Diverse Mechanisms of Regulating the Mitotic Cell Cycle

Author: Shou, Wenying

Year: 2001

Degree: Dissertation (Ph.D.)

Advisor: Dunphy, William G.

Committee Members: Deshaies, Raymond Joseph; Wold, Barbara J.; Sternberg, Paul W.; Meyerowitz, Elliot M.; Dunphy, William G.; Anderson, David J.

Option: Biology

DOI: 10.7907/TP2C-Q082

Abstract

The mitotic cell cycle - a process in which one cell divides into two - is carefully regulated in response to signals. Organisms as diverse as yeast and human all drive their cell cycles by turning cyclin-dependent kinases (Cdks) on and off. We have examined Cdk regulation in two systems. First, we isolated a relatively general Cdk inhibitor (CKI) p28Kix1 in Xenopus laevis. p28Kix1 binds to and directly inhibits multiple Cdks, and retards DNA replication and mitosis when added to Xenopus egg extracts. Remarkably, the protein level of p28Kix1 is dramatically upregulated around late gastrulation, suggesting that it is induced in response to a developmental signal and in turn functions to establish a somatic type of cell cycle. Second, we examined how Cdk inactivation is achieved during mitotic exit in the yeast S. cerevisiae. We found that mutants in at least six linkage groups bypassed the mitotic arrest in cdc15Δ cells. The net1-1 mutant was studied further. Net1 inhibits mitotic exit by inhibiting Cdc 14, an essential protein phosphatase, using two parallel mechanisms: by binding and inactivating Cdc14, and by tethering Cdc14 to the nucleolus. Correct orientation of the mitotic spindle activates Cdc15, which evicts Cdc14 from the nucleolus into the entire cell. Cdc14 subsequently inactivates Cdks by promoting cyclin degradation and CKI induction, and mitotic exit ensues. Unexpectedly, Netl also regulates the structure and function of the nucleolus: it tethers the transcriptional silencing protein Sir2 to the nucleolus, regulates proper localization of multiple nucleolar antigens and rDNA morphology, and directly binds to and stimulates the activity of RNA Pol I. In summary, we observe that different signals regulate cell cycle progression by controlling Cdk activity, that cell cycle regulators may play important roles in other biological processes, and that localization of a protein to a subcellular structure could imply that it is sequestered instead of employed there.

Files