Volume 14, Issue 5, Pages 787-797 (May 2008) Myosin Phosphatase-Targeting Subunit 1 Regulates Mitosis by Antagonizing Polo-like Kinase 1  Shigeko Yamashiro, Yoshihiko Yamakita, Go Totsukawa, Hidemasa Goto, Kozo Kaibuchi, Masaaki Ito, David J. Hartshorne, Fumio Matsumura  Developmental Cell  Volume 14, Issue 5, Pages 787-797 (May 2008) DOI: 10.1016/j.devcel.2008.02.013 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Identification of the Mitosis-Specific Phosphorylation Sites of MYPT1 (A) Phosphopeptide map analyses of wild-type (a), as well as single-point mutants of S432A (b), S473A (c), S601A (d), and a triple mutant (e). M2/3 spot disappeared by S432A (Ser432 replaced with Ala) mutation (b), M5 by S473A mutation (c), and M1 by S601 mutation (d). M1, M2/3, and M5 all disappeared by triple mutation (e). X is a Xenopus-specific spot. The directions of the first dimension (electrophoresis; EP) and the second dimension (thin layer chromatography; TLC) are indicated by arrows. (B) In vitro phosphorylation of MYPT1 by cdc2. Upper panel, autoradiography; lower panel, Coomassie brilliant blue staining. (C) Phosphopeptide map analyses of MYPT1 phosphorylated in vivo (a) and phosphorylated in vitro by cdc2 (b). Phosphorylation at the M2/3 site sometimes gave two spots as shown here (Totsukawa et al., 1999). (D) Diagram of rat MYPT1 indicating locations of the mitosis-specific phosphorylation sites. Developmental Cell 2008 14, 787-797DOI: (10.1016/j.devcel.2008.02.013) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Phosphorylation-Dependent Association of MYPT1 and PLK1 (A) Polo box domain binding motif and sequence alignment of various vertebrate MYPT1s at Ser473. (B) Coimmunoprecipitation of MYPT1 and PLK1. I, interphase (lanes 1 and 3); M, mitosis (lanes 2 and 4–6). MYPT1 immunoprecipitates (IP) (lanes 3 and 4) were blotted with anti-MYPT1, PLK1, PP1C, and Aurora B antibodies. PLK1 IP (lane 5) was blotted with anti-MYPT1, PP1C, and PLK1 antibodies. EB1 immunoprecipitates (lane 6) as a negative control. (C) Dot-blot analyses. Binding of GST-tagged PBD or mutant PBD to phosphopeptides was detected by an anti-GST antibody. (D) GST pull-down assay. The central domain (388–636) of MYPT (lanes 4 and 5) and its mutant (Ser473A, lane 6) were phosphorylated with (lanes 5 and 6) or without (lane 4) cdc2, and their binding to GST-PBD was examined by western blotting. Lanes 1–3, GST alone as a control. (E) Far-western ligand binding assay. Binding of GST-PBD to unphosphorylated, control MYPT1 (lane 1) and cdc2-phosphorylated MYPT1 (lane 2) was detected with a GST antibody (upper panel). Equal loading of MYPT1 was confirmed by western blotting (lower panel). (F) Western blot analyses of an antibody against S473-phosphorylated MYPT1 (pS473-Ab, lanes 1 and 2). M, mitosis; I, interphase. Lanes 3 and 4, western blot with a general MYPT1 antibody. Developmental Cell 2008 14, 787-797DOI: (10.1016/j.devcel.2008.02.013) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Immunofluorescent Localization of S473-Phosphorylated MYPT1 during Mitosis (A) HeLa cells at prophase (a–d), prometaphase (e–h), or telophase (i–l) were stained with the anti-S473-phosphorylated MYPT1 antibody (pS473) and an anti-PLK1 antibody. The scale bar represents 5 μm. (B) Localization of phosphorylated MYPT1 at the kinetochores. Mitotic chromosome spreads were stained with pS473 and PLK1 antibodies (a–d) or pS473 and CREST antibodies (e–h) or MYPT1 and CREST antibodies (i–l). The scale bar represents 5 μm. Developmental Cell 2008 14, 787-797DOI: (10.1016/j.devcel.2008.02.013) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 MYPT1 Depletion Rescues Mitotic Arrest Caused by PLK1 Depletion (A) Western blot analyses to detect levels of MYPT1 and PLK1 of control (lane 1), MYPT1-depleted (lane 2), PLK1-depleted (lane 3), or both MYPT1- and PLK1-depleted (lane 4) SW962 cells. (B) Phase-contrast images of control (a), MYPT1-depleted (b), PLK1-depleted (c), and double-depleted (d) SW962 cells. (C) Mitotic indexes of control, MYPT1-depleted, PLK1-depleted, and double-depleted cells (200–300 cells were counted, repeated three times). (D) Percentage of multinucleate cells in control, MYPT1-depleted, PLK1-depleted, and double-depleted cells (400–500 cells were counted, repeated three times). (E) Cell proliferation analyses of control, MYPT1-depleted, PLK1-depleted, and double-depleted HeLa cells. HeLa cells were transfected at day 1 with siRNA. Note that about two thirds of PLK1-depleted cells at day 2 were mitotically arrested. Error bars for (C)–(E) are standard deviation. Developmental Cell 2008 14, 787-797DOI: (10.1016/j.devcel.2008.02.013) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Live-Cell Imaging of Control, PLK1-Depleted, Double-Depleted, and MYPT1-Depleted Cells (A) HeLa cells stably expressing histone H2B-GFP were transfected with siRNAs to deplete PLK1, MYPT1, or both and imaged using a DeltaVision Image Restoration microscope system. Five Z section images were taken at 1 min intervals and deconvoluted, and projected images are shown. The scale bar represents 10 μm. Time in minutes. (B) Orthogonal images of MYPT1-depleted cells at 90 min and 100 min. Developmental Cell 2008 14, 787-797DOI: (10.1016/j.devcel.2008.02.013) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Double Depletion of MYPT1 and PLK1 Reinstates γ-Tubulin at Centrosomes (A) Localization of γ-tubulin (red), PLK1 (green), and DNA (blue) in control (a–d), MYPT1-depleted (e–h), PLK1-depleted (i–l), and double-depleted (m–p) SW962 cells. Arrowheads indicate centrosomes. The scale bar represents 5 μm. (B) Localization of γ-tubulin (red), MYPT1 (green), and DNA (blue) in control (a) and MYPT1-depleted (b) cells. The scale bar represents 5 μm. (C) Distributions of the number of γ-tubulin-containing foci per cell for control, MYPT1-depleted, PLK1-depleted, and both MYPT1- and PLK1-depleted mitotic SW962 cells. (D) Box plot of γ-tubulin intensities of individual centrosomes of control, MYPT1-depleted, PLK1-depleted, and double-depleted cells. The upper and lower edge of each box represents upper and lower quartiles, respectively. The median is indicated by a horizontal line. Developmental Cell 2008 14, 787-797DOI: (10.1016/j.devcel.2008.02.013) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Restoration of Cdc25c Phosphorylation by Double Depletion and Increase in Thr210 Phosphorylation by MYPT1 Depletion (A) Cdc25c phosphorylation in control (lane 3), MYPT1-depleted (lane 4), PLK1-depleted (lane 5), and double-depleted (lane 6) mitotic cells was immunoblotted with the anti-Cdc25c antibody. The mobility of interphase Cdc25c is shown in lane 1 for comparison with that of nocodazole-arrested, mitotic Cdc25c (lane 2). (B) Double-label immunofluorescence of control and MYPT1-depleted cells stained with anti-phospho-Thr210 (red) and anti-PLK (green) antibodies. The scale bar represents 5 μm. (C) Box plot revealing an increase in Thr210 phosphorylation by MYPT1 depletion. Ratios (pThr210/PLK1) of staining intensities on centrosomes (n = 52 for control, n = 88 for depleted cells) were analyzed. The upper and lower edge of each box represents upper and lower quartiles, respectively. The median is indicated by a horizontal line. (D) Western blot of total lysates from control and MYPT1-depleted cells. (E) Western blot of PLK immunoprecipitates from control and MYPT1-depleted cells. Kinase activities were assayed by autophosphorylation of PLK1 (Auto-P) or phosphorylation of casein (Casein-P). (F) In vitro phosphatase assay by MYPT1/PP1. Baculovirus-expressed PLK1 (20 ng) was incubated at 30°C for 20 min without (lane 1) or with 8 ng of MYPT1/PP1 (lane 2), and blotted with the anti-phospho-Thr210 antibody. Developmental Cell 2008 14, 787-797DOI: (10.1016/j.devcel.2008.02.013) Copyright © 2008 Elsevier Inc. Terms and Conditions