Focal adhesions (FA) are small dense structures in the plasma membrane where the cell makes close contact with the substratum and binds to the extracellular matrix (ECM) through clusters of integrins acting as ECM receptors. While basal keratinocytes (k'cytes) in normal intact epidermis use hemidesmosomes and not FA to adhere to the basement membrane, laterally migrating and proliferating basal k'cytes near the edges of repairing epidermal wounds and in cell culture use FA to adhere to the ECM. High concentrations of phosphotyrosine (PY) in the FA of spreading cells in culture coincide with increased activity of focal adhesion kinase (FAK), a recently described 119 kDa protein tyrosine kinase that localizes to FA, suggesting that the activation of FAK is required for FA formation. Several of our findings suggest an essential role for FAK in epidermal wound repair. First, FAK is localized to the dermal-epidermal junction in repairing human burn wounds but not in normal, unwounded epidermis. Second, cultured k'cytes which model the migrating and proliferating k'cytes at the edge of repairing wounds have abundant FA in which both PY and FAK are selectively concentrated Third, when isolated from attached and spreading k'cytes in culture, FAK has greatly increased PY levels compared to non-attached or attached but non-spreading k'cytes indicating that FAK is activated in spreading k'cytes. These findings suggest that the expression, localization and activation of FAK in k'cytes is regulated in order to allow their adhesion by FA as they spread, migrate and/or proliferate in culture and in wounded or hyperproliferative epidermis. Hence, the specific objectives of this study are: 1. to determine the localization and expression of FAK: a. in normal, hyperproliferative and repairing human epidermis b. in normal, hyperproliferative and repairing mouse epidermis treated with various cytokines 2. to determine the localization, expression and activity of FAK in cultured human k'cytes whose spreading, migration or proliferation has been altered by cell density, added growth factors and neuropeptides or available ECM components In particular, in situ hybridization using a FAK mRNA anti-sense probe and immunohistochemistry using affinity-purified polyclonal antibodies against FAK will be done on skin where epidermal resurfacing of wounds is occurring, where only hyperproliferation is occurring, or where the skin is intact and normal. These techniques for determining expression and localization of FAK will be supplemented with hybridization to Northern blots and immunodetection on Western blots for mouse skin samples. With cultured k'cytes, in situ hybridization, immunofluorescence for PY and FAK, and quantitative immunoprecipitation of FAK coupled with immunodetection of PY on Western blots will be used to determine the expression, localization and activity (based on its PY content) of FAK.
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