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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: 3.53). 04/2010; 5(4):e10251. DOI: 10.1371/journal.pone.0010251
Source: PubMed

ABSTRACT 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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    • "Combining inhibition of Mps1 with Chk1 depletion did not exhibit an additive effect on Aurora B activity, thus sug­ gesting that Mps1 and Chk1 are involved in the same step of Aurora B activation. Mps1 phosphorylates Borealin to enhance Aurora B activity (Jelluma et al., 2008; Sliedrecht et al., 2010). One possibility is that phosphorylation of Borealin by Mps1 facil­ itates Ser331 or TSS phosphorylation, for example by stabiliz­ ing interactions between CPC proteins (Vader et al., 2006). "
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    ABSTRACT: Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora B(S331A), in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation.
    The Journal of Cell Biology 10/2011; 195(3):449-66. DOI:10.1083/jcb.201104023 · 9.69 Impact Factor
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    • "Similarly, in mammalian cells, Mps1 is required for the error correction of the KT–MT attachment (Jelluma et al, 2008). It is currently a matter of debate whether Mps1 regulates the Aurora B kinase (Jelluma et al, 2008; Sliedrecht et al, 2010), or vice versa (Hewitt et al, 2010; Santaguida et al, 2010), or they work in parallel pathways (Maure et al, 2007; Maciejowski et al, 2010) to promote sister KT bi-orientation. "
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    ABSTRACT: Eukaryotic cells segregate their chromosomes accurately to opposite poles during mitosis, which is necessary for maintenance of their genetic integrity. This process mainly relies on the forces generated by kinetochore-microtubule (KT-MT) attachment. During prometaphase, the KT initially interacts with a single MT extending from a spindle pole and then moves towards a spindle pole. Subsequently, MTs from the other spindle pole also interact with the KT. Eventually, one sister KT becomes attached to MTs from one pole while the other sister to those from the other pole (sister KT bi-orientation). If sister KTs interact with MTs with aberrant orientation, this must be corrected to attain proper bi-orientation (error correction) before the anaphase is initiated. Here, I discuss how KTs initially interact with MTs and how this interaction develops into bi-orientation; both processes are fundamentally crucial for proper chromosome segregation in the subsequent anaphase.
    The EMBO Journal 12/2010; 29(24):4070-82. DOI:10.1038/emboj.2010.294 · 10.75 Impact Factor
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    • "Note that cells were also treated with MG132 to prevent mitotic exit caused by SAC override. Under these conditions, AZ3146 inhibited chromosome alignment dramatically affect Mad1 (Jelluma et al., 2008b; Kwiatkowski et al., 2010; Sliedrecht et al., 2010); e.g., inhibition of Mps1 using another small molecule, reversine, blocked recruitment of Mad1 and its kinetochore receptor, the Rod–Zw10–Zwilch (RZZ) complex (Santaguida et al., 2010). We reasoned that these differences may be a result of when Mps1 was first inhibited either before or during mitosis. "
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    ABSTRACT: Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1's catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1-C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.
    The Journal of Cell Biology 07/2010; 190(1):25-34. DOI:10.1083/jcb.201002133 · 9.69 Impact Factor
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