PP4c dephosphorylates NDEL1 at Cdk1 sites and suppresses Cdk1 activation. (A, top) We examined whether PP4c dephosphorylates phospho-NDEL1 (P-NDEL1) that was phosphorylated by GST-Cdk1 using recombinant proteins. Note the lower mobility of NDEL1 phosphorylated by GST-Cdk1. PP4c efficiently removed phosphate from one of the Cdk1 phosphorylation sites of NDEL1 (T219). (bottom) We tested whether GST-PP4c dephosphorylates P-NDEL1 phosphorylated by Aurora A kinase. GST-PP4c did not show any dephosphorylation activity at the GST–Aurora A phosphorylation site (S251). Western blotting pattern using an anti-NDEL1, an antiphospho-T219 antibody (Cdk1 site), or an antiphospho-S251 antibody (Aurora A site) is shown at the bottom of each. Note that signal by antiphospho-T219 antibody was diminished after dephosphorylation by PP4c. (B) Subcellular distribution of PP4c (left), NDEL1 (middle), and PP4R1 (right) in asynchronously growing HeLa cells at interphase or prophase (representative images of three independent experiments). Arrowheads indicate centrosomes. (C) Examination of PP4c distribution and phosphorylation of cyclin B1 (left) and NDEL1 (right). Synchronously growing HeLa cells were stained with the indicated antibodies for phosphorylated proteins. Arrowheads indicate the centrosomes (representative images of three independent experiments). (D) Persistent expression of PP4c at the centrosome prevented phosphorylation of cyclin B1 (top) and NDEL1 (middle). Synchronously growing HeLa cells transfected with constructs as indicated above the panels were costained with the indicated antibodies. Images were captured at G2 or prophase. The distances of separated centrosomes are summarized at the bottom (representative images of five independent experiments). (bottom) Statistical analysis of centrosomal distances. The p-value was calculated using an unpaired t test (*, P < 0.001; one example of three independent experiments; n = 200). Error bars represent SEM. Bars, 10 μm.