PP2A–B56γ is a KIF4A pT799 phosphatase. (A) Purified PP2A–B55α, –B56γ, and -β holoenzymes were tested for activity against recombinant KIF4A pT799, PRC1 pT481, or pT602 at 4°C for 0–60 min. Samples were blotted for the total and phosphorylated forms of the proteins. (B) Blots of purified PP2A holoenzymes and total cell lysate. (C) PP2A–B55α, –B56γ, and -β activity toward KIF4A pT799 is plotted on line graphs (means ± SEM, n = 2). Signal intensity for each time point (I_t) is plotted relative to t = 0 as I_t/I_t=0. (D) Kinesin ATPase assays were performed in the presence or absence of microtubules (±MT) using 100 nM KIF4A, Aurora B–phosphorylated KIF4A (KIF4AP), and KIF4AP treated with 1 nM PP2A–B56γ or B55α. Blotting confirmed the phosphorylation status of KIF4AP after 30-min treatment with PP2A–B55α or –B56γ in the presence (+) or absence (−) of okadaic acid (OA). White lines indicate that intervening lanes have been spliced out. (E) HeLa cells were treated with PP2A–B56 regulatory subunit siRNA for 24 h and then arrested with thymidine for 18 h. After 10 h, these cells were stained for DNA, mouse anti–Aurora B, and rabbit anti-KIF4A pT799. Arrows mark the central spindle pT799 signal. (F) Increased KIF4A pT799 staining relative to the control value is plotted in the bar graph (means ± SEM, n = 3 measuring >80 cells per condition).