Continual Production of Phosphatidic Acid by Phospholipase D Is Essential for Antigen-stimulated Membrane Ruffling in Cultured Mast Cells

Abstract

Phospholipase Ds (PLDs) are regulated enzymes that generate phosphatidic acid (PA), a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Mast cells, when stimulated with antigen, show a dramatic alteration in their cytoskeleton and also release their secretory granules by exocytosis. Butan-1-ol, which diverts the production of PA generated by PLD to the corresponding phosphatidylalcohol, was found to inhibit membrane ruffling when added together with antigen or when added after antigen. Inhibition by butan-1-ol was completely reversible because removal of butan-1-ol restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PA production during membrane ruffling, which was maintained for at least 30 min. PLD1 and PLD2 are both expressed in mast cells and green fluorescent protein-tagged proteins were used to identify PLD2 localizing to membrane ruffles of antigen-stimulated mast cells together with endogenous ADP ribosylation factor 6 (ARF6). In contrast, green fluorescent protein-PLD1 localized to intracellular vesicles and remained in this location after stimulation with antigen. Membrane ruffling was independent of exocytosis of secretory granules because phorbol 12-myristate 13-acetate increased membrane ruffling in the absence of exocytosis. Antigen or phorbol 12-myristate 13-acetate stimulation increased both PLD1 and PLD2 activity when expressed individually in RBL-2H3 cells. Although basal activity of PLD2-overexpressing cells is very high, membrane ruffling was still dependent on antigen stimulation. In permeabilized cells, antigen-stimulated phosphatidylinositol(4,5)bisphosphate synthesis was dependent on both ARF6 and PA generated from PLD. We conclude that both activation of ARF6 by antigen and a continual PLD2 activity are essential for local phosphatidylinositol(4,5)bisphosphate generation that regulates dynamic actin cytoskeletal rearrangements.

Phospholipases D1 and D2 Coordinately Regulate Macrophage Phagocytosis

“…Overexpressed PLD2 has been demonstrated in the plasma membrane as well as intracellular vesicles and the nucleus (18,64,67). Like PLD1, PLD2 has been demonstrated to undergo agonist-induced translocation between membrane compartments (25,67,71,72).…”

Activation of diacylglycerol kinase α is required for VEGF-induced angiogenic signaling in vitro

“…On the other hand, activation of Dgk-a has been reported to convey proliferative and motility signals, respectively, in IL-2-stimulated T lymphocytes and in HGF-stimulated endothelial cells (Flores et al, 1996;Cutrupi et al, 2000). In addition, generation of PA through phospholipase D (PLD)-mediated hydrolysis of phospholipid is required for vescicular trafficking, lamellipodia, cell migration and proliferation in different cellular systems (English, 1996;Honda et al, 1999;O'Luanaigh et al, 2002).…”

An Essential Role for Phospholipase D in the Activation of Protein Kinase C and Degranulation in Mast Cells

“…In addition, overexpression of tagged PLD1 and PLD2, the two known mammalian isoforms of PLD (25,26), in the RBL-2H3 mast cell line indicates that PLD1 associates with granule membranes and intracellular vesicles, whereas PLD2 associates with the plasma membrane (3,23,27). Both isoforms are activated upon Ag stimulation, and the expression of a catalytically inactive mutant of PLD1 blocks migration of granules to the cell periphery, as does 1-butanol.…”