INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3'-KINASE IN STEM-CELL-FACTOR-INDUCED PHOSPHOLIPASE-D ACTIVATION AND ARACHIDONIC-ACID RELEASE

We have shown previously that the stem cell factor (SCF) receptor-unde rgoes phosphorylation on serine residues following ligand stimulation, and that this phopshorylation is dependent mainly on the activity of protein kinase C (PKC). In the present study, we have further investig ated the molecular mechanisms behind SCF-stimulated activation of PKC, and found that SCF does not activate phosphatidylinositol-specific ph ospholipase C. In contrast, phospholipase D (PLD) is activated in resp onse to SCF in a dose-dependent manner. Activation of PLD was not inhi bited by calphostin C, an inhibitor of PKC. On the other hand, inhibit ors of phosphatidylinositol PtdIns 3'-kinase (PtdIns 3'-kinase), i.e. wortmannin and LY294002, inhibited SCF-induced PLD activation. Moreove r, a mutant SCF receptor in which Tyr721, which is responsible for act ivation of PtdIns 3'-kinase, is mutated to a phenylalanine residue was unable to mediate activation of PLD. Thus, PtdIns 3'-kinase appears t o be essential for SCF-induced PLD activation. Furthermore, we demonst rate that phosphatidic acid (PtdH), generated through the action of PL D in response to SCF, is metabolized to diacylglycerol by dephosphoryl ation. Diacylglycerol can then activate PKC, and, moreover, after deac ylation by a diacylglycerol lipase, yield arachidonic acid, an importa nt second messenger in cell signaling.