Abstract
In this study, we have explored the roles of ADP‐ribosylation factors (ARFs), phospholipase D (PLD) isozymes, and arfaptins in phorbol ester (PMA)‐induced membrane ruffling in HeLa cells. PMA stimulation induced ruffling and translocated cortactin to the plasma membrane. The cortactin translocation was inhibited by dominant negative (DN)‐ARF6, DN‐ARF1, and DN‐Rac1, but not by DN‐RhoA and DN‐Cdc42. The inability of DN‐forms of ARF6, ARF1, and Rac1 to affect PLD activity in response to PMA indicated that this enzyme was not activated via these small G proteins and that its activation was not essential for the induction of ruffling. Endogenous‐ARF1, ‐ARF6, and ‐Rac1 existed in the ruffling region along with cortactin after PMA stimulation. DN‐ARF1 had no effect on the ruffling induced by DA‐ARF6 or DA‐Rac1, and DN‐ARF6 had no effect on that induced by DA‐ARF1 or DA‐Rac1. On the other hand DN‐Rac1 suppressed the effect of DA‐ARF6 but not that of DA‐ARF1. These results suggest that PMA causes membrane ruffling via an ARF6‐Rac1 pathway and also an ARF1 pathway operating in parallel. Overexpression of PLD1 and PLD2 inhibited PMA‐induced cortactin translocation and actin–cortactin complex formation, supporting the view that these enzymes are not required for ruffling, but actually suppress it. We conclude that PMA‐induced membrane ruffling is caused via ARF6‐Rac1 and ARF1 pathways operating in parallel and that PLD may be inhibitory. © 2004 Wiley‐Liss, Inc.