Mitosis is the stage of cell division in which duplicated chromosomes are separated and partitioned to each daughter cell. The accurate segregation of chromosomes during mitosis requires the precise temporal and spatial coordination of multiple processes such as chromosome condensation, breakdown of the nuclear envelope, and the assembly and operation of the bipolar mitotic spindle. The spindle is the molecular machine that attaches to replicated sister chromatids and at the appropriate time pulls them to opposite poles of the dividing cell so that each daughter cell receives a complete set of chromosomes. Protein phosphorylation is a major mechanism for orchestrating the complex series of events required for mitosis. Our research focuses on the Cdc14 phosphatases, a conserved group of enzymes that play important roles in controlling protein phosphorylation during mitosis.
Cdc14 functions- Protein phosphorylation catalyzed by cyclin-dependent kinases (Cdk) initiates mitosis. In late mitosis, after chromosomes are segregated, this mitotic Cdk activity must be suppressed so that cells can terminate the mitotic program. In budding yeast, Cdc14 has an essential and well-defined role in triggering Cdk inactivation by dephosphorylating three crucial regulatory proteins. Our studies and those of other groups reveal that Cdc14 not only promotes Cdk inactivation at the end of mitosis but also targets many other proteins involved in coordinating chromosome segregation and initiating the onset of cytokinesis. Hence, a major objective of our work is to fully define the mitotic functions of yeast Cdc14 by identifying and characterizing additional substrates. We have recently discovered four putative Cdc14 targets that are components of the yeast spindle pole body, an organelle involved in forming the mitotic spindle and the functional equivalent of the metazoan centrosome. A major goal is to determine whether Cdc14 modulates spindle pole body function by targeting these four proteins, and if so, to define the underlying mechanism(s).
Two human Cdc14 phosphatases, hCdc14A and hCdc14B, have been identified, but little is known about their function or regulation. The lack of information about their physiologic substrates hinders efforts to determine the function of human Cdc14 phosphatases. We are developing a proteomics-based approach to screen for potential substrates using mutant forms of each human Cdc14 enzyme that specifically bind but do not hydrolyze substrates. We have successfully applied a similar substrate-trapping strategy in yeast.
Cdc14 Regulation- We have discovered that a nucleolar protein known as Net1 is a major cell cycle-dependent regulator of yeast Cdc14 activity. Net1 functions both as a docking protein and potent inhibitor that inactivates and sequesters Cdc14 in the nucleolus during interphase and early mitosis releasing it only at late anaphase. To elucidate the mechanism of Net1 inhibition, we are conducting a detailed study of the interaction between Net1 and Cdc14 using mutational analyses. In collaboration with Dr. Stauffacher’s group (Biological Sciences), we are attempting to determine the X-ray structure of the Cdc14/Net1 complex. We are also interested in the processes that control the cell-cycle dependent release of Cdc14 from Net1 and the translocation of Cdc14 in and out of the nucleus.