Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56

Abstract

Supplemental Figure legendsFigure S1. MPS1 autophosphorylation and localisation are regulated by PP2A-B56. (A) Control depleted or MPS1-depleted HeLa MPS1-GFP mitotic cells were treated with MPS1 inhibitor or DMSO for 5 min as indicated. Cells were stained with antibodies against MPS1 pT676 and CENP-C. Total MPS1-GFP was detected by GFP fluorescence. (B) Recombinant, insect cell expressed wild type (WT) or kinasedead (KD) His-MPS1 was treated with l-phosphatase or reaction buffer alone and analysed by SDS-PAGE and Coomassie Brilliant Blue staining (CBB) or Western blotting with antibodies against MPS1 pT676. (C) MPS1 T-loop phosphorylation and endogenous MPS1 levels were assessed in control depleted mitotic HeLa MPS1-GFP cells or HeLa MPS1-GFP cells depleted of PP1αβγ (siPP1), PP2CA (siPP2A), PPP4C (siPP4), PPP5C (siPP5) or PPP6C (siPP6) and treated with MPS1i as indicated. Mean kinetochore MPS1 pT676 phosphorylation (D) and MPS1-GFP kinetochore levels ± SEM (E) relative to CENP-C are plotted. Values are from three independent experiments with 15 kinetochores measured in at least 10 cells per condition. (F) Representative Western blot analysis of PP1, PP2A, PP4, PP5 and PP6 depletion efficiencies. Actin is shown as a loading control. (G) Representative Western blot of PP2A-B55 and PP2A-B56 depletion efficiencies. Actin is used as a loading control. (H) Western blot of HeLa-Flp-In/TREx GFP-BUBR1 WT or GFP-

Kinetochore phosphatases suppress autonomous kinase activity to control the spindle assembly checkpoint

“…Furthermore, this may be reinforced by an additional positive feedback loop to MPS1 itself, since PLK1 can phosphorylate the activation loop of MPS1 to stimulate its kinase activity 26,27,37 (MPS1→BUB:PLK1→MPS1, Figure 4B, P2). This may help to explain the recent observation that this 'autoactivation' site remains phosphorylated when MPS1 is inhibited in BUBR1 PP2A cells 38 . In this case, PP2A removal may increase phosphorylation indirectly via PLK1, instead of (or in addition to) preventing dephosphorylation directly, as the authors propose.…”

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

“…To test whether PP1 activity contributes to the accelerated destruction of cyclin B1 we performed a biochemical analysis of mitotic exit in the presence of the highly specific small molecule PP1 inhibitor tautomycetin which inhibits all three PP1 isoforms but not PP2A (Choy et al, 2017;Hayward et al, 2019c). Tautomycetin prevented dephosphorylation of PPP1CA-pT320 ( Figure 1A and 1B), and the half-life of pT320 increased from ~2 min in the control to >60 min ( Figure 1C).…”

“…CDC20 was depleted using siRNA oligo #14 5'-CGGAAGACCUGCCGUUACA-3' (ThermoFisher). siRNA oligos for PP2A-B55 and PP2A-B56 have been described (Hayward et al, 2019a;Hayward et al, 2019c).…”

Recent advances in understanding the role of Cdk1 in the Spindle Assembly Checkpoint

“…However, it has also recently been shown that Mad1 stably interacts with Cdk1 and that Mps1, through kinetochore recruitment of Mad1, in turn, promotes kinetochore localization of Cdk1 (Figure 2) 72,73 . At kinetochores, Cdk1 may further phosphorylate other substrates to sustain the SAC like Cdc20 or BubR1 and possibly also help error correction and SAC resolution by favoring BubR1 interaction with the protein phosphatase PP2A-B56 65, [78][79][80] . Recent evidence also indicated how the indirect downregulation of the protein phosphatase PP2A-B55 activity by Cdk1 is instrumental for the SAC-promoting action of Cdk1 itself 72,81 .…”