ras‐p21 Activates phospholipase D and A2, but not phospholipase C or PKC, in Xenopus laevis Oocytes

Abstract

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.

Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras- and tyrosine kinase (fps)-transformed fibroblasts

“…Constitutive activation of basal PLD activity and the relative labeling of PA has been demonstrated in rastransformed ®broblasts (Martin et al, 1993;Carnero et al, 1994b). This result is compatible with the activation of PLD by p21 ras (Carnero et al, 1994a), which probably involves pp60 src (Jiang et al, 1995a) and Ral (Jiang et al, 1995b). In contrast, the activation of PLD by serum, platelet-derived growth factor, and phorbol ester is attenuated in ras-transformed cells (Huang and Cabot, 1992;Fu et al, 1992;Martin et al, 1993).…”

“…Increased DAG concentration in ras-transformed cells could result from activation of PC-phospholipase D (PLD) and subsequent conversion of phosphatidate (PA) to DAG by phosphatidate phosphohydrolase (PAP). Micro-injection of Xenopus laevis oocytes with p21 ras caused rapid DAG production that was blocked by propranolol, an inhibitor of PAP (Carnero et al, 1994a). Choline kinase activity is elevated in ras-transformed cells (Macara, 1989;Lacal, 1990;Teegarden et al, 1990), and CTP-phosphocholine cytidylyltransferase is lower (Teegarden et al, 1990).…”

Specific Inhibition of Phorbol Ester-stimulated Phospholipase D by Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B-1470 in HEK-293 Cells

“…Finally, Ras itself has been reported to activate PLD in different cell types (31)(32)(33). Thus, GTPase cascades are apparently not only involved in cell proliferation and cytoskeleton organization, but they may also participate in PLD activation (34).…”

Ablation of Go α-Subunit Results in a Transformed Phenotype and Constitutively Active Phosphatidylcholine-specific Phospholipase C

“…As mentioned, it is indeed possible that the "apparent" PC-PLC activity observed in some studies was due to the combined increase in PC-PLD, PAPH, and/or CK (30,36,37). The data in this work, however, provide definitive evidence that a PC-PLC is present and constitutively active in the G o ␣-ablated cells.…”