Chemical Genetics of Mps1 Figure 1. Stable expression of LAP-Mps1as renders selective sensitivity to 23dMB-PP1. A: Mps1 and tubulin immunoblots of lysates of UTRM10 and HCT-TRM cells in which endogenous Mps1 is removed by doxycycline-induced expression of Mps1 shRNA and replaced with the indicated LAP-Mps1 mutants. B–D: pT676-Mps1 and Mps1 immunoblots of UTR-Mps1as (B) and HCT-Mps1as (B–D) cell lines treated with nocodazole and MG132 in combination DMSO/23dMB-PP1 for 1 hour (B), with nocodazole and MG132 for 1 hour in combination with an additional 23dMB-PP1 treatment for the indicated amounts of time (C), or with nocodazole and MG132 in combination with 23dMB-PP1 for 1 hour, followed by PBS wash after which cells were left for 15 or 30 minutes in media without 23dMB-PP1 (D). doi:10.1371/journal.pone.0010251.g001 D and S3A, C) and ,55% for at least 10 hours (data not shown). In stark contrast, addition of 23dMB-PP1 caused all HCT-Mps1as but not HCT-Mps1WT cells to exit mitosis within 60 minutes (Fig. 2D and S3A, C). These data show that the ability of cells to delay mitotic progression absolutely requires Mps1 kinase activity. Moreover, the finding that all 23dMB-PP1-treated HCT-Mps1as cells had lost this ability illustrated the clonality of this cell line. Mps1 kinase activity promotes localization of Mad1, Mad2, Cdc20 and Bub1 to unattached kinetochores Proper functioning of the mitotic checkpoint requires the recruitment of checkpoint proteins to the kinetochore. Kinase activity of Mps1 is required for the recruitment of Mad2 to unattached kinetochores, at least partly explaining its necessity for checkpoint signaling [9,12,21]. There is, however, discrepancy between reports on the requirement of Mps1 for the recruitment of Mad1 [11,12,21,24], Bub1 [11,12,24,25], BubR1 [11,12,18,23] and CENP-E [9,12,21,24,26] to kinetochores. To examine how Mps1 catalytic activity contributes to recruitment of these and other checkpoint proteins, we assayed their kinetochore association after inhibition of Mps1as in our cell lines. Like the ability to delay mitosis, kinetochore recruitment of Mad1, Mad2, BubR1, Bub1, CENP-E and Cdc20 was altered neither in DMSO-treated Mps1WT or Mps1as cells, nor in 23dMB- PP1-treated Mps12WT cells (Fig. 3 and S4). However, addition of 23dMB-PP1 to Mps1as cells significantly reduced the level of Mad1, Mad2, Bub1 and Cdc20 at unattached kinetochores, but only marginally affected BubR1 levels and did not influence CENP-E levels (Fig. 3). These data show that Mps1 kinase activity promotes kinetochore-binding of Mad1, Mad2, Bub1 and Cdc20. Mps1 inhibition strongly diminishes MCC levels in mitotic cells The mitotic checkpoint functions through the production of an inhibitory complex called the mitotic checkpoint complex (MCC). The MCC targets Cdc20, the main activator of the anaphase promoting complex or cyclosome (APC/C) in prometaphase, thereby preventing exit from mitosis [2]. The amplification of MCC assembly by unattached kinetochores is dependent on kinetochore localization of Mad1 and Mad2 [27,28]. Since inhibition of Mps1 resulted in loss of kinetochore-bound Mad1/ Mad2, we set out to investigate if Mps1 inhibition influences the amount of MCC in nocodazole-treated cells. As expected, Cdc20 immunoprecipitations from cells treated with DMSO contained the MCC subunits Mad2, BubR1 and Bub3 (Fig. 4A). Although inhibition of Mps1 did not alter the levels of coprecipitated BubR1 and Bub3, the amount of Cdc20-bound Mad2 in mitotic cells was reduced to interphase levels after treatment with 23dMB-PP1 PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10251