Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ral signaling cascade

J Biol Chem. 1999 Dec 3;274(49):34691-8. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins*
  • Bacterial Toxins / pharmacology
  • Carbachol / pharmacology
  • Cell Line
  • Cell Membrane / enzymology
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Epidermal Growth Factor / metabolism
  • GTP Phosphohydrolases / metabolism
  • Gene Expression Regulation, Enzymologic* / drug effects
  • Humans
  • Insulin / metabolism
  • MAP Kinase Signaling System
  • Phospholipase D / metabolism*
  • Protein Kinase C / metabolism*
  • Recombinant Proteins / metabolism
  • Signal Transduction* / drug effects
  • Tetradecanoylphorbol Acetate / metabolism
  • ral GTP-Binding Proteins*
  • ral Guanine Nucleotide Exchange Factor / metabolism*
  • ras Proteins / metabolism*


  • Bacterial Proteins
  • Bacterial Toxins
  • Insulin
  • Recombinant Proteins
  • ral Guanine Nucleotide Exchange Factor
  • toxB protein, Clostridium difficile
  • Epidermal Growth Factor
  • Carbachol
  • Protein Kinase C
  • Phospholipase D
  • GTP Phosphohydrolases
  • RALA protein, human
  • ral GTP-Binding Proteins
  • ras Proteins
  • Tetradecanoylphorbol Acetate