Figure 9.
Multisite phosphorylation of NDC80 by Aurora A is counteracted by PP1/PP2A. (A) Parental and PPP6C KO cell lines were treated with STLC to arrest cells in mitosis in the absence (Control) and presence of PP1/2A-i and then stained for NDC80 pS55, CENP-A, and DNA. (B) Level of NDC80 pS55 signal at kinetochores (KTs) was measured for the different conditions (n = 701–951 KTs). Each mean difference is depicted as a dot. Each 95% confidence interval is indicated by the ends of the vertical error bars; the confidence interval is bias-corrected and accelerated. (C) The number of NDC80 pS55 positive kinetochores was measured for the different conditions (mean ± SD; n = 12–15). Statistical significance was analyzed using a Brown-Forsythe ANOVA (**, P < 0.01). (D and E) Mitotic lysates of parental and PPP6C KO HeLa cells treated with Aurora A (AurA-i), Aurora B, and phosphatase (calyculin, PP1/2A-i) inhibitors in the combinations and order shown were blotted for the proteins listed in the figure. Overall NDC80 phosphorylation was monitored using a Phos-tag gel. (F) HeLa cells depleted of endogenous NDC80 using a 5′-UTR siRNA were transfected with NDC80-GFP WT and phospho-deficient mutant constructs as shown in the figure. Cells were stained for DNA, NDC80, and specific NDC80 phospho-antibodies. (G) Spindle size is plotted for the NDC80 WT and point mutant in panel F (mean ± SD; n = 42–58). Statistical significance was analyzed using a Brown-Forsythe ANOVA (****, P < 0.0001). (H) A schematic showing the proposed dynamic substoichiometric phosphorylation of the NDC80 N-terminus, and roles of Aurora A and PP1/PP2A. Source data are available for this figure: SourceData F9.