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

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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 acti- vate 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).
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