Functional Interaction Between Bubr1 and Securin in an Anaphase- Promoting Complex/Cyclosomecdc20–Independent Manner

Research Article

Functional Interaction between BubR1 and Securin in an Anaphase- Promoting Complex/CyclosomeCdc20–Independent Manner

Hyun-Soo Kim,1 Yoon-Kyung Jeon,2 Geun-Hyoung Ha,1 Hye-Young Park,1 Yu-Jin Kim,1 Hyun-Jin Shin,1 Chang Geun Lee,1 Doo-Hyun Chung,2 and Chang-Woo Lee1

1Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi, Korea and 2Department of Pathology, Seoul National University College of Medicine, Seoul, Korea

Abstract Cdc20 (APC/CCdc20). However, Cdc20 in MCC is unable to activate APC/C, as shown by several experiments in which the addi- Activation of the mitotic checkpoint requires the precise tion of Mad2 and/or BubR1 leads to the checkpoint-mediated timing and spatial organization of mitotic regulatory inhibition of APC/CCdc20 activity (2, 10, 11). Silencing of this events, and ensures accurate chromosome segregation. checkpoint signal is initiated by the release of the inhibitory Mitotic checkpoint proteins such as BubR1 and Mad2 bind mitotic checkpoint protein complex from APC/CCdc20; APC/CCdc20 to Cdc20, and inhibit anaphase-promoting complex/cyclo- can drive cells into anaphase by inducing the degradation of someCdc20–mediated securin degradation and the onset of securin (pituitary tumor-transforming gene, PTTG), a small protein anaphase. BubR1 mediates the proper attachment of micro- that inhibits the protease separase and mitotic cyclins (12–15). tubules to kinetochores, and links the regulation of chromo- In metaphase-to-anaphase transition, APC/CCdc20 initiates the some-spindle attachment to mitotic checkpoint signaling. process of chromosome segregation through ubiquitination of Therefore, disruption of BubR1 activity results in a loss securin. Once APC/CCdc20 is activated, separase cleaves the Scc1/ of the checkpoint control, chromosome instability, and/or Rad21 subunit of the cohesion complex. This complex holds sister early onset of malignancy. In this study, we show that BubR1 chromatids together, and its cleavage therefore dissolves cohesion directly interacts with securin in vitro and in vivo. In addi- between sister chromatids (12–16). Although cells, and even mice, tion, the BubR1 interaction contributes to the stability of can survive without securin (17), securin destruction mediated securin, and there is a significant positive correlation be- by APC/CCdc20 is essential for the activation of separase, which in tween BubR1 and securin expressions in human cancer. turn, proteolytically degrades cohesin molecules, leading to the Importantly, BubR1 competes with Cdc20 for binding to onset of anaphase and mitotic exit. A recent study showed that securin, and thereby the interaction between BubR1 and overexpression of securin aberrantly prolongs the progression of securin is greatly increased by the depletion of Cdc20. Our mitosis to anaphase (18). In addition, cells lacking securin findings may identify a novel regulation of BubR1 that can degradation or expressing a nondegradable mutant form of securin generate an additional anaphase-inhibitory signal through exhibit asymmetrical cytokinesis without chromosome segrega- the Cdc20-independent interaction of BubR1 with securin. tion, resulting in macronuclear formation and aneuploidy (19). [Cancer Res 2009;69(1):27–36] Securin is overexpressed in most human cancers and transforms cells both in vitro and in vivo (20–22). Therefore, periodic Introduction regulation of securin stability or degradation is critical for main- The mitotic spindle checkpoint ensures accurate segregation taining the balance between the mitotic checkpoint and chromo- of mitotic chromosomes by delaying anaphase onset until each some segregation. kinetochore has correctly attached to the mitotic spindle. Various Both BubR1 and Mad2 have been shown to interact directly with Cdc20 mitotic checkpoint proteins including Bub1, BubR1, Bub3, and APC/C in vivo and to inhibit its ubiquitination activity in vitro. Cdc20 Mad2 are recruited to kinetochores that lack attachments or Mad2 can associate with APC/C , but Mad2 does not prevent tension to generate a ‘‘wait anaphase’’ signal through the forma- the binding of Cdc20 to APC/C (2, 23, 24). BubR1 can associate tion of an inhibitory ternary complex known as the mitotic tightly with Cdc20 and/or with another protein that is essential for checkpoint complex (MCC; refs. 1–6). Thus, the mitotic spindle checkpoint function called Bub3. Binding of BubR1 to Cdc20 can checkpoint ensures that activation of the anaphase-promoting prevent the association of Cdc20 with APC/C (10, 11). It is not complex/cyclosome (APC/C), an E3 ubiquitin ligase, is delayed known, however, whether this is the physiologic function of BubR1 until all chromosomes have achieved bipolar kinetochore- because if the primary function of BubR1 were to sequester Cdc20 microtubule attachment (7–9). MCC formation is facilitated by from APC/C, activation of the mitotic checkpoint would also lead the binding of BubR1 and Mad2 to Cdc20, which is a crucial to association of BubR1 with APC/C. Furthermore, the mechanisms Cdc20 cofactor of the APC/C and is the main target of the mitotic spindle by which these mitotic checkpoint proteins inhibit APC/C checkpoint. Therefore, MCC can inhibit APC/C associated with activity towards some substrates, including securin, have not yet been investigated. BubR1 is enriched at kinetochores and also associates tightly Note: Supplementary data for this article are available at Cancer Research Online with Bub3 and another protein that is essential for the mitotic (http://cancerres.aacrjournals.org/). Requests for reprints: Chang-Woo Lee, Department of Molecular Cell Biology, checkpoint activation; BubR1 is then disassociated from the MCC Center for Molecular Medicine, Samsung Biomedical Research Institute, and destabilized following anaphase (10, 11). Importantly, BubR1 Sungkyunkwan University School of Medicine, Suwon 440-746, Korea. Phone: 82- monitors the proper attachment of microtubules to kinetochores 31299-6121; Fax: 82-31299-6269; E-mail: [email protected]. I2009 American Association for Cancer Research. and links the regulation of chromosome-spindle attachment to doi:10.1158/0008-5472.CAN-08-0820 mitotic checkpoint signaling (3, 25, 26). Several studies have shown www.aacrjournals.org 27 Cancer Res 2009; 69: (1). January 1, 2009

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2009 American Association for Cancer Research. Cancer Research that disruption of BubR1 activity results in a loss of checkpoint anti–cyclin B (Santa Cruz Biotechnology), anti-Cdh1 (Calbiochem), anti- control, chromosomal instability (caused by premature anaphase), actin (Sigma), anti-Myc (Abcam), and anti-His (Abcam) antibodies. and/or early onset of malignancy (3, 27–29). These findings GST pull-down assay and immunoprecipitation. For the GST pull- indicate that the BubR1 mitotic checkpoint protein plays an down assays, the fusion proteins were adsorbed onto glutathione-protein A/G-Sepharose beads (Amersham Biosciences) and incubated with whole- essential role in the maintenance of genomic integrity; therefore, cell extracts (3 mg) from asynchronized or nocodazole-treated HCT116 defects in BubR1-mediated signaling not only eliminate checkpoint cells for 4 h at 4jC. The bound proteins were separated by SDS-PAGE and control but are also linked to certain human diseases such as then analyzed by immunoblotting with the appropriate antibody. For cancer. In our initial efforts to understand the novel molecular immunoprecipitation from total cell extracts, asynchronized or nocoda- mechanisms of BubR1-mediated checkpoint signaling (30), we used zole-treated HCT116 cells or HCT116-BubR1KD cells were resuspended proteomic and subsequent matrix-assisted laser desorption in lysis buffer A (30, 32), incubated at 4jC for 30 min, and centrifuged ionization-time of flight analyses. In the present study, we used at 14,000 rpm for 15 min. The supernatants (cytoplasmic fractions) an anti-BubR1 antibody to immunoprecipitate proteins extracted were obtained and the cell pellets were resuspended in lysis buffer B from cells treated with nocodazole and identified securin as a novel [50 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 1% NP40, 1 mmol/L EDTA, BubR1-interacting protein (Supplementary Fig. S1). We found that 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L DTT, 0.2 mmol/L Na3Vo4, and 50 mmol/L NaF] containing a protease inhibitor cocktail, BubR1 can form different complexes with securin, as it does with incubated at 4jC for 30 min, lysed by passing the cell pellets through Cdc20, and that the interaction with BubR1 contributes to the a 27-gauge needle five times, centrifuged at 14,000 rpm for 15 min, and stability of securin, possibly by preventing Cdc20 binding. the supernatants of cell pellets (nuclear fractions) were obtained. The mixed extracts (cytoplasm plus nuclear fractions) were diluted with a no-salt buffer to reduce the salt concentration to 150 mmol/L, and then Materials and Methods centrifuged again before being analyzed by immunoprecipitation. For Cells and synchronization. The hSecurin+/+ (HCT116 securin+/+) and immunoprecipitation, each mixed extract was incubated with antibodies À À À À hSecurin / (HCT116 securin / ) cells were kindly provided by Bert against securin or normal immunoglobulin IgG(control) for 2 h at 4 jCas Vogelstein (31). Generation of the stable HCT116 BubR1 knock down described previously (32). (HCT116 BubR1KD) cell lines has been previously described (30). HeLa cells Recombinant protein purification and in vitro competition assay. were purchased from the American Type Culture Collection, and LL86, Cdc20 cDNAs were cloned into pFastBac vector (Invitrogen) and used L132, SW-900, HCC-95, SK-MES-1, HCC-1171, HCC-1833, HCC-2108, and to generate a recombinant baculovirus in Sf9 insect cells using the Bac- Calu-3 cells were purchased from the Korean Cell Line Bank. For to-Bac baculovirus system (Invitrogen). Three days after viral infection, synchronization, HCT116 or HCT116 BubR1KD cells were grown in the Sf9 cells were resuspended with STE buffer [10 mmol/L Tris-HCl (pH 8.0), presence of 200 ng/mL of nocodazole for 16 h, harvested, and then used in 150 mmol/L NaCl, 2% Triton X-100, 1 mmol/L EDTA, 1 mmol/L various assays. phenylmethylsulfonyl fluoride, 2 mmol/L DTT, 100 Ag/mL lysozyme, and Plasmid construction, small interfering RNA synthesis, and trans- 1.5% N-lauroylsarcosine sodium salt] containing a protease inhibitor fection. The full-length cDNA sequences of the human BubR1 and Securin cocktail (Sigma), and lysed by sonication. Recombinant Cdc20 proteins genes were amplified by PCR using templates generated by reverse were purified from Sf9 cell lysates using nickel-NTA agarose-bead columns transcription from HeLa cell total mRNA and using oligo-dT as a primer. (Qiagen). After washing with STE buffer containing 60 mmol of imidazole, Glutathione S-transferase (GST) fusion constructs for expression in purified proteins were eluted with STE buffer containing 500 mmol of Escherichia coli cells were generated by in-frame insertion of PCR fragments imidazole. To convert STE buffer into TNN buffer, pooled fractions were encoding BubR1 amino acid residues 1 to 300, 1 to 525, 401 to 700, and passed into the PD10 column (GE Healthcare Bio-Sciences). Eluted His- 526 to 1050 into the pGEX-KG vector (Pharmacia). cDNAs for BubR1 wild- Cdc20 proteins were frozen in aliquots and stored at À80jC. To increase the type and mutant versions of BubR1, residues 1 to 525 and K795R, were expression level of BubR1 gene in E. coli, the full sequence of the BubR1 subcloned into the Myc epitope–encoding pcDNA3.1 vector to generate gene was codon-optimized (OligoEngine). The optimized BubR1 gene was pcDNA-Myc-BubR1 wild-type, 1-525, and K795R, respectively. The coding cloned into the pSumo vector (Lifesensors) containing T7 promoter and sequences of the securin wild-type or deletion mutants (amino acid residues His-BubR1 proteins were expressed in E. coli BL21 cells. Purification of His- 1–67, 65–122, and 122–202) were cloned into the pGEX-KG vector. For tagged proteins is described above in detail. For in vitro competition assays, small interfering RNA (siRNA) synthesis, the following gene-specific asynchronized HCT116 cells were lysed in TNN buffer. Each cell extract sequences were used to generate siRNAs (Dharmacon): Cdc20 siRNA no. was preincubated with His-Cdc20 or His-BubR1 proteins (0.5, 1, and 2 Ag) 1, 5¶-ACCUGGCGGUGACCGCUAU-3¶; Cdc20 siRNA no. 2, 5¶-UGUGUGGC- for 1 h at 4jC, followed by incubation with GST-securin protein (4 Ag) for CUAGUGCUCCU-3¶; Cdh1 siRNA, 5¶-UGAGAAGUC-UCCCAGUCAG-3¶; a further 4 h. BubR1 siRNA no. 1, 5¶-AAGGGUUCAGAGCCAUCAG-3¶; BubR1 siRNA no. Immunohistochemistry and statistical analyses. Tissue samples from 2, 5¶-GGAGAUCCUCUACAAAGGG-3¶; control siRNA, 5¶-CCUACGCG- a total of 117 cases of lung squamous cell carcinomas (SqCC) diagnosed at GAAUACUUCGA-3¶. cDNAs for mutant versions of securin, residues 1 to Seoul National University Hospital were histologically examined. BubR1 and 67 and 65 to 202, were subcloned into the Myc epitope–encoding pcDNA3.1 securin expressions were evaluated by immunohistochemistry using anti- vector to generate pcDNA-Myc-securin (1–67) and (65–202), respectively. BubR1 and anti-securin antibodies, respectively, with the labeled strepta- For transient transfections, electroporation of HCT116 or HeLa cells was vidin-biotin complex staining method (LSAB kit, DAKO). BubR1 and securin performed using a Microporator (Digital Biotechnology, South Korea) expressions were analyzed semiquantitatively by summing the scores that according to the manufacturer’s instructions. had been obtained by multiplying the staining intensity and proportion of Immunoblot assays and antibodies. For immunoblot assays, the cells positive tumor cells. Statistical analysis was performed using SPSS 11.5 were synchronized as described above or left asynchronized, harvested by (Stanford version). Pearson’s m2 test used to examine the relationships scraping, and then washed twice in cold PBS and lysed in a TNN buffer (30) between BubR1 and securin. containing a protease inhibitor cocktail (Sigma). Equal amounts of protein Live cell imaging and fluorescence intensity measurement of RFP- (quantified by Bio-Rad assay) from each sample were separated by SDS- securin. To estimate the duration of mitosis, HCT116 or HCT116 BubR1KD PAGE, transferred to a nitrocellulose filter, blocked, and analyzed with anti- cells were cotransfected with an expression plasmid encoding green BubR1 (BD Biosciences PharMingen), anti-Bub3 (BD Biosciences Phar- fluorescent protein (GFP)-H2B and red fluorescent protein (RFP)-securin Mingen), anti-Mad2 (BD Biosciences PharMingen), anti–Aurora A (BD and then imaged in yT 0.15-mm dishes in McCoy’s supplemented medium Biosciences PharMingen), anti-securin (Zymed), anti-separase (Novus), anti- containing 10% fetal bovine serum. For 4 h, 0.5-s exposures were taken every Cdc20 (Santa Cruz Biotechnology), anti-p53 (Santa Cruz Biotechnology), 3 min using a 20Â NA0.75 objective lens and an LSM500 META confocal

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2009 American Association for Cancer Research. Functional Interaction between BubR1 and Securin microscope (Carl Zeiss). Fluorescence intensity of RFP-securin was mea- nuclear envelope breakdown (Fig. 2A). In HCT116 cells, the time to sured using AxioVision LE software (Carl Zeiss). completion of chromosome separation was about 73 minutes. However, HCT116 BubR1KD cells showed a significantly faster exit from mitosis at about 61 minutes, as has recently been reported Results (Fig. 2B and C). As expected, both HCT116 and HCT116 BubR1KD BubR1 interacts with securin in vitro and in vivo. To cells transfected with plasmids encoding RFP-securin took longer determine whether BubR1 interacts directly with securin, we to reach anaphase compared with the control nontransfected generated full-length GST-BubR1 and GST-securin fusion proteins, HCT116 and HCT116 BubR1KD cells, indicating that securin blocks incubated these proteins with cellular proteins extracted from the progression of mitosis to anaphase. Interestingly, our time- HCT116 cells, and performed immunoblot analysis with anti- lapse analysis at the single-cell level revealed that the levels of RFP- securin or anti-BubR1 antibodies (Fig. 1A, lanes 1–6). Pull-down securin were constant for more than 230 to 290 minutes, but were assays revealed that GST-BubR1 binds to securin and GST-securin then degraded after anaphase onset in HCT116 cells (Fig. 2B and binds to BubR1. Similarly, we generated full-length His-BubR1 and C). However, the relative duration of RFP-securin in HCT116 His-securin proteins, which were incubated with beads bound BubR1KD cells was markedly shorter at 85 to 125 minutes after to either GST-securin (Fig. 1A, lanes 7–9) or GST-BubR1 (Fig. 1A, nuclear envelope breakdown. In both cell types, RFP-securin lanes 10–13). Again, we found that BubR1 and securin were pre- degradation begins after anaphase onset; however, during mitosis, sent in the complex formed in vitro. Consistent with these find- both the duration and inductive level of RFP-securin were markedly ings, immunoprecipitation with anti-securin antibody from cellular reduced in HCT116 BubR1KD cells compared with those in HCT116 À À extracts of either HCT116 securin+/+ or HCT116 securin / (as a cells. The extent of securin destabilization in HCT116 BubR1KD cells negative control) cells and subsequent immunoblotting with an seemed to correlate with the BubR1 expression levels. Further- anti-BubR1 antibody showed that securin and BubR1 form a more, we measured the endogenous levels of securin in HCT116 and complex in vivo (Fig. 1B). HCT116 BubR1KD using immunostaining analysis. As expected, the To define the domains responsible for the BubR1-securin expression of securin in HCT116 BubR1KD cells was significantly interaction, we incubated a series of GST-BubR1 and GST-securin decreased in both interphase and mitotic (prometaphase) cells, deletion mutants with extracts from HCT116 cells expressing wild- when compared with that in control HCT116 cells (Supplementary type BubR1 and securin. As shown in Fig. 1C, a fragment Fig. S3B). We confirmed the effects of BubR1 on the stability of containing the BubR1 NH2-terminal homology domain (amino securin by transiently transfecting HeLa cells with siRNAs against acids 1–525) formed a complex with securin, whereas the central BubR1 or luciferase. Again, cells transfected with two separate (amino acids 401–700) and COOH-terminal (amino acids 526–1050) BubR1 siRNAs (which substantially depleted BubR1 protein levels) regions of BubR1 did not. Similarly, a fragment containing the contained markedly lower levels of endogenous securin in a BubR1 central (amino acids 65–122) and COOH-terminal (amino acids dose-dependent manner (Fig. 2D). These results strongly indicate 122–202) regions of securin interacted with BubR1, whereas that BubR1 contributes to the stability of securin. the NH2-terminal (amino acids 1–67) region of securin did not In vivo correlation between BubR1 and securin expressions (Fig. 1D). In positive-control experiments, we confirmed that in human cancer. To further examine the stability of securin, BubR1 and securin interact with their well-defined binding HCT116 and HCT116 BubR1KD cells were released from nocodazole partners, Bub3 and p53, respectively, under these experimental control and exposed to the protein translation inhibitor, cyclo- conditions. To exclude the possibility that the interaction between heximide. Securin levels in these cells were then examined over BubR1 and securin is mediated by the formation of a supercomplex time by immunoblotting assay (Fig. 3A). Following exposure to with Cdc20, HCT116 cell extracts were immunoprecipitated with cycloheximide for 30 minutes, securin levels decreased by almost either normal IgGor anti-securin antibody (Supplementary Fig. S2). 80% in HCT116 BubR1KD cells, but by only f5% in parental cells. Subsequent immunoblotting with anti-BubR1 or anti-Mad2 anti- At 60 minutes posttreatment, HCT116 BubR1KD cells showed body revealed that securin forms a complex with BubR1 but not almost no detectable securin, whereas securin proteins could still with Mad2; under the same conditions, securin strongly interacted be detected at relatively significant levels in parental HCT116 cells. with separase and Cdc20 as positive controls (Supplementary Similarly, these cells were synchronized at the boundary between Fig. S2). Together, these data indicate that BubR1 directly interacts G1 and S phases using a double thymidine block (Thy-DB) and then with securin in vitro and in vivo. released into the cell cycle. As shown in Supplementary Fig. S3C BubR1 contributes to the stability of securin in unperturbed and D, cells depleted of BubR1 (HCT116 BubR1KD) did not mitotic cells. The degradation of securin, which is mediated by significantly differ from the control cells (HCT116 con) in terms Cdc20 APC/C , is required for anaphase onset. Our data indicate that of progression through the S and G2 phases, mitosis, or exit from BubR1 directly interacts with securin in vitro and in vivo. Therefore, mitosis. However, in the absence of microtubule inhibitor, the it is likely that BubR1 not only inhibits the ubiquitination activity of levels of securin were also dramatically decreased in HCT116 APC/CCdc20 (Supplementary Fig. S3A) but also regulates the BubR1KD, whereas steady state levels were observed in HCT116 con stability of securin through a direct interaction. To investigate cells. To further characterize this relationship, we tested the effects whether the stability of securin is regulated by BubR1 in of introducing exogenous BubR1 wild-type, a kinase-insufficient unperturbed mitotic cells, we transfected parental HCT116 and mutant [BubR1 (K795R)], or a BubR1-deletion mutant [BubR1 HCT116 BubR1KD cells, which exhibit a 60% to 70% reduction of (1–525)]. As expected, both BubR1 wild-type and mutants markedly BubR1 levels as described previously (30), with plasmids encoding increased the stability of securin (Fig. 3B). These findings suggest GFP-tagged H2B (GFP-H2B) to visualize chromosomes and/or RFP- that the interaction between BubR1 and securin could play an tagged securin (RFP-securin; Fig. 2A–C). First, time-lapse micro- alternative role in the stability of securin. scopic analyses were used to determine the relative times for the Next, we investigated the correlation between BubR1 and securin individual cell types to complete chromosome separation from the expression levels in lung cells. We prepared cell extracts from two www.aacrjournals.org 29 Cancer Res 2009; 69: (1). January 1, 2009

Figure 1. BubR1 interacts with securin in vitro and in vivo. A, HCT116 cell lysates were incubated with beads bound to either GST or GST-securin (top) and GST-BubR1 (bottom). After binding, beads were resolved by SDS-PAGE and analyzed by immunoblotting using anti-BubR1 or anti-securin antibody (lanes 1–6). Purified His-BubR1 and His-securin proteins were incubated with beads bound to either GST-securin (lanes 7–9) or GST-BubR1 (lanes 10–13). The beads were resolved by SDS-PAGE and analyzed by immunoblottingwith anti-BubR1 ( lanes 7–9) and anti-securin (lanes 10–13) antibodies. B, cellular extracts from HCT securin+/+ or HCT116 securinÀ/À cells were immunoprecipitated with normal immunoglobulin IgG (control) or anti-securin antibody, and immunoprecipitates were immunoblotted with anti-BubR1 antibody. C, structural schematic of BubR1 showingthe NH 2-terminal homology and Bub3-binding, Cdc20-binding, and kinase domains. HCT116 cell lysates were incubated with beads bound to GST alone or to a series of BubR1-deletion mutants fused to GST. Bound proteins were resolved and immunoblotted with anti-Bub3 (positive control) or anti-securin antibody. D, structural schematic of securin showingthe KEN-box and D-box motifs and four proline-rich (PXXP) motifs. HCT116 cell lysates were incubated with beads bound to GST alone or to a series of securin-deletion mutants fused to GST. After binding, beads were resolved and immunoblotted with anti-BubR1, anti–Aurora A, or anti-p53 (positive control) antibody. immortalized nontumor cell lines (LL86 and L132), three SqCC cell securin levels in 117 human SqCC by immunohistochemical lines (SW-900, HCC-95, and SK-MES-1), and five adenocarcinoma analyses with anti-BubR1 and anti-securin antibodies. Typical cell lines (HCC-1171, HCC-1833, HCC-2108, SK-LU-1, and Calu-3) immunohistochemical staining of either tumor specimens or adja- and performed immunoblot analyses. Interestingly, the levels of cent normal tissues is shown in Fig. 3C. BubR1 was significantly securin expression in lung cells markedly correlated with those of expressed in 34% of tumor specimens with positive staining defined BubR1 (Supplementary Fig. S4). We further examined BubR1 and as more than one cell among five tumor cells showing intense

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Figure 2. BubR1 contributes to the stability of securin. A, HCT116 control (HCT116 con) and HCT116 BubR1 knockdown (HCT116-BubR1KD ) cells were transfected with expression plasmids encodingH2B-GFP and RFP-securin fusion proteins, respectively. Cells were then cultured and imagedby time- lapse microscopy duringmitotic progression. Times from nuclear envelope breakdown ( NEBD) to complete chromosome separation are indicated. Representative time-lapse images of HCT116 control and HCT116-BubR1KD cells expressingboth H2B-GFP and RFP-securin. B, mitotic progression data from randomly selected HCT116 control and HCT116-BubR1KD cells transfected with H2B-GFP alone or with both H2B-GFP and RFP-securin. Columns, average mitotic duration in each cell line. C, as in A, HCT116 control and two different clones of HCT116-BubR1KD cells with different BubR1 expression levels (no. 1, 60% reduction; no. 2, 40% reduction of BubR1 expression) were transfected with H2B-GFP and RFP-securin expression plasmids. The intensity of RFP fluorescence in these cells was measured as described in Materials and Methods. D, HeLa cells were transiently transfected with control luciferase siRNA, BubR1 siRNA no. 1, or BubR1 siRNA no. 2, as described in Materials and Methods. Transfected cells were harvested for immunoblottingusinganti-securin and anti-actin antibodies. www.aacrjournals.org 31 Cancer Res 2009; 69: (1). January 1, 2009

Figure 3. Functional correlation between BubR1 and securin expressions in human cancers. A, HCT116 con and HCT116-BubR1KD cells were treated with nocodazole for 12 h (Noco, 200 ng/mL), treated with cycloheximide (CHX), and then released into the cell cycle. At the times indicated, cells were harvested for immunoblottingusinganti-BubR1, anti-securin, or anti-actin antibody. B, HeLa cells were transfected with an empty vector (pCMV-Myc), Myc-tagged BubR1 (1–525), Myc-BubR1 K795R, or Myc-BubR1 wild-type (WT). Transfected cells were harvested for immunoblotting usinganti-Myc, anti-securin, and anti-actin antibodies. C and D, lung SqCC and neighboring normal follicle samples were collected and subjected to immunohistochemical stainingwith antibodies against BubR1 and securin. BubR1 and securin expressions in 117 human lungSqCC were analyzed semiquantitatively by summingthe scores obtained by multiplyingthe staining intensity and the proportion of positive tumor cells; more than one positive cell among5 tumor cells was defined as positive (+ve), and less than one positive cell from 10 tumor cells was defined as negative (Àve). Pearson’s m2 test was used to examine the relationships between BubR1 and securin (P > 0.001).

staining for BubR1 protein; 66% of tumor specimens were BubR1- Although Cdc20 siRNA no. 2 slightly induced the sub-G1 phase negative, defined as less than one BubR1-positive cell among 10 (apoptosis) population, neither Cdc20 siRNAs triggered a marked tumor cells. Interestingly, the securin-negative staining rate was 54% change in the cell cycle profile compared with the negative con- in BubR1-negative tumor specimens, whereas 12% were securin- trol (Supplementary Fig. S5). Therefore, we immunoprecipitated positive, revealing a significant positive correlation between BubR1 securin using anti-securin antibody or normal IgG(negative con- and securin expressions. Thus, the stability of securin seems to be trol) and performed immunoblot assays (Fig. 4A). The amount of directly associated with the levels of BubR1 protein. separase bound to the securin immunocomplex was almost Interaction between BubR1 and securin is independent of constant regardless of the Cdc20 expression levels. However, Cdc20. BubR1 binds to the central and COOH-terminal domains of the levels of BubR1 in this complex were significantly increased securin, but interacts strongly with full-length securin. However, in Cdc20-depleted cells (Fig. 4A and B). Interestingly, the amount Cdc20 binds to full-length securin but not with the NH2-terminal, of BubR1 bound to securin seemed to correlate with the degree central, or COOH-terminal domains of securin, indicating that the to which Cdc20 expression was inhibited. Therefore, these data domains of securin responsible for the interactions with BubR1 indicate that the interaction between BubR1 and securin is and/or Cdc20 overlap (Fig. 1C; data not shown). To examine the independent of the APC/CCdc20 pathway. effect of Cdc20 on the interaction between BubR1 and securin, As previously reported, both Cdc20 and Cdh1 have essential HCT116 cells were transfected with two different siRNAs against roles in stimulating the ubiquitin ligase activity of the APC/C. APC/ Cdc20, which reduced Cdc20 expression levels by f65% and 40%, CCdc20 promotes the degradation of securin at the metaphase- respectively, or with siRNA against luciferase (negative control). to-anaphase transition, whereas APC/CCdh1 activity in late mitosis

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and G1 is required for the degradation of mitotic cyclins (5, 9, 16), significantly blocked the interaction between securin and BubR1. indicating that Cdc20 is not redundant with Cdh1 for the transition Increasing amounts of recombinant His-Cdc20 gradually reduced from metaphase-to-anaphase. However, securin not only interacts the relative levels of BubR1 bound to securin (Fig. 5A, bottom). with Cdc20, but also with Cdh1 (16). Therefore, we were interested This inhibition was specific because the levels of unrelated pro- to determine whether Cdh1 could affect the interaction between teins, such as GST-p53 and GST alone, were not affected by BubR1 and securin. We transfected HCT116 cells with a set of coincubation with His-Cdc20 (data not shown). Additionally, we siRNAs against Cdh1, Cdc20, or luciferase and performed incubated HCT116 cell extracts with GST-securin to investigate immunoprecipitation assays using anti-securin antibody or normal the levels of Cdc20 that bind to securin in the presence of puri- IgG(Fig. 4 C). Interestingly, in cells deplete of Cdh1, the relative fied recombinant His-BubR1 as a competitor (Fig. 5B). Consistent levels of BubR1 that coprecipitated with securin were similar to with this, increasing levels His-BubR1 gradually reduced the level those in the control luciferase siRNA-transfected cells, whereas of Cdc20 that bound to securin in a dose-dependent manner cells lacking Cdc20 expression had increased levels of BubR1 (Fig. 5B, bottom), indicating that BubR1 competes with Cdc20 for (Fig. 4C and D). Again, these results suggest that the interaction binding to securin. Next, we generated a NH2-terminal deletion between BubR1 and securin is independent of Cdc20. mutant of securin that contained the COOH-terminal BubR1- BubR1 competes with Cdc20 for binding to securin. Cdc20 binding region (amino acids 65–202) to use as a competitor can bind BubR1 and Mad2 simultaneously and also interact with for the interactions between endogenous BubR1 and securin securin. Moreover, our results indicate that BubR1 may compete proteins (Fig. 5C). HCT116 cells were transfected with Myc-tagged with Cdc20 for securin binding, thereby providing an alternative securin (1–67) as a control or with Myc-tagged securin (65–202) mechanism for controlling the metaphase-to-anaphase transition. expression plasmids. Lysates from the transfected cells were To assess whether BubR1 competes with Cdc20 for binding to immunoprecipitated with control IgGor anti-securin antibody securin, we performed in vitro competition assays using purified and immunoblotted with anti-securin, anti-BubR1, or anti-separase GST-securin in E. coli cells and using His-BubR1 and His-Cdc20 antibody. Interestingly, the amount of BubR1 bound to the securin in Sf9 cells (Fig. 5A). Recombinant GST-securin, but not GST immunocomplex was significantly decreased in cells transfected alone, formed a complex with endogenous BubR1 extracted from with Myc-securin (65–202). However, the levels of BubR1 in HCT116 cells. However, the addition of recombinant His-Cdc20 this complex were almost constant in cells transfected with

Figure 4. Interaction between BubR1 and securin is independent of Cdc20. A, HCT116 cells were transiently transfected with 12 Agof control luciferase siRNA or Cdc20-targeting siRNA. Transfected cells were harvested for immunoprecipitation usingnormal immunoglobulin IgG or anti-securin antibody. Immunoprecipitates were immunoblotted with anti-securin (as a control), anti-BubR1, or anti-separase antibodies. L, longexposure; S, short exposure. B, relative amounts of BubR1 protein, immunoprecipitated with the anti-securin antibody, based on the result presented in A. C, HCT116 cells were transfected with control luciferase siRNA, Cdc20 siRNA, or Cdh1 siRNA. Cells were harvested for immunoprecipitation with normal IgG or anti-securin antibody. Immunoprecipitates were analyzed by immunoblottingusinganti-BubR1 or anti-separase antibody. The amounts of Cdc20 and Cdh1 after each siRNA transfection were visualized by immunoblotting. D, the relative amount of BubR1 coprecipitatingwith anti-securin antibody in C was visualized by immunoblot analysis usinganti-BubR1 antibody.

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Figure 5. BubR1 competes with Cdc20 for bindingto securin. A, purified GST or GST-securin was incubated with HCT116 cell lysates in the presence of increasing concentrations of recombinant His-Cdc20, as indicated. After binding, the reaction mixtures were separated by SDS-PAGE and then analyzed by immunoblottingwith anti-His antibody (lanes 1–6). His-Cdc20 protein was expressed and purified Sf9 cells. Recombinant His-Cdc20 was analyzed by immunoblottingusing anti-Cdc20 and anti-His antibodies (lanes 7 and 8). The graph shows the relative amounts of BubR1, which bound to GST-securin in the presence of His-Cdc20 as a competitor (bottom), based on the results presented above (lanes 1–6). B, purified GST or GST- securin was incubated with HCT116 cell lysates in the presence of increasing concentrations of recombinant His-BubR1, as indicated. After binding, the reaction mixtures were separated by SDS-PAGE and then analyzed by immunoblottingwith anti-His antibody (lanes 1–6). His-BubR1 protein was expressed and purified from E. coli. Recombinant His-BubR1 was analyzed by immunoblottingwith anti-BubR1 and anti-His antibodies (lanes 7 and 8). The graph shows the relative amounts of Cdc20, which bound to GST-securin in the presence of His-BubR1 as a competitor (bottom), based on the results presented above (lanes 1–6). C, HCT116 cells were transfected with pCMV-Myc (Myc), pCMV-Myc-securin (1–67), or pCMV- Myc-securin (65–202) plasmid. Cells were lysed and immunoprecipitated with anti-Myc antibody or control IgG, and the resultingimmunoprecipitates were immunoblotted with anti-BubR1, anti-separase, or anti-securin antibodies. Input, the endogenous securin, BubR1, and separase proteins, respectively.

Myc-securin (1–67) expression plasmid compared with the control Discussion Myc vector-transfected cells. These results indicate that COOH- BubR1 inhibits the activity of APC/C by blocking formation of terminal securin (65–202) competes with endogenous securin for the active APC/CCdc20 complex. Mad2 also seems to inhibit binding to endogenous BubR1. APC/C by a similar mechanism (5, 9). Therefore, Cdc20 can be In summary, BubR1 and securin are intimately associated in a inhibited by either BubR1 or Mad2, but both BubR1 and Mad2 can stable complex, and interaction with BubR1 is therefore likely to synergistically inhibit Cdc20 (2, 10, 11). Cells that overexpress Cdc20 contribute to the stability of securin by competing with APC/CCdc20. greatly reduced the mitotic arrest in response to spindle damage,

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2009 American Association for Cancer Research. Functional Interaction between BubR1 and Securin presumably because increased levels of Cdc20 activate APC/C and Cdc20 and p53 (30, 37, 39). BubR1 is autophosphorylated and allow cells to bypass the mitotic checkpoint (10, 32, 33). Interestingly, phosphorylated by Aurora B and Plk1 in response to mitotic spindle coexpression of BubR1 restored the mitotic arrest of the Cdc20- damage (40, 41). Phosphorylation of BubR1 seems to be important for overexpressing cells (10), indicating that BubR1 directly inhibits the the regulation of mitotic progress because substitution or deletion of activity of APC/CCdc20. However, it is possible that BubR1 may BubR1 kinase residues generates a dominant-negative mutant that function independently of APC/CCdc20 to inhibit the onset of competes with endogenous wild-type BubR1. Therefore, we investi- anaphase. It has recently been reported that loss of Cdc20 causes a gated whether the introduction of a BubR1-deletion mutant (BubR1 securin-dependent metaphase arrest in mouse embryos, but a Cdc20 1–525), which contains the NH2-terminal securin-binding region, or a and securin double mutant could not maintain the metaphase arrest kinase-insufficient mutant (BubR1 K795R) contribute to the stability (34), suggesting that securin has both Cdc20-independent and - of securin, possibly by sequestration of Cdc20 (Fig. 3B). HeLa cells dependent roles in preventing mitotic exit. In this study, our data were transfected with an empty vector (pCMV-Myc), Myc-tagged provide compelling evidence that the mitotic checkpoint protein BubR1 1 to 525 (Myc-BubR1 1–525), Myc-BubR1 K795R or Myc-BubR1 BubR1 forms a complex with securin, and that BubR1 contributes to wild-type, and the cells were collected for immunoblot analysis with the stability of securin by a Cdc20-independent interaction. anti-Myc and anti-securin antibodies. Importantly, cells expressing the Abnormality in the cellular Cdc20 level or in its function may BubR1 mutants BubR1 1 to 525 or BubR1 K795R significantly deregulate APC/C activation and promote premature anaphase. increased the stability of securin to levels that were similar to those Moreover, recent reports have provided evidence that several expressing BubR1 wild-type, indicating that BubR1 stabilizes securin human tumors show aberrant overexpression of Cdc20 (33, 35), by competitive inhibition of Cdc20 binding. However, our in vitro indicating that Cdc20 overexpression might be characteristic of kinase assay results using BubR1 immunocomplex and purified GST- tumorigenesis. Nevertheless, human cancer cells displaying aber- securin fusion proteins (as a substrate) showed that BubR1 kinase rant Cdc20 levels still activate the checkpoint in response to does not contribute to the phosphorylation of securin (Supplementary C mitotic spindle damage. Inhibition and/or inactivation of BubR1 in Fig. S6 ), indicating that the BubR1 kinase activity may not be these cancer cells override and/or compromise the mitotic required for the stability of securin. However, we do not exclude the checkpoint rather than completely destroy checkpoint control possibility that the BubR1 kinase activity may regulate securin levels (3, 30, 36). These results indicate that BubR1 may generate an through means other than directly phosphorylating securin. additional signal to induce mitotic arrest independently of the A recent report has suggested that hyperphosphorylated forms of Cdc20 signaling pathway. However, the role of BubR1 is unlikely to securin are unstable and that protein phosphatase 2A regulates securin levels by preventing securin phosphorylation (42). Unex- be restricted to checkpoint control. We have recently shown that pectedly, our in vitro kinase assay failed to show the direct phos- BubR1 functions as a potent apoptotic molecule for preventing the phorylation of securin by BubR1 kinase (Supplementary Fig. S6; data adaptation of abnormal, chromosomally unstable mitotic cells, not shown). However, the hyperphosphorylation of securin was indicating that BubR1 plays an important role in subsequent post- markedly reduced in BubR1-depleted cells in response to mitotic mitotic adaptation (37). Here, we also propose that BubR1 spindle damage (Supplementary Fig. S6), indicating that BubR1 may contributes to the generation of another mitotic checkpoint signal be indirectly involved in the checkpoint-mediated phosphorylation by forming a complex with securin, which has an inhibitory effect of securin. Otherwise, interaction with BubR1 may lead to changes in on separase. the biochemical properties or conformational stoichiometry of Both BubR1 and Cdc20 are phosphorylated during mitosis and the securin as a phosphorylation substrate by an undetermined kinase. phosphorylation of these proteins is required for function of the In summary, BubR1 can form different complexes with securin, as mitotic checkpoint. It has been reported that Cdc20 associates with it does with Cdc20, and that the interaction with BubR1 contributes the mitotic APC/C more strongly than it does with the interphase to the stability of securin, possibly by preventing Cdc20 binding. APC/C (10, 11, 38). This is likely to be because binding of Cdc20 to Therefore, we propose that silencing of mitotic checkpoint signaling APC/C during mitosis may be mediated by phosphorylation. How- Cdc20 activates the Cdc20-mediated APC/C E3 ubiquitin ligase, and then ever, interaction with and inhibition of APC/C is independent of activated APC/C may ubiquitinate BubR1 and securin (Supplemen- the BubR1 kinase activity because a kinase-dead mutant of BubR1 tary Fig. S7). Given that defects in or inactivation of BubR1- can still bind and inhibit Cdc20 (10). In addition, phosphorylation of mediated checkpoint signaling could cause fatal errors in mitotic Cdc20 is not required for inhibition by BubR1 (11). Moreover, it seems progression, BubR1-dependent securin stability may be an alterna- that small portions of BubR1 form a complex with Cdc20; therefore, tive mechanism in regulating the anaphase-inhibitory signal. a significant population of free BubR1 that is not bound to Cdc20 is able to interact with securin. Although differences in affinity cannot be determined under our experimental conditions, free BubR1 interacts Disclosure of Potential Conflicts of Interest with and stabilizes securin by preventing APC/CCdc20-mediated No potential conflicts of interest were disclosed. ubiquitination. Interestingly, it has recently been suggested that if Cdc20 the APC/C complex is formed, it becomes more resistant to the Acknowledgments action of BubR1 (10). In addition to BubR1, Mad2 is also thought to Received 3/4/2008; revised 9/14/2008; accepted 10/5/2008. bind directly to Cdc20 and inhibit APC/C. Thus, there may be Grant support: Korea Health 21 R&D Project, Ministry of Health and Welfare stoichiometric binding between the checkpoint proteins and securin, (03-PJ10-PG13-GD01-0002), the Korea Research Foundation (KRF-2006-312-C00625), creating supercomplexes such as BubR1-Cdc20-securin and/or Mad2- and the 21C Frontier Functional Human Genome Project from the Ministry of Science & Technology in Korea (FG07-21-01). Cdc20-securin. However, our data clearly showed that securin forms The costs of publication of this article were defrayed in part by the payment of page a complex with BubR1 but not with Mad2 (Supplementary Fig. S2). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. BubR1 is a serine/threonine kinase and phosphorylates proteins We thank Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD) for that are involved in the regulation of mitosis and postmitosis, such as securin-null HCT116 cells. www.aacrjournals.org 35 Cancer Res 2009; 69: (1). January 1, 2009

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