Structural centrosome abnormalities have been implicated as a potential cause of loss of cell and tissue architecture seen in cancer (i.e., anaplasia) as a result of altered centrosome function in microtubule nucleation and organization. This would result in chromosome missegregation during mitosis as a consequence of multipolar spindle formation. Previous studies of mutant mice or cell culture experiments have shown that both BRCA1 and Aurora-A are involved in the regulation of mitosis. Recently, we found that phosphorylation of BRCA1 Ser308 by Aurora-A is required for mitosis entry. The eukaryotic cell cycle is a cascade of highly complex processes that must occur with striking temporal and spatial precision. Regulatory networks called checkpoints are capable of recognizing and correcting mistakes occurring during these processes. Cells respond to DNA damage and replication blocks in two ways: They arrest the cell cycle to allow time for repair and they induce the transcription of genes that facilitate repair. In the proposed research, the infrastructure of these checkpoint circuits will be probed in mammalian cells using BRCA1 to determine how cells sense problems in mitosis. The general aim is to explore more deeply than heretofore the mechanisms of progression of mitosis regulated by BRCA1, originally uncovered by us. First, we will investigate the roles of BRCA1 phosphorylation by Aurora-A in regulation of the cyclin B1-cdk1 complex, whose activation on the centrosome is essential to enter mitosis. Second, we will study the roles of phosphorylation of BRCA1 Ser308 by Aurora-A in centrosome separation and maturation. Accumulating studies have shown that centrosome maturation in mitosis is crucial for the precise segregation of chromosomes. Biochemical and biological investigations will be formulated to understand the functional interaction of BRCA1 with Aurora-A in the centrosome cycle. Third, we will determine the mechanisms of regulation of Aurora-A by p53 and BRCA1. We plan to utilize BRCA1(-/-) mouse embryo fibroblasts (MEFs) for the proposed experiments. These studies are all aimed at a deeper understanding of both processes of oncogenic transformation and novel approaches to cancer therapy.
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