ZO 1 AND SIGNAL TRANSDUCTION IN TIGHT JUNCTIONS

这些研究的长期目标是了解 人类的紧密连接。这些结构形成了连续的细胞间 上皮细胞和内皮细胞之间的接触，密封 水、溶质和免疫细胞运动的细胞旁路径。 屏障的通透性在不同的上皮细胞之间差异很大，是 生理调节的，受疾病过程影响的因此， 了解屏障调节对人类具有重大意义 上皮病理生理学、治疗学和药物输送。目前， 调节组装和密封的细胞信号机制是 人们对此知之甚少。两种紧密连接蛋白Z0-1的基因序列分析 和Z0-2揭示了两者都是膜相关鸟酸盐的成员 激酶同源家族(MAGUK)是一类可能的信号转导蛋白， 这个家族的原始成员是椎间盘--大肿瘤抑制因子 果蝇。我们已经证明，瞬时转基因和过度- Z0-1在MDCK细胞中的表达诱导交界区肌动蛋白 积累。此外，我们在A431细胞系中观察到，表皮 生长因子诱导肌动蛋白和Z0-1进入紧密连接 Z0-1/2在酪氨酸残基上进行瞬时磷酸化。在MDCK中 细胞HGF诱导连接通透性和肌动蛋白增加 重新安排。我们假设Z0-1诱导的酪氨酸磷酸化 通过生长因子创建与SH2结构域的瞬时关联- 含有参与调控的信号蛋白 交界周围肌动蛋白。眼下的目标是确定SH2结构域- 含有与酪氨酸磷酸化的Z0-1/2相关的蛋白质 培养MDCK和A431细胞。磷酸化酪氨酸将在 在体内表达的Z0-1抗原表位标记片段 MDCK和A431细胞免疫共沉淀。基因的定点突变 标记了Z0-1的表位在MDCK和A431细胞中表达，将用于 检测诱导过程中对特定酪氨酸磷酸化的要求 Sh2-蛋白结合，结构和通透性的变化 EGF和HGF。所有MAGUK在Z0-1中都含有保守的酪氨酸Y621，并且 我们将进行体外结合研究，试图鉴定SH2- 含有结合这个部位的蛋白质。Z0-1的蛋白质结构域 负责诱导肌动蛋白积聚的定义将使用 MDCK细胞中的转基因技术。结构性变化将是 以光和超微结构为特征。的作用 酪氨酸激酶c-src、c-yes和c-fyn在Z0-1酪氨酸调节中的作用 将使用MDCK测试磷酸化、连接形态和封闭性 稳定转染并表达不同水平的这些激酶的细胞。 MAGUK间磷酸化依赖信号的保守性 将与其他计划项目调查人员一起进行调查。

The long-range goal of these studies is to understand the regulation of human tight junctions. These structures form a continuous intercellular contact between both epithelial and endothelial cells, sealing the paracellular path to the movement of water, solutes and immune cells. Permeability of the barrier varies widely among epithelia, is physiologically regulated and effected by disease processes. Thus, understanding barrier regulation has significant implications for human epithelial pathophysiology, therapeutics and drug delivery. Presently, cellular signalling mechanisms which regulate assembly and sealing are poorly understood. cDNA sequencing of two tight junction proteins, Z0-1 and Z0-2 reveals both are members of the Membrane-Associated Guanylate Kinase Homolog (MAGUK) family of putative signal transduction proteins, the original member of this family is the discs-large tumor suppressor of Drosophila. We have shown that transient transfection, and over- expression, of Z0-1 in MDCK cells induces perijunctional actin accumulation. Further, we observe in the A431 cell line that Epidermal Growth Factor induces actin and Z0-1 to move into tight junctions and both Z0-1/2 to undergo transient phosphorylation on tyrosine residues. In MDCK cells HGF induces increased junctional permeability and actin rearrangements. We hypothesize tyrosine phosphorylation of Z0-1 induced by growth factors creates transient association with an SH2 domain- containing signalling protein which is involved in regulating perijunctional actin. The immediate goals are to identify SH2 domain- containing proteins which associate with tyrosine-phosphorylated Z0-1/2 in cultured MDCK and A431 cells. Phosphorylated tyrosines will be mapped in vivo, on epitope-tagged fragments of Z0-1 expressed in and immunoprecipitated from MDCK and A431 cells. Site directed mutagenesis of epitope tagged Z0-1, expressed in MDCK and A431 cells, will be used to test the requirement for phosphorylation of specific tyrosines in inducing SH2-protein binding, and structural and permeability changes induced by EGF and HGF. All MAGUKs contain a conserved tyrosine, Y621 in Z0-1, and we will perform in vitro binding studies attempting to identity an SH2- containing protein which binds this site. The protein domains of Z0-1 responsible for inducing actin accumulation will be defined using transfection techniques in MDCK cells. Structural changes will be characterized at the light and ultrastructural levels. The role of tyrosine kinases, c-src, c-yes and c-fyn, in regulating Z0-1 tyrosine phosphorylation, junction morphology and sealing will be tested using MDCK cells stably transfected and expressing different levels of these kinases. The conserved nature of phosphorylation-dependent signaling among MAGUKs will be investigated with other Program Project investigators.