Aurora B kinase targets Cdt1 for phosphorylation in vitro and affects Cdt1-MT binding. (A) Sequence alignment of Cdt1 from the indicated species using Clustal Omega. Highlighted in the red font are the potential Ser/Thr Aurora B sites. On the top, mentioned are the residues and their positions with respect to the full-length human Cdt1 (1–546 aa). (B) In vitro kinase assay with Aurora B alone or kinase plus ZM447439 inhibitor (10 µM) on HEK 293–purified Cdt1 or hVimentin (as a positive control). The autoradiogram (top) and Coomassie-stained gel (bottom) are shown. M, migration of molecular mass standards in kilodaltons on a 12% SDS-PAGE gel. (C) Pulldown of Aurora B by HA/His-tagged Cdt1-WT or cy-Cdt1 mutant that blocks cyclin A/Cdk binding (RRL [68–70] AAA), from thymidine synchronized and nocodazole arrested mitotic HeLa cell extracts. The pulldown was performed using Ni⁺²-NTA agarose beads followed by immunoblotting with either anti-HA or anti–Aurora B (AurB-K) antibodies. 1% of the lysate was loaded as total protein. HEK cells expressing GFP were used as a control. (D) HeLa cells were either treated with Aurora B inhibitor, ZM447439 (5 µM), for 1 h or left untreated (control) followed by release into MG132 (10 µM) containing medium in each case. The cells were immunostained with anti-Cdt1 (red), anti-tubulin (green) antibodies and counterstained with DAPI to mark the chromosomes (blue); scale bar, 5 µm. (E) n = 10 cells were quantified to obtain normalized Cdt1 fluorescence intensities on spindle MTs as depicted in D. P < 0.0001 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. (F) Schematic representation showing the full-length Cdt1 (546 aa) and its deletion variant devoid of N-terminal 92 aa generated in fusion with GFP tag for expression in bacteria. The following two proteins generated represent Aurora B phosphomimetic (8D, Ser/Thr substituted with Asp) and phospho-deficient (8A, Ser/Thr substituted with Ala) Cdt1 mutants. Black asterisk, confirmation of phosphosites by mass spectrometry; red asterisk, residues that were inherently phosphorylated in Cdt1 obtained from HEK 293 cells. (G) Representative Western blot is shown. Samples fractionated as supernatant (S) and pellet (P) analyzed by Western blot probed with antibody against 6×-His tag to detect Cdt1 and stained with Ponceau S for tubulin. (H) Quantification (mean ± SD, n = 3) showing MT cosedimentation of purified Cdt1^92–546 or the mutant proteins (1 µM each) with the indicated concentrations of taxol-stabilized MTs (in μM). The 95% CI values obtained for each fit were: B_max = 0.34–0.65, K_d = 0.43–10.57 for 8D; B_max = 0.36–0.67, K_d = 0–3.22 for 8A.