Localization and regulation of phospholipase D2 by ARF6

AbstractPhospholipase D (PLD) and ADP‐ribosylation factor 6 (ARF6) have been implicated in vesicular trafficking and rearrangement of the actin cytoskeleton. We have explored the co‐localization of rat PLD1b and rat PLD2 with wild type and mutant forms of ARF6 in HeLa cells and studied their activation by ARF6 and the role of the actin cytoskeleton. GFP‐tagged PLD1 had a similar pattern to multivesicular and late endosomes and the trans‐Golgi apparatus, but not to other organelles. When wild type or dominant negative ARF6 and PLD1 or PLD2 were co‐expressed, they had a similar localization in cytosolic particles and at the cell periphery. In contrast, dominant active ARF6 caused cell shrinkage and had a similar localization with PLD1 and PLD2 in dense structures, containing the trans‐Golgi apparatus and actin. Disruption of the actin cytoskeleton with cytochalasin D did not induce the formation of these structures. To determine, if ARF6 selectively activated PLD1 or PLD2, wild type and mutant forms of the ARF isoform were transfected together with PLD1 or PLD2. Wild type ARF6 did not affect either PLD isozyme, but dominant active ARF6 selectively activated PLD2 and dominant negative