Aurora B–dependent phosphorylation of Kif2A during cytokinesis. (A and B) Immunoblotting of GFP-Kif2A wild-type (WT) and mutant proteins that were immunoprecipitated (IP) from cells synchronized at the indicated stages of the cell cycle. Immunoprecipitated samples were subjected to Phos-tag SDS-PAGE followed by immunoblotting using anti-GFP antibody. Note that in the Phos-tag gel, mobility of GFP-Kif2A significantly decreased compared with that in a gel without Phos-tag, and the relative band position to those of molecular markers varied among experiments (see Materials and methods); however, the identical band pattern was reproducibly observed for GFP-Kif2A (n = 2 for each). Arrows indicate upper bands corresponding to phosphorylated GFP-Kif2A. (C) Immunostaining of MTs in cells expressing GFP-Kif2A mutants. (D) Normalized fluorescence intensity of GFP at the central spindle of the cells expressing GFP-Kif2A mutants. (E) Central spindle length in cells expressing GFP-Kif2A mutants. The mean ± SE of at least six cells from two independent experiments is shown in D and E. Asterisks indicate statistically significant differences from the control cells expressing wild-type GFP-Kif2A (P < 10−3, t test), or between the cells expressing GFP-Kif2A (T97A) and GFP-Kif2A (T97A/S157C; P < 0.05). (F and G) Destabilization and oscillation of the central spindle and cytokinesis failure upon GFP-Kif2A (T97A/S157C) or mKate2-Kif2A (T97A/S157C) expression. Arrows and arrowheads indicate the oscillating central spindle and the furrow edge, respectively. (H–J) Frequency of central spindle destabilization, furrow regression, or uneven chromosome distribution in cells expressing GFP-Kif2A mutants. For each mutant, ≥18 cells from seven independent experiments were analyzed. Cells expressing GFP-Kif2A mutants at a comparable level were used for quantification (mean GFP intensity of 100–300 or 100–400 arbitrary units for D and E or H–J, respectively; see also Fig. S3, C and D). Bars, 5 µm.
