PP2A-B56 modulates MPS1 kinetochore localization by opposing both MPS1 and Aurora B. (A) MPS1 pT33/S37 phosphorylation and MPS1-GFP and CENP-C localization were compared before and after MPS1i (2 µM) in control, PP1αβγ, or PP2A catalytic α subunit–depleted HeLa MPS1-GFP cells. (B and C) Mean kinetochore levels ± SEM of pT33/S37 (B) and total MPS1-GFP (C) relative to CENP-C are plotted. (D) MPS1 pT33/S37 phosphorylation and MPS1-GFP localization were detected in control, PP2A-B55-, or PP2A-B56-depleted cells. (E and F) Mean pT33/S37 (E) and total MPS1-GFP (F) kinetochore levels ± SEM relative to CENP-C are plotted. (G) MPS1-GFP and CENP-C localization before and after 10-min treatment with 2 µM AURBKi in HeLa MPS1-GFP cells (control, PP1αβγ, PP2A catalytic subunit α, or PP2A-B55 or PP2A-B56 regulatory subunit depleted). (H) Mean kinetochore levels ± SEM of MPS1-GFP relative to CENP-C were plotted. (I) MPS1-GFP localization in control depleted or PP2A-B56 depleted HeLa MPS1-GFP cells treated with DMSO or a combination of AURKB1 (2 µM) and MPS1i (2 µM) for 10 min. (J) Mean kinetochore levels ± SEM of MPS1-GFP relative to CENP-C were plotted. (K) Schematic drawing illustrating kinase and phosphatase regulation of key sites on MPS1 and the kinetochore.