Investigation of CDC2-independent mitotic protein kinases

Mitosis is a highly coordinated event which ensures the equal segregation of the cytoplasmic and genetic material into two daughter cells. A key regulatory process in ensuring the temporal fidelity of mitosis involves reversible protein phosphorylation mediated by protein kinases and phosphatases. The major known protein kinase required for triggering mitosis is the cyclindependent kinase, Cdc2. Recent evidence suggests that Cdc2-independent protein kinase signaling pathways are also involved in mitosis. The murine mammary tumour cell line, FT210, has a temperature-sensitive mutation in the cdc2 gene that causes cell cycle arrest in late G2 at the non-permissive temperature. This arrest can be overcome, and mitotic events induced, by protein phosphatase inhibitors. This experimental system allows distinction between Cdc2-independent and -dependent pathways at mitosis. This allows for identification and investigation of the roles of protein kinases in Cdc2-independent mitotic pathways. I have used an in-gel protein kinase renaturation assay to identify two kinases that are activated independently of Cdc2 at mitosis: a novel 45 kDa mitotic protein kinase and a cell cycle-regulated novel 60 kDa histone-specific mitotic kinase. The 45 kDa protein kinase, p45, was active only at mitosis. p45 kinase activity was detected in all cell lines tested suggesting an essential role in cell division. The p45 kinase was determined to be unrelated to the known 46 kDa kinases, JNK1 and MAPKAP kinase-2. The histone kinase was also active only at mitosis. The identification of this histone-specific mitotic kinase confirms the existence of Cdc2-independent pathways for chromosome condensation at mitosis. I also investigated the regulation of the mitotic kinase, polo-like kinase-1 (Plk1), in Cdc2 mutant cells. Plk1 was inactive at the non-permissive temperature and therefore requires Cdc2-dependent signaling pathways for activity. Plk1 is involved in regulating chromosome segregation during mitosis. These results further strengthen the role of Cdc2 in the regulation of mitotic microtubule dynamics.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat