BubR1 interacts with PCAF and is acetylated at prometaphase kinetochores. (A) BubR1 acetylation was compared between asynchronously growing cells and prometaphase cells. HeLa cells were treated with nocodazole for 13 h (or left untreated) and subjected to mitotic shake-off. IP was carried out with anti-Ac-K antibodies and subsequent WB with anti-BubR1 antibodies. The slower migrating form of BubR1, phospho-BubR1 (as shown in WB of input control), was detected with the anti-Ac-K antibodies. (B) BubR1 and PCAF interact in nocodazole-treated cells. Cells were treated with nocodazole for 13 h (or left untreated), and lysates were analysed by IP with anti-PCAF, followed by WB using anti-BubR1. The blot was then re-probed with anti-PCAF to normalize the IP. (C) BubR1 interacts with PCAF at prometaphase kinetochores. Metaphase chromosome spreads were immunostained with anti-PCAF (red) and anti-BubR1 (green) antibodies. PCAF localization at prometaphase kinetochores was verified by siRNA experiments (Supplementary Figure 2). White scale bar, 5 μm. (D) Images in (C) were enlarged to visualize the localization of BubR1 and PCAF with the chromosome. (E) BubR1 purified from insect cells (left) was subjected to an in vitro acetylation assay in the presence of purified PCAF and acetyl-CoA. The reaction was analysed by WB with anti-Ac-K antibodies (right). One μg each of BubR1 (8.4 pmol) and PCAF (10.8 pmol) was employed for the assay, which corresponds to a molar ratio of 1:1.3. The same blot was re-probed with anti-BubR1 or anti-PCAF antibodies. (F) BubR1 IPs from 1 mg of HeLa cell lysates were subjected to acetylation assays with or without recombinant PCAF (1 μg), followed by WB with anti-Ac-K antibodies.

Identification of the BubR1 acetylation site. (A) The BubR1 N-terminal region (ΔBR2; amino acids 1–514) was subdivided into five regions to precisely map the acetylation site. (B) GST fusions of the recombinant proteins (ΔBR2-1–5) were purified from Escherichia coli and subjected to in vitro acetylation with or without PCAF in the reaction. (C) K250 in ΔBR2–3 was subjected to in vitro mutagenesis with Arg (ΔBR2–3 K250R), purified from E. coli, and subjected to an in vitro acetylation assay. Samples were analysed for acetylation by WB with anti-Ac-K antibodies. (D) Mass spectrometry analysis to detect in vivo acetylation of BubR1. HeLa cells were treated with nocodazole and prometaphase cells were collected by mitotic shake-off. Attached cells (interphase) and prometaphase cells (M phase) were immunoprecipitated with anti-BubR1 antibodies and SDS–PAGE. Excised BubR1 bands were analysed for the acetylated site by mass spectrometry. An additional peak at 1239.853, which was shifted by acetylation (42-Da) from 1197.847 at M phase, confirms that K250 of BubR1 is acetylated at prometaphase in vivo. (E) Comparison of BubR1 sequences surrounding K250 among vertebrates. Conserved Destruction Box (D-box)-like sequences and two KEN boxes are marked.

BubR1 acetylation is required for checkpoint activity. (A–C) BubR1 acetylation is required for checkpoint activity. (A) Schematic illustration of the experiment. HeLa cells were infected with 100 MOI of GFP-fused BubR1-WT (Ad-WT), K250R (Ad-K250R), or K250Q (Ad-K250Q)-expressing adenovirus. Adenovirus expressing GFP only was employed for control. Five hours later, cells were depleted of endogenous BubR1 by synthetic siBubR1 targeting the 3′UTR (siBubR1) or siMad2 targeting Mad2, as marked. Nineteen hours post siRNA transfection, cells were treated with nocodazole (200 ng/ml) and aliquots of cells were collected at the indicated time points for WB (B) and measuring mitotic index in (C). Western analysis was used to determine the levels of BubR1 and Mad2 after siRNA transfection and adenovirus infection as indicated. (C) Comparison of the mitotic index of cells indicated after nocodazole treatment, measured by MPM2–FACS analysis (Davis et al, 1983). (D) Acetylation of BubR1 does not directly affect its phosphorylation. HeLa cells were transfected with the indicated plasmids and treated with nocodazole for 13 h. After mitotic shake-off, cells were washed with PBS five times and incubated in the presence or absence of 15 μM of MG132 for 1.5 h. Attached cells (Attach) were included for control. Lysates were prepared and subjected to IP (9E10), followed by WB with anti-p-S676 BubR1, which detects Plk-1-mediated phosphorylated BubR1. The same blot was re-probed with 9E10 (middle panel) and anti-Ac-K250 antibodies. (E) BubR1 acetylation at K250 does not alter Cdc20 binding. Wild-type-, K250R-, and K250Q-encoding constructs were translated in vitro in the presence of [³⁵S]-methionine and incubated with in vitro-translated Cdc20. Cdc20 complexes were immunoprecipitated using an anti-Cdc20 antibody and analysed by SDS–PAGE. ΔBR1 (amino acids 1–322), which lacks the Cdc20-interacting domain, a pcDNA3–myc empty vector, and reticulocyte lysate alone were included as negative controls. 5% of the in vitro-translated products were included as input controls. (F) 293T cells transfected with various BubR1 constructs were arrested with nocodazole, treated with MG132 for 3 h, and analysed for complex formation with Cdc20 by IP with 9E10 and by WB with anti-Cdc20 antibodies. In the lysis buffer, 10 μM of TSA was added. The same blot was re-probed with an anti-Ac-K250 antibody. Cells transfected with an empty vector (pcDNA3–myc) were included as a negative control.

BubR1 acetylation inhibits its degradation and regulates the timing of anaphase onset. The HeLa cells stably expressing histone H2B–GFP (HeLa–H2B) were co-transfected with synthetic siBubR1 targeting the 3′UTR and WT, K250R, or K250Q expression plasmids, respectively, tagged with DsRed at the N-termini using lipofectamine™ 2000 and subjected to time-lapse microscopy. (A) Schematic illustration of the experiment. At 13 h after transfection, cells were synchronized at the S phase through the addition of 2 mM thymidine to the culture. After a further 13 h, cells were washed from the thymidine block and released into the cell cycle. Images were taken starting at 8 h after release and were processed for 18 h. (B) Fluorescence intensities (red) and mitotic timing (segregation of green) were measured and scored from NEBD. Mean red fluorescence was measured and plotted on the Y-axis as pixel values using Image J. The X-axis represents the time from NEBD in minutes. The curves shown are representative examples of several transfected cells from at least three separate experiments. Arrows indicate the timing of anaphase onset. (C) Captured images of (B) at indicated time points (marked at lower right). NEBD is marked as 00:00. BubR1 fluorescence (DsRed) images are shown in the bottom panels in grayscale to facilitate the measurement of fluorescence intensities for WT BubR1 (Supplementary movie 1), K250R (Supplementary movie 2), and K250Q (Supplementary movie 3), respectively. In the case of K250Q-rescued cells, cells were arrested in prometaphase for hours, then the cells frequently died. In the K250R-rescued cell, anaphase began without alignment of chromosomes in metaphase plane, and the mitotic timing was shortened to ∼25 min. Stages in mitosis were determined from chromosome morphology. It should be noted that the fluorescence intensity around kinetochores declined before the onset of anaphase in WT- and K250R-expressing cells. Fluorescence intensity around kinetochores in K250Q-expressing cell did not decline for hours. (D) Box plot distributions of statistical mitotic timings from NEBD to anaphase onset for control HeLa cells (n=60); after depletion of BubR1 (siBubR1) (n=65); WT BubR1-transfected cells rescued from BubR1 depletion (n=21); K250R transfection after siRNA of BubR1 (n=43); and K250Q transfection after siBubR1 (n=40). The bars in the box are the median values. Outliers (open circle) and suspected outliers (asterisk) as determined by statistical analysis are shown. Box plots were drawn again on a smaller Y-axis scale, without K250Q-rescued data, in the right panel to facilitate the comparisons of mitotic timing. Data are from three independent experiments analysing 20–65 transfected cells using SPSS software.

Treatment with TSA during mitosis inhibits the degradation of BubR1 and a delay in anaphase entry. (A) HeLa cells were synchronized at prometaphase by tyminidine–nocodazole arrest followed by mitotic shake-off, and then either released into the cell cycle (left panel) or maintained in nocodazole for 5 h (right panel). To assess the effect of HDAC inhibition on BubR1, cells were treated with 100 nM of TSA or left untreated for the indicated time points and subjected to WB; a total of 100 μg/ml of cycloheximide (CHX) was included as a control to suppress de novo protein synthesis. Blots were re-probed with an anti-Lamin A/C antibody to normalize protein levels. Attached cells (Attach) remaining after mitotic shake-off served as controls. (B) The cell-cycle stage was assessed by propidium iodide staining and flow cytometry at the same time points as in (A); 2 N and 4 N DNA contents are indicated.

Antibodies raised against acetylated BubR1 detect BubR1 exclusively at prometaphase. The anti-Ac-K250 antibodies specifically recognize BubR1 acetylated at K250. (A) Recombinant proteins ΔBR2–3 and ΔBR2–3 K250R were subjected to in vitro acetylation and WB. Similar to the anti-Ac-K antibody, anti-Ac-K250 antibodies detect only acetylated BubR1 and not K250R. Antibodies raised against acetyl-K250 do not recognize autoacetylated PCAF, whereas the anti-Ac-K antibody does recognize it. The same blot was re-probed with anti-GST antibodies as loading controls. (B) HeLa cells in interphase (I) and M phase (M) were subjected to IP with anti-BubR1, anti-Ac-K, or anti-Ac-K250 antibodies and then immunoblotted with anti-BubR1 antibodies. BubR1 acetylation at K250 was detected in the M phase. (C) Levels of BubR1 and BubR1 acetylation after nocodazole release in the presence of CHX. HeLa cells were synchronized (time point 0), then washed and released into the cell cycle in the presence of 100 μg/ml of CHX. Lysates were prepared at the indicated time points after release and analysed by WB with anti-BubR1 and anti-Ac-K250 antibodies. The same blot was re-probed with anti-Actin antibodies. (D) BubR1 acetylation in mitosis is sustained by treating the cells with the pan-HDAC inhibitor TSA. WB with anti-Ac-K250 detects acetylated BubR1 exclusively at prometaphase-arrested cells (left panel). The right panel shows cells released from nocodazole arrest in the presence of 100 nM TSA, which resulted in the attenuation of BubR1 acetylation (Ac-K250). The same blot was re-probed with anti-BubR1 and anti-Lamin A/C.

BubR1 acetylation inhibits its polyubiquitination-dependent proteolysis. (A) Two μg of wild-type Myc-BubR1-, -K250R-, or -K250Q-encoding expression plasmids was transfected into HeLa cells. The cells were then synchronized and arrested in mitosis by continuous nocodazole treatment. The cells were then treated with MG132 or CHX for 3 h in the presence of nocodazole before lysis. Total cell lysates were analysed by WB with 9E10 to detect the ectopically expressed BubR1. The same blot was re-probed with an anti-Lamin A/C antibody. (B) BubR1 acetylation results in the inhibition of polyubiquitination-dependent proteolysis. 293T cells were cotransfected with the plasmids encoding wild type, K250R, or K250Q with HA–Ubiquitin, and proteins were immunoprecipitated with 9E10 and analysed by WB using a 12CA5 anti-HA antibody to detect polyubiquitination of BubR1 [(Ub)n–BubR1]. The blot was re-probed with 9E10 to control for the amount of immunoprecipitation. (C) HeLa cells were transfected as in (A). At 16 h post transfection, the cells were treated with 100 μM of monastrol to arrest the cells in prometaphase without disturbing microtubule structures. After 5 h, cells were treated with MG132 (+MG132) or left untreated. Cells were then fixed and co-stained with 9E10 (red) and CREST (green). Other phases of mitosis are illustrated in Supplementary Fig 8. Bars, 5 μm. (D) Quantification of results in (C). Histograms summarize three different BubR1 intensities at kinetochores in the absence or presence of MG132, measured by staining with 9E10 and normalized for CREST staining. Results are from two independent experiments (mean±s.e.m.; n⩾8 prometaphase cells per experiment, P<0.01).

K250 acetylation functions as a switch between APC/C inhibition and APC/C-dependent BubR1 destruction. (A) 293T cells were co-transfected with Myc-tagged BubR1 (1 μg each) and HA-tagged CDC20 expression constructs in a dose-dependent manner (0, 1, and 2 μg). Lysates were then subjected to IP with 9E10 and to WB with anti-ubiquitin antibody. The blot was re-probed with 9E10 to control for the amount of IP. (B) BubR1 protein levels were compared after knockdown expressions of APC3 to assess whether the APC/C complex was responsible for BubR1 degradation. HeLa cells were transfected with synthetic siRNAs. At 60 h later, cells were treated with 200 ng/ml nocodazole for 12 h, and the mitotic cells were collected by mitotic shake-off. Lysates were prepared before and after CHX addition for 1 h. Note that after nocodazole release for 60 min in the presence of CHX, APC3 appears as a faster migrating form, indicating that APC3 is highly modified in prometaphase. (C) WB analysis of Cdc20 and Cdh1 after transfection of siRNAs, as indicated. These cells were used to measure BubR1 levels in (D). (D) Experiments were carried out in a manner similar to (B), except that, to block the exit from mitosis, a Cyclin B expression construct was transfected simultaneously with siRNAs. The lysates for WB were collected at 0, 15, 30, and 60 min after nocodazole release and CHX treatment. (E) Intensities of fluorescence, which reflects the level of DsRed–BubR1 in mitotic cells, were recorded against time relative to NEBD. The D-box or the KEN boxes were destroyed by in vitro mutagenesis in DsRed–K250R by substituting AAA for KEN boxes (left panel) and RXXL to AXXA for the D-box (right panel). The curves shown are representative of at least 20 individual cells from at least two separate experiments. A fluorescence intensity curve for K250R was included in all experiments as a control. (F) Our working model for BubR1 acetylation and the regulation of APC/C activity. BubR1 is in a complex with Cdc20 and APC/C through its KEN1, KEN2, and D-box domains. BubR1 acetylation/deacetylation changes the surface structure of degrons in relation to APC/C–Cdc20 complex. When kinetochores are not yet attached to spindles (prometaphase), BubR1 is acetylated by PCAF. BubR1 acetylation prevents it from being a substrate of the APC/C complex, and acetylated BubR1 is a potent inhibitor of the APC/C–Cdc20 complex. When all kinetochores are stably attached to spindles, BubR1 is no longer acetylated because of a change in the stoichiometry of the BubR1 complex or its deacetylation. The conformational change allows the D-box and KEN box degrons of BubR1 to be recognized as a substrate of the APC/C-Cdc20 complex. In our model, the mode of Cdc20 binding to APC/C complex and BubR1 changes, depending on the status of BubR1 acetylation.