Regulation of Kinetochore Recruitment of Two Essential Mitotic Spindle Checkpoint Proteins by Mps1 Phosphorylation

Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Molecular biology of the cell (Impact Factor: 4.47). 11/2008; 20(1):10-20. DOI: 10.1091/mbc.E08-03-0324
Source: PubMed


Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.

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    • "Mps1 undergoes autophosphorylation during mitosis [26], [29]–[31]. For example, autophosphorylation on Thr676 within the activation loop of Mps1 occurs in mitosis and this phosphorylation is required for its kinase activity in vitro and for the SAC in vivo [32]–[33]. "
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    ABSTRACT: The spindle assembly checkpoint (SAC) is a surveillance mechanism monitoring cell cycle progression, thus ensuring accurate chromosome segregation. The conserved mitotic kinase Mps1 is a key component of the SAC. The human Mps1 exhibits comprehensive phosphorylation during mitosis. However, the related biological relevance is largely unknown. Here, we demonstrate that 8 autophosphorylation sites within the N-terminus of Mps1, outside of the catalytic domain, are involved in regulating Mps1 kinetochore localization. The phospho-mimicking mutant of the 8 autophosphorylation sites impairs Mps1 localization to kinetochore and also affects the kinetochore recruitment of BubR1 and Mad2, two key SAC effectors, subsequently leading to chromosome segregation errors. Interestingly, the non-phosphorylatable mutant of the 8 autophosphorylation sites enhances Mps1 kinetochore localization and delays anaphase onset. We further show that the Mps1 phospho-mimicking and non-phosphorylatable mutants do not affect metaphase chromosome congression. Thus, our results highlight the importance of dynamic autophosphorylation of Mps1 in regulating accurate chromosome segregation and ensuring proper mitotic progression.
    PLoS ONE 09/2014; 9(9):e104723. DOI:10.1371/journal.pone.0104723 · 3.23 Impact Factor
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    • "The mitotic cell is characterized by dramatic changes to cell state, which include a temporary reduction in cell volume and a concomitant condensation of the cytosol [48], [49], a selective inhibition of receptor-mediated endocytosis [30], [34], a mitotic stage-specific abrogation of endosomal recycling [30], a reorganization of tubulin to the mitotic spindle, the activation of mitotic kinases such as Mps1 [44], [53], and of kinases such as ERK [54], [55]. Notably, endocytosis [22], [23], [24], [51], [56], [57], [58], recycling [59], Mps1 [37], ERK [38], [60], microtubules and microtubule-associated proteins [4], [46], have all been implicated in the regulation of TGF-β/Smad signaling; suggesting that multiple aspects of the regulation of the TGF-β signal may be altered in mitosis. "
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    ABSTRACT: The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis.
    PLoS ONE 08/2012; 7(8):e43459. DOI:10.1371/journal.pone.0043459 · 3.23 Impact Factor
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    • "Apart from hypothetical differences in conformational behavior, the discordant results concerning dependence of kinetochore localization on Mps1 kinase activity might perhaps also reflect different assay conditions. Nocodazole and MG132 were applied in all the experiments in which increased kinetochore localization of Mps1 kd or pharmacologically inhibited Mps1 was observed but not in those in which kinetochore localization was absent (Figure 1D; Xu et al., 2009). Another difference between human and Drosophila Mps1 concerns the consequences of overexpression. "
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    ABSTRACT: Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1`s significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.
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