Studies of the roles of ADP-ribosylation factors and phospholipase D in phorbol ester-induced membrane ruffling.
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.
Hiroyama
Masami
M
Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Exton
John H
JH
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D051356:Cortactin; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D010739:Phospholipase D / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014018:Tissue Distribution; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000089982:ADP-Ribosylation Factor 6; C105088:ARFIP1 protein, human; C101158:ARFIP2 protein, human; D048868:Adaptor Proteins, Signal Transducing; C495868:CTTN protein, human; D051356:Cortactin; D008840:Microfilament Proteins; D010739:Phospholipase D; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020830:rac1 GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate