Chemical Genetic Inhibition of Mps1 in Stable Human Cell Lines Reveals Novel Aspects of Mps1 Function in Mitosis

Department of Physiological Chemistry, Cancer Genomics Centre, University Medical Center Utrecht, Utrecht, The Netherlands.
PLoS ONE (Impact Factor: 3.23). 04/2010; 5(4):e10251. DOI: 10.1371/journal.pone.0010251
Source: PubMed


Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by the mitotic checkpoint that allows chromosome segregation only when all chromosomes are stably attached. Proper functioning of the attachment and checkpoint processes is thus important to prevent chromosomal instability. Both processes rely on the mitotic kinase Mps1.
We present here two cell lines in which endogenous Mps1 has been stably replaced with a mutant kinase (Mps1-as) that is specifically inhibited by bulky PP1 analogs. Mps1 inhibition in these cell lines is highly penetrant and reversible. Timed inhibition during bipolar spindle assembly shows that Mps1 is critical for attachment error-correction and confirms its role in Aurora B regulation. We furthermore show that Mps1 has multiple controls over mitotic checkpoint activity. Mps1 inhibition precludes Mad1 localization to unattached kinetochores but also accelerates mitosis. This acceleration correlates with absence of detectable mitotic checkpoint complex after Mps1 inhibition. Finally, we show that short-term inhibition of Mps1 catalytic activity is sufficient to kill cells.
Mps1 is involved in the regulation of multiple key processes that ensure correct chromosome segregation and is a promising target for inhibition in anti-cancer strategies. We report here two cell lines that allow specific and highly penetrant inhibition of Mps1 in a reproducible manner through the use of chemical genetics. Using these cell lines we confirm previously suggested roles for Mps1 activity in mitosis, present evidence for novel functions and examine cell viability after short and prolonged Mps1 inhibition. These cell lines present the best cellular model system to date for investigations into Mps1 biology and the effects of penetrance and duration of Mps1 inhibition on cell viability.

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    • "RESEARCH ARTICLE Journal is in line with several other studies showing that the checkpoint inhibits APC/C catalytic activity (Maciejowski et al., 2010; Mansfeld et al., 2011; Sliedrecht et al., 2010). Next, we tested whether inability to activate the spindle checkpoint at the prophase-to-prometaphase transition would further increase APC/C Cdc20 activity towards Nek2A, as also shown for cyclin A (Collin et al., 2013). "
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    ABSTRACT: Nek2A is a presumed APC/C(Cdc20) substrate, which, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid stabilization by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A while Nek2A disappeared at normal rate. Secondly, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction critically depended on Cdc20 binding to the APC/C. Thirdly, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity.
    Full-text · Article · Feb 2015 · Journal of Cell Science
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    • "Downstream events and mitotic exit are prevented by simultaneous addition of the proteasome inhibitor MG132. In control cells, Mps1 inhibition resulted in the loss of BubR1 and Bub1 from the kinetochore within 5 min (Fig. S1 A), which confirms the dependence of these SAC proteins on Mps1 activity for localization (Maciejowski et al., 2010; Sliedrecht et al., 2010). Mps1 inhibition in the presence of the general phosphoprotein phosphatase (PPP) inhibitor calyculin A resulted in retention of BubR1 and Bub1 at kinetochores (Fig. 1, B and C). "
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    ABSTRACT: The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.
    Preview · Article · Sep 2014 · The Journal of Cell Biology
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    • "MAD1-MAD2 catalyzes production of an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), resulting in maintenance of sister chromatid cohesion and of the mitotic state (De Antoni et al. 2005; Kulukian et al. 2009; Simonetta et al. 2009). A current model of SAC signaling is as follows: various activities at kinetochores, including BUB1, MPS1, and Rod-ZW10-Zwilch, contribute to recruitment of the MAD1-MAD2 complex (Basto et al. 2000; Brady and Hardwick 2000; Chan et al. 2000; Martin-Lluesma et al. 2002; Meraldi et al. 2004; Kops et al. 2005; Liu et al. 2006; Klebig et al. 2009; Santaguida et al. 2010; Sliedrecht et al. 2010; Maciejowski et al. 2010; Kim et al. 2012; London and Biggins 2014; Moyle et al. 2014). This complex in turn binds soluble MAD2 molecules and converts these into a form that allows association with CDC20, an essential mitotic cofactor of the APC/C (Mapelli and Musacchio 2007). "
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    ABSTRACT: Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase. Electronic supplementary material The online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users.
    Preview · Article · Apr 2014 · Chromosoma
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