Supervisor: vanemteadur Mart Loog (Tartu Ülikool)
Opponent: Dr. Jan M. Skotheim, PhD, Department of Biology, Stanford University, Stanford, California, USA.
The cell cycle is the process by which cells duplicate their contents and then divide to produce a pair of daughter cells. The master regulators of the cell cycle are cyclin dependent kinases (CDKs). CDKs are activated by their perio¬dically accumulating regulatory partners, the cyclins. The enzymatic activity of cyclin-Cdk complexes is tightly controlled by a variety of mechanisms. Sub¬strate targeting by a given cyclin-Cdk complex is mediated by the active site on the CDK and docking sites on the cyclin subunits. Additionally, the presence of a phosphate-binding pocket on the CDK adaptor subunit Cks1 promotes inter¬action with targets containing multiple phosphorylation sites. In simple euka¬ryotes, such as budding yeast, a single CDK, Cdk1, enzyme associates with se¬veral different cyclins. The combination of rising levels of CDK activity and the distinct substrate specificities of different cyclin-Cdk complexes enables the tem¬porally ordered phosphorylation of the many target proteins that regulate cell cycle events. Robust inhibition of S-phase CDK activity in the G1 phase of the cell cycle is the major mechanism preventing uncontrolled onset of DNA replication. In budding yeast, S phase is switched on after the rapid proteolytic degradation of the Cdk1 inhibitor Sic1. Sic1 is a stoichometric inhibitor of Clb-Cdk1 comple¬xes. It appears at the end of mitosis, and its destruction at the G1/S boundary is induced by Cdk1-mediated multisite phosphorylation. The first part of the present dissertation provides an overview of cell cycle control systems, focusing on the different substrate specificities of the various cyclin-Cdk complexes. Next, the CDK inhibitors in yeast and mammalian cells are introduced. Finally, the role of Cks1 as a phosphate binding adaptor mole¬cule for CDK, and the functional implications of this role are reviewed. The original results presented here cover the following areas: a) studies and discus-sions on the changes in cyclin-Cdk1 substrate specificity during the cell cycle b) in vivo and in vitro characterization and analysis of multisite phosphorylation of Sic1, and c) characterization of the parameters promoting Cks1-mediated multi-site phosphorylation of Cdk1 targets.