RalA interacts directly with the Arf-responsive, PIP₂-dependent phospholipase D1

RalA GTPase associates with a phospholipase D (PLD) that is activated in v-Src- and v-Ras-transformed cells. Two mammalian PLDs were recently cloned: PLD1, which is activated by Arf family GTPases and dependent upon phosphatidylinositol-4,5-bisphosphate (PIP₂), and PLD2, which is also dependent upon PIPE, but not stimulated by Arf. Another PLD has been described that is stimulated by oleate. Evidence is provided that the RalA-assiciated PLD is PLD1. First, the PLD precipitated by RalA from murine fibroblasts was stimulated by Arf, dependent upon PIP₂, and inhibited by oleate. Second, immobilized RalA precipitated PLD1 from sf9 insect cells overexpressing PLD1. Third, a series of RalA mutants precipitated PLD activity from both PLD1-expressing insect cells and murine fibroblasts with the same efficiency. And finally, immobilized RalA precipitated PLD1 from a purified PLD1 preparation. These data argue that RalA associates directly with the Arf-responsive, PIP₂-dependent PLD1.