Fig 2: CENP-R is a bona fide substrate of Aurora B. (A) Mass spectrometric identification of CENP-R Ser28 phosphorylation by Aurora B kinase. (B) In vitro kinase assay using Aurora B kinase to phosphorylate GST-CENP-R-WT and the S28A mutant. An autoradiograph showing incorporation of radioactive γ-32P-ATP (top) and CBB staining of purified proteins (bottom). (C) Real-time imaging of chromosome movements in HeLa cells co-transfected with mCherry-H2B and CENP-R-GFP (WT, S28A, and S28D) constructs in the absence of endogenous CENP-R. Chromosomes were marked by mCherry-H2B. Arrow indicates misaligned chromosome. Time is indicated in minutes. Scale bar, 10 μm. (D) Bar graph illustrating percentage of cells with chromosome alignment defects in C. An average of 50 GFP- and mCherry-positive mitotic cells from three separate experiments were surveyed. Statistical significance was tested by two-sided t-test and represented by *P < 0.05 and ****P < 0.0001; n = 3. Bars indicate mean ± SD.

Fig 3: Working model accounting for function of Aurora B phosphorylation of CENP-R in the kinetochore–microtubule attachment. During prometaphase, when the aberrant kinetochore microtubule attachment is detected, Aurora B phosphorylates CENP-R. Then, phosphorylated CENP-R detaches from kinetochore–microtubule binding interface. The wrong attachments are corrected and the cell re-establishes correct bipolar attachment with proper tension. On the contrary, when phospho-mimetic CENP-R-S28D mutant is persistently expressed, the kinetochore–microtubule attachments are unstable. The correct attachment with tension cannot be established. In the scenario of CENP-R depletion, the kinetochore–microtubule attachments are also unstable. Both expression of CENP-R-S28D and depletion of CENP-R cause chronic activation of the SAC.

Fig 4: Phosphorylation of CENP-R by Aurora B regulates mitotic spindle plasticity. (A) CENP-R is essential for inter-centromere tension during mitotic chromosome segregation. shControl-treated cells and ShCENP-R-treated cells were immuno-stained for centromere (ACA; red), CENP-R (green), and DNA (blue). Note that shRNA suppressed the expression level of CENP-R. Scale bar, 10 μm. (B) Quantitation of inter-centromere distance marked by ACA staining in A. Centromere distance is measured between kinetochores that are marked by ACA staining and localized in the same focal plane as described under ‘Materials and methods’. Each value from treated samples was calculated from >100 kinetochores selected from at least 10 different cells. Statistical significance was tested by two-sided t-test and represented by **P < 0.01; n = 3. Bars indicate mean ± SD. (C) Representative images of cells treated with shCENP-R and rescued with CENP-R-GFP (WT, S28A, and S28D). Before fixation, cells were treated with cold DMEM at 4°C for 10 min. Then, cells were fixed and stained for microtubule (red), ACA (cyan), and DNA (blue). Scale bar, 10 μm. (D) Quantification of the level of unaligned kinetochores in cells treated as in C (arrow). Bars represent mean ± SD of three independent experiments. In each experiment, 10 cells were measured (>30 kinetochores per cell). Statistical significance was tested by Student's t-test and represented by **P < 0.01 and ****P < 0.0001. (E) In vitro pull-down assay of GST-CENP-R (WT, S28A, and S28D) with MBP-CENP-U-His. The gel was stained with CBB (upper panel). Lower panel shows western blotting analysis with anti-His antibody.

Fig 5: Phosphorylation of CENP-R by Aurora B destabilizes CENP-R localization to the centromere. (A) Phosphorylation of CENP-R by Aurora B attenuates the localization of CENP-R to the centromere. HeLa cells were transfected with ShCENP-R along with CENP-R (WT, S28A, and S28D) constructs. At 48 h post transfection, cells were fixed and stained with CENP-U (red), centromere (ACA; cyan), and DAPI (blue). Scale bar, 10 µm. (B) Evaluation of CENP-R, CENP-U, and Hec1 protein expression levels in the experiment shown in A. (C) Aurora B localization to the centromere is independent of CENP-R, according to the statistical analysis of Aurora B localization in the CENP-R-deficient cells. Statistical significance was tested by two-sided t-test and represented by ****P < 0.0001; n.s., not significant; n = 3. Bars indicate mean ± SD. (D) Statistical analyses of CENP-U and Hec1 intensity at the centromere/kinetochore of CENP-R-GFP-expressing cells (WT, S28A, and S28D). Note that the level of centromere-associated CENP-R-GFP is low in the S28D-expressing cells. An average of 60 mitotic cells from three separate experiments were surveyed. Statistical significance was tested by two-sided t-test and represented by ***P < 0.001; n = 3. Bars indicate mean ± SD.