Fig. 7: Bora binds AURKA through two Tpx2-like motifs that position a phospho-motif in the kinase active site.
a Zoom-in view of the binding interface between Tpx2 and AURKA (PDB: 1OL5). The T-loop phospho-residue Thr288, sulfate ion (yellow), and the phospho-T-loop and sulfate ion coordinating residues of AURKA (H176, R180, R255, and R286) are highlighted in stick representation. b Binding of fluorescein-labeled Tpx21–43 polypeptide to AURKA mutants assessed by monitoring the fluorescence polarization signal in the presence of increasing concentrations of AURKA mutants. Kd values represent mean value with standard deviations of the mean as error bars (n = 3 independent experiment samples). c Western blot analysis of 2-step kinase reactions carried out with Bora18-120 [S27A T52A] phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Plk1K82R KD (substrate) and AURKAWT or mutated versions as indicated (step 2). Blots were probed with antibodies to Bora, AURKA, and phosphoT210 Plk1 or pan Plk1 as indicated (from top to bottom). d Competitive displacement of fluorescein-labeled Tpx21–43 from the indicated AURKA mutants by increasing concentrations of pBora1–224 as monitored by fluorescence polarization. Displayed data points and the half maximal inhibitory concentration (IC50) value represent the average normalized fluorescence polarization signal for each reaction conditions (starting binding signal = 100%, end titration binding signal =0%) (n = 3 independent experiment samples). e Theoretical/illustrative model of pBora binding to the kinase domain of AURKA. AURKA is shown in gray surface with helix αC highlighted in yellow. Bora motifs M1 (blue) and M2 (purple) were modeled by two corresponding motifs from Tpx2 and the phospho-motif M3 (pink) was modeled by the TSS motif from phospho-INCENP.
