Figure 3. PP1-87B/PP1-γ dephosphorylates Mps1 T-loop in vitro and in vivo.

(A,B) Western blot analysis of Mps1/TTK T676 autophosphorylation. Recombinant human Mps1 was incubated during 30 min with increasing concentrations of recombinant human PP1-γ (A) or PP1-γ (42.7 nM) was added to previously active Mps1 and phosphorylation of Mps1 T676 (Mps1^T676Ph) monitored over time (B). (C) Western blot analysis of Drosophila Mps1 T490 autophosphorylation. Recombinant Drosophila Mps1 was incubated during 30 min with 10 nM of recombinant human PP1-γ or λ-PP and phosphorylation of Mps1 T490 (Mps1^T490Ph) monitored. (D) Clustal W alignments of amino acid residues of indicated Mps1 orthologues. Red boxes highlight putative PP1-docking motifs identified in silico by the Eukaryotic Linear Motif (ELM) resource. (E) MBP-PP1-87B pull-downs from total cell lysates of S2 cells expressing EGFP-Mps1^WT or EGFP-Mps1^K231A/F234A or EGFP-Mps1^WT upon depletion of Sds22. Immobilized MBP was used as negative control. Input, beads and flow-through (FT) were probed by western blotting for the indicated proteins. (F) Quantification of Mps1 binding to PP1-87B from the pull-downs in (E). The chemiluminescence signal intensity of EGFP-Mps1 was determined relative to the signal of MBP-PP1-87B beads. The graph represents the quantification of relative levels of EGFP-Mps1 in MBP-PP1-87B pull-downs from at least two independent experiments. The values obtained for EGFP-Mps1^WT from control cells were set to 1. (G) MBP-87B pull-downs of purified recombinant Mps1 N-terminus region (104–330 amino acids) harboring the wild-type (6xHis-N-Mps1^WT) or mutated PP1-docking motif (6xHis-N-Mps1^K231A/F234A). Immobilized MBP was used as negative control. Input, beads and flow-through (FT) were probed by western blotting for the indicated proteins. (H) Western blot analysis of EGFP-Mps1^WT and EGFP-Mps1^K231A/F234A T-loop dephosphorylation by PP1-γ. EGFP-Mps1^WT and EGFP-Mps1^K231A/F234A were immunoprecipitated from mitotic S2 lysates and incubated with ATP for 30 min to allow Mps1 T-loop autophosphorylation. Increasing concentrations of recombinant GST-PP1-γ were subsequently added to the reaction mixture and Mps1 T490 phosphorylation (Mps1^T490Ph) assessed after 30 min. Immunoprecipitates were probed by immunoblotting (IB) for the indicated proteins. (I) Quantification of Mps1^T490Ph levels from the dephosphorylation assay in (H). The chemiluminescence signal intensity of Mps1^T490Ph was determined relative to the corresponding signal of EGFP-Mps1 immunoprecipitates. The values obtained for each control reaction were set to 100%. The graph represents the quantification of Mps1^T490Ph relative levels from two independent experiments. (J–L) Representative immunofluorescence images (J,K) and corresponding quantifications (L) of Mps1 T490 phosphorylation (Mps1T^490Ph) at kinetochores of unaligned (J) and aligned (K) chromosomes from S2 cells expressing EGFP-Mps1^WT or EGFP-Mps1^K231A/F234A and from PP1-87B depleted S2 cells expressing EGFP-Mps1^WT. EGFP-Mps1 transgenes were expressed under the control of Mps1 cis-regulatory region (Althoff et al., 2012). Mps1T^490Ph fluorescence intensities were determined relative to EGFP-Mps1 signal (N ≥ 150 unaligned kinetochores from at least 8 cells for each condition and N ≥ 111 aligned kinetochores from at least 7 cells for each condition). Scale bar: 5 μm. Data information: in (F), (I) and (L) data are presented as mean ± SD. Asterisks indicate that differences between mean ranks are statistically significant, ****p<0.0001 (Kruskal-Wallis, Dunn’s multiple comparison test). Numerical source data for this figure are provided in Figure 3—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.25366.017

Figure 3—figure supplement 1. Specificity of Mps1 T-loop dephosphorylation in vitro.

(A,B) Western blot analysis of Mps1T676 autophosphorylation. Recombinant human Mps1 was incubated during 30 min with increasing concentrations of recombinant human λ-phosphatase (λPP) (A) or λ-PP (42.7 nM) was added to previously active Mps1 and phosphorylation of Mps1 T676 (Mps1^T676Ph) monitored over time (B).

DOI: http://dx.doi.org/10.7554/eLife.25366.019

Figure 3—figure supplement 2. Western blotting analysis of S2 cells expressing EGFP-Mps1^WT and EGFP-Mps1^K231A/F234A.

Western blot of analysis of BubR1 depletion and of EGFP-Mps1^WT and EGFP-Mps1^K231A/F234A expression in Drosophila S2 cells used in the studies described in Figure 3J–L. α-Tubulin was used as loading control. Expression of EGFP-Mps1 transgenes was under control of Mps1 cis-regulatory region.

DOI: http://dx.doi.org/10.7554/eLife.25366.020

Figure 3—figure supplement 3. Mps1^WT and Mps1^K231A/F234A dynamics at kinetochores.

(A,B) FRAP analysis of EGFP-Mps1^WT and EGFP-Mps1^K231A/F234A at prometaphase and metaphase kinetochores. (A) Graph displays recovery-time curves of EGFP-Mps1 fluorescence (lines) and their time point means (dots) after bleaching of individual kinetochores in the indicated conditions. (B) Table showing the recovery half-lives and the % of mobile EGFP-Mps1 populations obtained after fitting the average fluorescence intensity at each time point to a single-exponential one-phase association curve (N ≥ 10 cells for each condition).

DOI: http://dx.doi.org/10.7554/eLife.25366.021