Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Acetylation of BubR1 at K250 identifies unattached kinetochores. ( A ) Immunofluorescent staining of metaphase chromosome spreads. Mitotic HeLa cells were enriched by treatment with colcemid (100 ng/ml) for 18 h, followed by a 5-min release, and then subjected to co-immunostaining using anti-BubR1 and anti-acetylated BubR1 (AcK250) monoclonal antibodies (mAbs). Kinetochores were categorized into three groups based on anti-AcK250 mAb immunostaining: those immunostained at both sister kinetochores (Paired); one kinetochore (Single); or neither (No). Scale bar, 5 μm. ( B ) Proportion of anti-AcK250 mAb immunostaining patterns in panel (A). Results are from two independent experiments (mean ± s.e.m.; n = 40 chromosomes). P- values were obtained using a t -test. ( C ) Representative images of microtubule attachment at kinetochores using SIM. Mitotic HeLa cells were treated with 10 μM MG132 to enrich the metaphase population, fixed, and then co-immunostained using anti-α-tubulin, anti-AcK250, and CREST antibodies. CREST-marked kinetochores were divided into three groups based on 3D-reconstructed images: unattached kinetochore (No attachment), monotelic kinetochore (Single attachment), and amphitelic kinetochore (Bi-attachment). Scale bar, 5 μm (top). Bottom, 3D-reconstructed images of anti-AcK250 mAb staining. Scale bar, 0.5 μm. The SIM images were acquired using an AP DeltaVision OMX Ultra High-Resolution Fluorescence Microscope (GE Healthcare). Image reconstruction and alignment were performed using SoftWoRx software. Three-dimensional reconstruction was performed using ImageJ software (3D projection). ( D ) Relative intensity of anti-AcK250 immunostaining normalized to CREST. Number of kinetochores scored: No attachment (No), n = 20; monotelic attachment (Single), n = 22; bipolar attachment (Bi), n = 16. Numbers of kinetochores scored are from 30 different cells, each from two independent experiments. Statistical differences between groups were assessed using the Mann–Whitney U test; P -values are indicated. (E–H) HeLa cells treated with 200 ng/ml nocodazole (Noc), 2 μM paclitaxel/taxol (Tax), 100 μM monastrol (Mon), or 10 μM MG132, respectively, were co-immunostained with CREST, anti-α-tubulin, and anti-AcK250 ( E, F ) or anti-BubR1 ( G, H ). Scale bar, 5 μm. ( F, H ) Graphs are from two independent experiments (mean ± s.e.m.). Number of cells scored for (F): Noc, n = 7; Tax, n = 15; Mon, n = 21; MG312, n = 8 and for (H): Noc, n = 28; Tax, n = 17; Mon, n = 33; MG132, n = 12.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Staining, Immunostaining, Bioprocessing, Fluorescence, Microscopy, Software, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Lysine 250 acetylation of BubR1 (BubR1–AcK250) is associated with kinetochore expansion. ( A ) Representative images of M-phase HeLa cells immunostained with anti-BubR1, CREST, and α-tubulin antibodies. Cells were treated with 10 μM MG132 to enrich the metaphase population. Kinetochores were categorized into three groups based on 3D-reconstructed SIM images: No attachment, Single attachment, or Bi-attachment. Scale bar, 5 μm. Bottom, 3D reconstruction. Scale bar, 0.5 μm. ( B ) Volume of BubR1 in cells exhibiting no attachment (No), monotelic attachment (Single), and amphitelic attachment (Bi). Number of kinetochores scored: No, n = 131; Single, n = 89; Bi, n = 115. Results are from 155 cells in three independent experiments (mean ± s.e.m.). ( C ) Representative SIM images of FLAG-tagged BubR1-WT, -K250R, and -K250Q in mitotic kinetochores. HeLa-FRT-TO cells expressing a single copy of FLAG-BubR1, -K250R , or -K250Q were depleted of endogenous BubR1 via siRNA transfection . Simultaneously, doxycycline was used to induce the expression of BubR1-WT, -K250R , and -K250Q , respectively. Cells were treated with nocodazole (Noc) alone or with MG132 (Noc + MG132) to assess the effect without perturbing the stability of K250R. Cells were treated with nocodazole (200 ng/ml, 20 h) alone or in combination with MG132 (10 μM, final 2 h). Scale bar, 5 μm. ( D ) Volume (μm 3 ) of FLAG (left) and relative intensities of FLAG to CENP-A (right) in nocodazole-treated cells with or without MG132. Number of kinetochores scored in nocodazole-only treatment: WT, n = 12; K250R, n = 14; and K250Q, n = 14. For nocodazole + MG132 treatment: WT, n = 72; K250R, n = 72; and K250Q, n = 72. The graphs are from two independent experiments (mean ± s.e.m.).

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: K250-BubR1 acetylation is involved in the maintenance of crescent-shaped fibrous corona. ( A ) Representative images of immunostaining with anti-MAD2 and anti-ZW10 antibodies after induction of BubR1-WT, -K250R , and -K250Q expression in HeLa-FRT-TO cells. Nocodazole (200 ng/ml) alone or with MG132 was applied before fixation. Endogenous BubR1 was depleted via siRNA. Scale bar, 5 μm. ( B ) Representative SIM images of immunostained ZW10 at kinetochores of HeLa cells expressing FLAG-tagged BubR1-WT, -K250R , or -K250Q following double thymidine block and release. FRT/TO HeLa cells were treated with doxycycline for 55 h to induce expression of ectopic BubR1 variants. Endogenous BubR1 was depleted using siRNA targeting the 3′ UTR. Cells were fixed for immunostaining 10 h after the final thymidine release to enrich the mitotic population. ( C ) Effects of BubR1 acetylation status on CENP-E and RZZ complex formation. Cells were treated with nocodazole alone (+Noc) or with MG132 (+Noc + MG132). Relative protein levels were measured using a densitometer, and the ratios are indicated at the bottom. ( D ) Representative images showing immunostained ZW10 and anti-CENP-A antibodies in FRT/TO HeLa cells expressing BubR1-WT, K250R , or K250Q with (siCtrl) or without CENP-E (siCENP-E). Cells were transfected with siRNA targeting luciferase (siCtrl) or CENP-E (siCENP-E) for 48 h prior to fixation. Nocodazole was administered for 20 h before analysis.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Immunostaining, Expressing, Blocking Assay, Transfection, Luciferase

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Aurora kinase B activity is required for BubR1 acetylation and maintenance of fibrous corona. ( A ) Immunostaining with anti-AcK250 mAb after treatment of nocodazole-arrested cells with the indicated mitotic kinase inhibitors: 10 nM of BI 2536 (PLK1 inhibitor), 2 μM of Reversine (MPS1 inhibitor), 200 nM of Hesperadin (Aurora B inhibitor), and 2 μM of ZM447439 (Aurora B inhibitor). Kinase inhibitors were applied during the final 2 h of a 20-h nocodazole treatment. Scale bar, 5 μm. ( B ) Relative intensities of AcK250 at kinetochores from panel (A): Nocodazole-arrested cells without kinase inhibitor treatment (NT), and nocodazole + BI 2536-treated (BI 2536), reversine-treated (Rev), hesperadin-treated (Hes), or ZM447439-treated (ZM) cells (mean ± s.e.m.). Each dot represents the relative AcK250 signal intensity per cell. Number of cells scored: NT, n = 31; BI, n = 44; Rev, n = 18; Hes, n = 21; ZM, n = 28. Results are from two independent experiments. ( C ) Assessing the effect of Aurora B kinase activity in MCC maintenance. HeLa cells were transfected with Myc-tagged Aurora B-WT or kinase-dead mutant K106R -expressing constructs. Endogenous Aurora B was depleted using siRNA targeting the 3′ UTR. BI 2536 (10 nM), Reversine (2 μM), Hesperadin (200 nM), or ZM447439 (2 μM) were added during the final 2 h of a 20-h nocodazole treatment (left). Immunoprecipitation was performed using an anti-CDC27 (APC3) antibody, followed by WB with indicated antibodies. To assess MCC stability, MG132 was added with the indicated kinase inhibitors in nocodazole-arrested cells (right). WB with indicated antibodies in total cell lysate (TCL, 5%) is shown at the bottom. WB with 9E10 shows the expression of Myc-tagged AurkB-WT or AurkB-K106R . Note the high level of AcK250–BubR1 in WT-AurkB -expressing mitotic cells, but not in AurkB-K106R -expressers (AcK250) or interphase cells (lane 2). Both the BubR1 level in anti-CDC27 immunoprecipitate and AcK250 BubR1 in TCL are high in nocodazole-treated mitotic cells, which are inhibited by AurkB inhibitors but not by BI 2536 or Reversine (lanes 4–7). Phospho-AurkB is detected only in WT-AurkB -expressers (AurkB-pT232). ( D ) Representative images of anti-AcK250 immunostaining in cells expressing wild-type Aurora B ( AurkB-WT ) or kinase-dead Aurora B ( AurkB-K106R ). Scale bar, 5 μm. ( E ) Intensity of anti-AcK250 immunofluorescence measured from panel (D). Number of cells counted: WT, n = 11; AurkB-K106R, n = 7 (mean ± s.e.m.). Each point represents the mean AcK250 signal intensity per cell. ( F ) Effect of AurkB activity in the expansion of BubR1 (left) and ZW10 (right) at kinetochores in response to nocodazole treatment. Scale bar, 5 μm. ( G ) Volume of BubR1 and ZW10 at the kinetochores from panel (F). Number of kinetochores: BubR1 in WT, n = 105; BubR1 in K106R, n = 121; ZW10 in WT, n = 153; ZW10 in K106R, n = 85 (mean ± s.e.m.). The results are from 131 cells in three independent experiments. ( H ) Interaction between BubR1, Aurora B, and phosphorylated Aurora B (AurkB-pT232) was assessed by IP with anti-BubR1 and WB with indicated antibodies. Mitotic HeLa cells were collected by mitotic shake-off after 20 h of nocodazole treatment (M phase, M). Interphase-attached cells (Attached, A) were employed for control.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Activity Assay, Immunostaining, Transfection, Mutagenesis, Expressing, Construct, Immunoprecipitation, Immunofluorescence, Control

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Aurora B phosphorylates serine 16 and serine 39 of BubR1 upon nocodazole treatment, which is required for K250 acetylation. ( A ) Identification of Aurora B-mediated phosphorylation sites of BubR1 in response to nocodazole treatment. (Top) Schematic illustration of BubR1 marked with AurkB-binding region and phosphorylation sites. Potential phosphorylation sites (Ser16 and Ser39) by AurkB and K250 acetylation site are marked with red. N-terminus of BubR1 (1–150) binds to AurkB (results from Fig. ). (Bottom) IP-WB analysis to identify phosphorylation sites that crosstalk with K250 acetylation. mCherry-tagged BubR1 expression constructs were mutagenized in vitro to substitute the phosphorylation sites with alanine, based on proteome analysis . They were then transfected into HeLa cells and treated with nocodazole (200 ng/ml) for 20 h. Mitotic cells were collected by shake-off, and attached cells (Attach) were employed as a control. Mitotic cell lysates, along with lysates from attached cells (WT, Attach), were subjected to immunoprecipitation with anti-mCherry antibody, followed by WB with anti-AcK250 mAb. The same blot was reprobed with anti-BubR1 and anti-mCherry antibodies for normalization. Total cell lysates were subjected to WB with anti-phospho-H3 and anti-cyclin B antibodies to assess the mitosis stage. WB with anti-mCherry and anti-β-actin antibodies in TCL were used as loading controls. Relative band intensities (AcK250/BubR1) were measured using a densitometer and are indicated. ( B ) Identification of the Aurora B-binding region in BubR1. IP with 9E10 (anti-Myc) and WB with anti-AurkB (Aurora B kinase) were performed. All BubR1-expressing constructs were Myc-tagged. The same blot was reprobed with 9E10 for normalization. Two percent of TCL was subjected to WB with the indicated antibodies. ( C ) Effect of Ser16 or Ser39 phosphorylation in K250 acetylation. Immunofluorescence assay in cells expressing the indicated mCherry-tagged BubR1 variants. Nocodazole-treated cells were formaldehyde-fixed and subjected to immunostaining with anti-AcK250, anti-ACA (CREST), and anti-mCherry antibodies. ( D ) Graph showing the intensities of anti-AcK250 immunofluorescence from panel (C). Anti-AcK250 immunofluorescence signals were normalized to anti-mCherry. The results are from two independent experiments. Number of cells: WT, n = 30; K250R, n = 20; K250Q, n = 24; S16A, n = 28; S39A, n = 29 (mean ± s.e.m.). ( E ) WB analysis showing the effects of mitotic kinase inhibitors on phosphorylation of BubR1 (1–150) in unattached kinetochore (nocodazole treatment). HeLa cells were subjected to nocodazole treatment and mitotic shake-off. The mitotic lysates were then resuspended in in vitro phosphorylation buffer containing 100 μM ATP (see the “Materials and methods” section). MBP-tagged recombinant BubR1 (amino acids 1–150) was employed as the substrate. The mitotic lysate and recombinant BubR1 were incubated in the buffer for 2 h with/without indicated inhibitors (10 nM BI 2536, 2 μM reversine, 200 nM hesperadin, and 2 μM ZM447439). In parallel, the lysates were subjected to WB with anti-AurkB-pT232, -AurkB, and -β-actin. ( F ) In vitro phosphorylation assay showing that phosphorylation of S39 and S16 is AurkB-dependent. Recombinant BubR1 (amino acids 1–150) was incubated with anti-AurkB immunoprecipitate of nocodazole-arrested HeLa cells. Reactions were carried out for 2 h in the presence or absence of the AurkB inhibitor ZM447439 (2 μM). [γ- 32 P]-ATP label detects the phosphorylation of the substrate BubR1 (1–150). WB with anti-MBP and anti-AurkB in the same lysate was performed for control.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Construct, In Vitro, Transfection, Control, Immunoprecipitation, Immunofluorescence, Immunostaining, Recombinant, Incubation

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Phosphorylation of Ser39 (and Ser16) by AurkB is required for K250 acetylation in SAC signaling. ( A ) IP-WB analysis to assess the effect of Ser16 and Ser39 phosphorylation in MCC maintenance upon nocodazole treatment. The indicated mCherry-tagged BubR1 variants were transfected, and endogenous BubR1 was depleted by co-transfection of siRNA for BubR1 targeting the 3′ UTR. Immunoprecipitation was performed with an anti-mCherry antibody, followed by WB with the indicated antibodies. Results for nocodazole-arrested HeLa cells treated with MG132 for 2 h before harvest are shown at right (+Noc + MG132). WB of 2% TCL is shown at the bottom. (B, C) Effects of phosphorylation and acetylation in MCC maintenance. ( B ) Effects of AurkB-mediated phosphorylation at serines 39 (and Ser16) in MCC maintenance. Phospho-deficient mutants S16A-BubR1 and S39A-BubR1 , as well as the S16A;S39A double mutant, and phospho-deficient mutations introduced into the acetylation-mimetic form ( S16A;K250Q, S39A;K250Q , and S16A;S39A;K250Q ) were transfected into HeLa cells, along with siBubR1 to deplete endogenous BubR1. The cells were treated with nocodazole and subjected to IP with anti-mCherry antibody, followed by WB with the indicated antibodies. ( C ) Effect of the phospho-mimetic S16D-BubR1 or S39D-BubR1 on MCC maintenance. The acetylation-deficient K250R mutation was introduced into S16D or S39D ( S16D;K250R and S39D;K250R ) variants, and their capabilities for maintaining MCC after nocodazole treatment were assessed. All BubR1 variants were tagged with mCherry, transfected into HeLa cells, and subjected to IP and WB using the indicated antibodies. ( D ) Effect of AurkB-mediated phosphorylation and K250 acetylation on mitotic timing. Endogenous BubR1 was depleted using siRNA, and the indicated mCherry-tagged BubR1 variants were transfected into HeLa cells stably expressing H2B-GFP . Images were captured at 5-min intervals. Arrows mark anaphase onset.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Phospho-proteomics, Transfection, Cotransfection, Immunoprecipitation, Mutagenesis, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: AurkB-AcK250–BubR1 pathway coordinates the unattachment signal to the maintenance of fibrous corona and SAC. (A, B) Effects of AurkB-mediated phosphorylation and subsequent K250 acetylation in fibrous corona expansion. ( A ) SIM images of anti-Bub1 or anti-MAD2 immunostaining in nocodazole-treated cells are shown. The indicated BubR1 constructs were transfected with siBubR1 targeting the 3′ UTR 48 h before fixation, and nocodazole (200 ng/ml) was applied for 20 h. Volumes were measured from the immunofluorescence assays shown. Number of cells scored: Bub1 in WT, n = 63; Bub1 in S16A, n = 64; Bub1 in S39A, n = 70; MAD2 in WT, n = 196; MAD2 in S16A, n = 196; MAD2 in S39A, n = 69 (mean ± s.e.m.). ( B ) SIM images of anti-ZW10 immunostaining in nocodazole-treated cells. Endogenous BubR1 was depleted via co-transfection with siBubR1. Nocodazole (200 ng/ml) was administered for 20 h, two days post transfection. Insets show enlarged images of ZW10 and CREST staining. Scale bar, 5 μm. ( C ) Volume of ZW10 measured from the immunofluorescence assay shown in panel (B). Number of cells scored: WT, n = 27; S16A, n = 29; S39A, n = 27; S16A;K250Q, n = 31; S39A;K250Q, n = 32; K250Q, n = 29; S16D, n = 31; S39D, n = 32; S16D;K250R, n = 27; S39D;K250R, n = 27; K250R, n = 27 (mean ± s.e.m.). ( D ) Model illustrating the coordination of AurkB-mediated phosphorylation and K250 acetylation in spindle checkpoint signaling. At unattached kinetochores, AurkB phosphorylates BubR1 at S39 (and S16 as well), promoting BubR1 acetylation at K250 by PCAF. This acetylation ensures the maintenance of the RZZ complex and MCC. It also sustains CENP-E at the kinetochore, preparing lateral to end-on attachment (left). Upon successful end-on capture, PP2A activity recruited by AcK250–BubR1 antagonizes AurkB activity, stabilizing KT-MT attachment. Simultaneously, BubR1 immediately deacetylates and degrades through APC/C-mediated ubiquitination, resulting in MCC disassembly and SAC silencing.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Phospho-proteomics, Immunostaining, Construct, Transfection, Immunofluorescence, Cotransfection, Staining, Activity Assay, Ubiquitin Proteomics