FIGURE 4.

Phosphorylation of Sds22 by PLK1 is essential for Aurora B kinase activity. A, schematic illustration of experimental design. B, HeLa cells expressing the centromere-targeted Aurora B kinase sensor were imaged at metaphase. Aurora B kinase sensor is responsive to Aurora B inhibitor Hesperadin. PLK1 inhibitor BI 2536 also modulates Aurora B kinase activity in mitosis. Scale bar, 10 μm. C, HeLa cells expressing the centromere-targeted Aurora B kinase sensor and mCherry-Sds22^WT and its mutants were imaged at metaphase. Aurora B kinase sensor is responsive to mCherry-Sds22^5A. Scale bar, 10 μm. D, statistical analyses of the FRET/CFP emission ratio as in B and C indicate that mCherry-Sds22^5A decreases Aurora B activity. Error bars represent means ± S.E.; >20 cells of each categories from three different preparations. Student's t test was used to calculate p value for comparison. *, p < 0.05; **, p < 0.01. E, Western blotting analysis of the level of Ser(P)⁷-CENP-A indicates that expression of Sds22^5A suppresses Aurora B kinase activity. F, statistical analyses of band intensity of CENP-A Ser(P)⁷ as in E indicate that expression of Sds22^5A decreases Aurora B activity. Western blot signal intensities were quantified using ImageJ software. The average protein band intensities were measured, and the background intensities were subtracted. The CENP-A Ser(P)⁷ intensities were then normalized against CENP-A values to account for any variations in immunoblot acquisition. Error bars represent means ± S.E. from three different preparations. Student's t test was used to calculate p value for comparison. *, p < 0.05; **, p < 0.01; ***, p < 0.001.