Laura Trinkle-Mulcahy , Ph.D.
Department of Cellular and Molecular Medicine
Faculty of Medicine
University of Ottawa
451 Smyth Road, Ottawa, ON K1H 8M5
Phone: (613) 562-5800 (8068)
FAX: (613) 562-5434
The reversible addition of phosphate groups to proteins is the major general mechanism that regulates most physiological processes in mammalian cells, and over one-third of the thousands of proteins expressed by the typical cell are believed to be phosphoproteins. The enzymes that regulate protein phosphorylation are termed protein kinases (add phosphates) and protein phosphatases (remove phosphates). Abnormal phosphorylation, resulting from misregulation of kinase and/or phosphatase activity, has been linked to the progression of diseases such as cancer and diabetes. Our research is aimed at characterizing the specific targeting of phosphatase complexes within living cells, to better understand the phosphorylation-mediated processes that can give rise to these diseases in humans and identify steps that can targeted for disruption in therapeutic approaches.
Progress in phosphatase research has been hindered by a lack of robust and specific reagents, such as inhibitors and isoform-specific antibodies. We addressed this discrepancy by developing techniques that allow us to study the dynamic regulation of phosphatases in live cells. Protein Phosphatase 1 (PP1) is one of the major serine/threonine protein phosphatases, and exists as three distinct isoforms in mammalian cells. The catalytic subunit is always found in complex with other proteins, termed “targeting subunits”, from which it derives both its localization and substrate specificity. Dissecting the in vivo roles of specific PP1 complexes thus requires detailed molecular analysis of their composition.
In order to study both the localization and the composition of PP1 complexes in vivo, we established human cell lines that stably express PP1 isoforms tagged with the fluorescent reporter molecule GFP (green fluorescent protein). Live cell imaging of GFP-PP1alpha and GFP-PP1gamma, for example, reveals distinct and dynamic patterns of localization throughout the cell cycle and provides valuable clues to the intracellular regulatory pathways in which they’re involved. In addition, dynamic properties such as turnover rate and direct interactions with targeting subunits can be measured using microscopy techniques such as FRAP (fluorescence recovery after photobleaching) and FRET (fluorescence resonance energy transfer).
GFP can also be used as an affinity tag, to immunoprecipitate the same proteins analyzed by microscopy and identify the proteins that co-purify with them in protein complexes. We have used this powerful “what you see is what you get” approach to identify several novel PP1 complexes and to compare the affinities of targeting subunits for a particular isoform. Having noted an isoform-specific recruitment to segregating chromosomes during mitosis, for example, we adapted a powerful quantitative proteomics technique based on stable isotope labeling (SILAC) to identify the targeting subunit responsible, which we termed “RepoMan” (Recruits PP1 Onto Mitotic chromosomes at Anaphase).
Ongoing projects in the lab include identifying and characterizing PP1 targeting subunits and determining isoform specificity, if any. We are also continuing to study the targeting of RepoMan/PP1 to chromosome-associated substrates throughout the cell cycle, and that of other novel PP1 complexes identified in our SILAC IP screens of GFP-PP1 in nuclear and cytoplasmic extracts. As one of only a handful of labs using this combined approach of cell biology, biochemistry and quantitative proteomics, we feel confident that we will continue to general novel and exciting data that will help to unravel the intricacies of PP1 regulation in mammalian cells.
For more information about our work on targeted PP1:
Trinkle-Mulcahy, L., Andersen, J., Lam, Y.W., Moorhead, G., Mann, M. and Lamond, A.I. Repo-Man recruits PP1γ to chromatin and is essential for cell viability. J. Cell Biol. 172:679-92.
Vagnarelli, P., Hudson, D.F., Ribeiro, S.A., Trinkle-Mulcahy, L., Spence, J.M., Lai, F., Farr, C.J., Lamond, A.I. and Earnshaw, W.C.. Condensin and Repo-Man/PP1 co-operate in the regulation of chromosome architecture during mitosis. Nat. Cell Biol. 8:1133-42.
Trinkle-Mulcahy, L., Andrews, P.D., Wickramasinghe, S., Sleeman, J., Prescott, A., Lam, Y.W., Lyon, C., Swedlow, J.R. and Lamond, A.I. Time-lapse imaging reveals dynamic relocalization of PP1γ throughout the mammalian cell cycle. Mol. Biol. Cell, 14:107-117, 2003.
Trinkle-Mulcahy, L., Sleeman, J.E. and Lamond, A.I. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells. J. Cell Science. 114:4219-4228, 2001.
For general information about the role of protein phosphatases in the cell nucleus and during cell division:
Moorhead , G.B.G., Trinkle-Mulcahy, L. and Ulke-Lemee, A. Emerging roles of nuclear protein phosphatases. Nat. Rev. Mol. Cell Biol. 3:234-44, 2007.
Trinkle-Mulcahy, L. and Lamond, A.I. Mitotic phosphatases: no longer silent partners. Current Opinion in Cell Biology. 18:623-31, 2006.
For information about the impact of new fluorescence imaging and proteomics technologies on studies of nuclear structure and function:
Trinkle-Mulcahy, L., Boulon, S., Lam, Y.W., Urcia, R., Boisvert, F.-M., Vandermoere, F., Morrice, N.A., Swift, S., Rothbauer, U., Leonhardt, H. and Lamond, A.I. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. In Press.
Trinkle-Mulcahy, L. and Lamond, A.I. Toward a high-resolution view of nuclear dynamics. Science. 318:1402-07, 2007.
Trinkle-Mulcahy, L. and Lamond, A.I. Nuclear functions in space and time: Gene expression in a dynamic, constrained environment. FEBS Lett. 582:1960-70, 2008.
For more information about fluorescent microscopy techniques and working with FP-tagged PP1 (stable cell lines, FRAP, FRET, etc):
Trinkle-Mulcahy, L., Chusainow, J., Lam, Y.W., Swift, S. and Lamond, A.I. Visualization of intracellular PP1 targeting through transiently and stably expressed fluorescent protein fusions. Methods in Molecular Biology. Vol.. 365:133-54; Protein Phosphatase Protocols, Ed. G. Moorhead, Humana Press, 2006.
Trinkle-Mulcahy, L. and Swift, S. Caught on camera with another protein - Just good friends or something more? A Guide to Localization, Colocalization and Interaction on the Light Microscope. In Focus (Proceedings of the Royal Microscopy Society), 8:4-15.
Swift, S.R., Thomson, C., Appleton, P., Lam, Y.W. and L. Trinkle-Mulcahy. Basic principles deconvolution microscopy. Proc. Royal Mic. Soc., 40, 2005.
Swift, S.R. and L. Trinkle-Mulcahy. Basic principles of FRAP, FLIM and FRET. Proc. Royal Mic. Soc., 39:3-10, 2004.