, RE Hammer3 and TL Megraw1 1Department of Pharmacology, The Cecil and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA; 2Department of Molecular Genetics, The Cecil and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA and 3Department of Biochemistry, The Cecil and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA Viruses of the DNA tumor virus family share the ability to cell-cycle and apoptosis regulation, viral oncoproteins transform vertebrate cells through the action of virus- transform cells. Investigation of DNA tumor virus encoded tumor antigens that interfere with normal cell oncoproteins has led to the identification of many physiology. They accomplish this very efficiently by fundamental mechanisms of tumor suppression (Lavia inhibiting endogenous tumor suppressor proteins that et al., 2003; Ahuja et al., 2005). By altering the activity control cell proliferation and apoptosis. Simian virus 40 of p53, retinoblastoma protein and serine/threonine (SV40) encodes two oncoproteins, large tumor antigen, protein phosphatase 2A (PP2A), three key tumor which directly inhibits the tumor suppressors p53 and Rb, suppressors, SV40 can cause tumor formation in and small tumor antigen (ST), which interferes with transgenic mouse models (Ahuja et al., 2005; Arroyo serine/threonine protein phosphatase 2A (PP2A). We and Hahn, 2005). have constructed a Drosophila model for SV40 ST A single transcript expressed from the early region of expression and show that ST induces supernumerary the SV40 genome encodes three proteins by alternative centrosomes, an activity we also demonstrate in human splicing: large tumor antigen (LT), small tumor antigen cells. In early Drosophila embryos, ST also caused (ST)and 17kT (Sullivan and Pipas, 2002).ST coop- increased microtubule stability, chromosome segregation erates with LT to transform cells (Skoczylas et al., 2004). errors, defective assembly of actin into cleavage furrows, ST has two domains and two known binding partners: a cleavage failure, a rise in cyclin E levels and embryonic domain with homology to dnaJ proteins, or ‘J domain’ lethality. Using ST mutants and genetic interaction that binds Hsc70, and a PP2A-binding domain. The experiments between ST and PP2A subunit mutations, oncogenic activity of ST requires PP2A binding, but not we show that all of these phenotypes are dependent on the J domain (Mungre et al., 1994; Saenz-Robles et al., ST’s interaction with PP2A. These analyses demonstrate 2001; Skoczylas et al., 2004). Because of this property, the validity and utility of Drosophila as a model for viral ST has been used as a tool to assess PP2A’s role in oncoprotein function in vivo. transformation (Chen et al., 2004; Skoczylas et al., Oncogene (2008) 27, 6334–6346; doi:10.1038/onc.2008.254; 2004). By inhibiting PP2A, ST disrupts dephosphoryla- published online 28 July 2008 tion of targets for transformation, including c-myc and RalA (Mumby, 2007). The PP2A holoenzyme consists Keywords: SV40 ST; centriole; centrosome; PP2A; of three subunits, a scaffolding subunit (A), a regulatory aneuploidy; cyclin E subunit (B)and a catalytic subunit (C).In addition, substrate specificity is conferred by four classes of regulatory subunits: B, B0,B00 and B000 (Janssens and Goris, 2001; Sontag, 2001). ST binds directly to the Introduction PP2A A subunit (PP2AA), displacing B subunits from the holoenzyme but retaining the C subunit (Yang et al., Viruses of the DNA tumor virus family induce cell 1991; Chen et al., 2007b). The structure of PP2AA bound proliferation to promote the replication of the viral to ST revealed a direct association between several genome. Extensively investigated members of this family PP2AA HEAT repeats with the second of two zinc- include human papillomavirus (HPV), adenovirus, binding domains in ST, an association that is similar polyomavirus and simian virus 40 (SV40). By hijacking to PP2AB0 bound to PP2AA (Xu et al., 2006; Chen et al., 2007b; Cho and Xu, 2007; Cho et al., 2007). Thus, ST Correspondence: Dr TL Megraw, Department of Pharmacology, The might function as a PP2A B subunit (Cho et al., 2007). Cecil and Ida Green Center for Reproductive Biology Sciences, The Therefore, in addition to inhibiting activity toward University of Texas Southwestern Medical Center at Dallas, 6001 normal substrates through competition with B subunits, Forest Park Road, Room ND11.120, Dallas, TX 75390-9051, USA. E-mail: [email protected] ST may confer new substrate specificities to PP2A. Received 21 March 2008; revised 23 June 2008; accepted 25 June 2008; Although DNA tumor virus oncoproteins disrupt published online 28 July 2008 pathways controlling the cell cycle and apoptosis, they SV40 ST promotes centrosome overduplication S Kotadia et al 6335 also compromise cell division, potentially leading to organize or maintain chromosomes within the spindle aneuploidy (Lavia et al., 2003). For example, HPV E7 (Figure 2b, arrow). Often, the extra centrosomes in ST and adenovirus E1A induce centrosome overduplica- embryos were free, and not associated with a mitotic tion, impacting genomic stability by increasing the spindle (for example, Figure 1d). Thus, ST disrupts incidence of multipolar mitotic spindles (De Luca centrosome duplication and chromosome segregation et al., 2003; Duensing and Munger, 2003; Duensing from the earliest embryonic cleavage cycles. et al., 2004). Although the transforming properties of ST As induction of supernumerary centrosomes is a novel have been extensively investigated, there is little under- phenotype associated with ST, we asked whether ST standing of the impact that disruption of normal elicited this activity in mammalian cells. To test for signaling caused by ST and other viral oncoproteins centrosome duplication effects of ST in human cells, we have on animal development. To address this question, transiently transfected U2OS cells with a plasmid that we have constructed a Drosophila melanogaster model expresses native ST protein. Figures 2e–g show that ST- for SV40 ST pathogenesis; the first Drosophila model for expressing U2OS cells also have increased centrosome expression of a viral oncoprotein. We show that ST numbers, with 23.2±3.9% (mean±s.d.)having >2 causes increased centrosome numbers, chromosome centrosomes, compared to 9.8±2.9% of control cells. segregation defects, aberrant cytoskeleton assembly, To examine the effects of ST on centrosome duplica- cytokinesis failures and a rise in cyclin E levels. Using tion within the time frame of a single cell cycle, we ST mutations and Drosophila mutant analysis, we show established a stable Drosophila cell line that expresses that disruption of embryogenesis by ST requires PP2A ST-FLAG under inducible control from the metal- subunits, confirming the ST-PP2A connection from lothionein promoter (Figure 2h). The doubling time of vertebrate studies. Kc cells is approximately 24 h at 25 1C, yet after only 20 h of induction, ST-FLAG caused a significant increase in centrosome number within a single cell cycle from 21.2±1.6% (mean±s.d.)(uninduced)to Results 44.8±3.8% of cells that contain X4 centrosomes (Figures 2i and j). The excess centrosomes coalesced at ST induces centrosome overduplication, and cytoskeletal the spindle poles in mitotic cells, resulting in bipolar and cleavage defects that lead to embryonic lethality spindles (Figure 2i). For this analysis, we restricted Expression of the SV40 early region, which encodes the centrosome counts to mitotic cells, making it unlikely LT, ST and 17kT proteins through alternative splicing that tallied cells experienced cytokinesis during the 20-h (Figure 1a)(Sullivan and Pipas, 2002),in Drosophila induction period. Flow cytometry was used to examine early embryos caused an increase in centrosome cell-cycle effects of ST expression in Kc cells, yet no numbers and lethality (Figures 1c and d; Supplementary significant differences in cell-cycle parameters or in the Results; Supplementary Figure S1). ST was expressed polyploid cell population were seen at 20 and 42 h of exclusively from this construct (Figure 1e). To test induction of ST compared to the controls (Supplemen- whether ST indeed caused the increased centrosome tary Figure S2). Therefore, centrosome overduplication numbers and other phenotypes (see below)observed did not arise as an indirect perturbation of the cell cycle with the early region construct, and to eliminate a by ST. Moreover, double staining of Kc cells for Cid, a potential contribution from undetectable expression of kinetochore marker, and CNN showed that there was LT and 17kT, transgenic flies that express an ST cDNA no increase in chromosome number coinciding with fused to a 3 Â FLAG tag at the C terminus were increased centrosome numbers (Supplementary Figure generated (ST-FLAG). As a control, transgenic flies S3). Thus, supernumerary centrosomes induced by ST containing the FLAG tag vector were generated are achieved by overduplication rather than through a (vector). Comparison