Simian virus 40 large T antigen targets the spindle assembly checkpoint protein Bub1

Abstract

The mitotic spindle checkpoint protein Bub1 has been found to be mutated at low frequency in certain human cancers characterized by aneuploidy. Simian virus 40 large T antigen efficiently immortalizes rodent cells and occasionally transforms them to tumorigenicity. T antigen can also cause genomic instability, inducing chromosomal aberrations and aneuploidy. Here, we report an interaction between Bub1 and T antigen. T antigen coimmunoprecipitates with endogenous Bub1 and Bub3, another component of the spindle checkpoint complex. Genetic analysis demonstrates that the interaction of T antigen with Bub1 is not required for immortalization but is closely correlated with transformation. T antigen induces an override of the spindle checkpoint dependent on Bub1 binding. This interaction with proteins of the spindle checkpoint machinery suggests another role for T antigen and provides insight into its ability to cause chromosomal aberrations, aneuploidy, and transformation.

Fig. 1.

The yeast two-hybrid screen. (a) Schematic representation of T antigen indicating the domains for the interaction with Hsc70, pRB, and p53. The amino-terminal 136 aa of T antigen fused to the DNA-binding domain of LexA was used as the bait. (b) Bub1 has two domains highly conserved between human, mouse, and yeast: CD1 (amino acids 22–147) and CD2 (amino acids 786–1065), which encodes the kinase domain of the protein. Also depicted is the domain required for kinetochore localization and binding to Bub3 (amino acids 201–300) (29). The cDNA isolated from the screen encodes the C-terminal 485 aa of Bub1. (c) Bub1 interacts specifically with T antigen and not with a control bait in the yeast two-hybrid system. The RPB7 and RPB4 RNA polymerase II subunits are fused to LexA and B42, respectively, and were used as positive controls for the reporter activation assays and as negative controls for the interaction with T antigen or Bub1. XGAL, 5-bromo-4-chloro-3-indolyl β-d-galactoside.

Fig. 2.

T antigen coimmunoprecipitates (IP) with Bub1 and Bub3 in various cell lines. (a) T antigen/Bub1 and T antigen/Bub3 coimmunoprecipitations in the tsa cell lines. tsa8 and tsa14 are two cell lines derived by using the thermolabile tsA58 T antigen, and SV4 is a control cell line derived by using WT T antigen. WCL denotes whole-cell lysates from tsa8, tsa14, or SV4 cells. (b) T antigen/Bub1 and T antigen/Bub3 coimmunoprecipitations in NIH 3T3 cells. NIH 3T3/V is a pooled culture of clones transfected with pBluescript plasmid, and NIH 3T3/T is a pooled culture of clones transfected with SV40 early region DNA. In the NIH 3T3/T cells, T antigen has two additional higher molecular mass forms probably corresponding to different modification states of the protein. (c) An siRNA specifically targeting Bub1 confirms the specificity of the Bub1 antibody in Western blotting. A control siRNA or a specific siRNA oligo duplex targeting Bub1 was transfected into U2OS cells, and the cell lysates were analyzed by immunoblotting with Bub1 antibody. Equal amounts of total protein from the lysate were loaded in each lane as demonstrated by the actin immunoblot. (d) T antigen/Bub1 coimmunoprecipitation in U2OS cells. Stable U2OS lines expressing an empty vector, T antigen (two independent cell clones), K1, or dl89–97 mutant T antigens were used for a coimmunoprecipitation assay. The cell lines were also assessed for T antigen expression levels by immunoblotting.

Fig. 3.

Genetic analysis of the T antigen/Bub1 interaction. (a) Sequence alignment of the polyomavirus large T antigens reveals a conserved sequence motif WEXWW. The region between T antigen amino acids 80–110 of SV40, human BK polyomavirus (BK), human JC polyomavirus (JC), Kilham strain of polyomavirus (Kilham), amino acids 40–65 of bovine polyomavirus (BPV), and amino acids 100–125 of murine polyomavirus (PyV) was aligned by using the dnastar program. The sequence motif W(D/E)XWW (amino acids 91–95) is conserved between SV40, BKV, JCV, BPV, and Kilham T antigens, but no obvious homologous sequence is found in the same region of PyV. (b) T antigen mutants W91A, W94A, and W95A are reduced in Bub1 binding. Rat-1 stable lines expressing T antigen point mutants between amino acids 90 and 95 were generated by retroviral infection, and Bub1 binding was assessed in a coimmunoprecipitation assay. As controls, cell lines expressing an empty vector, WT T antigen, or dl89–97 T antigen were included. (Upper) T antigen expression levels. (c) All T antigen mutants analyzed are proficient in immortalization of REFs. An immortalization assay in REFs was conducted by using retroviruses that transduced empty vector, WT T antigen, or each of the mutant T antigens D44N or dl89–97. The immortalization efficiency was calculated relative to that of WT T antigen. Filled and open bars represent duplicate experiments.

Fig. 4.

T antigen mutants defective in Bub1 binding fail to transform. cDNA expression vectors encoding either WT or mutant T antigen were transfected into low-passage Rat-1 cells. An empty expression vector was included as a negative control. Three weeks later, dense foci were visualized by crystal violet staining. Similar results were obtained in at least three experiments.

Fig. 5.

T antigen compromises the spindle checkpoint. (a) The mitotic index of U2OS, U2OS/dl89–97, or U2OS/T cells was assessed after 12, 15, or 19 h of nocodazole treatment. A minimum of 300 cells was scored for each time point in each of three independent experiments. Error bars indicate the SD. (b) T antigen decreases the frequency of phospho-histone H3-positive cells, but the dl89–97 mutants do not. (Left) The mitotic cells as visualized by phosphohistone H3 staining. (Right) Nuclear DNA visualized by Hoechst 33342 staining.