The major focus of our research efforts is signal transduction via the Focal Adhesion Kinase, or FAK, a protein tyrosine kinase. FAK discretely localizes within cells to focal adhesions, regions where the cell comes in close contact with the extracellular matrix. FAK is a major component of a signaling pathway that is controlled by integrins, which are receptors for proteins in the extracellular matrix. Other stimuli, like growth factors, can also stimulate FAK activity and FAK may play an important role in cross talk between the integrins and growth factor receptors. Cell motility and cell survival are two important biological processes that are controlled by FAK. Since cancerous/metastatic cells exhibit altered cell motility and survival, altered FAK signaling may promote some of the phenotypes exhibited in cancer cells. Indeed, experimental manipulation of FAK signaling can alter some phenotypes associated with cancer in both normal and cancer cell lines. Thus elucidation of the fundamental mechanisms of signaling via FAK may have important therapeutic applications to the treatment of human disease.

Project 1: The N-terminal domain of FAK is critical for linking FAK signaling to growth factor receptor signaling pathways. The molecular basis for the regulation of FAK by growth factor signaling pathways has not been established, but may involve direct or indirect physical interactions between FAK and other components of growth factor signaling pathways. In fact, the N-terminal domain of FAK contains a FERM domain, which is a domain that mediates protein-protein interactions. A current project in the lab is to develop a theoretical three-dimensional model of the structure of the N-terminal domain of FAK by computer homology modeling. This model will be utilized in project 1 to identify regions that may function as docking sites for other proteins, e.g. acidic patches or basic pockets. These regions will be altered by site-directed mutagenesis. The mutants will then be characterized for interactions with growth factor receptors and other potential binding partners in vitro. In the longer term, selected mutants will be expressed in the normal mammary gland cell line, MCF10A, and the T47D breast cancer cell line to determine if the mutants exhibit an altered ability to promote cell motility and cell survival.

Project 2: The C-terminal domain of FAK is critical for the correct localization of FAK to focal adhesions and for linking FAK to the integrin-dependent signaling pathway. Several focal adhesion-associated proteins, including paxillin, bind to the C-terminal domain of FAK, but mutagenesis strategies have not definitively established which binding partner mediates FAK localization. As a parallel approach, we are exploring a peptide competition approach to further test the role of paxillin binding in targeting FAK to focal adhesions. We have identified a minimal FAK binding site on paxillin as a short peptide motif and demonstrated that a synthetic peptide based upon this sequence inhibits the interaction between FAK and paxillin in vitro. In project 2, a membrane permeable synthetic peptide will be introduced into cells. The ability of the peptide to impair the interaction between FAK and paxillin will be assessed by coimmunoprecipitation and Western blotting. The effect of the peptide upon localization of FAK will be examined by immunofluorescence and the consequences to FAK signaling by immunoprecipitation and Western blotting.