Phospholipase D (PLD) activity, as measured by the transphosphatidylation of cellular phospholipids, is elevated in BALB/c 3T3 cells transformed by v-Src. Phorbol esters that activate protein kinase C (PKC) also increase PLC activity in BALB/c 3T3 cells. v-Src-induced PLD activity could be distinguished from phorbol ester-induced PLD activity by differential radiolabelling of phospholipids, which are the substrates of PLD. Both v-Src- and phorbol ester-induced PLD activity could be detected when phospholipids were prelabelled with either radiolabelled myristate or palmitate; however, only phorbol ester-induced PLD activity could be detected when either arachidonate or 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (alkyl-lysoPC) was used to prelabel the phospholipids. The increased PLD activity in v-Src-transformed cells was not detected when the cells were prelabelled with either arachidonic acid or alkyl-lysoPC, which contains an ether linkage at sn-1 of the glycerol backbone. As both arachidonic acid and alkyl-lysoPC are incorporated into phosphatidylcholine (PC), the substrate for v-Src-induced PLD activity, these data suggest that the PLD activated by v-Src can distinguish PCs lacking arachidonic acid and ether linkages. Consistent with v-Src activating a PLD activity that is distinct from that activated by phorbol esters that activate PKC directly, neither depleting cells of PKC nor treatment with the protein kinase inhibitor, staurosporine, had any effect on v-Src-induced PLD activity, whereas both PKC depletion and staurosporine inhibited phorbol ester-induced PLD activity. Taken together, these data suggest that v-Src activates a PKC-independent PLD activity that is specific for a subpopulation of PC and distinct from the PLD activity induced by PKC activity induced by phorbol esters. The diacylglycerol produced from PC by the action of the v-Src-induced PLD may therefore be responsible for the activation of PKC by v-Src.

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