Fig. 4: Aurora B switches substrate specificity to phosphorylate TopoIIα.

a Immunofluorescent images of DLD1 cells stained for TopoIIα phospho-S29 (magenta), a-tubulin (green) and DNA (blue). Images are representative of three independent experiments. Scale bar = 5 μm. b Representative still images of DLD1 cells stained with TopoIIα pS29 (magenta), a-tubulin (green) and DNA (blue) after 30 min treatment with the PKCε inhibitor BLU577 (500 nM) or 15 min Aurora B inhibition with ZM447439 (2 μM). Quantification of integrated pixel intensity measurements of TopoIIα pS29. Graph shows mean ± SEM of three experiments where 20 cells were scored per condition (one-way ANOVA; **P = 0.0034 ****P = ≤0.0001; Control 1 ± 0, BLU577 0.5967 ± 0.08, ZM447439 0.18 ± 0.04) Scale bar = 5 μm. c Peptide array (n = 1) of full length TopoIIα with recombinant Aurora B wild type (top panel) or Aurora B S227A (lower panel). d Quantification of TopoIIα array spot intensity for S29 and T1460 peptides. Graph represents mean ± SD of the intensity of spots highlighted in red in c (n = 3 spots for S29, n = 4 spots for T1460). (two-tailed Students’ t test; ****P = 5.17 × 10⁻⁶(S29), P = 1.4 × 10⁻⁸ (T1460); S29: Aurora B WT 1.45 ± 0.34, S227A 0.12 ± 0.03, T1460: WT 0.2 ± 0.05, S227A 1.47 ± 0.13). Spot densitometry analysed by ImageJ software. e In vitro kinase assay of TopoIIα with Aurora B wild type or S227A kinase. TopoIIα was immunoblotted for phosphorylation at residues S29 and T1460. Immunoblot is representative of four independent experiments. f Representative images of DLD1 cells induced to express GFP-PKCε WT or D383/451N mutant stained for TopoIIα pS29 (magenta) and tubulin (green). Quantification of integrated pixel intensity measurements of TopoIIα pS29. Graph shows mean ± SEM of three experiments where ≥20 cells were scored per condition (two-tailed Students’ t test; **P = 0.009) Scale bar = 5 µm. g Representative images of DLD1 cells induced to express GFP-Aurora B WT or S227A mutant stained for TopoIIα pS29 (magenta) and GFP-Aurora B (green). Quantification of integrated pixel intensity measurements of TopoIIα pS29. Graph shows mean ± SEM of three experiments where ≥10 cells were scored per condition (two-tailed Students’ t test; ***P = 0.0008) Scale bar = 5 µm.