Role of villin in epithelial cell signal transduction

描述(申请人提供)：绒毛蛋白，一种上皮细胞微绒毛蛋白，在钙离子、磷脂酰肌醇4，5-二磷酸和酪氨酸磷酸化的浓度变化的反应下，形成肌动蛋白细丝、核、帽或断丝，这些通常是细胞激活的直接结果。我们之前的研究已经证实，Villin的配体结合特性对其在细胞迁移中的作用具有重要的机械意义。在这项拟议的继续研究中，我们将研究绒毛蛋白酪氨酸磷酸化调节细胞迁移的生化和分子机制。本研究的具体目标是：(1)在分子水平上了解酪氨酸磷酸化绒毛蛋白的结构和功能之间的关系。(2)鉴定和鉴定调控(S)绒毛蛋白磷酸化和细胞迁移的酪氨酸激酶(S)。(3)鉴定和鉴定调控(S)绒毛蛋白去磷酸化和细胞迁移的酪氨酸磷酸酶(S)。最终，我们通过这项研究努力了解两个关键功能：第一，了解绒毛蛋白的酪氨酸磷酸化及其配体结合特性如何调节上皮细胞的信号转导和运动；第二，肌动蛋白结合蛋白如何与其他第二信使相互作用，调节细胞结构和功能。我们将使用以下方法来研究绒毛蛋白的酪氨酸磷酸化及其与配体结合的特性：使用重组的绒毛蛋白进行体外重组；建立肠上皮细胞系Caco-2；在Tet-off可诱导细胞系HeLa或MDCK中过表达绒毛蛋白和调节绒毛蛋白酪氨酸磷酸化的酶；以及建立(使用腺病毒载体)感染绒毛蛋白野生型和磷酸化位点突变的绒毛蛋白基因敲除小鼠的肠细胞原代培养。这些研究有可能改变运动性，以增强正常生理和改善疾病。上皮细胞运动的抑制在几种疾病中可能是显著的，包括炎症性肠病、乳糜泻和结肠癌。

DESCRIPTION (provided by applicant): Villin, an epithelial cell microvillar protein bundles, nucleates, caps, or severs actin filaments in response to changes in the concentration of calcium, phosphatidylinositol 4,5-bisphosphate, and tyrosine phosphorylation, which are often immediate consequences of cell activation. Our previous studies have identified that villin's ligand-binding properties are mechanistically important to its role in cell migration. In this proposed continued study, we will examine the biochemical and molecular mechanisms by which tyrosine phosphorylation of villin regulates cell migration. The specific goals of the proposed research are: (1) To understand at the molecular level the relationship between the structure and function of tyrosine phosphorylated villin. (2) To identify and characterize the tyrosine kinase(s) that regulate(s) villin phosphorylation and villin-induced cell migration. (3) To identify and characterize the tyrosine phosphatase(s) that regulate(s) villin dephosphorylation and villin-induced cell migration. Ultimately, we strive by this study to understand two critical functions: first, to understand how tyrosine phosphorylation of villin and its ligand-binding properties regulate epithelial cell signal transduction and motility; second how actin-binding proteins interact with other second messengers to regulate cell structure and function. We will characterize tyrosine phosphorylation of villin and its ligand-binding properties using the following approaches: in vitro reconstitution using recombinant villin proteins; the intestinal epithelial cell line, Caco-2; over expression of villin and enzymes, that regulate tyrosine phosphorylation of villin, in the Tet-Off inducible cell lines, HeLa or MDCK; and develop primary cultures of enterocytes from villin knock-out mice infected (using adenoviral vectors) with wild-type and phosphorylation site mutants of villin. These studies have the potential to allow modification of motility for enhancement of normal physiology and for amelioration of disease. An inhibition of epithelial cell motility can be significant in several diseases, including inflammatory bowel disease, celiac disease, and colon cancer.