My research interests are in oncogenic transformation and cell signaling via cellular focal adhesions, which are sites of close attachment between cells and the surrounding extracellular matrix. Several components of focal adhesions, including the focal adhesion kinase (FAK) and paxillin, were originally isolated as substrates for the Src protein tyrosine kinase in cells transformed by the v-src oncogene. FAK can also be amplified in some human tumors suggesting it might also play a role in human disease. My research efforts have focused upon two protein tyrosine kinases, FAK and the related protein CAKß/Pyk2/CadTK. These two kinases share some properties, e.g. both bind to a number of focal adhesion-associated proteins, but also exhibit some distinct properties, e.g. subcellular localization and regulation by different stimuli. Thus FAK and CAKß are likely to serve partially overlapping, but not redundant, functions in cells. The major goal of the research program is to elucidate the basic mechanisms by which these enzymes transmit biochemical signals in normal cells and the biological consequences of aberrant signaling by these kinases.

Three main aspects of FAK/CAKß signaling are currently under investigation in the laboratory. First, experiments are being performed to elucidate the basic mechanisms by which FAK and CAKß function. These include studies exploring interactions between FAK/CAKß and other proteins. Recent experiments have identified a binding site for the SH3 domain of the Src tyrosine kinase in FAK, and biochemical analyses revealed that this interaction may be important for signaling downstream of FAK. Other experiments have defined sites of interaction between FAK and paxillin and biochemical analyses using mutant proteins suggest that this interaction promotes tyrosine phosphorylation of paxillin, but that the interaction is dispensable for the localization of FAK to focal adhesions. One approach to study the different properties exhibited by FAK and CAKß has been to generate chimeric molecules. By analyzing the properties of the chimeras, it has been possible to determine which regions of these kinases are responsible for their different subcellular localizations, differential phosphorylation of common substrates and for activation of the two kinases by different stimuli. Thus, these experimental approaches have provided insight into the mechanisms of regulation by upstream signals, engagement of components of downstream signaling pathways and targeting of FAK/CAKß to discrete cellular locations. The second aspect of FAK/CAKß signaling under exploration is the role of these tyrosine kinases in human disease, specifically in breast cancer. To facilitate the analysis of aberrant FAK signaling, a hyperactive mutant of FAK has been engineered and characterized. A model cell line has been derived from the T47D breast cancer cell line. FAK/CAKß signaling has been enhanced by overexpression of wild type or activated variants of these kinases and signaling through the endogenous FAK protein has been blocked by expression of a dominant negative FAK mutant. Under both conditions, altered biological properties are observed. This system will be exploited to determine the mechanisms by which FAK can alter the phenotype of breast cancer cells when transmitting aberrant signals. The third area of investigation is the negative regulation of FAK/CAKß signaling by protein tyrosine phosphatases. One phosphatase, PTP-PEST, contains a binding site within its noncatalytic domain for paxillin, a focal adhesion-associated FAK and CAKß substrate. This interaction promotes dephosphorylation of paxillin in vivo. Recent evidence suggests that CAKß, but not FAK, may also be a PTP-PEST substrate. These results suggest that PTP-PEST may regulate signaling by targeting multiple components of the CAKß signaling pathway for dephosphorylation. Currently, biochemical and biological approaches are being used to determine the consequences of dephosphorylation of CAKß and paxillin by PTP-PEST.

Thomas, Ellis, B., Boerner, R.J., Knight, W.B., White III, G.C., & Schaller, M.D. (1998). SH2 and SH3 mediated interactions between FAK and Src. J Biol Chem, 273, 577-583.

Y. Shen, G. Schneider, J-F. Cloutier, A. Veillette and M.D. Schaller. (1998). Direct association of protein-tyrosine phosphatase PTP-PEST with paxillin. J Biol Chem 273, 6474-6481.

Y. Shen and M.D. Schaller. (1999). Focal adhesion targeting: the critical determinant of FAK regulation and substrate phosphorylation. Mol Biol Cell 10, 2507-2518.

J.W. Thomas, M.A. Cooley, J.M. Broome, R. Salgia, J.D. Griffin, C.R. Lombardo and M.D. Schaller. (1999). The role of FAK binding in the regulation of tyrosine phosphorylation of paxillin. J Biol Chem 274, 36684-36692.

Y. Shen, P. Lyons, M.A. Cooley, D. Davidson, A. Veillette, R. Salgia, J.D. Griffin and M.D. Schaller. (2000). The noncatalytic domain of PTP-PEST targets paxillin for dephosphorylation in vivo. J Biol Chem 275, 1405-1413.

M.A. Cooley, J.M. Broome, C. Ohngemach, L.H. Romer and M.D. Schaller. (2000). Paxillin binding is not the sole determinant of focal adhesion localization or dominant negative activity of FAK/FRNK. Mol. Biol. Cell 11:3247-3263.

P.L. Lyons, J.M. Dunty, J.M. Schaefer and M.D. Schaller. 2001. Inhibition of the catalytic activity of CAKb by PTP-PEST mediated dephosphorylation. J. Biol. Chem. 276:24422-24431.