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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.
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