PTP--PEST AND FOCAL ADHESION SIGNALING

细胞从细胞外基质接收环境信号，为生长和生存提供指令，这种现象被称为锚定依赖生长。许多癌细胞表现出锚定无关的生长，可能是由于细胞黏附依赖的信号通路的正常调节被篡夺所致。细胞通过称为整合素的受体与细胞外基质黏附，产生细胞内信号，包括蛋白质在酪氨酸上的磷酸化。目前已鉴定出几种受细胞黏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，蛋白酪氨酸磷酸酶(PTPase)也可能在调节细胞黏附依赖的酪氨酸磷酸化方面发挥作用。PTP-PEST已成为此类PTPase的候选蛋白，因为它直接与巴西林结合，并能结合/去磷酸化p130Cas，这两种蛋白都是含有磷酸酪氨酸的蛋白，在局灶性粘连中与FAK共存。这些观察结果表明，Paxlin和PTP-PEST之间的联系是将PTPase靶向底物的一种机制，PTP-PEST可能通过整合素调节的PTKs来拮抗信号。这一假设将通过解决四个具体目标来检验。首先，PTP-PEST将在细胞中过表达，以确定它是否可以通过FAK、CAKβ(一种类似FAK的PTK)和Src干扰生化和生物信号转导。其次，内源PTP-PEST将被显性的负和/或反义策略干扰，并评估其对FAK、CAKβ和Src信号的影响。第三，将进行实验以确定FAK、CAKbeta、Src和paxlin是否是PTP-PEST底物，以及PTP-PEST诱导每种底物去磷酸化的后果。最后，对PTP-PEST的突变株进行改造，以确定PXLIN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.