Song J, Foster D A
Institute for Biomolecular Structure and Function, Hunter College, City University of New York, NY 10021.
Biochem J. 1993 Sep 15;294 ( Pt 3)(Pt 3):711-7. doi: 10.1042/bj2940711.
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.
通过细胞磷脂的转磷脂酰基作用测定的磷脂酶D（PLD）活性，在经v-Src转化的BALB/c 3T3细胞中升高。激活蛋白激酶C（PKC）的佛波酯也会增加BALB/c 3T3细胞中的PLC活性。v-Src诱导的PLD活性可通过对作为PLD底物的磷脂进行差异放射性标记，与佛波酯诱导的PLD活性区分开来。当磷脂用放射性标记的肉豆蔻酸或棕榈酸预标记时，v-Src和佛波酯诱导的PLD活性均可被检测到；然而，当用花生四烯酸或1-O-烷基-sn-甘油-3-磷酸胆碱（烷基溶血磷脂酰胆碱）预标记磷脂时，仅能检测到佛波酯诱导的PLD活性。当细胞用花生四烯酸或烷基溶血磷脂酰胆碱预标记时，未检测到v-Src转化细胞中PLD活性的增加，烷基溶血磷脂酰胆碱在甘油主链的sn-1位含有醚键。由于花生四烯酸和烷基溶血磷脂酰胆碱均可掺入磷脂酰胆碱（PC）中，而磷脂酰胆碱是v-Src诱导的PLD活性的底物，这些数据表明，由v-Src激活的PLD可以区分缺乏花生四烯酸和醚键的PC。与v-Src激活一种不同于由直接激活PKC的佛波酯激活的PLD活性一致，耗尽细胞中的PKC或用蛋白激酶抑制剂星形孢菌素处理，对v-Src诱导的PLD活性均无任何影响，而PKC耗尽和星形孢菌素均抑制佛波酯诱导的PLD活性。综上所述，这些数据表明，v-Src激活一种不依赖PKC的PLD活性，该活性对PC的一个亚群具有特异性，且不同于由佛波酯诱导的PKC活性所诱导的PLD活性。因此，由v-Src诱导的PLD作用于PC产生的二酰基甘油可能是v-Src激活PKC的原因。
