Roles of the PHO85 cyclin-dependent kinase in budding yeast

grantor: University of TorontoCyclin-dependent kinase (Cdk) complexes are essential activators of cell cycle progression in all eukaryotes. The activity of Cdks is regulated in part by their association with essential positive regulatory subunits called cyclins. In contrast to mammalian cells, in which multiple Cdks contribute to cell cycle progression, the budding yeast cell cycle is largely controlled by the activity of a single Cdk, Cdc28. In this thesis, I describe the identification of a second Cdk, Pho85, to have a role in cell cycle control in the budding yeast 'Saccharomyces cerevisiae' ('S. cerevisiae'). The Pho85 kinase, which is homologous to Cdc28, was first identified as a negative regulator of acid phosphatase gene expression in association with the Pho80 cyclin. Entry into the budding yeast mitotic cell cycle, or Start, requires Cdc28 and one of its three G1 cyclins, Cln1, Cln2, and Cln3. I show that in the absence of Cln1 and Cln2, the Pcl1 and Pcl2 G1 cyclins, in association with Pho85, become essential for the Start transition. Pcl2 associated with a Pho85-dependent kinase activity in G1 phase and phosphorylated a known target of the Pho85 kinase, Pho4, 'in vitro'. In addition, the Pcl2-Pho85 kinase had a different substrate specificity than Cdc28 and was not dependent on the Cdc28 kinase. Two-hybrid screening and data base searching identified seven new members of the Pho85 cyclin family. The putative Pho85 cyclins were placed into the Pcl1,2 or Pho80 subfamily based on sequence similarity in the cyclin box region which is a region conserved amongst all cyclins. Analysis of strains lacking members of the Pcl1,2 subfamily suggests a role for these cyclins in regulating cell morphogenesis. Finally, I present evidence to suggest a role for Pho85 in influencing G1-specific gene expression. 'In vitro', Pho85 interacted with the Swi5 transcription factor which controls expression of genes in G1 phase. ' In vivo', Pho85 was required for the asymmetric localization of an inhibitor of gene expression called Ash1 to daughter cell nuclei. Studies of the role for Pho85 in Ash1 localization are likely to reveal a novel function for a cyclin-Cdk complex in maintaining asymmetry in the cell.Ph.D