PTP--PEST AND FOCAL ADHESION SIGNALING

细胞从细胞外基质接收环境信号，这些信号提供生长和存活的指令，这种现象称为锚定依赖性生长。 许多癌细胞表现出锚定非依赖性生长，这可能是由于细胞粘附依赖性信号通路的正常调节被篡夺。 细胞通过称为整合素的受体粘附到细胞外基质，产生细胞内信号，包括酪氨酸上蛋白质的磷酸化。已经鉴定了几种受细胞粘附调节的蛋白酪氨酸激酶（PTK），包括粘着斑激酶（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已经成为这种PTP 3的候选者，因为它直接结合桩蛋白并可以结合/去磷酸化p130 cas，这两者都是与粘着斑中的FAK共定位的含磷酸酪氨酸的蛋白质。 这些观察结果表明，桩蛋白和PTP-PEST之间的关联是将PTK靶向其底物的机制，并且PTP-PEST可能通过整联蛋白调节的PTK拮抗信号传导。 这一假设将通过解决四个具体目标进行检验。首先，PTP-PEST将在细胞中过表达，以确定它是否可以通过FAK，CAKbeta（FAK样PTK）和Src扰乱生物化学和生物学信号传导。 第二，内源性PTP-PEST将使用显性负和/或反义策略干扰，并评估对FAK、CAKbeta和Src信号传导的影响。第三，将进行实验以确定FAK、CAK β、Src和桩蛋白是否为PTP-PEST底物以及PTP-PEST诱导的每种底物的去磷酸化的结果。最后，将对桩蛋白结合缺陷的PTP-PEST突变体进行工程化以确定桩蛋白结合在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.