Fig. 4: K63-linked ubiquitination at Lys202 is required for Aurora B kinase activity.

a, b K202 is the main ubiquitination site on Aurora B. HEK293T cells were co-transfected with the indicated plasmids and treated with 30 ng/mL NOC for 16 h. Mitotic cells were subjected to Ni-NTA pull-down under denaturing conditions. V5-His-Aurora B pull-downs were analyzed by immunoblotting with the indicated antibodies. c Immunoprecipitations used in the in vitro kinase assay. FH-Aurora B or FH-Aurora B-K202R was purified from mitotic HEK293T cells using Flag-M2 agarose beads, resolved by SDS–PAGE and silver staining (upper), and immunoblotted with anti-K63-Ub antibody (bottom). d In vitro kinase assay was performed using the purified Aurora B complex shown in c, in the presence of ATP. H3 was used as the substrate of Aurora B. e–i Aurora B-K202R has lower kinase activity in vivo. Representative confocal images showing Aurora B-pT232 (e) or H3-pS10 (g) intensity in cells transfected with pEGFP-Aurora B or pEGFP-Aurora B-K202R. Endogenous Aurora B was silenced by siAurora B in HeLa cells, followed by co-transfection with the indicated plasmids. Then cells were synchronized with 30 ng/mL NOC, fixed, and stained with the indicated antibodies. Aurora B-pT232, red; CREST, purple; DAPI, blue; H3-pS10, red. Bar, 5 μm. f, h Box-and-whiskers plots showing the quantification of the relative intensity of Aurora B-pT232 or H3-pS10 as shown in e and g, respectively. Intensities were normalized to the general kinetochore marker CREST. Boxes show the upper and lower quartiles with a line at the median. Whiskers as the extent of 100% of the data. Data are shown as means ± SD (n = 15 cells, and six areas for each cell were counted). **p < 0.01 versus CT, one-way ordinary ANOVA test, analyzed by GraphPad Prism 8. Experiments were repeated at least three times. i Protein samples from cells treated as shown in e and g were analyzed by western blotting. Actin was used as a loading control.