Functional and Structural Characterization of Bub3·BubR1 Interactions Required for Spindle Assembly Checkpoint Signaling in Human Cells

• DOI: 10.1074/jbc.m115.702142
• Publication Date: 2016-04-03T01:17:24Z
• Authors: Florian Prinz; Vera Puetter; Simon J. Holton; Dorothee Andres; Christian M. Steg...; Dennis Kwiatkowski; Stefan Prechtl; Kirstin Petersen; Georg Beckmann; Bertolt Kreft; Dominik Mumberg; Amaury Fernández-...
• Keywords: Models, Molecular; 0301 basic medicine; Apoptosis; Cell Cycle Proteins; Spindle Apparatus; Protein Serine-Threonine Kinases; Cell Line; 3. Good health; Kinetics; 03 medical and health sciences; Cell Line, Tumor; Gene Knockdown Techniques; MCF-7 Cells; Humans; M Phase Cell Cycle Checkpoints; Thermodynamics; Protein Interaction Domains and Motifs; RNA, Messenger; Poly-ADP-Ribose Binding Proteins; Cell Proliferation; HeLa Cells

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, 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 knockdown of Bub3 abrogates the SAC, promotes apoptosis, and inhibits the proliferation of human cancer cells. We also show that the integrity of the human mitotic checkpoint complex depends on the specific recognition between BubR1 and Bub3, for which the BubR1 Gle2 binding sequence 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 Gle2 binding 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.