Roles of Bub1 and BubR1 in human checkpoint signalling

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

The accurate and equal segregation of genetic material to daughter cells requires attachment of sister chromatids to dynamic spindle microtubules from opposite spindle poles. This occurs through the kinetochore, which not only provides the mechanical linkage between microtubules and chromosomes, but which also acts as the platform for a surveillance system, known as the spindle assembly checkpoint (SAC), that ensures sister chromatids do not separate until all chromosomes are correctly bi-oriented. Components of the checkpoint include the Mad1, Mad2, Bub3 proteins and the Bub1, BubR1, Mps1(Mph1) and Aurora B kinases. SAC components bind kinetochores and initiate signalling when individual kinetochores are either not bound to spindle microtubules or not under tension. It has recently been found that phosphorylation of multiple repetitive motifs (MELT) in the kinetochore protein KNL1 by the Mps1 protein kinase, creates a binding site for the Bub3-Bub1 complex. Further phosphorylation of the central non-catalytic region of Bub1 by Mps1 promotes the association of Mad1-Mad2 complex to Bub1. The Mad1-Mad2 complex, once bound to kinetochores, catalyses the conversion of soluble Mad2 (O-Mad2) into a form (C-Mad2), which binds both Cdc20 and Bub3-BubR1, to form the mitotic checkpoint complex (MCC), a potent inhibitor of the anaphase promoting complex (APC/C), an essential E3 ubiquitin ligase. When the checkpoint is satisfied the spindle checkpoint signal is silenced and the Cdc20-APC/C is activated. This triggers the poly-ubiquitination of securin and cyclin, which allows the dissolution of sister chromatid cohesion and mitotic progression, respectively. In human cells the Rod-Zw10-Zwilch (RZZ) complex, which is absent in yeast, provides a second binding site for the Mad1-Mad2 complex at kinetochores. This PhD project is designed to elucidate the roles of the Bub1 and BubR1 kinases in checkpoint signalling in both normal and transformed human cells.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1643079 Studentship BB/M01116X/1 05/10/2015 30/09/2019 Cerys Currie
 
Description Accurate chromosome segregation in mitosis is essential for human life, health and development. A process called the spindle assembly checkpoint (SAC) acts as a surveillance mechanism to prevent errors in chromosome segregation. In the past the SAC has been studied in fission yeast, but recent data indicates that the process is more complex in human cells. In yeast there is one pathway ensuring faithful segregation, but in human cells there are at least two separable pathways. My project provides further proof of these two pathways in human cells. I have been able to directly edit the human genome in RPE1 cells using CRISPR-Cas9 technology, and knockout a gene which was thought to be essential for the SAC previously (BUB1). I have shown that this gene is not essential for the SAC in human cells, and there is some evidence that the gene may become more important in cancer cells (HeLa) compared to a healthy cell (RPE1). In the future, this could become a useful drug target for therapeutics. I will now be carrying out further experiments to identify the exact role of this gene in ensuring proper chromosome segregation.
Exploitation Route There is some evidence that the gene I am studying (BUB1) is more important for chromosome segregation in cancer cell lines (HeLa) opposed to a healthy human cell line (hTERT-RPE1 cells). This is important, as Bayer has developed small molecule inhibitors which inhibit Bub1 kinase activity. These drugs cause big problems for HeLa cells, but a much less penetrant phenotype in RPE1 cells. Also, knocking out Bub1 in RPE1 cells is proof that Bub1 is not essential for these cells which are more representative of healthy human tissue. In the future I envisage that Bub1 could become an attractive target for chemotherapy, as the healthy cells may be unaffected while cancer cells will not be able to segregate their chromosomes correctly. This will cause the cancer cells to die, and reduce off target effects of chemotherapy. However, much more work needs to be done before this is possible.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology