title: Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway.
abstract: The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulin-dependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles.
authors.affiliation: Department of Pharmacology, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Megan A
authors.initials: MA
authors.lastname: Rizzo
authors.forename: K
authors.initials: K
authors.lastname: Shome
authors.forename: C
authors.initials: C
authors.lastname: Vasudevan
authors.forename: D B
authors.initials: DB
authors.lastname: Stolz
authors.forename: T C
authors.initials: TC
authors.lastname: Sung
authors.forename: M A
authors.initials: MA
authors.lastname: Frohman
authors.forename: S C
authors.initials: SC
authors.lastname: Watkins
authors.forename: G
authors.initials: G
authors.lastname: Romero
mesh_terms: D000818:Animals; D001692:Biological Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D010712:Phosphatidic Acids / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D051381:Rats
chemical_list: D010712:Phosphatidic Acids; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010739:Phospholipase D