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，即细胞膜上的受体，它协调粘附细胞与底物的接触，并不断重组肌动蛋白细胞骨架。整合素参与启动细胞内信号传导，驱动多蛋白复合物的组装，称为局灶粘连（FAs）。FA信号既控制细胞骨架重塑，也控制其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的积极调节因子。ptpa缺陷小鼠胚胎成纤维细胞表现出整合素刺激的迁移缺陷，其特征是细胞骨架重排延迟和FA形成受损。通过对这些缺陷的研究，我们发现PTPa在整合素介导的信号传导中发挥两种作用，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，以及（ii）作为Src-FAK复合物的下游靶点的非催化作用。PTPa的第二个作用是在其c端末端Tyr789的酪氨酸残基上被活性Src-FAK磷酸化。这对于促进细胞迁移的FA形成和信号传导至关重要，然而PTPa-phosphoTyr789 （PTPa-pTyr789）的信号传导作用在很大程度上是未知的。****我们已经开始确定整合素刺激的调节PTPa磷酸化的分子机制和PTPa-和PTPa- ptyr789信号的新靶点。我们的证据表明，PTPa是FAs形成和功能不可或缺的分子相互作用的协调者。我们假设PTPa，特别是PTPa- phosphotyr789，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- ptyr789分子复合物的性质和调控形成，并阐明它们在FA信号传导和转换（组装和拆卸）中的功能作用来研究这一假设。为此，我们将使用综合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- ptyr789在调节FA动力学中的作用：组装，周转和分子稳定性。****目标2：定义PTPa的分子相互作用，使用蛋白质组学，调节其FA定位和信号传导。****总的来说，我们提出的研究将阐明PTPa和PTPa- ptyr789在整合素调节的局点粘附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协调的分子网络的理解。******

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.******