PTP--PEST AND FOCAL ADHESION SIGNALING

细胞接受来自细胞外基质的环境信号，提供生长和生存的指令，这种现象被称为锚定依赖性生长。许多癌细胞表现出锚定独立生长，可能是由于篡夺了细胞粘附依赖信号通路的正常调节。细胞通过被称为整合素的受体粘附到细胞外基质上，产生细胞内信号，包括酪氨酸蛋白的磷酸化。已经确定了几种受细胞粘附调节的蛋白酪氨酸激酶（PTKs），包括局灶粘附激酶（FAK）和Src。除了这些激酶外，蛋白酪氨酸磷酸酶（PTPases）也可能在调节细胞粘附依赖性酪氨酸磷酸化中起作用。PTP-PEST已经成为这样一种PTPase的候选物，因为它直接与paxillin结合，并且可以结合/去磷酸化p130cas，这两种蛋白都是含有磷酸酪氨酸的蛋白，在局灶粘附中与FAK共定位。这些观察结果表明，paxillin和PTP-PEST之间的关联是一种将PTPase靶向其底物的机制，PTP-PEST可能通过整合素调控的PTKs拮抗信号传导。这一假设将通过解决四个具体目标来验证。首先，PTP-PEST将在细胞中过表达，以确定它是否会干扰FAK、CAKbeta（一种类似FAK的PTK）和Src的生化和生物信号传导。其次，内源性PTP-PEST将使用显性负性和/或反义策略进行干扰，并评估对FAK、CAKbeta和Src信号传导的影响。第三，实验将确定FAK、CAKbeta、Src和paxillin是否是PTP-PEST的底物，以及PTP-PEST诱导它们去磷酸化的后果。最后，将设计PTP-PEST的paxillin结合缺陷突变体，以确定paxillin结合在PTP-PEST介导的底物去磷酸化中的作用以及PTP-PEST的生物学功能。

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.