BUBR1-bound PP2A-B56 dephosphorylates MPS1 auto-activatory site T676. (A) Mitotic HeLa MPS1-GFP cells were pretreated with DMSO (Control [Con]), 5 µM tautomycetin (PP1i), or 25 nM calyculin (PP1/PP2Ai), and then 2 µM MPS1i was added for 5 min where indicated (+MPS1i). MPS1 pT676 and CENP-C were detected using antibodies and total MPS1 by GFP fluorescence. (B and C) Mean kinetochore levels ± SEM of MPS1 pT676 (B) and MPS1-GFP (C) relative to the −MPS1i control are plotted. (D) MPS1 pT676 phosphorylation and MPS1-GFP localization in control or PP2A-B55– or PP2A-B56–depleted mitotically arrested HeLa MPS1-GFP cells treated with 2 µM MPS1i. MPS1 pT676 and CENP-C were detected using antibodies, and MPS1 by GFP fluorescence. (E and F) Mean kinetochore levels ± SEM of MPS1 pT676 (E) and total MPS1-GFP (F) are plotted. (G) HeLa-Flp-In/TREx GFP-BUBR1^WT or GFP-BUBR1^L669A/I672A-expressing cells depleted of endogenous BUBR1 were mitotically arrested and treated with DMSO (Control) or 2 µM MPS1i for 5 min. MPS1 pT676 and CENP-C were detected using antibodies, and BUBR1 by GFP fluorescence. (H and I) MPS1 pT676 phosphorylation (H) and mean GFP-BUBR1 kinetochore levels (I) ± SEM are plotted. (J) Model illustrating how BUBR1-associated PP2A-B56 regulates MPS1 T-loop phosphorylation.