The role of BUBR1 in the maintenance of chromosomal stability

Abstract

The majority of solid tumours contain an incorrect number of chromosomes. This state, called aneuploidy, can be caused by defects in chromosome segregation during the division of cells. Prevention against aneuploidy is dependent on BUBR1. This protein is an essential component of a surveillance mechanism, known as the Mitotic Checkpoint, and is needed for the formation of stable attachments between chromosomes and the mitotic spindle. In addition, mutations in the gene encoding BUBR1 are linked to the inherited disease Mosaic Variegated Aneuploidy. The research described in this thesis focuses on the contribution of BUBR1 in the maintenance of chromosomal stability in normal and disease-related mitosis. We showed that mutations found in patients with Mosaic Variegated Aneuploidy result in chromosome missegregation, primarily by reduction of BUBR1 protein abundance. We furthermore found that BUBR1 kinase activity is dispensable for proper mitosis, but that integrity of the domain is essential to prevent degradation of the complete protein. These insights were inspired by the finding that BUBR1/Mad3 and BUB1 were formed by gene duplication followed by subfunctionalisation, which allowed evolution of vertebrate BUBR1 towards a pseudokinase. Together these data shed new light on the catalysis-independent role of vertebrate BUBR1 in the mitotic checkpoint and formation of kinetochore-microtubule attachments and showed that the kinase domain has a structural instead of catalytic purpose. Subfunctionalisation was not restricted to the kinase domain, but has also caused specialisation another protein domain of BUBR1. We showed that this part of BUBR1, known as the TPR domain, is needed for the role of BUBR1 in the mitotic checkpoint by contributing to the binding to other essential mitotic checkpoint proteins. Lastly, we have identified novel phosphorylations on BUBR1 by the mitotic protein kinase PLK1. One of these modifications regulates the localisation of BUBR1 to chromosomes in mitotic cells. The other modification helps BUBR1 in its role in formation of attachments between chromosomes and the mitotic spindle by facilitating binding to a phosphatase. Together the work described in this thesis provides mechanistic insight into the role of BUBR1 in guarding error-free mitosis.