BubR1 T600/T608 phosphorylation promotes BubR1-B56 interaction

It is well characterized that PP2A/B56 bound to BubR1 plays important roles for both chromosome alignment and checkpoint silencing which resembles the phenotype observed in BubR1 2A^22–24. To test whether the observed mitotic defects caused by BubR1 2A is related to PP2A/B56, we first examined the kinetochore signals of the PP2A/B56 substrate Mps1 pT33^20,28,32 by quantitative immunofluorescence since B56 on kinetochores is beyond detection by fluorescence microscopy^22,23. The kinetochore signals of Mps1 pT33 was increased more than 30% in cells expressing BubR1 2A while reduced around 30% in cells with BubR1 2E compared with the cells with wild type BubR1 (Supplementary Fig. 1e, f) indicating PP2A/B56 could not be efficiently recruited to BubR1 2A. We then examined the kinetochore signals of BubR1 pT680 which is phosphorylated by Plk1 and required for efficient binding of PP2A/B56 to BubR1. Intriguingly, there was around 80% reduction of pT680 kinetochore signals in BubR1 2A cells compared with wild-type cells while in cells expressing BubR1 2E, the kinetochore signals of pT680 were partially rescued (Fig. 2a, b).

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Fig. 2

Double phosphorylation of BubR1 T600/T608 promotes BubR1-B56 and BubR1-Plk1 interaction.

a Representative images of mitotic cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1. The cells were released from RO3306 into the medium with nocodazole (200ng/ml) for 45min before fixation and staining with corresponding antibodies. Scale bar is 10µm. b Quantification of of kinetochore signals of BubR1 pT680 against YFP-BubR1 from (a). The values of 150 kintochores from 10 cells for each condition were presented. The red line indicates the mean value which was set to 1 for wild type BubR1 sample (WT) and the rest was normalized to it. Bar is standard error of the mean. Mann–Whitney U-test was applied. *P<0.1; ****P<0.0001. c Similar treatment as (a). d Quantification of kinetochore signals of Plk1 against YFP-BubR1 from (c). The values of 150 kintochores from 10 cells for each condition were presented. The red line indicates the mean value which was set to 1 for BubR1 ΔKARD sample and the rest was normalized to it. Bar is standard error of the mean. Mann–Whitney U-test was applied. ns means not significant; ****P<0.0001. e YFP-tagged BubR1 was expressed in HeLa cells and immunoprecipitated by GFP-trap beads. Plk1 were detected by western blot from the immunoprecipitate. Quantification of Plk1 intensity against YFP intensity was plotted on the right from two repeats. The column represents median value. The ratio from ΔKARD 2A was normalized against the ratio from ΔKARD which was set to 1. f ITC showing the binding between BubR1 peptides and recombinant Plk1.

The above results clearly show that BubR1 pT600/pT608 is required for efficient phosphorylation of BubR1 T680 and its interaction with PP2A/B56 as a result.

Phosphorylation of T600/T608 is required for stable interaction of Plk1 with BubR1

How could the phosphorylation on BubR1 T600/T608 enhances the phosphorylation of BubR1 T680? Since T600 and T608 are close to the cannonical Plk1 binding motif (619-SpTP-621), we reasoned that the double phosphorylation might be involved in the BubR1-Plk1 interaction. We decided to test whether BubR1 pT600/pT608 is required for kinetochore localization of Plk1. Based on the fact that BubR1 has only minor contribution to kinetochore recruited Plk1, it is difficult to precisely measure the signal variations of BubR1-associated Plk1 within such a small window. On the other hand, BubR1 phosphorylation is negatively regulated by PP2A/B56^29–31. Thus, it is possible to increase the kinetochore signals of BubR1-associated Plk1 by disrupting BubR1 binding to PP2A/B56 which allows more accurate quantification of the Plk1 signals. We first examined the kinetochore localization of Plk1 in BubR1ΔKARD background which is not able to bind B56 due to two alanine mutations within the core binding motif (LxxIxE into AxxAxE). Indeed, loss of PP2A/B56 binding increased ~2-fold the kinetochore levels of Plk1 (Fig. 2c, d). In this background, we examined the kinetochore localization of Plk1 in cells expressing BubR1 2A and found the Plk1 signals was significantly reduced to similar extent as T620A mutant while single alanine mutation T600A or T608A caused mild Plk1 reduction (Fig. 2c, d). Similar result was achieved by immunoprecipitating YFP-tagged BubR1 in cells expressing corresponding constructs. BubR1-associated Plk1 was strongly reduced in YFP-BubR1 2A sample compared with wild type BubR1 sample in ΔKARD background (Fig. 2e). To investigate if the phosphorylation of T620 was affected by the T600A/T608A mutation we analyzed the phosphorylation pattern of purified YFP-BubR1 and BubR1 2A by quantitative mass spectrometry. We did not directly identify the phosphorylated pT620 peptide but measured the unphosphorylated FVSTPFHEIMSLK peptide levels which was strongly reduced in BubR1 2A purifications suggesting increased T620 phosphorylation (Supplementary Fig. 1g). We also detected pS670 which was similar in both BubR1 WT and 2A (Supplementary Fig. 1h).

We then tested if double phosphorylation of T600/608 creates a second Plk1 binding site besides the canonical one. We synthesized peptide BubR1^596-614 with T600/T608 phosphorylated. As a control, we also synthesized a peptide BubR1^616-624 with T620 phosphorylated. Using isothermal titration calorimetry (ITC), we measured the binding affinity between recombinant Plk1 PBD and the peptides. The assay clearly showed binding of BuBR1^616-624, but not of BubR1^596-614 to the recombinant protein indicating no extra binding site generated on BubR1 by the double phosphorylation (Fig. 2f; Supplementary Fig. 2a–c).

Both the T600 and T608 phosphorylation sites fit the consensus recognized by Plk1, [D/N/E/Y]-X-S/T-[F/Φ; no P]-[Φ/X], where Φ is hydrophobic amino acid and X indicates any amino acid¹⁷. To test whether these two phosphorylation relies on Plk1 kinase activity, we generated a BubR1 pT600 phosphor antibody and characterized it by quantitative immunofluorescence. This antibody strongly decorated kinetochores of the cells treated with nocodazole and this decoration was fully abolished after lambda phosphatase treatment (Supplementary Fig. 3a). In cells depleted of BubR1 or treated with Plk1 inhibitor BI2536, the kinetochore signals recognized by the antibody were largely reduced (Supplementary Fig. 3b–d). There was around 30% of the kinetochore signals remained in both cases indicating this antibody cross-reacts with other phosphorylation modification on kinetochores. We further characterized this antibody in cells complemented with RNAi-resistant constructs expressing YFP-tagged wild type or mutant BubR1 with endogenous BubR1 depleted by RNAi. To eliminate the negative regulation by PP2A/B56, all these BubR1 proteins harbor defective B56 binding motif. The quantification showed a significant reduction of pT600 kinetochore signals in either T600A or T620A mutant to the same extent compared with BubR1ΔKARD indicating the phosphorylation of T600 requires the phosphorylation of T620 (Supplementary Fig. 3e). Using this antibody, we also examined the occurance of BubR1 T600 phosphorylation at distinct phases of mitosis. The phosphorylations happens as early as in prophase when BubR1 starts localizing onto kinetochores. The signals peak at prometaphase and declines continuously till anaphase (Supplementary Fig. 3f).

These data suggest that BubR1 binds Plk1 through the cannonical STP motif after T620 gets phosphorylated by Cdk1. Plk1 in turn, mediates the phosphorylation of T600 and T608 to enhance its binding to BubR1. Once Plk1 stably binds BubR1, it further phosphorylates amino acids S676 and T680 to promote the binding of PP2A/B56 with BubR1. Thus, kinetochore localized PP2A/B56 ensures accurate chromosome segregation.

The double phosphorylation can be bypassed by enhancing PBD binding or B56 binding to support accurate mitotic progression

According to the above model, we predict that enhancing the binding of Plk1 with BubR1 may bypass the requirement of the double phosphorylation on T600 and T608 for accurate mitosis. To test this, we engineered BubR1 2A mutant by replacing the BubR1 PBD BM (RFVSTPFHE) with WDR47 PBD BM (IHTSTPRNP) whose affinity towards Plk1 PBD is almost 25 times stronger (BubR1 2A+WDR47 hereafter) (Fig. 3a; Supplementary Figs. 2d, e and 4a). We first examined the ability of the engineered protein BubR1 2A+WDR47 to recruit Plk1 onto kinetochores by quantitative immunofluorescence. As expected, BubR1 2A+WDR47 efficiently recruited Plk1 to the same extent as BubR1 itself (Fig. 3b, c). We then tested the ability of the engineered protein to support accurate mitotic progression by live cell imaging. The results showed the mitotic delay caused by mutating T600/T608 to alanine (80min) was fully restored to normal (40min) in the presence of WDR47 PBD BM (Fig. 3d, e). Live cell imaging testing the SAC activity also showed efficient silencing of the SAC in cells complemented with BubR1 2A+WDR47 (Supplementary Fig. 4b).

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Fig. 3

Enhancing BubR1-Plk1 or BubR1-B56 binding could bypass the requirement of the double phosphorylation.

a K_D measured by ITC of recombinant Plk1 with phospho-modified peptides. b Representative images of mitotic cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1. The cells were released from RO3306 into the medium with nocodazole (200ng/ml) for 45min before fixation and staining with corresponding antibodies. Scale bar is 10µm. c Quantification of kinetochore signals of Plk1 against YFP-BubR1 from (b). Mean values of at least 150 kintochores from at least 10 cells for each condition were presented. The red line indicates the mean value which was set to 1 for BubR1 ΔKARD sample and the rest was normalized to it. Bar is standard error of the mean. Mann–Whitney U-test was applied. ns means not significant; ****P<0.0001. d Representative stills of unperturbed mitosis recorded by live cell imaging. The cells were transfected with siRNA oligos against luciferase as control or siRNA oligos against BubR1 with RNAi-resistant constructs expressing YFP-BubR1. mCherry-H3 expressing plasmid was co-transfected for chromosome indication. Scale bar is 10µm. e Quantification of the mitotic time from NEBD to anaphase from (d). Each circle represents the time from NEBD to anaphase of a single cell. Red line indicates the median time. The number of cells analyzed per condition is indicated above (n = X). Mann–Whitney U-test was applied. *P<0.1; ***P<0.001; ****P<0.0001.

It is known that BubR1-bound Plk1 promotes BubR1-PP2A/B56 interaction. We wondered whether enhancing the binding of PP2A/B56 with BubR1 could bypass the requirement of the pT600/pT608. To test the idea, we engineered a BubR1 2A mutant with D675E/S676E/R677E (BubR1 2A+3E hereafter) which efficiently binds B56 as described before³³ (Supplementary Fig. 4a). Quantitative immunofluorescence showed the engineered protein could not efficiently recruit Plk1 onto kinetochores similar as BubR1 2A mutant (Fig. 3b, c). However, live cell imaging showed BubR1 2A+3E was as efficient as wild type BubR1 in promoting accurate mitotic progression as well as SAC silencing (Fig. 3d, e; Supplementary Fig. 4b).

The above data further demonstrates BubR1-Plk1 interaction requires both PBD BM and the double phosphorylation in front. However, for BubR1 with optimal PBD BM, the phosphorylation is not necessary anymore for accurate mitosis. The results also show the BubR1-Plk1 interaction is largely dispensible for accurate mitosis in the presence of enhanced binding between BubR1 and PP2A/B56.

Efficient Bub1-Plk1 binding also requires extra phosphorylation besides the PBD BM

Bub1, the paralogue of BubR1, is one of the two main Plk1 receptors on outer kinetochores. Whether Bub1 shares similar principle for Plk1 interaction as BubR1 is not clear. At least five amino acids (T589, S593, S596, T601, and S602) N terminal to the canonical Plk1 binding site (608-SpTP-610) of Bub1 are phosphorylated (phosphosite.org). Two of them, T589 and T601 fit the consensus motif for Plk1. Another two S593 and S596 are likely phosphorylated by Cdk1. We mutated all the four residues into alanine (Bub1 4A) or into glutamic acid (Bub1 4E) to mimic the unphosphorylated and phosphorylated Bub1. To avoid the signal interference from BubR1-associated Plk1, the BubR1 binding domain (amino acids 266-311)³⁴ was removed from all the above mutants. We measured the kinetochore signals of Plk1 in HeLa cells depleted of endogenous Bub1 and complemented RNAi-resistant YFP-tagged Bub1 constructs. Bub1 4A reduced the kinetochore Plk1 level to less than 50% while Bub1 4E maintained Plk1 around 80% of Bub1ΔBubR1 (Fig. 4a, b).

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Fig. 4

Bub1-Plk1 interaction requires multiple phosphorylation.

a Representative images of mitotic cells transfected with siRNA oligos against Bub1 and RNAi-resistant constructs expressing YFP-Bub1. The cells were released from RO3306 into the medium with nocodazole (200ng/ml) for 45min before fixation and staining with corresponding antibodies. Scale bar is 10µm. b Quantification of kinetochore signals of Plk1 against YFP-Bub1 from (a). Mean values of 150 kintochores from 10 cells for each condition were presented. The red line indicates the mean value which was set to 1 for wild type Bub1 (WT) sample and the rest was normalized to it. Bar is standard error of the mean. Mann–Whitney U-test was applied. ****P<0.0001. c Representative images of mitotic cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1. The cells were released from RO3306 into the medium with nocodazole (200ng/ml) for 45min before fixation and staining with corresponding antibodies. Scale bar is 10µm. d Quantification of kinetochore signals of Plk1 against YFP-BubR1 from (c). Mean values of 150 kintochores from 10 cells for each condition were presented. The red line indicates the mean value which was set to 1 for BubR1 ΔKARD sample and the rest was normalized to it. Bar is standard error of the mean. Mann–Whitney U-test was applied. *P<0.1; ****P<0.0001.

Since the Bub1 Plk1 binding module (the region containing the phosphorylation sites and PBD BM) could efficiently recruit Plk1 to kinetochores, we wonder whether this module could replace the BubR1 Plk1 binding module for efficient Plk1 binding. We grafted Bub1 Plk1 binding module (575–614aa) to the corresponding region on BubR1 (595–625aa) and measured the kinetochore signals of Plk1 in HeLa cells expressing the engineered protein after depletion of endogenous BubR1. The results showed BubR1 containing Bub1 Plk1 binding module but not the 4A mutant could recruit Plk1 as efficiently as BubR1 itself (Fig. 4c, d).

These data suggest self-priming may be a general principle for Plk1 interaction with PBD BM-containing proteins.

Molecular cloning

The constructs used in this study were generated by standard cloning method and mutagenesis was conducted by mutation PCR. Briefly, wild-type Bub1 or BubR1 was cloned into pcDNA5/FRT/TO N-YFP vector by KpnI and NotI. For BubR1 engineered protein containing distinct PBD BM, BamHI site was first introduced by mutation PCR to replace the DNA sequence at designed positions. Afterward, the DNA encoding distinct PBD BM was amplified and inserted by BamHI restrictive enzyme and T4 DNA ligase. Gene amplification or mutation PCR was performed with KOD DNA polymerase (Toyobo). All the restriction enzymes and T4 DNA ligase were purchased from Thermo Fisher Scientific.

Live cell imaging

HeLa cells were first seeded in 6-well plate and synchronized with thymidine. RNA interference and plasmid transfection was performed as described in the above section. 900ng of RNAi-resistant YFP-BubR1 or YFP-Bub1 construct and 30ng of mCherry-H3 were co-transfected together with 50nM of RNAi oligos in each well for undisburbed mitosis imaging. For SAC strength assays, only YFP-BubR1 and RNAi oligos were co-transfected. 24h after transfection, the cells were re-seeded into an 8-well chamber slide (Ibidi) during the second thymidine block. 18h later, cells were released from the second thymidine block and recorded by live cell imaging 6h later. Leibovitz’s L-15 medium (Thermo Fisher Scientific) supplemented with 10% FBS was applied into each chamber before the slide was mounted onto microscopy. For SAC assays, nocodazole (30ng/ml or 100ng/ml as specified in figure legends, Selleck) was added into the L15 medium. Nikon A1HD25 imaging system (Nikon) was used for live cell imaging. DIC, YFP, and RFP signals were collected every 10min for a total of 18h. NIS-Elements AR Analysis (Nikon) was used for data analysis. At least two repeats for each assay were performed with more than 30 cells in each condition quantified.

Immunoprecipitation and western blot

HeLa cells were synchronized by double thymidine and transfected with YFP-BubR1 constructs after the first thymidine arrest. 36h later, the cells were released from the second thymidine arrest into medium containing nocodazole (200ng/ml) for an additional 12h. Mitotic cells were collected by shake off and lysed in buffer containing 10mM Tris pH 7.5, 150mM NaCl, 0.5mM EDTA, and 0.5% NP40. After centrifugation at 16,000×g for 15min, the supernatant was incubated with GFP-Trap agarose beads (Proteintech) and shaken at 1100rpm for 120min at 4°C on thermomixer (Eppendorf). After three washes, the bound protein was eluted in 2 x SDS sample buffer and separated by SDS-PAGE. Target proteins were detected by western blot. Antibodies used in this study include YFP antibody (homemade in JN lab, 1:2000), Plk1 (Santa Cruz sc-17783, 1:500).

Immunofluorescence

Cells growing on coverslips in 6-well plate were treated as described in the above. Cells were fixed by 4% paraformaldehyde in PHEM buffer (60mM PIPES, 25mM HEPES, pH 6.9, 10mM EGTA, and 4mM MgSO₄) at room temperature for 20min. The fixed cells were permeabilized by 0.5% Triton X-100 in PHEM for 10min at room temperature before being stained by corresponding antibodies. The antibodies used in this study include Bub1 (Abcam, ab54893, 1:200), BubR1 (home made in JN lab, 1:200), CENP-C (MBL, PD030, 1:800), BubR1 pT680 (Abcam200061, 1:500), BubR1 pT600 (Abclonal, 1:200), α tubulin (Sigma, F2168, 1:400), Mad2 (home made in JN lab, 1:200), Mps1 pT33 (home made in JN lab, 1:200) and Plk1 (Santa Cruz, sc-17783, 1:100). The BubR1 phospho antibody was raised in rabbit against C-RNV[pT]ISPNPE-amide peptide coupled to carrier and the antibody was affinity purified (Abclonal). To avoid interferring the coupling, the original C at 602 was replaced by S. Fluorescent secondary antibodies are Alexa Fluor Dyes (Invitrogen, 1:1000) except GFP booster Alexa Fluor 488 was used for YFP detection (Proteintech, gb2AF488-10, 1:500). Z-stacks with 200nm interval were taken by DMi8 fluorescent microscopy (Leica) using a 100× oil objective followed by deconvolution with Thunder cleaning function. Signal quantification was performed by drawing a circle around each kinetochore marked by CENP-C staining. The three continuous peak values within the circle on the interested channel were averaged and subtracted of the background values from a neighboring circle. Two repeats were performed for each immunofluorescence assay with at least 150 kinetochores from 10 cells in one repeat quantified.

Recombinant protein production

Plk1 367-603aa was expressed in E. coli at 18°C overnight. Cells were collected by centrifugation and were resuspended in lysis buffer (50mM NaP pH 7.5, 300mM NaCl, 10mM imidazole, 10% glycerol, 0.5mM TCEP, protease inhibitors). After sonication, cell lysate was centrifuged at 17,000×g at 4°C for 30min to get rid of cell debris. The lysate was loaded onto a 5ml HiTrap column and washed with IMAC binding buffer (50mM NaP pH 7.5, 300mM NaCl, 10mM imidazole, 10% glycerol, 0.5mM TCEP) and eluted with a gradient of IMAC elution buffer (50mM NaP 7.5, 300mM NaCl, 500mM imidazole, 10% glycerol, 0.5mM TCEP). The tag was removed by TEV cleavage and following dialysis the tag and TEV was removed by running the sample on a 5ml HiTrap column. The flow through was collected. The protein was concentrated and run on a Superdex 200 column equilibrated with GF buffer (50mM NaP pH 7.5, 150mM NaCl, 10% glycerol, 0.5mM TCEP). The identity of the protein was confirmed by MS analysis.

Peptide binding assay

Peptides were ordered from Peptide 2.0 Inc (Chantilly, VA, USA). The purity was 95–98% as determined by high performance liquid chromatography (HPLC) and by mass spectrometry. The protein and the peptides were extensively dialyzed against 50mM NaP, 150mM NaCl, 0.5mM TCEP, pH 7.5. All ITC experiments were performed on Auto-iTC200 (Microcal, Malvern Instruments Ltd.) at 25°C. The concentrations of peptide and protein were determined by measuring the absorbance at 280nm using a spectrometer and applying values for the extinction coefficients computed for the corresponding sequences by the ProtParam program (http://web.expasy.org/protparam/). The peptides at ~450μM or 120μM (for submicromolar affinities) were loaded into the syringe and titrated into the calorimetric cell containing the recombinant Plk1, respectively, at 35μM or 10μM. The titration sequence consisted of a single 0.4μl injection followed by 19 injections, 2μl each, with 150s spacing between injections to ensure that the thermal power returns to the baseline before the next injection. The stirring speed was 750rpm. Control experiments with the peptides injected in the sample cell filled with buffer were carried out under the same conditions. These control experiments showed heats of dilution negligible in all cases. The heats per injection normalized per mole of injectant versus the molar ratio [peptide]/[protein] were fitted to a single-site model. A 1:1 stoichiometry complex was assumed for the fitting of the ITC binding isotherms in the case of the low c-assays. Data were analyzed with MicroCal PEAQ-ITC (version 1.1.0.1262) analysis software (Malvern Instruments Ltd.).

Mass spectrometry

Eluates were reduced with 5mM DTT at 55°C for 30min, cooled to room temperature, and alkylated with 15mM iodoacetamide at room temperature for 45min in the dark. Alkylation reactions were quenched with an additional 5mM of DTT. Proteins were enriched from eluates using the SP3 method³⁵ and digested overnight in 25mM ammonium bicarbonate with trypsin for mass spectrometric analysis. Peptides were analyzed on an Orbitrap Lumos mass spectrometer (ThermoFisher Scientific) equipped with a Vanquish Neo UHPLC system (ThermoFisher Scientific). Raw data were searched using COMET in high-resolution mode³⁶ against a target-decoy (reversed)³⁷ version of the human proteome sequence database (UniProt; downloaded 2/2020, 40704 entries of forward and reverse protein sequences), requiring fully tryptic peptides (K, R; not preceding P) with up to three mis-cleavages. Static modifications included carbamidomethylcysteine and variable modifications included oxidized methionine and S/T/Y phosphorylation. Searches were filtered using orthogonal measures, and quantification of LC-MS/MS spectra was performed using MassChroQ³⁸.

We thank the Protein Production and Characterization Platform at the Novo Nordisk Foundation Center for Protein Research for the production of Plk1 protein. We also thank Dr. Adrian Saurin at the University of Dundee for constructive communications. This work was supported by the National Natural Science Foundation of China (31970666) and Taishan Scholar Project (tsqn201812054) from Shandong, China. Work at the Novo Nordisk Foundation Center for Protein Research was supported by grant NNF14CC0001 and J.N. is supported by grants from the Danish Cancer Society (R269-A15586-B17), Independent Research Fund Denmark (8021-00101B and 0134-00199B) and Novo Nordisk Foundation (NNF20OC0065098). A.N.K. is supported by grant from NIH (R35GM119455).