Fig 2: Aurora B phosphorylates CIT-K. (a) Schematic diagram of CIT-K structure illustrating the phosphorylated sites identified by MS. The GST- tagged fragments used for the in vitro phosphorylation assays shown in (b), (c) and (d) are depicted at the bottom. (b) GST-tagged CIT-K polypeptides, GST alone and the positive control MBP were incubated with (+) or without (-) recombinant Aurora B in the presence of [?-32P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at -80°C. The Ponceau S staining of the protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. (c) GST-tagged wild-type CIT-K-CC1 (WT) and S699A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (-) recombinant Aurora B in the presence of [?-32P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at -80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. (d) The GST-tagged wild-type CIT-K-C1+PH peptide (WT), along with the S1385A-S1386A-T1387A (TripleA) and S1474A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (-) recombinant Aurora B in the presence of [?-32P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at -80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. (e) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with an siRNA directed against the CIT-K 3'-UTR for 48 h, blocked in metaphase by thymidine/nocodazole block, released for 90 min and then treated with 10 µM RO3306 for further 15 min. Proteins were extracted and used in a pull-down assay with anti-Flag antibodies. The extracts and pull downs were analysed by western blot to detect KIF14, KIF23, Aurora B and Flag::CIT-K. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. (f) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with siRNAs directed against either a random sequence (control) or 3'-UTR CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). All images are maximum intensity projections of the three most central z sections; z step = 0.25 µm. Scale bars, 10 µm. (g) Quantification of CIT-K midzone localization from the experiments showed in (f). No less than 50 early–mid-telophase cells were counted in each experiment, n = 4. Bars indicate standard errors.

Fig 3: Cartoon illustrating the cross-regulation between Aurora B and CIT-K in midbody assembly. The red arrows indicate the phosphorylation of the CIT-K CC1 and C1 domains by Aurora B and the phosphorylation of the INCENP TSS motif by CIT-K. In the presence of CIT-K (a), midbody components including the CPC, centralspindlin, KIF20A and KIF14 are properly aligned along the midbody. By contrast, after depletion of CIT-K (b) KIF14 is not recruited to the midbody [12,14], the CPC and KIF20A are dispersed and centralspindlin is mis-positioned on one side of the midbody.