This proposal concerns the molecular mechanism of the spindle assembly checkpoint in the mammalian system. This mitotic checkpoint ensures the fidelity of chromosome segregation; inactivation of this checkpoint causes mis-segregation of chromosomes and aneuploidy, leading to tumorigenesis. The checkpoint mechanism prevents premature anaphase initiation by inhibiting the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase that controls sister chromatid separation. The activity of APC at metaphase is controlled by its activator CDC20 and by its inhibitor MAD2, a checkpoint protein. We propose experiments here to address the function of two checkpoint proteins, MAD2 and BUBR1. Our long-term goal is to identify all the components in the mitotic checkpoint pathway, to understand the biochemistry of each signaling step and to reconstitute the signaling pathway in vitro using purified metaphase chromosomes. 1. To detect a checkpoint-dependent chance in MAD2 structure. We will develop a mammalian cell-free checkpoint system using purified metaphase chromosomes and determine the biochemical basis of the checkpoint signaling by the MAD2 protein. 2. To define the role of BUBR1 in control of CDC20 activity. CDC20 is a substrate of the checkpoint kinase BUBR1. We will analyze the biochemical effect and physiological function of phosphorylation of CDC20 by BUBR1, both in vitro and in vivo. 3. To investigate the regulation of BUBR1 by checkpoint pathway. BUBR 1 is activated by the checkpoint pathway. We will study the molecular mechanism that regulates BUBR1 kinase activity in response to checkpoint activation. Results from proposed studies will provide a molecular pathway for the mitotic checkpoint control in mammalian cells. Since the checkpoint pathway is inactivated in several types of cancer, a better understanding of the biochemical pathway for the checkpoint signaling is not only essential to our understanding of the basic workings of the cell cycle machinery and regulated proteolysis in all eukaryotic cells, but also central for the development of novel strategies for cancer diagnosis and treatment.
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