Chemical Genetic Inhibition of Mps1 in Stable Human Cell Lines Reveals Novel Aspects of Mps1 Function in Mitosis Tale Sliedrecht1,2, Chao Zhang 3 , Kevan M. Shokat3, Geert J. P. L. Kops1,2* 1 Department of Physiological Chemistry, Cancer Genomics Centre, University Medical Center (UMC) Utrecht, Utrecht, The Netherlands, 2 Netherlands Proteomics Centre, Utrecht, The Netherlands, 3 Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, United States of America Abstract Background: 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. Principal Finding: 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. Conclusions/Significance: 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. Citation: Sliedrecht T, Zhang C, Shokat KM, Kops GJPL (2010) Chemical Genetic Inhibition of Mps1 in Stable Human Cell Lines Reveals Novel Aspects of Mps1 Function in Mitosis. PLoS ONE 5(4): e10251. doi:10.1371/journal.pone.0010251 Editor: Daniela Cimini, Virginia Tech, United States of America Received February 12, 2010; Accepted March 26, 2010; Published April 22, 2010 Copyright: 2010 Sliedrecht et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Netherlands Genomics Initiative of the Netherlands Organization for Scientific Research and ERC-StG-242617 to GJPLK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: g.j.p.l.kops@umcutrecht.nl Introduction To maintain a stable genome, cells have evolved a variety of processes that ensure accurate chromosome segregation. In early mitosis, kinetochores of sister chromatids attach to microtubules emanating from opposite spindle poles. Correct end-on attach- ment of microtubules to kinetochores relies on the error-correction machinery that destabilizes improper attachments through the actions of the Aurora B kinase [1]. As long as unattached kinetochores persist, the onset of anaphase is prevented by a surveillance mechanism called the mitotic checkpoint that will halt cell cycle progression until all chromosomes are stably attached to the mitotic spindle [2]. The mitotic checkpoint will be satisfied upon stable biorientation of all chromosomes, after which chromosome segregation is allowed to proceed. Proper execution of chromosome biorientation and mitotic checkpoint signaling relies on a set of multifunctional kinases, one of which is the dual specificity kinase Mps1 [3]. First discovered to regulate spindle pole body duplication in budding yeast [4], Mps1 was subsequently found to additionally regulate the mitotic checkpoint [5] and spindle assembly [6]. Regulation of the mitotic checkpoint by Mps1 is evolutionary conserved and has been shown in fission yeast, fruit flies, Xenopus egg extracts and human cells [7–11]. Mps1 exerts this control, at least in part, through regulating kinetochore localization of several checkpoint proteins including Mad1 and Mad2 [9,11,12]. Recently, Mps1 was also reported to regulate sister chromatid biorientation in both budding yeast and humans [12,13]. In human cells, Mps1 promotes biorientation by regulating Aurora B activity through phosphorylation of the chromosomal passenger complex (CPC) member Borealin [12,14]. Due to its central role in mitosis, misregulation of Mps1 kinase activity results in chromosomal instability (CIN) and subsequent aneuploidy, a hallmark shared by cells from solid tumors [15,16]. Inefficient activation of Mps1 results in weakened mitotic checkpoint activity and the persistence of falsely attached PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10251