title: Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ral signaling cascade.
abstract: Stimulation of phospholipase D (PLD) in HEK-293 cells expressing the M(3) muscarinic receptor by phorbol ester-activated protein kinase C (PKC) apparently involves Ral GTPases. We report here that PKC, but not muscarinic receptor-induced PLD stimulation in these cells, is strongly and specifically reduced by expression of dominant-negative RalA, G26A RalA, as well as dominant-negative Ras, S17N Ras. In contrast, overexpression of the Ras-activated Ral-specific guanine nucleotide exchange factor, Ral-GDS, specifically enhanced PKC-induced PLD stimulation. Moreover, recombinant Ral-GDS potentiated Ral-dependent PKC-induced PLD stimulation in membranes. Epidermal growth factor, platelet-derived growth factor, and insulin, ligands for receptor tyrosine kinases (RTKs) endogenously expressed in HEK-293 cells, apparently use the PKC- and Ras/Ral-dependent pathway for PLD stimulation. First, PLD stimulation by the RTK agonists was prevented by PKC inhibition and PKC down-regulation. Second, expression of dominant-negative RalA and Ras mutants strongly reduced RTK-induced PLD stimulation. Third, overexpression of Ral-GDS largely potentiated PLD stimulation by the RTK agonists. Finally, using the Ral binding domain of the Ral effector RLIP as an activation-specific probe for Ral proteins, it is demonstrated that endogenous RalA is activated by phorbol ester and RTK agonists. Taken together, strong evidence is provided that RTK-induced PLD stimulation in HEK-293 cells is mediated by PKC and a Ras/Ral signaling cascade.
authors.affiliation: Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Voss
authors.forename: P A
authors.initials: PA
authors.lastname: Weernink
authors.forename: S
authors.initials: S
authors.lastname: Haupenthal
authors.forename: U
authors.initials: U
authors.lastname: Möller
authors.forename: R H
authors.initials: RH
authors.lastname: Cool
authors.forename: B
authors.initials: B
authors.lastname: Bauer
authors.forename: J H
authors.initials: JH
authors.lastname: Camonis
authors.forename: K H
authors.initials: KH
authors.lastname: Jakobs
authors.forename: M
authors.initials: M
authors.lastname: Schmidt
mesh_terms: D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000378:metabolism; D020935:MAP Kinase Signaling System; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D020725:ral GTP-Binding Proteins; D020726:ral Guanine Nucleotide Exchange Factor / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007328:Insulin; D011994:Recombinant Proteins; D020726:ral Guanine Nucleotide Exchange Factor; C031815:toxB protein, Clostridium difficile; D004815:Epidermal Growth Factor; D002217:Carbachol; D011493:Protein Kinase C; D010739:Phospholipase D; D020558:GTP Phosphohydrolases; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate