Protein Tyrosine Phosphatase Alpha in Integrin Signaling and Cell Movement

细胞运动是一个基本的生理过程。细胞运动的主要决定因素是整合素，细胞膜上的受体，其协调粘附性细胞-基质接触与肌动蛋白细胞骨架的连续重组。整合素结合启动细胞内信号传导，其驱动称为粘着斑（FA）的多蛋白复合物的组装。FA信号既控制细胞骨架重塑，也控制它们自身的动态组装和拆卸，以调节细胞的形状和运动。 受体蛋白酪氨酸磷酸酶α（PTPa）是促进细胞运动的整合素信号传导的正调节剂。PTPa缺陷的小鼠胚胎成纤维细胞表现出有缺陷的整合素刺激的迁移，其特征在于延迟的细胞骨架重排和受损的FA形成。通过研究这些缺陷，我们发现PTPa在整合素介导的信号传导中起两种作用，以促进FA组装并最终促进细胞迁移：（i）作为Src-FAK激酶复合物的上游激活剂的催化作用，和（ii）作为Src-FAK复合物的下游靶标的非催化作用。在其第二个作用中，PTPa被其C-末端尾Tyr 789中的酪氨酸残基上的活性Src-FAK磷酸化。这对于FA形成和促进细胞迁移的信号传导是关键的，然而PTPa-phosphoTyr 789（PTPa-pTyr 789）的信号传导作用在很大程度上是未知的。 我们已经开始鉴定调节PTPa磷酸化的整合素刺激的分子机制以及PTPa-和PTPa-pTyr 789信号传导的新靶点。我们的证据表明，PTPa是一个协调员的分子相互作用的形成和功能的脂肪酸。我们假设PTPa，特别是PTPa-phosphoTyr 789，是FA组装和重塑的关键调节因子，并作为协调分子相互作用的枢纽，指导整合素信号传导并决定细胞运动。我们将通过确定PTPa和PTPa-pTyr 789分子复合物的性质和调节形成，并阐明其在FA信号传导和营业额（组装和拆卸）中的功能作用来研究这一假设。为此，我们将使用整合的细胞和分子生物学方法来研究表达或缺乏野生型和突变型PTPa的独特模型细胞系统。 目的1：研究PTPa和PTPa-pTyr 789在调节FA动力学中的作用：组装，周转和分子稳定性。 目的2：利用蛋白质组学技术，确定PTPa的分子相互作用，调节其FA定位和信号传导。 总之，我们提出的研究将阐明PTPa和PTPa-pTyr 789在整合素调节的粘着斑动力学和信号传导中的调节和作用。这将进一步加深我们对控制细胞运动基本过程的精确编排的分子网络的理解。

Cell movement is a fundamental physiological process. Principal determinants of cell motility are the integrins, receptors on the cell membrane that co-ordinate adhesive cell-substrate contacts with continual re-organization of the actin cytoskeleton. Integrin engagement initiates intracellular signaling that drives the assembly of multiprotein complexes known as focal adhesions (FAs). FA signaling controls both cytoskeletal remodeling and also their own dynamic assembly and disassembly to regulate cell shape and movement. Receptor protein tyrosine phosphatase alpha (PTPa) is a positive regulator of integrin signaling that promotes cell movement. PTPa-deficient mouse embryo fibroblasts exhibit defective integrin-stimulated migration that is characterized by delayed cytoskeletal rearrangement and impaired FA formation. Through investigation of these defects, we have discovered that PTPa plays two roles in integrin-mediated signaling to promote FA assembly and, ultimately, cell migration: (i) a catalytic role as an upstream activator of the Src-FAK kinase complex, and (ii) a non-catalytic role as a downstream target of the Src-FAK complex. In its second role, PTPa is phosphorylated by active Src-FAK on a tyrosine residue in its C-terminal tail, Tyr789. This is pivotal for FA formation and signaling that promotes cell migration, however the signaling actions of PTPa-phosphoTyr789 (PTPa-pTyr789) are largely unknown. We have begun to identify integrin-stimulated molecular mechanisms that regulate PTPa phosphorylation and novel targets of PTPa- and PTPa-pTyr789 signaling. Our evidence indicates that PTPa is a coordinator of molecular interactions integral to the formation and function of FAs. We hypothesize that PTPa, specifically PTPa-phosphoTyr789, is a critical regulator of FA assembly and remodeling and acts as a hub to co-ordinate molecular interactions that direct integrin signaling and determine cell movement. We will investigate this hypothesis by determining the nature and regulated formation of PTPa- and PTPa-pTyr789-containing molecular complexes and elucidating their functional roles in FA signaling and turnover (assembly and disassembly). For this, we will use integrated cell and molecular biology approaches to investigate unique model cell systems that express or lack wild-type and mutant forms of PTPa. Aim 1: Investigate the role of PTPa and PTPa-pTyr789 in regulating FA dynamics: assembly, turnover, and molecular stability. Aim 2: Define the molecular interactions of PTPa, using proteomics, that regulate its FA localization and signaling. Overall, our proposed research will elucidate the regulation and roles of PTPa and PTPa-pTyr789, in integrin-regulated focal adhesion dynamics and signaling. This will further our understanding of the precisely orchestrated molecular network that controls the essential process of cell movement.