Author: Tiffany A. Bumbarger

Activity and Regulation of the Mitotic Phosphatase Cdc14p
Chelsi Kaiser, Whitney Franklin, Dr. Kim Simons
Departments of Physical Sciences, Emporia State University, Emporia, KS 66801

Abstract: The end of mitosis is marked by a duplication of cellular DNA, segregation of chromosomes to opposite sides of the mitotic spindle, and proper alignment of the spindle within the dividing cell. If any of these events are not completed properly, the mitotic exit network delays cytokinesis. Most cancerous cells and some other human diseases are marked by aneuploidy, an abnormal number of chromosomes in a cell. Questions remain in cell biology: how does the cell prevent aneuploidy, and how can these features be exploited to cure cancer? Likewise, a similar question is asked in the area of chemical biology: how do regulatory proteins detect and prevent these errors from occurring? As key player in cell cycle control, Cdc14p is the regulatory protein that once activated, promotes the cell to exit from mitosis and begin cytokinesis. The goals of the research project are to express the CDC14 gene to make the mitotic phosphatase Cdc14p, to purify the protein using traditional and affinity purification techniques, and to test the activity of the protein in a fluorescence assay.

Figure 1: The M (Mitotic) phase of the cell cycle1.

Figure 2: The Mitotic Exit Network. Cdc14p plays a key role in how a cell exits mitosis and enters cytokinesis2.

Research Plan:

Clone Cdc14p for overexpression
in E. coli

Transfect inducible cells with the plasmids and express the genes

Sequence constructs

Purify proteins using affinity resin and standard chromatography techniques on a FPLC
column

Assay phosphatase activity using fluorescently induced substrates.

Figure 3: Below left: DNA Agarose Gel of PCR product. The bands were cut out of the gel for purification purposes. CDC14 is the gene that codes for Cdc14p (protein) and pGEX is the vector in which a ligation reaction was performed to combine the two. Below right: Visual representation of pGEX vector containing the CDC14 gene.

Cartoon of the Crystal Structure of the Dual Specificity Protein Phosphatase Cdc14p
(pdb code 1OHE)

Future Goals :
We will study Cdc14p using a Fluorescent Phosphatase Assay. Using a spectrofluorimeter we will be able to observe Cdc14p activity in a reaction with a fluorescent molecule called MUP (shown below).
To help us to further understand the roles of Cdc14p in the mitotic exit network of S. cerevisiae, other regulatory proteins will be added to the assay to see the effects on Cdc14p activity. Research on Cdc14p may act as a stepping stone to further understand cancer since human CDC14 is known to be defective in some types of cancer.

Figure 4: MUP– When the phosphate group is hydrolyzed, the product becomes fluorescent.

1Image modified from: http://www.cbu.edu/~seisen/CellCycle.htm
2Image by: Bosl, William and Rong Li. Mitotic Exit Control as an Evolved Complex System. Cell, Vol. 121, 325–333, May 6, 2005
This poster made possible by a NASA Space Grant and EPSCoR funding through the Kansas Space Grant Consortium.
A Kathrine White Faculty Incentive Grant (Emporia State University Foundation), an Undergraduate Research Grant (Office of the Graduate School and Distance Education at ESU), and K-INBRE (Kansas IDeA Network of Biomedical Research Excellence) supported this research project.