The spindle assembly checkpoint (SAC) is an essential safeguarding mechanism devised to ensure equal chromosome distribution in daughter cells upon mitosis. The proteins Bub3 and BubR1 are key components of the mitotic checkpoint complex (MCC), an essential part of the molecular machinery on which the SAC relies. In the present work we have performed a detailed functional and biochemical characterization of the interaction between human Bub3 and BubR1 in cells and in vitro. Our results demonstrate that genetic knock down of Bub3 abrogates the SAC, promotes apoptosis and inhibits the proliferation of human cancer cells. We also show that the integrity of the human MCC depends on the specific recognition between BubR1 and Bub3, for which the BubR1 GLEBS motif is essential. This 1:1 binding event is high affinity, enthalpy-driven and with slow dissociation kinetics. The affinity, kinetics and thermodynamic parameters of the interaction are differentially modulated by small regions in the N- and C-termini of the GLEBS domain sequence, suggesting the existence of "hotspots" for this protein-protein interaction. Furthermore, we show that specific disruption of endogenous BubR1-Bub3 complexes in human cancer cells phenocopies the effects observed in gene targeting experiments. Our work enhances the current understanding of key members of the SAC and paves the road for the pursuit of novel targeted cancer therapies based on SAC inhibition.
Copyright © 2016, The American Society for Biochemistry and Molecular Biology.

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