Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a gua nine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamelli podia, to separate from neighboring cells, and to exhibit a dramatic increa se in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF 1, using a novel pull-down assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activa tion is required for ARNO-induced cell motility. Finally, ARNO-induced acti vation of ARF6 also results in increased activation of phospholipase D (PLD ), and inhibition of PLD activity also inhibits motility. However, inhibiti on of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leadin g to Rac activation, the other to changes in membrane phospholipid composit ion, and that both pathways are required for cell motility.