A family of protein kinases involved in cell cycle checkpoints

Cells require the precise coordination of events to execute the cell cycle, even under normal conditions. To complicate matters, additional circumstances such as damaged DNA, incomplete replication, or nutrient deprivation can inflict significant damage if the cell cycle is not temporarily arrested to allow for recovery. Arresting the cell cycle under these conditions requires a cell cycle checkpoint, a signaling pathway that recognizes damage or deficiencies and activates the necessary responses, including the cell cycle arrest. The examination of many different checkpoint pathways have identified in each a distinctive high molecular weight protein with some homology to protein and lipid kinases, yet overall these checkpoint genes form a discrete family of its own. This thesis will discuss three of these checkpoint kinases, including their identification and studies of their mechanism of action in their respective pathways.;The identification of the mammalian target of the drug rapamycin, FRAP (FKBP12-rapamycin associated protein), was followed by the molecular cloning of its cDNA. This sequence was discovered to belong to a novel family of genes, which included two similar yeast kinases involved in the checkpoint response to nutrient deprivation. A molecular cloning based on finding additional homologues of FRAP identified a second novel human gene, FRP1 (FRAP-related protein). Its sequence showed similarity not only to FRAP but more closely to other members of this family that were involved in checkpoint responses to DNA damage.;Studies on a third checkpoint gene and another member of this kinase family, the Saccharomyces cerevisiae MEC1, showed that its kinase domain and highly conserved C-terminal tail domain were required for its checkpoint activity. Furthermore, overexpression of an inactive allele of MEC1 can cause a dominant-negative effect on the cell. Genetic studies identified two targets of this inhibition, one related to its checkpoint function and the other to a function essential for normal growth.