PTP--PEST AND FOCAL ADHESION SIGNALING

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Cells receive environmental cues from the extracellular matrix that provide instructions for growth and survival, a phenomena known as anchorage-dependent growth. Many cancerous cells exhibit anchorage independent growth presumably due to the usurpation of the normal regulation of cell adhesion-dependent signaling pathways. Cell adhesion to the extracellular matrix via receptors called the integrins generates intracellular signals, including the phosphorylation of proteins on tyrosine. Several protein tyrosine kinases (PTKs) that are regulated by cell adhesion have been identified and include the focal adhesion kinase (FAK) and Src. In addition to these kinases, protein tyrosine phosphatases (PTPases) might also function in regulating cell adhesion-dependent tyrosine phosphorylation. PTP-PEST has emerged as a candidate for such a PTPase since it binds directly to paxillin and can bind/dephosphorylate p130cas, both of which are phosphotyrosine-containing proteins that colocalize with FAK in focal adhesions. These observations suggest the hypothesis that the association between paxillin and PTP-PEST is a mechanism for targeting the PTPase to its substrates and that PTP-PEST might antagonize signaling through integrin regulated PTKs. This hypothesis will be tested by addressing four specific aims. First, PTP-PEST will be overexpressed in cells to determine if it can perturb biochemical and biological signaling by FAK, CAKbeta (a FAK-like PTK) and Src. Second, endogenous PTP-PEST will be perturbed using dominant negative and/or antisense strategies and the consequences upon FAK, CAKbeta and Src signaling assessed. Third, experiments will be performed to determine if FAK, CAKbeta, Src and paxillin are PTP-PEST substrates and the consequences of PTP-PEST induced dephosphorylation of each. Finally, mutants of PTP-PEST that are defective for paxillin binding will be engineered to determine the role of paxillin binding in PTP-PEST mediated dephosphorylation of substrates and the biological function of PTP-PEST.