Carnero A, Dolfi F, Lacal J C
Instituto de Investigaciones Biomédicas, Madrid, Spain.
J Cell Biochem. 1994 Apr;54(4):478-86. doi: 10.1002/jcb.240540415.
Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.
非洲爪蟾卵母细胞是用于表征导致DNA合成诱导的信号转导途径的强大工具。由于已假定磷脂酶A2（PLA2）、磷脂酶C（PLC）或磷脂酶D（PLD）的激活是ras功能的介质，我们使用卵母细胞系统来研究ras-p21与这些磷脂酶之间假定的功能关系。在显微注射ras-p21后，观察到磷脂酸（PA）和二酰基甘油（DAG）迅速生成，这表明PLC和PLD酶均被激活。然而，DAG的生成对普萘洛尔抑制PA水解酶敏感，表明PLD是负责生成PA和DAG的酶。显微注射PLD或ras-p21以依赖p42丝裂原活化蛋白激酶（p42MAPK）的方式诱导溶血磷脂酰胆碱的后期生成，这是PLA2激活的一个迹象。奎纳克林对该酶的抑制并不抑制PLD或ras诱导的生发泡破裂（GVBD），这表明ras或PLD功能不需要PLA2激活。与3T3成纤维细胞相反，在3T3成纤维细胞中，ras-p21的促有丝分裂活性在功能上依赖于佛波酯（TPA）和星形孢菌素敏感的蛋白激酶C（PKC）亚型，在非洲爪蟾卵母细胞中，ras-p21诱导的GVBD独立于PKC，而PLC诱导的GVBD对PKC抑制敏感。因此，我们的结果证明了ras-p21蛋白可激活PLD和PLA2，而未观察到对PLC有影响。
