Phospholipase D Stimulation by Receptor Tyrosine Kinases Mediated by Protein Kinase C and a Ras/Ral Signaling Cascade

• Published in: The Journal of biological chemistry Vol. 274; no. 49; pp. 34691 - 34698
• Main Authors: Voss, M, Weernink, P A, Haupenthal, S, Möller, U, Cool, R H, Bauer, B, Camonis, J H, Jakobs, K H, Schmidt, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 03.12.1999
• Subjects: Bacterial Proteins; Bacterial Toxins - pharmacology; Carbachol - pharmacology; Cell Line; Cell Membrane - enzymology; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Growth Factor - metabolism; Gene Expression Regulation, Enzymologic - drug effects; GTP Phosphohydrolases - metabolism; guanine nucleotide exchange factor; Humans; Insulin - metabolism; MAP Kinase Signaling System; Phospholipase D - metabolism; Protein Kinase C - metabolism; ral GTP-Binding Proteins; ral Guanine Nucleotide Exchange Factor - metabolism; Ral protein; Ras protein; ras Proteins - metabolism; Recombinant Proteins - metabolism; Signal Transduction - drug effects; Tetradecanoylphorbol Acetate - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.49.34691

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.