Ales Vancura, Ph.D.

Nuclear Signaling by Phospholipase C

Our Laboratory focuses on understanding the role of phospholipase C (PLC) in regulation of chromatin structure, kinetochore activity, and chromosome segregation. PLC generates two prominent eukaryotic second messengers: 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Traditionally, PLC activity has been thought to be associated with the plasma membrane. However, studies from our Laboratory and others have shown that PLC operates independently in the nucleus as well.

Using yeast Saccharomyces cerevisiae as a model, we have recently found that PLC (Plc1p protein encoded by the PLC1 gene) associates with kinetochores and affects their ability to bind microtubules. Kinetochores are protein complexes, which assemble on centromeric regions of chromosomes, bind microtubules, and are essential for chromosome segregation during mitosis. We found that cells with deletion of PLC1 gene display higher frequency of chromosome loss, nocodazole sensitivity, and mitotic delay. Furthermore, chromatin extracts from these cells exhibit reduced microtubule binding to minichromosomes. Both enzymatic activity of Plc1p and its ability to bind kinetochores are required for its mitotic function(s).

Currently we are testing hypothesis that hydrolysis of phosphatidylinositols by PLC regulates chromatin structure at centromeres, telomeres, and certain promoters. We are using multiple approaches, including chromatin immunoprecipitation to determine protein-DNA interactions in vivo, two-hybrid assay to identify interacting proteins and to screen for inhibitory peptides, and site-directed mutagenesis to characterize regulatory proteins.

The work in this Laboratory has been significantly enhanced by the diligent and creative work of our doctoral students, who after graduating assumed postdoctoral positions at premier research institutions (Rockefeller University - Laboratory of Dr. Greengard, Nobel Prize Laureate and Washington University Medical School).

This work has been continuously supported since 1995 by the American Cancer Society and the National Institutes of Health.

Recent publications

Lin, H., Choi, J.H., Hasek, J., DeLillo, N., Lou, W., and Vancura, A. 2000. Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae. Mol. Cell. Biol. 20: 3597-3607.

Vancurova, I., Choi, J.H., Lin, H., Kuret, J., and Vancura, A. 1999. Regulation of phosphatidylinositol (4)P 5-kinase from Schizosaccharomyces pombe by casein kinase I. J. Biol. Chem. 274: 1147-1155.

Chakraborty, T.R., Vancura, A., Balija, V.S., and Haldar, D. 1999. Phosphatidic acid synthesis in mitochondria. Topography of formation and transmembrane migration. J. Biol. Chem. 274: 29786-29790.

Choi, J.H., Lou, W., and Vancura, A. 1998. A novel membrane bound glutathione S-transferase functions in the stationary phase of the yeast Saccharomyces cerevisiae. J. Biol. Chem. 273: 29915-29922.

Lin, H., Choi, J.H., and Vancura, A. 1998. Phosphoinositide-specific phospholipase C interacts with phosphatidylinositol kinase homolog TOR2. Biochem. Biophys. Res. Commun. 252: 285-289.