Phosphorylation of MPS1 at S281 regulates its kinetochore localization and checkpoint signaling. (A) Domain organization, functional binding sites, and mapped CDK sites of Homo sapiens MPS1. HeLa Flp-In/TREx GFP-MPS1^WT, GFP-MPS1^S281A, MPS1S^436A, or MPS1^S821A cells were depleted of endogenous MPS1. GFP-MPS1 transgenes were induced for a total of 54 h, and then the cells were treated with 0.1 µM Taxol for 2 h. To prevent exit from mitosis, 20 µM MG132 was added for the last 30 min. Cells were stained with antibodies for kinetochores (CREST). (B) HeLa Flp-In/TREx GFP-MPS1^WT, GFP-MPS1^S281A, or GFP-MPS1^S281D cells were depleted of endogenous MPS1. GFP-MPS1 transgenes were induced, and after 24 h, the cells were arrested for 2.5 h with 20 µM MG132, treated with 3 µM nocodazole for 5 min, and then stained with antibodies for MAD1 and kinetochores (CREST). Kinetochore-associated GFP-MPS1 or MAD1 normalized to the mean of the MPS1^WT condition are indicated as the mean ± SEM (n ≥ 25 cells and 15 kinetochores measured per cell in four independent experiments). (C) HeLa Flp-In/TREx GFP-Mis12-MPS1^WT or GFP-Mis12-MPS1^S281A cells were depleted of endogenous MPS1. GFP-Mis12-MPS1 transgenes were induced (+) for 24 h and the cells treated with 0.1 µM Taxol for 2 h and either DMSO (Control) or 2 µM AZ3146 (MPS1-i) for 10 min. Cells were stained with antibodies for BUBR1 and kinetochores (CREST). Mis12-MPS1 was visualized by GFP fluorescence. (D) The bar graphs show the mean levels of Mis12-MPS1^WT, Mis12-MPS1^S281A, and BUBR1 at kinetochores in both control and MPS1-inhibited cells (MPS1-i). Bars indicate the SEM (15 kinetochores per cell and 12 cells per condition in three independent experiments). (E) GFP-Mis12-MPS1 transgenes were induced for 48 h in HeLa Flp-In/TREx GFP-Mis12-MPS1^WT or GFP-Mis12-MPS1^S281A cells depleted of endogenous MPS1, and the mitotic index was scored. The graph shows the mean mitotic index from three independent experiments. Error bars indicate SEM. (F) HeLa cells expressing MPS1 GFP-MPS1^WT, MPS1^S281A, or MPS1^S281D were imaged every 2 min as they passed through mitosis. GFP-MPS1 and DNA visualized using SiR-Hoechst are shown. Arrows mark DNA bridges. (G) NEBD-anaphase duration is plotted; each point represents an individual cell with mean NEBD-anaphase time and SD shown. ****, P < 0.0001 (Student’s t test). (H) Cumulative mitotic exit frequency of cells expressing either GFP-MPS1^WT (n = 30), GFP-MPS1^S281A (n = 28), or GFP-MPS1^S281D (n = 25). (I) Simulation of checkpoint regulated mitotic exit for MPS1^WT and the MPS1^S281A/D mutants, plotted as for the experimental data in H.