Current Biochemistry Faculty - Harry Charbonneau

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Harry Charbonneau

Professor of Biochemistry
439C Hansen, (765) 494-4754
Ph.D. University of Georgia, 1981
charb@purdue.edu

Investigators:
Graduate Students:
Steven Bremmer
Ying Wang

Area: Mitotic Function and Regulation of the Cdc14 Protein Phosphatase

We are interested in understanding how protein phosphorylation is used to orchestrate the complex and dynamic processes of mitosis, the stage in somatic cell division when duplicated chromosomes are physically separated and transmitted to each daughter cell. The successful transmission of a complete genome to each daughter cell during mitosis requires the precise coordination of multiple processes such as chromosome condensation, breakdown of the nuclear envelope, and the assembly and movement of the mitotic spindle apparatus, the molecular machinery that physically separates chromosomes. Protein phosphorylation is a key regulatory mechanism for the temporal and spatial coordination of these crucial mitotic events.

To help define the role of phosphorylation in mitotic control we are investigating the Cdc14 protein phosphatase of budding yeast. In this organism, Cdc14 is required for cells to complete mitosis and initiate cytokinesis. Protein phosphorylation catalyzed by cyclin-dependent kinases (Cdk) initiates mitosis. Later in mitosis, after chromosomes are segregated, Cdc14 dephosphorylates three regulatory proteins to trigger events leading to Cdk inactivation. This function of Cdc14 is essential because mitotic Cdk activity must be suppressed for all eukaryotic cells to terminate mitosis.

	Cdc14 Functions- A major goal of our research is to identify additional Cdc14 substrates in order to define all the cell cycle functions of this phosphatase. Our studies and those of other groups reveal that Cdc14 not only promotes Cdk inactivation at the end of mitosis but also targets proteins involved in other crucial processes that coordinate the end of mitosis, return the nucleus to a pre-mitotic state, and prepare the cell for cytokinesis. 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).
Yeast Cdc14 is the prototype for a subfamily of phosphatases that is highly conserved among eukaryotes. We are also examining the role of Cdc14 orthologs in human cell division. Two human Cdc14 phosphatases, hCdc14A and hCdc14B, have been identified, but little is known about their function or regulation. One approach to defining the functions of the human Cdc14 phosphatases is to identify their targets using substrate-trapping strategies that we have successfully applied in yeast. Mutations in genes encoding proteins that regulate cell division are frequently found in human tumors and it is clear that the resulting loss of growth control is involved in cancer. Defining how mitosis and other complex events of cell division are regulated by protein phosphorylation is a major goal in basic cancer research.

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.

Selected Publications:

Wang, W.Q., Bembenek, J., Gee, K. R., Yu H., Charbonneau, H., and Zhang, Z. Y. Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14. J. Biol. Chem. 279:30459-30468 (2004).
Charbonneau, H. Cell cycle functions and regulation of Cdc14 phosphatases. The Handbook of Cell Signaling, (Bradshaw, R.A. & Dennis, E.A., eds.) Academic Press. Vol. I: Chpt. 116; 697-701 (2003).
Traverso, E. E., Baskerville, C., Liu, Y., Shou, W., James, P., Deshaies, R., J., and Charbonneau, H. Characterization of the Net1 Cell Cycle-dependent Regulator of the Cdc14 Phosphatase from Budding Yeast. J. Biol. Chem. 276:21924-21931 (2001).
Shou, W., Sakamoto, K.. M., Keener, J., Morimoto, K. W., Traverso, E. E., Azzam, R., Hoppe, G. J., Feldman, R. R. M., DeModena, J., Moazed, D., Charbonneau, H., Nomura, M., and Deshaies, R. J. Net1 stimulates RNA Polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit. Mol. Cell 8:45-55 (2001).
Shou, W. Seol, J.H., Shevchenko, A., Baskerville, C. Moazed, D., Chen, Z. W. S., Jang, J., Shevchenko, A., Charbonneau, H., and Deshaies, R. J. Exit from Mitosis Is Triggered by Tem1-Dependent Release of the Protein Phosphatase Cdc14 from Nucleolar RENT Complex. Cell 97:233–244 (1999).