1, A and B; and Fig. assembly checkpoint (SAC) response depends on the activity of a conserved protein kinase, monopolar spindle 1 (MPS1; TAK-779 Stucke et al., 2002; Liu and Winey, 2012; Pachis and Kops, 2018). MPS1 localizes to unattached kinetochores and initiates the multisite phosphorylation of the kinetochore protein KNL1 and the SAC proteins BUB1 and MAD1 (Ciliberto and Hauf, 2017; Faesen et al., 2017; Ji et al., 2017). This promotes the recruitment of SAC proteins to unattached kinetochores and thus the generation of a checkpoint response (Musacchio, 2015). Clustering of several MPS1 molecules at unattached kinetochores is usually thought to promote trans autophosphorylation and hence kinase activity (Kang et al., 2007; Dodson et al., 2013; Combes et al., 2018). This activation step involves autophosphorylation of its T-loop on threonine 676 (T676; Kang et al., 2007; Mattison et al., 2007; Jelluma et al., 2008a). MPS1 is usually released upon microtubule binding to kinetochores (Jelluma et al., 2010), leading to the termination of the checkpoint response and presumably removal of these activating phosphorylations. Despite this understanding, the phosphatases acting on MPS1 and other checkpoint proteins still need to be clarified. Both PP2A-B56 and PP1 have been implicated in KNL1 dephosphorylation and SAC silencing (Espert et al., 2014; Nijenhuis et al., 2014). PP1 has been shown to dephosphorylate the MPS1 T-loop in flies (Moura et al., 2017), but it is not clear whether this mechanism is usually conserved in mammals. PP2A-B56 exists in several spatially distinct populations in mammalian mitotic cells (Qian et al., 2013; Vallardi et al., 2019). One pool is bound to the C-terminal domain name of BUBR1 via a conserved LxxIxE motif (Hertz et al., 2016). This pool of PP2A-B56 has been shown to oppose both Aurora B and MPS1 in chromosome alignment and SAC signaling, respectively (Suijkerbuijk et al., 2012; Kruse et al., 2013; Xu et al., 2013; Espert et al., 2014). In addition to orchestrating SAC signaling, MPS1 also contributes directly to the turnover of erroneous microtubuleCkinetochore attachments by phosphorylating the Ska complex at microtubuleCkinetochore junctions. This activity of MPS1 is also opposed by PP2A-B56 (Maciejowski et al., 2017). Here we investigate this complex network of phosphatases and find that this BUBR1-dependent pool of PP2A-B56 is the key MPS1 T-loop phosphatase. Furthermore, we demonstrate that dynamic turnover of MPS1 T-loop phosphorylation by PP2A-B56 is crucial for both TAK-779 the SAC and error correction pathways. Results and discussion MPS1 T-loop phosphorylation is usually controlled by PP2A MPS1 activity is usually dynamically regulated by autophosphorylation at T676 in the T-loop of the kinase domain name (Kang et al., 2007; Mattison et al., 2007; Jelluma et al., 2008a). To identify the class of phosphatase acting at this site, mitotic HeLa cells expressing endogenously tagged MPS1-GFP were pretreated with PPP family phosphatase inhibitors, and then briefly incubated with MPS1 inhibitor (MPS1i) to stop T-loop autophosphorylation (Ishihara et al., TAK-779 1989; Mitsuhashi et al., 2001; Hewitt et al., 2010; Choy et al., 2017; Alfonso-Prez et al., 2019). In control cells, MPS1i resulted in loss of the MPS1 pT676 signal (Fig. 1, A and MED B; and Fig. S1, A and B). The level of total MPS1-GFP increased, as reported before (Hewitt TAK-779 et al., 2010; Jelluma et al., 2010; Santaguida et al., 2010; Fig. 1, A and C). Addition of a dual PP1/2A inhibitor (PP1/2Ai; calyculin A) but not PP1 inhibitor (PP1i; tautomycetin) prevented the loss of the pT676 signal (Fig. 1, A and B). Neither treatment affected the increase of MPS1-GFP levels at kinetochores upon MPS1 inhibition (Fig. 1, A and C). Comparable results were obtained in untransformed human telomerase reverse transcriptaseCimmortalized retinal pigment epithelial cells (RPE-1; Fig. S1, CCE), indicating that these findings were independent of the transformation status of the cells. Taken together, these data suggest that in mammalian cells, in contrast to = 2,490 cells), MPS1KD (= 2,451 cells), MPS1AA (= 2,689 cells), or MPS1DD (=.