How to Assemble a Functional Mitotic Checkpoint Complex

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism that preserves genomic integrity through regulating proper timing of the metaphase-to-anaphase transition during mitosis. In response to SAC activation, a "wait anaphase" signal is generated to halt mitotic progression by promoting formation of the SAC effector. The SAC effector functions to inhibit the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase whose activity is critical for the metaphase-to-anaphase transition and mitotic exit. Closed MAD2 (C-MAD2), one of the two native conformations adopted by the checkpoint protein MAD2, is a well-recognized component of the "wait anaphase" signal generated following SAC activation. However, it has been controversial whether MAD2 is an integral component of the mitotic checkpoint complex (MCC), the suggested SAC effector that potently inhibits the APC/C. We show here that MCC assembly is not only subject to cell cycle regulation, but is driven by intracellular levels of C-MAD2. We have also provided strong evidence demonstrating that C-MAD2 (but not the inactive O-MAD2) is indeed incorporated into the MCC. The selective incorporation of C-MAD2 into the MCC during mitosis arises not only from the previously characterized CDC20:C-MAD2 interaction, but also from a novel direct interaction between C-MAD2 and BUBR1 that was first reported by us. The C-MAD2:BUBR1 interaction has been observed both in vitro and in vivo, and is fundamental for the inhibitory activity of the MCC. We also examined the requirement of several mitotic kinases for MCC assembly, including the core SAC component MPS1 kinase. Through the use of small molecule inhibitors or siRNA-mediated knockdown, we found that the kinase activity of MPS1 is required for MCC assembly and subsequent APC/C inhibition. In contrast, MPS1 appears to have no role in maintaining the stability of pre-assembled MCC. Inhibition of MPS1 kinase by the small molecule reversine prevents MAD2 incorporation into the MCC, simultaneously impairing both CDC20:MAD2 and BUBR1:MAD2 interactions but having no apparent effect on the BUBR1:CDC20 interaction. Strikingly, the impairment caused by MPS1 inhibition can be rescued by expressing a C-conformation locked MAD2 mutant in mitotic cells. In the presence of reversine, C-MAD2 incorporation into the MCC strongly correlates with MCC:APC/C binding, APC/C inhibition and SAC function. This work has expanded our understanding of SAC signal transducer formation, and has established a direct connection between the SAC signal transducer and SAC effector during checkpoint activation.