The objective of the proposed study is to elucidate the role of cAMP dependent protein kinase A (PKA) in cell cycle control in the yeast Saccharomyces cerevisiae. With the elucidation of the mechanisms by which cell growth and division are controlled, the development of cancer cures will flourish. This project in particular will specifically expand our understanding of the way in which cell division occurs in response to internal and external stimuli. To examine the role of PKA in cell cycle control, the following questions will be asked. First, what are the normal fluctuations in PKA activity during the cell cycle? Second, what are the effects of altering the levels of PKA on progression through the cell cycle? Third, do yeast regulate PKA through cAMP independent mechanisms? And fourth, what are the substrates of PKA involved in normal cell cycle progression? To examine the normal levels of PKA activity throughout the cell cycle, cell extracts will be made at different points in the cell cycle, and fluctuations in PKA activity and protein subunit levels can be determined. The effects of altering the levels of PKA activity during the cell cycle can be addressed by overexpressing one of the catalytic subunits. PKA can be immunoprecipitated out of labeled cell extracts made a different points in the cell cycle to identify any associated proteins that are correlated with the presence or absence of kinase activity, and can be characterized further. In order to identify substrates of PKA, a genetic screen can be employed in Saccharomyces cerevisiae to identify those proteins that are involved in the G1 to S phase transition and also in mitosis.
