Cancer is a disease of the cell cycle. The aberrant proliferation of cancerous cells is caused by the loss of the normal inhibitory actions of cell cycle control mechanisms. This laboratory has recently identified possible components of a negative cell cycle control system in the yeast Saccharomyces cerevisiae. The action of this """"""""checkpoint"""""""" system prevents cells from exiting the mitotic phase of the cycle prematurely. It is reasonable to suggest that defects in similar systems operating in mammalian cells will lead to aberrant proliferation. We propose an extensive analysis of this checkpoint system with our objective being a molecular description of its function. This laboratory has identified three S. cerevisiae gene-products (Bub1p - Bub3p) required to prevent exit from M-phase when mitotic spindle function is inhibited. Biochemical, cell biological and genetic techniques will be used to investigate the functional relationships within the Bub pathway and the connection between the BUB gene-products and previously characterized cell cycle regulators. Although the existence of cell cycle checkpoints is well documented, little is known of their modes of action at the molecular level. The finding that Bub1p may be a protein kinase suggests specific hypotheses related to the mechanism of action of this checkpoint system. These hypotheses will be tested in the proposed experiments. An in vitro assay for Bub1p activity will be developed and experiments will be performed to identify its in vivo substrate. This laboratory has previously established that M-phase promoting factor (MPF), the protein kinase that drives the mitotic cycles of all eukaryotic cells, is improperly regulated in bub mutants. The possibility that MPF is activated by direct bub1p-mediated phosphorylation will be tested. In addition, the assay for Bub1p kinase activity will be utilized to study its regulation in cycling and arrested cells. The interaction of Bub1p with other cellular components, including the genetically associated Bub3p, will be explored. The potential functional similarity of Bub1p to the mos oncogene product will also be investigated.
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