Monopolar Spindle 1 (MPS1) Kinase Promotes Production of Closed MAD2 (C-MAD2) Conformer and Assembly of the Mitotic Checkpoint Complex

Background: How MPS1 kinase functions during mitotic checkpoint signaling is not fully understood. Results: C-MAD2 expression rescues premature mitotic exit caused by MPS1 inhibition without localizing at kinetochores. Conclusion: MPS1 functions primarily to enhance C-MAD2 production and subsequent formation of the mitotic checkpoint complex. Significance: The results underscore the importance of MPS1 kinase in maintaining kinetochore localization and catalytic efficiency of the MAD1C-MAD2 core. MPS1 kinase is an essential component of the spindle assembly checkpoint (SAC), but its functioning mechanisms are not fully understood. We have shown recently that direct interaction between BUBR1 and MAD2 is critical for assembly and function of the human mitotic checkpoint complex (MCC), the SAC effector. Here we report that inhibition of MPS1 kinase activity by reversine disrupts BUBR1-MAD2 as well as CDC20-MAD2 interactions, causing premature activation of the anaphase-promoting complex/cyclosome. The effect of MPS1 inhibition is likely due to reduction of closed MAD2 (C-MAD2), as expressing a MAD2 mutant (MAD2(L13A)) that is locked in the C conformation rescued the checkpoint defects. In the presence of reversine, exogenous C-MAD2 does not localize to unattached kinetochores but is still incorporated into the MCC. Contrary to a previous report, we found that sustained MPS1 activity is required for maintaining both the MAD1C-MAD2 complex and open MAD2 (O-MAD2) at unattached kinetochores to facilitate C-MAD2 production. Additionally, mitotic phosphorylation of BUBR1 is also affected by MPS1 inhibition but seems dispensable for MCC assembly. Our results support the notion that MPS1 kinase promotes C-MAD2 production and subsequent MCC assembly to activate the SAC.