Abstract
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.
Highlights
11252 JOURNAL OF BIOLOGICAL CHEMISTRY dle equator before the commencement of anaphase separation
Human Bub3 Knockdown Abrogates the Spindle Assembly Checkpoint, Promotes Apoptosis, and Inhibits the Proliferation of Tumor Cells—In our first experiments we wanted to analyze the effects of Bub3 genetic knockdown in several human tumor and non-transformed cell lines
Our results demonstrate that the interaction between Bub3 and the BubR1 Gle2 binding sequence (GLEBS) motif is of high affinity, has a long halflife, and is thermodynamically characterized by a strong enthalpy component
Summary
Cell Culture—Cell lines were obtained from different providers: HeLa, PC-3, and MCF-7 from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany); U2-OS, Hs68, MDA-MB-231, and MCF 10A cells from the ATCC; primary human fibroblasts from Provitro. Apoptosis was determined using the Cell Death Detection ELISAPLUS (Roche Applied Science) according to the manufacturer’s protocol, and optical densities were measured at 405 nm with reference wave length of 490 nm Both assays were measured in an Infinite M200 Plate Reader, TECAN. 2.5 l of Bub and 2.5 l of BubR1 variants with the corresponding detection reagents were mixed and incubated for 60 min before measuring the fluorescence signals. In a different experiment setup, 10 M biotinylated BubR1 GLEBS peptides were captured on SA-coated chips (GE Healthcare) following standard protocols [25], and the surface was flooded with a 2-fold dilution of GST-Bub (0 –100 nM, 6-point) using a single cycle kinetics protocol [26] with 120-s association and 600-s dissociation times. All calculations were carried out using the Discovery Studio Suite 4.1 software suite (Accelrys)
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