title: Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling.
abstract: Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.
authors.affiliation: Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea.
authors.forename: Jung Min
authors.initials: JM
authors.lastname: Han
authors.forename: Yong
authors.initials: Y
authors.lastname: Kim
authors.forename: Jun Sung
authors.initials: JS
authors.lastname: Lee
authors.forename: Chang Sup
authors.initials: CS
authors.lastname: Lee
authors.forename: Byoung Dae
authors.initials: BD
authors.lastname: Lee
authors.forename: Motoi
authors.initials: M
authors.lastname: Ohba
authors.forename: Toshio
authors.initials: T
authors.lastname: Kuroki
authors.forename: Pann-Ghill
authors.initials: PG
authors.lastname: Suh
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Ryu
mesh_terms: D000818:Animals; D019556:COS Cells; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
chemical_list: C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C087857:protein kinase C gamma; D066246:ErbB Receptors; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C106801:phospholipase D1; D013755:Tetradecanoylphorbol Acetate