Fig. 11.

Phosphorylation of S894 on INCENP is involved in activation of Aurora-B kinase. (A and B) HeLa-S3 clones inducibly expressing 6Myc-INCENP-WT (clone #13), -S894A (clone #8), -S894D (clone #13), or vector alone were fixed and co-immunostained with anti-pS894 (red) and anti-Myc (green) antibodies. DNA was stained with Hoechst 33258 (blue). Representative images of metaphase cells (A) and telophase cells (B) were captured by confocal laser scanning microscopy. Images with enhanced signal intensity are shown below the original images. Arrowheads and asterisks in A show cells successfully expressing 6Myc-INCENP-WT, -S894A, -S894D, or vector alone and cells failing to express them, respectively. Endogenous INCENP-pS894 signals were suppressed by the expression of the 6Myc-INCENP-S894A or -S894D mutant localized to chromosomes (note that neither the INCENP-A894 nor the -D894 mutant are recognized by the pS894 antibody, whereas both 6Myc-INCENP-WT-pS894 and endogenous INCENP-pS894 are recognized) (A, left panels). The replacement rate of ectopic 6Myc-INCENPs to endogenous INCENP at midbody was lower than that at kinetochores (B, left panels, insets). Insets in B show magnified images of the midbody. Scale bar, 10 μm. (C) HeLa-S3 clones inducibly expressing 6Myc-INCENP-WT, -S894A, -S894D, or vector alone were fixed and co-immunostained with anti-pT232 (red) and anti-Myc (green) antibodies. DNA was stained with Hoechst 33258 (blue). Arrowheads indicate the position of the metaphase plate. Arrows and asterisks show cells successfully expressing 6Myc-INCENP-S894A and cells failing to express this protein, respectively. Scale bar, 10 μm. (D) HeLa-S3 cells inducibly expressing 6Myc-tagged INCENP-WT, -S894A, -S894D, or vector alone were transfected with INCENP siRNA and fixed 1 h after release from nocodazole arrest, followed by co-immunostaining with anti-pT232 (red) and anti-Myc antibodies (green). DNA was stained with Hoechst 33258 (blue). Scale bar, 10 μm.