Lee S , Thebault P , Freschi L , Beaufils S , Blundell TL , Landry CR , Bolanos-Garcia VM , Elowe S .

GMX-191597 Canadian Institutes of Health Research , RG44650 Wellcome Trust

	FIGURE 1. Identification of a TPR motif in the N-terminal region of Mps1. A, amino acid sequence alignment of human N-terminal Mps1 and BUB1 and BUBR1 from different species. Secondary structure elements are mapped onto the crystal structure of N-terminal BUBR1 (Protein Data Bank code 2WVI). Figure was generated with ESPript (68). B, structure model of N-terminal Mps1 predicts this region is organized as a triple tandem of the TPR motif. C, model highlighting the Mps1 residues that are conserved and located in positions that define a canonical TPR motif. Most of these conserved residues are predicted to be engaged in stabilizing stacking interactions. Purple indicates the phosphorylation site serine 80 is highlighted. D, far-UV CD confirms N-terminal Mps1 is organized as a predominantly α-helical region. Inset, the thermal denaturation of this domain is highly cooperative and follows a two-state unfolding process. E, size-exclusion chromatogram of molecular mass markers only (●): peak 1, bovine serum albumin; peak 2, ovalbumin; peak 3, chymotrypsinogen A; peak 4, ribonuclease A. For the second chromatogram (−), the same molecular mass markers were combined with Mps1(1–239) prior to gel filtration. Mps1(1–239) retention time was closed to that of ovalbumin (43 kDa) thus revealing the former self-associates to form stable dimers.
	FIGURE 2. Biophysical characterization of the N-terminal fragment Mps1(1–239). Interfacial properties of Mps1(1–239), TPR Bub1, and TPR BubR1 are shown. Surface pressure (A) and surface concentration (Γ = 0.2 Δ) (B) together with the corresponding ellipsometric angle at air/water interfaces were determined by null ellipsometry measurements. Solutions of Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●) are shown. Protein solutions were prepared at 30 μg/ml in 20 mm phosphate buffer, pH 7. C, surface pressure versus surface concentration of Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●). θ corresponds to the slope dπ/dΓ, and Γ0 is the surface concentration at which the surface pressure becomes different from zero. Γ0 is calculated from the intersect of the slope π versus Γ. Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●) at 1 μg/ml in 20 mm phosphate buffer, pH 7, are shown. D, rheology measurements of Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●). The graph shows the evolution of the shear elastic constant, μ, versus time measured at the fixed frequency of 5 Hz, during protein adsorption at the interface. Protein solutions were prepared at 30 μg/ml in 20 mm phosphate buffer, pH 7. The error bar on μ is ± 5 mN/m. Inset, at the end of the kinetic (around 9 h, indicated by the arrow in the graph) the angular deviation θ(ω) versus the pulsation was measured. The curves correspond to Mps1(1–239). An elastic layer model (harmonic oscillator) fit the imaginary and real part of the response. For clarity, the imaginary part has been plotted versus −ω.
	FIGURE 3. Mps1 TPR domain appeared after the emergence of deuterostomes. A, human Mps1 overlapping fragments (length, 160 amino acids; offset, 10 amino acids) were blasted (PSI-BLAST) against the entire NCBI sequence database (nonredundant). For each listed organism for which we could find a hit for one or more fragments, we show if the hit was highly significant (red rectangles, E-value <1e-5), close to the threshold (orange rectangles, ∼1e-5 < E-value < 1), or not significant (yellow rectangles, E-value ≥1). If we could not find a hit for a specific fragment, the corresponding rectangle is gray. The column highlighted between white lines corresponds to the exact hMps1 TPR region. B, secondary structure prediction of the TPR region of hMps1 and the corresponding region in Mps1 orthologues. Alignment and prediction were performed using PSIPRED (33).
	FIGURE 4. Localization of Mps1(1–239). A, localization of 3×MYC-Mps1 constructs. HeLa cells were transfected with 3×MYC-Mps1 WT (row i), -Mps1-KD (row ii) or Mps1(1–239) (rows iii and iv) and synchronized in mitosis by release from a single thymidine arrest before being fixed and stained for immunofluorescence with antibodies against MYC (green) and CREST (blue). The panel on the right shows the colocalization profile between MYC and CREST signal across sister kinetochore pairs (3–4 pairs). A, row iii, colocalization of one sister kinetochore pair is included, and the pair is detailed in the inset. B, immunofluorescence of cells expressing mCherry alone (row i, medium; row iii, highly overexpressing) or mCherry-Mps1(1–239) (row ii, medium; row iv, highly overexpressing) were synchronized in mitosis and fixed for immunofluorescence as in A. Cells were stained with anti-Mps1 antibodies (green) and CREST autoimmune serum (blue). C, endogenous Mps1/CREST signal ratio (arbitrary units; a.u.) in cells highly overexpressing mCherry or mCherry-Mps1(1–239), n = 9 cells each. D, cells were transfected, synchronized, and fixed as in A and were then stained for the indicated proteins. Kinetochore localization was scored for these proteins in cells overexpressing Mps1(1–239) relative to control cells.
	FIGURE 5. Functional analysis of Mps1(1–239). A, HeLa cells were transfected with mCherry or mCherry-Mps1A and then synchronized in STLC followed by release into MG132 according to the schematic. After 1.5 h of MG132 treatment, cells were fixed and processed for immunofluorescence with antibodies against α-tubulin (green) and CREST (Cy5, illustrated here in magenta). Hoechst 33342 was used to visualize the DNA (blue). Chromosome alignment at metaphase plates was examined in cells expressing either mCherry or mCherry-Mps1(1–239) and grouped into the three indicated categories based on the extent of the alignment. Only cells in which both spindle poles were visible in the same plane were counted (n = 3, ≥100 cells/experiment). B, cells transfected with either mCherry or mCherry-Mps1 were treated for 16 h either with DMSO as control or 3.3 μm nocodazole (Noco), fixed, and stained with Hoechst 33342 to visualize the DNA. The mitotic index of transfected cells was counted (n = 5, ≥100 cells/experiment). C, cells were transfected as in B but were also simultaneously transfected with siRNA oligonucleotides targeting Gl2 (as control) or CenpE. 48 h after transfection, the cells were fixed and stained as in B before mitotic index counts were performed (n = 3, ≥100 cells/experiment). D, cells were transfected with either mCherry or mCherry-Mps1(1–239) and treated with 3.3 μm nocodazole for 16 h before being fixed and stained for Mad2 (green) and CREST (Cy5, shown here in blue). E, quantification of the Mad2/CREST ratio from cells in D. F, 293T cells were transfected with 3×MYC Mps1-WT or 3×MYC Mps1ΔN and treated with nocodazole for 16 h before being harvested. Immunoprecipitated Mps1 fragments were resolved by SDS-PAGE and immunoblotted with anti-Thr(P)-676, anti-Thr(P)-686, and anti-Ser(P)-S821 Mps1 antibodies. The membranes were stripped and reprobed for MYC to demonstrate equal input.
	FIGURE 6. Model for Mps1 TPR function. A, Mps1-WT can both dimerize and dock at KTs, thus increasing its local concentration and activity. Mps1 autophosphorylation or phosphorylation of a KT protein allows for its release into the cytoplasm. B, inactive Mps1 can dimerize and localize but cannot phosphorylate itself or other targets and is thus not released from the KT as efficiently as the WT protein. C, Mps1 lacking a functional TPR cannot localize to the KT and cannot dimerize resulting in reduced concentration of Mps1 protein and reduced kinase activity. This is manifest as reduced autophosphorylation and reduced phosphorylation of potential targets. Ultimately this results in suboptimal Mps1 activity.

Abrieu, Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. 2001, Pubmed

Abrieu, Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. 2001, Pubmed
Araki, N-terminal regions of Mps1 kinase determine functional bifurcation. 2010, Pubmed
Baumann, PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint. 2007, Pubmed
Beaufils, Characterization of the tetratricopeptide-containing domain of BUB1, BUBR1, and PP5 proves that domain amphiphilicity over amino acid sequence specificity governs protein adsorption and interfacial activity. 2008, Pubmed
Beddoe, A biophysical analysis of the tetratricopeptide repeat-rich mitochondrial import receptor, Tom70, reveals an elongated monomer that is inherently flexible, unstable, and unfolds via a multistate pathway. 2004, Pubmed
Bolanos-Garcia, The conserved N-terminal region of the mitotic checkpoint protein BUBR1: a putative TPR motif of high surface activity. 2005, Pubmed
Bolanos-Garcia, BUB1 and BUBR1: multifaceted kinases of the cell cycle. 2011, Pubmed
Chan, Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly. 2009, Pubmed
Cheeseman, Molecular architecture of the kinetochore-microtubule interface. 2008, Pubmed
D'Andrea, TPR proteins: the versatile helix. 2003, Pubmed
D'Arcy, Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition. 2010, Pubmed
Das, The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions. 1998, Pubmed
de Bakker, HOMSTRAD: adding sequence information to structure-based alignments of homologous protein families. 2001, Pubmed
Dou, Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1. 2011, Pubmed
Dubreil, Aggregation of puroindoline in phospholipid monolayers spread at the air-liquid interface. 2003, Pubmed
Elowe, Tension-sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore microtubule interactions. 2007, Pubmed
Fava, Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint. 2011, Pubmed
Gouet, ESPript: analysis of multiple sequence alignments in PostScript. 1999, Pubmed
Gromiha, Inter-residue interactions in protein folding and stability. 2004, Pubmed
Hached, Mps1 at kinetochores is essential for female mouse meiosis I. 2011, Pubmed
He, Mph1, a member of the Mps1-like family of dual specificity protein kinases, is required for the spindle checkpoint in S. pombe. 1998, Pubmed
Hewitt, Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1-C-Mad2 core complex. 2010, Pubmed
Holland, Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis. 2009, Pubmed
Hübner, Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs. 2010, Pubmed
Jelluma, Chromosomal instability by inefficient Mps1 auto-activation due to a weakened mitotic checkpoint and lagging chromosomes. 2008, Pubmed
Jelluma, Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment. 2008, Pubmed
Jelluma, Release of Mps1 from kinetochores is crucial for timely anaphase onset. 2010, Pubmed
Jones, Protein secondary structure prediction based on position-specific scoring matrices. 1999, Pubmed
Jones, Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis. 2005, Pubmed
Kang, Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint. 2007, Pubmed
Kiyomitsu, Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1. 2007, Pubmed
Kiyomitsu, Protein interaction domain mapping of human kinetochore protein Blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins Bub1 and BubR1. 2011, Pubmed
Klebig, Bub1 regulates chromosome segregation in a kinetochore-independent manner. 2009, Pubmed
Laskowski, Validation of protein models derived from experiment. 1998, Pubmed
Liu, Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores. 2003, Pubmed
Lüthy, Assessment of protein models with three-dimensional profiles. 1992, Pubmed
Maciejowski, Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling. 2010, Pubmed
Martin-Lluesma, Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. 2002, Pubmed
Mattison, Mps1 activation loop autophosphorylation enhances kinase activity. 2007, Pubmed
Mizuguchi, JOY: protein sequence-structure representation and analysis. 1998, Pubmed
Niault, Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I. 2007, Pubmed
Olsen, Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. 2010, Pubmed
Renault, Surface-induced polymerization of actin. 1999, Pubmed
Rieder, Stuck in division or passing through: what happens when cells cannot satisfy the spindle assembly checkpoint. 2004, Pubmed
Sali, Comparative protein modelling by satisfaction of spatial restraints. 1993, Pubmed
Santaguida, Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. 2010, Pubmed
Santaguida, The life and miracles of kinetochores. 2009, Pubmed
Saurin, Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis. 2011, Pubmed
Scheufler, Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. 2000, Pubmed
Shi, FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. 2001, Pubmed
Stucke, Kinetochore localization and microtubule interaction of the human spindle checkpoint kinase Mps1. 2004, Pubmed
Tanudji, Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay. 2004, Pubmed
Thompson, Examining the link between chromosomal instability and aneuploidy in human cells. 2008, Pubmed
Tighe, Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores. 2008, Pubmed
Tyler, Phosphoregulation of human Mps1 kinase. 2009, Pubmed
Vénien-Bryan, Characterization of the growth of 2D protein crystals on a lipid monolayer by ellipsometry and rigidity measurements coupled to electron microscopy. 1998, Pubmed
Vié, Specific anchoring modes of two distinct dystrophin rod sub-domains interacting in phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS. 2010, Pubmed
Vigneron, Kinetochore localization of spindle checkpoint proteins: who controls whom? 2004, Pubmed
Weiss, The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint. 1996, Pubmed
Welburn, Toward a molecular structure of the eukaryotic kinetochore. 2008, Pubmed
Xu, Regulation of kinetochore recruitment of two essential mitotic spindle checkpoint proteins by Mps1 phosphorylation. 2009, Pubmed
Yang, Cells satisfy the mitotic checkpoint in Taxol, and do so faster in concentrations that stabilize syntelic attachments. 2009, Pubmed
Zhao, Mps1 phosphorylation by MAP kinase is required for kinetochore localization of spindle-checkpoint proteins. 2006, Pubmed