Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ra1 signaling cascade

Abstract

Stimulation of phospholipase D (PLD) in HEK-293 cells expressing the M3 muscarinic receptor by phorbol ester-activated protein kinase C (PKC) apparently involves Ra1 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 Ra1A, G26A Ra1A, as well as dominant-negative Ras, S17N Ras. In contrast, overexpression of the Ras- activated Ra1-specific guanine nucleotide exchange factor, Ra1-GDS, specifically enhanced PKC-induced PLD stimulation. Moreover, recombinant Ra1- GDS potentiated Ra1-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/Ra1-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 Ra1A and Ras mutants strongly reduced RTK-induced PLD stimulation. Third, overexpression of Ra1-GDS largely potentiated PLD stimulation by the RTK agonists. Finally, using the Ra1 binding domain of the Ra1 effector RLIP as an activation- specific probe for Ra1 proteins, it is demonstrated that endogenous Ra1A 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/Ra1 signaling cascade.