Role of the GTPase ARF6 in Epithelial Cell Migration

描述（由申请人提供）：上皮细胞迁移发生在上皮肿瘤的组织形态发生、伤口愈合和转移过程中。迁移的开始需要整合一组复杂的信号，这些信号来源于与细胞外基质、邻近细胞以及与激素或其他可溶性因子的相互作用。已显示GTP酶的Rho家族的成员是这些信号的重要整合者，将它们翻译成细胞运动所需的肌动蛋白细胞骨架的变化。我们和其他人最近表明，第二类小GTP酶，ADP-核糖基化因子（ARF）也可以调节肌动蛋白细胞骨架的组装，并似乎与Rho家族成员协调进行。使用MOCK细胞作为模型上皮，我们发现ARF 6的激活（通过核苷酸交换因子ARNO的表达）足以诱导与用散射因子/HG F处理的细胞相似的迁移表型。相反，由上皮单层损伤或HGF处理诱导的迁移被显性负性ARF 6构建体抑制。迁移需要由ARF 6启动的两个下游事件，即Rho GT3 Raci的激活和ARF效应物磷脂酶D的激活。重要的是，Raci激活不依赖于PLD，PLD激活不依赖于Raci，表明各自代表ARF 6下游的不同信号传导途径。这些数据表明，ARF 6在上皮细胞运动的调节中起着核心作用。本提案的总体目标是确定ARF 6调节运动的分子机制。本研究的目的有三：1）通过鉴定PLD的特异性异构体及其代谢产物，明确PLD在细胞迁移中的作用; 2）我们将确定ARF 6激活Raci的分子机制，以及ARF 6对由ARF GAP（GIT），Rac核苷酸交换因子PIX，Rac效应激酶PAK和粘着斑组分桩蛋白; 3）我们将确定GIT酪氨酸磷酸化在通过GIT/PIX/PAK/桩蛋白复合物传递ARF 6衍生信号中的作用。

DESCRIPTION (provided by applicant): Migration of epithelial cells occurs during tissue morphogenesis, wound healing and metastasis of epithelial tumors. The onset of migration requires integration of a complex set of signals derived from interactions with the extracellular matrix, neighboring cells and with hormonal or other soluble factors. Members of the Rho family of GTPases have been shown to be important integratrs of these signals, translating them into changes in the actin cytoskeleton that are required for cell motility. We and others have recently shown that a second class of small GTPases, the ADP-ribosylation factors (ARFs) can also regulate assembly of the actin cytoskeleton and appear to do so coordinately with Rho family members. Using MOCK cells as a model epithelium, we found that activation of ARF6 (by expression of the nucleotide exchange factor ARNO) is sufficient to induce a migratory phenotype similar to that of cells treated with scatter factor/HG F. Conversely, migration induced by either wounding of epithelial monolayers or by HGF treatment is inhibited by a dominant negative ARF6 construct. Migration requires two downstream events initiated by ARF6, activation of the Rho GTPase Raci and activation of the ARF effector, phospholipase D. Importantly, Raci activation is not dependent on PLD, and PLD activation is not dependent on Raci, suggesting that each represents a distinct signaling pathway downstream of ARF6. These data suggest that ARF6 plays a central role in the regulation of epithelial cell motility. The overall goal of this proposal is to determine the molecular mechanisms through which ARF6 regulates motility. Three specific aims are proposed: 1) We will define the role of PLD in cell migration by identifying the specific PLD isoform(s) and the specific metabolic products involved; 2) We will determine the molecular mechanism by which ARF6 activates Raci, as well as the regulation by ARF6 of a muttiprotein complex consisting of an ARF GAP (GIT), the Rac nucleotide exchange factor PIX, the Rac effector kinase PAK and the focal adhesion component paxillin; 3) We will define the role of GIT tyrosine phosphorylation in the transmission of ARF6-derived signals through the GIT/PIX/PAK/paxillin complex.