ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly.

ARF6-regulated endocytosis of E-cadherin is essential during the disassembly of adherens junctions in epithelial cells. Here, we show that activation of ARF6 promotes clathrin-dependent internalization of E-cadherin and caveolae at the basolateral cell surface. Furthermore, we demonstrate that ARF6-GTP, a constitutively activate form of ARF6, interacts with and recruits Nm23-H1, a nucleoside diphosphate (NDP) kinase that provides a source of GTP for dynamin-dependent fission of coated vesicles during endocytosis. Finally, we show that ARF6-mediated recruitment of Nm-23-H1 to cell junctions is accompanied by a decrease in the cellular levels of Rac1-GTP, consistent with previous findings that Nm23-H1 down-regulates activation of Rac1. These studies provide a molecular basis for ARF6 function in polarized epithelia during adherens junction disassembly.

Arfaptin 2 regulates the aggregation of mutant huntingtin protein.

Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.

Elevated phospholipase D activity in H-Ras- but not K-Ras-transformed cells by the synergistic action of RalA and ARF6.

Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of H-Ras but not K-Ras. H-Ras and K-Ras have been reported to localize to different membrane microdomains, with H-Ras localizing to caveolin-enriched light membrane fractions. We reported previously that PLD activity elevated in response to mitogenic stimulation is restricted to the caveolin-enriched light membrane fractions. PLD activity in H-Ras-transformed cells is dependent upon RalA, and consistent with a lack of elevated PLD activity in K-Ras-transformed cells, RalA was not activated in K-Ras-transformed cells. Although H-Ras-induced PLD activity is dependent upon RalA, an activated mutant of RalA is not sufficient to elevate PLD activity. We reported previously that RalA interacts with PLD activating ADP ribosylation factor (ARF) proteins. In cells transformed by H-Ras, we found increased coprecipitation of ARF6 with RalA. Moreover, ARF6 colocalized with RalA in light membrane fractions. Interestingly, ARF6 protein levels were elevated in H-Ras- but not K-Ras-transformed cells. A dominant-negative mutant of ARF6 inhibited PLD activity in H-Ras-transformed NIH 3T3 cells. Activated mutants of either ARF6 or RalA were not sufficient to elevate PLD activity in NIH 3T3 cells; however, expression of both activated RalA and activated ARF6 in NIH 3T3 cells led to increased PLD activity. These data suggest a model whereby H-Ras stimulates the activation of both RalA and ARF6, which together lead to the elevation of PLD activity.