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

ABSTRACT 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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    • "Recently, Tyler et al. reported numerous hMps1 autophosphorylation sites [32], but several of these could not be confirmed here as in vivo hMps1 phosphorylation sites. On the other hand, 7 additional sites (S15, T288, T360, S362, T564, S682, S742) not identified in our SILAC analysis were also reported as in vivo autophosphorylation sites in recent publications [31], [32], [46]. A Web logo representation of all autophosphorylation sites identified in vivo, either here or previously (see Table S3), confirms that hMps1 phosphorylation sites demonstrate a propensity for E/D/N/Q at the −2 position (11 of 19 sites), very similar to the Plk1 consensus motif (Fig. 4C) [36]. "
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    ABSTRACT: Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.
    PLoS ONE 04/2011; 6(4):e18793. DOI:10.1371/journal.pone.0018793 · 3.23 Impact Factor
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