A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B

Abstract

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Figure 1.

Crystal structure of the MPS1 TPR domain. (a) Crystal structure of the TPR domain. A cartoon diagram of the three TPR1–3 helical doublets forming the concave surface is shown in blue shades that fade toward gray form the N toward the C terminus; the C-terminal helix is in gray, and the α2′ short helix between TPR1 and TPR2 is in cyan. (b) A side view of the TPR domain. (c) A surface representation of the TPR domain colored by sequence conservation among vertebrate MPS1 TPR domains; the top view emphasizes the conservation of the concave inner surface, and the bottom view shows some conserved patches on the generally unconserved outer surface. (d) The TPR domains of MPS1, BUBR1, and BUB1 are shown in the same orientation after structural superposition, as cartoon diagrams within a transparent surface. (e) The sequence alignment resulting from the structural superposition of the three TPR domains above is shown together with secondary structure elements. Dots indicate gaps. Loops indicate helices.

Figure 2.

MPS1 kinetochore localization is mediated by the NTE-TPR module. (a) Schematic representation of the domain organization of various MPS1 proteins used throughout this study. (b) Immunoblot of whole-cell lysates from mitotic HeLa Flp-in LAP-MPS1 cell lines that were transfected with mock or MPS1 siRNA and induced (+ doxycycline) to express the indicated LAP-MPS1 proteins; band intensity of MPS1/tubulin relative to mock is indicated. (c) Immunolocalization of LAP-MPS1^1–192 and centromeres (CREST) in nocodazole-treated, MPS1-depleted HeLaK FRT TetR cells. Cells were imaged for prophase figures. DNA (DAPI) is shown in blue. Insets show magnification of the boxed regions. (d and e) Representative images (d) and quantification (e) of immunolocalization of the various LAP-MPS1 proteins and centromeres (CENP-A) in nocodazole, 500 nM reversine, and MG132-treated, MPS1-depleted Flp-in HeLa cells. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in e displays total kinetochore intensities (±SD) of the indicated LAP-MPS1 proteins relative to centromeres (CENP-A) in cells treated as in d. Data are representative of three experiments. Ratios for LAP-MPS1^WT are set to 1. One dot represents one cell. Line indicates means ± SD. ***, P < 0.0001; significant (Student’s t test, unpaired). Bars, 5 µm. WT, wild type; Tub, tubulin.

Figure 3.

The NTE-TPR module is essential for mitotic checkpoint activity. (a) Mitotic index from flow cytometric analysis of MPM-2 positivity within a population of cells transfected with mock or MPS1 shRNA plasmids along with the indicated RNAi-resistant MPS1 alleles and treated with nocodazole for 16 h. Graph represents means of at least five independent experiments (±SEM); mean for LAP-MPS1^WT reconstitution is set to 1. (b) Time-lapse analysis of duration of mitotic arrest in nocodazole-treated Flp-in HeLa cells transfected with mock or MPS1 siRNA and expressing the indicated LAP-MPS1 proteins (induced). Data indicate cumulative percentage of cells (from a total of ≥100 cells) that exit mitosis (scored as cell flattening) at the indicated times after nuclear envelope breakdown (NEB) and are representative of at least two independent experiments. Data for mock siRNA–treated cells and MPS1 siRNA–treated cells expressing LAP-MPS1^WT overlap. (c) Immunolocalization of the indicated LAP-MIS12-MPS1 proteins and centromeres (CREST) in nocodazole-treated HeLa cells transfected with MPS1 siRNA for 48 h. M12, MIS12. DNA (DAPI) is shown in blue. Bar, 5 µm. A schematic representation of the LAP-MIS12-MPS1 protein is depicted. (d) Mitotic index from flow cytometric analysis as in a. Graph represents means of at least two independent experiments (±SEM); mean for LAP-MPS1^WT reconstitution is set to 1. WT, wild type; KD, kinase dead.

Figure 4.

NTE-mediated MPS1 localization depends on the NDC80 complex. (a, b, and e–h) Representative images (a, e, and g) and quantification (b, f, and h) of immunolocalization of LAP-MPS1^WT or LAP-MPS1^ΔTPR and centromeres (CREST) in Flp-in HeLa cells transfected with siRNAs to MPS1 and luciferase (mock), HEC1, NUF2, or KNL1 and treated with nocodazole and reversine. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graphs display total kinetochore intensities (±SEM) of the indicated proteins relative to centromeres (CREST). Data are from ≥21 cells from at least two independent experiments. Ratios for mock RNAi–treated cells are set to 1. (c and d) Representative images (c) and quantification (d) of immunolocalization of HEC1, KNL1, and centromeres (CREST) in HeLa cells transfected with mock or KNL1 siRNAs and treated with nocodazole. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in b shows total kinetochore intensities (±SD) of HEC1 and KNL1 relative to centromeres. Data are from ≥13 cells and are representative of three experiments. Ratios for mock RNAi–treated cells are set to 1. Bars, 5 µm. WT, wild type.

Figure 5.

The microtubule-binding domain of HEC1 directs MPS1 localization and function. (A) Time-lapse analysis of duration of mitotic arrest in nocodazole- and ZM447439 (ZM)-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA and expressing the indicated GFP-HEC1 proteins. Data indicate cumulative percentages of cells (from a total of ≥125 cells per treatment) that exit mitosis (scored as cell flattening) at the indicated times after NEB and are representative of three independent experiments. (b–d) Representative images (b) and quantification (c and d) of immunolocalization of MPS1, the indicated GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in c displays total kinetochore intensities (±SEM) of the indicated proteins relative to centromeres (CREST). Data are from a total of ≥103 cells per treatment from two experiments. Ratios are set to 1 for mock RNAi–treated cells (MPS1) and for GFP-HEC1^WT–expressing cells (GFP-HEC1). Graph in d displays total kinetochore intensities of the indicated proteins relative to centromeres (CREST) for all cells of a single experiment. (e and f) Representative images (e) and quantification (f) of immunolocalization of MPS1, the indicated LacI-GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated U2OS-LacO cells. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in f displays total intensities (±SEM) of MPS1 at LacO arrays relative to LacI-GFP-HEC1 (GFP) and total intensities of LacI-GFP-HEC1. Data are from a total of ≥17 cells from two experiments. Ratios for LacI-GFP-HEC1^WT–expressing cells are set to 1. Bars, 5 µm. WT, wild type; a.u., arbitrary unit.

Figure 6.

Aurora B regulates MPS1 kinetochore localization by controlling function of the TPR domain. (a and b) Representative images (a) and quantification (b) of immunolocalization of LAP-MPS1^1–192 and centromeres (CENP-A) in prophase HeLaK FRT TetR cells depleted of MPS1 and treated with ZM447439, as indicated. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in b shows total kinetochore intensities (±SEM) of MPS1 relative to centromeres. Data are from ≥38 cells from two experiments. Ratios for mock-treated cells are set to 1. (c and d) Representative images (c) and quantification (d) of immunolocalization of the indicated LAP-MPS1 proteins and centromeres (CENP-A) in MPS1-depleted HeLaK FRT TetR cells treated with nocodazole and reversine, with or without ZM447439. DNA (DAPI) is shown in blue. Insets are magnifications of the boxed regions. Graph in d shows total kinetochore intensities (±SEM) of MPS1 relative to centromeres in DMSO-treated (gray bars) or ZM447439-treated (blue bars) cells. Data are from ≥32 cells from two experiments. Ratios for mock-treated, LAP-MPS1^WT–expressing cells are set to 1. (e) Time-lapse analysis of the duration of mitotic arrest in HeLaK FRT TetR cells transfected with mock or MPS1 siRNA and expressing the indicated LAP-MPS1 proteins and treated with nocodazole and DMSO (top) or nocodazole and ZM447439 (ZM; bottom). Data indicate cumulative percentage of cells (from a total of ≥70 cells) that exit mitosis (scored as chromosomal decondensation) at the indicated times after NEB and are representative of at least two independent experiments. (F) Model of regulated MPS1 localization at unattached kinetochores. See Discussion for details. Bars, 5 µm. WT, wild type.