Role of the GTPase ARF6 in Epithelial Cell Migration

描述(申请人提供)：上皮细胞在上皮性肿瘤的组织形态发生、伤口愈合和转移过程中发生迁移。迁移的开始需要整合一组复杂的信号，这些信号来自与细胞外基质、邻近细胞以及与激素或其他可溶性因子的相互作用。Rho家族的GTP酶家族成员被证明是这些信号的重要整合者，将它们转化为细胞运动所需的肌动蛋白细胞骨架的变化。我们和其他人最近发现，第二类小的GTP酶，ADP核糖化因子(ARF)也可以调节肌动蛋白细胞骨架的组装，并且似乎与Rho家族成员协调地完成这一过程。以模拟细胞为模型上皮细胞，我们发现ARF6的激活(通过核苷酸交换因子Arno的表达)足以诱导类似于分散因子/HGF处理的细胞的迁移表型。相反，由上皮单层损伤或HGF处理所诱导的迁移被显性的负ARF6结构所抑制。迁移需要两个由ARF6启动的下游事件，Rho GTP酶RACI的激活和ARF效应器磷脂酶D的激活。重要的是，RACI的激活不依赖于PLD，PLD的激活也不依赖于RACI，这表明每一个事件都代表着ARF6下游的一条不同的信号通路。这些数据表明，ARF6在调节上皮细胞的运动性中起着核心作用。这项建议的总体目标是确定ARF6调节运动性的分子机制。我们提出了三个具体的目标：1)我们将通过鉴定特定的PLD亚型(S)和相关的特定代谢产物来确定PLD在细胞迁移中的作用；2)我们将确定ARF6激活RACI的分子机制，以及ARF6对由ARF GAP(GIT)、RAC核苷酸交换因子PIX、RAC效应蛋白激酶PAK和焦点黏附成分paxlin组成的多蛋白复合体的调节；3)我们将确定Git酪氨酸磷酸化在通过Git/PIX/PAK/paxlin复合体传递Arf6衍生的信号中的作用。

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.