A cell's decision to divide ultimately rests on its ability to respond correctly to environmental cues. Extracellular signals are interpreted by the cell through a complex system of positive and negative signalling pathways that connect to the machinery that controls cell division. When these regulatory pathways behave correctly, normal controlled cell division is possible. However, when these systems fail as they do in human cancer, uncontrolled division can lead to disastrous results. Cell division is controlled by a carefully orchestrated cycle of events. Perhaps the most intriguing finding of cell cycle research is that all eukaryotic cells appear to use a similar mechanism to regulate progression through important control points of the cell cycle. This is most evident when comparing the transition of cells from the G2 phase into mitosis. Cells regulate this transition by activating a kinase complex, composed of a catalytic subunit, cdc2, and regulatory subunit, cyclin B. The properties of this enzyme complex now form the paradigm of cell cycle regulation. All eukaryotic cells that have been studied contain homologs for cdc2 and cyclin B, and they regulate entry into mitosis in similar ways. It is now clear that other key regulatory points in cell cycle progression are controlled by interesting variations of this pattern. For example, in yeast the transition from G1 into S phase is controlled by the same catalytic subunit but in association with other cyclins known as CLN's. In mammalian cells this pattern is even more complicated, as eight different cyclins have now been discovered. In general, little is known about their correct catalytic partners or about their regulation. Recently, we have identified eight novel human kinases, six of which show greater than 50% amino acid identity with cdc2. Although the analysis of these kinases is at an early stage, it is already clear that some of these kinases have the hallmarks of cell-cycle-regulating kinases; they associate with cyclins and display temporally regulated kinase activity. This grant proposes to investigate the function of these new kinases in the control of mammalian cell cycle progression. Our first goal will be to complete the initial characterization of these kinases. This work is currently underway and encompasses the physical and immunochemical properties of the kinase polypeptides, the timing of their kinase activation, and their patterns of expression. Initial studies show that some of these kinases are closely related to the cdc2 kinase, and these proteins will be come the focus of more detailed cell cycle studies. We will determine whether these kinases are essential for cell cycle progression, how their kinase activities are regulated, what substrates they phosphorylate, and the structural features that distinguish their functional differences from cdc2. The kinases that are more distantly related to cdc2 are the focus of our last goal. Here we will examine the significance of these different properties, paying particular attention to features that are not characteristic of cdc2 or its closely related family members.
