Baker DJ, etal., J Cell Biol. 2006 Feb 13;172(4):529-40.
Aging is a highly complex biological process that is believed to involve multiple mechanisms. Mice that have small amounts of the mitotic checkpoint protein BubR1 age much faster than normal mice, but whether other mitotic checkpoint genes function to prevent the early onset of aging is unknown. In
this study, we show that several aging-associated phenotypes appear early in mice that are double haploinsufficient for the mitotic checkpoint genes Bub3 and Rae1 but not in mice that are single haploinsufficient for these genes. Mouse embryonic fibroblasts (MEFs) from Bub3/Rae1 haploinsufficient mice undergo premature senescence and accumulate high levels of p19, p53, p21, and p16, whereas MEFs from single haploinsufficient mice do not. Furthermore, although BubR1 hypomorphic mice have less aneuploidy than Bub3/Rae1 haploinsufficient mice, they age much faster. Our findings suggest that early onset of aging-associated phenotypes in mice with mitotic checkpoint gene defects is linked to cellular senescence and activation of the p53 and p16 pathways rather than to aneuploidy.
Lo KW, etal., J Biol Chem. 2007 Apr 13;282(15):11205-12. Epub 2007 Feb 7.
Cytoplasmic dynein is the motor protein responsible for the intracellular transport of various organelles and other cargoes toward microtubule minus ends. However, it remains to be determined how dynein is regulated to accomplish its varied roles. The dynein complex contains six subunits, including
three classes of light chains. The two isoforms of the DYNLT (Tctex1) family of light chains, DYNLT1 and DYNLT3, have been proposed to link dynein to specific cargoes. However, no specific binding partner had been found for the DYNLT3 light chain. We find that DYNLT3 binds to Bub3, a spindle checkpoint protein. Bub3 binds exclusively to DYNLT3 and not to the other dynein light chains. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that Bub3 interacts with the cytoplasmic dynein complex. DYNLT3 is present on kinetochores at prometaphase, but not later mitotic stages, demonstrating that this dynein light chain, like Bub3 and other checkpoint proteins, is depleted from the kinetochore during chromosome alignment. Knockdown of DYNLT3 with small interference RNA increases the mitotic index, in particular, the number of cells in prophase/prometaphase. These results demonstrate that dynein binds directly to a component of the spindle checkpoint complex through the DYNLT3 light chain. Thus, DYNLT3 contributes to dynein cargo binding specificity. These data also suggest that the subpopulation of dynein, containing the DYNLT3 light chain, may be important for chromosome congression, in addition to having a role in the transport of checkpoint proteins from the kinetochore to the spindle pole.
Prinz F, etal., J Biol Chem. 2016 May 20;291(21):11252-67. doi: 10.1074/jbc.M115.702142. Epub 2016 Mar 30.
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 th
e 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.
