NIH-3T3 cells expressing high levels of the c-ras proto-oncogene display reduced platelet derived growth factor-stimulated phospholipase activity

NIH-3T3 cells expressing elevated levels of the normal human c-Ha-ras proto-oncogene (c-ras) exhibit reduced platelet derived growth factor-stimulated phospholipase activity. Three clonal cell lines of NIH-3T3 cells expressing different levels of c-ras have been isolated and characterized. The level of c-ras expression correlates inversely with PDGF-stimulated phospholipase activity as monitored by prostaglandin E2 (PGE2) production. In addition, high levels of c-ras expression produce cells with morphological and biochemical characteristics indistinguishable from NIH-3T3 cells transformed by EJ-ras. These data suggest that abnormal c-ras expression can attenuate growth factor-stimulated phospholipase activity in NIH-3T3 cells, in a manner analogous to that observed in cells transformed by EJ-ras.     

Calcium oscillation associated with reduced protein kinase C activities in ras-transformed NIH3T3 cells

We show here novel intracellular Ca2+ oscillation in v-K-ras-transformed NIH3T3 cells induced by mitogenic peptide hormones, bradykinin and bombesin, as well as fetal calf serum. Induction of the Ca2+ oscillation is strongly correlated with the malignant properties and inversely with PKC activities in vitro and in vivo. These results suggest that the mitogen-induced Ca2+ oscillation is negatively regulated by PKC, which modulates Ca2+ influx in v-K-ras-transformed NIH3T3 cells.     

Geranylgeranylated, but not farnesylated, RhoB suppresses Ras transformation of NIH-3T3 cells

RhoB is a low molecular weight GTPase that is both farnesylated (RhoB-F) and geranylgeranylated (RhoB-GG) in cells. Based on data from rodent cell models, it has been suggested that RhoB displays differential effects on cell transformation, according to the nature of its prenylation. To test directly this hypothesis, we generated GTPase-deficient RhoB mutants that are exclusively either farnesylated or geranylgeranylated. We show that in Ras-transformed murine NIH-3T3 cells, RhoB-F enhances, whereas RhoB-GG and RhoB (F/GG) suppresses anchorage-dependent and -independent cell growth as well as tumor growth in nude mice. We then demonstrate that Ras constitutive activation of the tumor survival pathways Akt and NF-kappa B are blocked by RhoB-GG, but not by RhoB-F, providing further support for the opposing role of RhoB-F and RhoB-GG in Ras malignant transformation in NIH-3T3 cells. In addition, both RhoB (F/GG) and RhoB-GG induce apoptosis in Ras-transformed NIH-3T3 cells whereas RhoB-F has no effect. Our data demonstrate that RhoB-F and RhoB-GG which differ only by a 5-carbon isoprene behave differently in rodent cells highlighting the important role of prenyl groups in protein function and emphasize the potency of RhoB to regulate negatively the oncogenic signal.     

Enhanced phospholipase C stimulation and transformation in NIH-3T3 cells expressing Q209LGq-alpha-subunits.

NIH-3T3 cells were transfected with cDNA encoding the native alpha-subunit of the G protein Gq(alpha q) or a mutant (Q209L) form of alpha q. Cells expressing Q209L-alpha q showed greatly enhanced basal phospholipase C activity. Stimulation of phospholipase C activity by prostaglandin F2 alpha or fetal calf serum was increased up to 10-fold in Q209L-alpha q-transfected cells. Continuous expression of Q209L-alpha q or overexpression of alpha q in NIH-3T3 cells resulted in formation of foci after 3 weeks. The number of foci was proportional to the number of transfected cells and was greater in cells expressing the Q209L-alpha q than in cells that overexpressed the wild type alpha q. Q209L-alpha q-transfected NIH-3T3 cells also formed colonies in soft agar indicating their capacity to grow in an anchorage-independent manner. Expression of Q209L-alpha q in Rat-1 cells resulted in enhanced basal and fetal calf serum-stimulated phospholipase C activity, but these cells were not transformed as assessed by either the focus formation or the soft agar colony formation assays. These results indicate that expression of continuously activated Gq-alpha can result in transformation in a cell type-specific manner.     

Anandamide stimulates phospholipase D activity in PC12 cells but not in NIH 3T3 fibroblasts

The endogenous cannabinoid arachidonoylethanolamide was previously reported to have no effects on the phospholipase D activity in Chinese hamster ovary cells expressing the human brain-specific cannabinoid receptor, while in mouse peritoneal cells, delta9-tetrahydrocannabinol stimulated this enzyme. In this work, arachidonoylethanolamide (0.1-1 microM) was found to stimulate the phospholipase D-mediated phospholipid hydrolysis in rat adrenal pheochromocytoma PC12 cells, but not in mouse NIH 3T3 fibroblasts. The phospholipase D-activating effects of arachidonoylethanolamide were comparable to those elicited by phorbol ester and nerve growth factor, while arachidonic acid (1 microM) had no effects. The results show that, depending on the cell type, arachidonoylethanolamide can be an activator of the phospholipase D system.     

Localization of phospholipase A2 in normal and ras-transformed cells

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.     

Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown.

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.     

Inhibitors of secreted phospholipase A2 suppress the release of PGE2 in renal mesangial cells

The upregulation of PGE₂ by mesangial cells has been observed under chronic inflammation condition. In the present work, renal mesangial cells were stimulated to trigger a huge increase of PGE₂ synthesis and were treated in the absence or presence of known PLA₂ inhibitors. A variety of synthetic inhibitors, mainly developed in our labs, which are known to selectively inhibit each of GIVA cPLA₂, GVIA iPLA₂, and GIIA/GV sPLA₂, were used as tools in this study. Synthetic sPLA₂ inhibitors, such as GK115 (an amide derivative based on the non-natural amino acid (R)-γ-norleucine) as well as GK126 and GK241 (2-oxoamides based on the natural (S)-α-amino acid leucine and valine, respectively) presented an interesting effect on the suppression of PGE₂ formation.     

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.     

Reconstitution of a solubilized membrane but not cytosolic phospholipase C with membrane-associated Gp from GH3 cells

Hormones have been demonstrated to activate phosphoinositide hydrolysis in plasma membranes in a manner dependent upon or potentiated by GTP. For thyrotropin-releasing hormone activation in GH3 cell membranes, stimulation persisted in membranes from pertussis toxin-treated cells. These observations indicate the presence of a membrane phospholipase C (PL C) and a novel GTP-binding protein (Gp); however, neither of these proteins has been characterized. In this paper, we report studies of GH3 membrane PL C utilizing [3H]phosphatidylinositol 4,5-bisphosphate liposome substrate. Guanosine 5'-O-(3-thiotriphosphate) (GTP[S]), but not other nucleotides, was found to stimulate PL C activity and required greater than 1 nM Ca2+. High concentrations of Ca2+ (10 microM) also activated the membrane PL C. Treatment of membranes with N-ethylmaleimide inhibited Ca2+-activated but not GTP[S]-activated PL C. Extraction of membranes with 1 M KCl solubilized the membrane PL C; however, the solubilized PL C was not GTP[S]-stimulated. N-ethylmaleimide-treated, KCl-extracted membranes were markedly deficient in GTP[S]-stimulated PL C activity; however, activity could be restored by incubation with the desalted extracted PL C. Reconstitution appeared to involve the recoupling of membrane-associated Gp with soluble 330- and 110-kDa forms of the PL C. Cytosolic PL Cs failed to substitute for the solubilized membrane PL C. These results indicate that the Gp-regulated PL C in GH3 cell membranes is an extrinsic membrane protein that can be extracted reversibly at high ionic strength. Moreover, the membrane PL C can be distinguished from cytosolic PL C isoenzymes.     

G-protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin-Darby canine kidney cells

Membranes prepared from clone D1 of Madin-Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5'-(beta-thiodiphosphate). While Ca2+ at 1 microM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2(+)-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4- incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.     

Arachidonic acid release from NIH 3T3 cells by group-I phospholipase A2: Involvement of a receptor-mediated mechanism

Group I pancreatic phospholipase A₂ (PLA₂ I) is primarily a digestive enzyme. Recently, however, in addition to its catalytic activity a receptor-mediated function has been described for this enzyme. PLA₂ I binding to its receptor induces cellular chemokinesis, proliferation, and smooth muscle contraction. This enzyme also induces the production of prostaglandin E₂ in certain cells and may have a proinflammatory role. However, despite its ability to hydrolyze phospholipids in in vitro assays, PLA₂-I does not efficiently catalyze release of AA from intact cells. Here, we demonstrate that while short-term exposure of NIH 3T3 cells to PLA₂-I is ineffective, exposure of 6 h or longer significantly increases the basal release of AA. Dose-response curve of PLA₂-I-induced AA release was saturable with an EC₅₀ of 14.01 ± 1.36 nM (n = 3). [³H]-AA was preferentially released over [³H]-oleic acid by PLA₂-I, inactivated with 4-bromophenacyl bromide, was fully capable of mediating AA release. These data suggest that a non-catalytic, receptor-mediated mechanism is involved in PLA₂-I-induced AA release in NIH-3T3 cells. This relase of AA is not dependent on protein kinase C or Ca²⁺ concentration. Comparison of the effect of PLA₂-I with those of ATP and platelet-derived growth factor indicates that each of these agonists regulates AA release via independent pathways. Neither the basal enzymatic activity of the 85-kDa cytosolic PLA₂ nor the protein level of this enzyme was affected by treatment of cells with PLA₂-I. However, the increase in basal enzymatic activity of 85 kDa PLA₂ due to protein kinase C activation was further enhanced by pretreatment of cells with PLA₂-I. We conclude that: (1) short-term exposure of cells to PLA₂ I does not cause measurable AA release; (2) release of AA from intact cells by this enzyme requires long-term exposure; (3) AA release is not mediated by a direct catalytic effect of PLA₂ I; and (4) AA release by PLA₂ I is accomplished via a receptor-mediated process. Taken together, these results raise the possibility that PLA₂ I, in addition to its digestive function, may also contribute to aggravate preexisting inflammatory processes and/or to initiate new ones when chronic exposure of cells to this enzyme occurs. © 1995 Wiley-Liss Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Soluble form of the ST2 gene product exhibits growth promoting activity in NIH-3T3 cells

The ST2 gene is induced in murine fibroblast cells at the start of cell proliferation. Although IL-33 has been identified as a ligand for one of the two major gene products of ST2 – namely, the transmembrane receptor form ST2L – prompting immunological research on inflammation, the roles of the ST2 gene products in cell proliferation remain to be elucidated.Using a cell proliferation assay system with NIH-3T3 cells, a normal murine fibroblast cell line, we found that treatment with recombinant ST2 caused an acceleration of cell proliferation, suggesting that ST2 acts in an autocrine/paracrine fashion. Strikingly, shRNA-induced knockdown of both ST2 gene products, ST2 and ST2L, reduced cell proliferation. This effect was effectively canceled by the expression of shRNA-resistant ST2, but not shRNA-resistant ST2L.The novel enhancement of cell proliferation by ST2 appears to involve positive feedback. Since the ST2 level is increased in various diseases involving inflammation, future investigations into the role of ST2 gene products in relation to various diseases, including malignancies, may be warranted.     

Fluid flow induced PGE2 release by bone cells is reduced by glycocalyx degradation whereas calcium signals are not

It has been hypothesized that bone cells have a hyaluronic acid (HA) rich glycocalyx (cell coat or pericellular matrix) and that this contributes to bone cell mechanotransduction via fluid flow. The glycocalyx of bone cells of the MC3T3-E1 osteoblastic cell line and the MLO-Y4 osteocytic cell line were characterized. Alcian blue staining and lectin binding experiments suggested that these cells have a glycocalyx rich in HA. Sulphated proteoglycans were not detected. Staining with hyaluronic acid binding protein and degradation by hyaluronidase confirmed that HA was a major component of the glycocalyx. We subjected cells, with and without hyaluronidase treatment, to oscillating fluid flow under standardized in vitro conditions. There was no effect of glycocalyx degradation on the intracellular calcium signal, in either cell type, in terms of the percentage of cells responding (40-80%) or the magnitude of the response (2-5 times baseline). However, a 4-fold fluid flow induced increase in PGE2 was eliminated by hyaluronidase pre-treatment in MLO-Y4 cells. We conclude that under these conditions the calcium and PGE2 responses occur via different pathways. An intact glycocalyx is not necessary in order to initiate a calcium signal in response to oscillating fluid flow. However, in osteocyte-like cells the PGE2 pathway is more dependent on mechanical signals transmitted through the glycocalyx.     

ATP-dependent regulation of phospholipase C in permeabilized 3T3 cells

Regulation of phospholipase C (PLC) coupled with a G-protein was studied with Swiss 3T3 cells permeabilized by digitonin. In permeabilized cells, activation of phospholipase C required millimolar concentrations of ATP in addition to a G-protein activator, AlF4- or nonhydrolysable GTP analogues. To determine the mechanism of the action of ATP, we examined the effects of ATP analogues. ATP gamma S directly activated phospholipase C in the presence or absence of AlF4-. On the other hand, neither beta,gamma-methylene ATP nor adenyl-5'-yl imidodiphosphate nor ADP beta S could support the AlF4(-)-dependent activation of phospholipase C. The action of ATP gamma S was not through the substrate supply for phospholipase C, because ATP gamma S did not augment the levels of PIP2 or PIP in permeabilized cells. These results suggested the significance of the gamma-phosphate group of ATP and/or phosphorylation by ATP in the activation of phospholipase C by a putative G-protein.     

Diacylglycerol stimulates phospholipase A2 from Swiss 3T3 fibroblasts

We recently demonstrated that diacylglycerol induced arachidonate release and prostaglandin E2 synthesis in 3T3 fibroblasts, and greatly augmented prostaglandin E2 synthesis in response to submaximal and maximal concentrations of bradykinin. We have now partially purified a phospholipase A2 from the cells. When phosphatidyl[3H]choline was used as substrate, several diacylglycerols augmented phospholipase A2 activity. Diacylglycerol was effective at concentrations as low as 30 nM. Protein kinase C inhibition did not affect diacylglycerol's stimulation of phospholipase A2. Diacylglycerol did not alter the calcium requirement for phospholipase A2 or its pH optimum. The present study demonstrates that the effect of diacylglycerol to augment arachidonate metabolism is at the level of phospholipase A2, itself.     

Porcine pancreatic phospholipase A2 stimulates secretin release from secretin-producing cells

We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C.     

CD2 triggering stimulates a phospholipase A2 activity beside the phospholipase C pathway in human T lymphocytes

In previous studies we demonstrated the triggering of the phospholipase C (PLC) pathway during the activation of an Ag-specific human CD4+ T lymphocyte clone by a mitogenic pair of CD2 (X11,D66) mAb. Similar conditions were applied to investigate a possible involvement of a phospholipase A2 (PLA2) acting as an additional alternative pathway during human T cell activation. Our results show that arachidonic acid or its derivatives are released after CD2 triggering. This release is largely independent of PLC activation and is mediated by a PLA2 because: 1) phosphatidylcholine is the preferential source of [3H]arachidonate release; 2) [3H]arachidonic acid release and phosphatidylcholine hydrolysis are blocked by two inhibitors of solubilized PLA2, mepacrine, and 4-p-bromophenacylbromide; and 3) we evidenced a PLA2 activity in cell homogenates. Extracellular calcium appears to play a critical role because the effects of CD2 mAb were inhibited in a Ca2(+)-depleted medium. In contrast, protein kinase C is not implicated since PMA, a protein kinase C activator, neither stimulated arachidonic acid release nor modulated CD2-induced arachidonic acid release. Cyclic AMP which has been proved to regulate the activity of the PLC in T lymphocytes does not appear to play an important role in the regulation of PLA2 activity since PGE2 has only a minimal effect on [3H]-arachidonate release. Altogether, these findings suggest that CD2 triggering stimulates a PLA2 activity in T lymphocytes via an extracellular Ca2(+)-dependent PLC protein kinase C independent mechanism.     

Ornithine decarboxylase activity is associated with proliferation but not with T3-induced differentiation of Caco-2 cells

Omithine decarboxylase (ODC) activity and polyamine (putrescine, spermidine, spermine) concentrations were measured in parallel in enterocyte-like Caco-2 cells maintained under various culture conditions. ODC activity was maximal at the begining of the exponential growth phase, decreasing dramatically thereafter to a negligible level at confluency (day 9). Kinetic studies performed on day 3 revealed the presence of a single enzyme with a K_m around 200 μM and a V_max of about 2 nmol CO₂ released/h/mg protein. Similar values were obtained in both serum-supplemented and transferrin/selenium (TS)-defined culture media, indicating that ODC kinetic parameters are not affected by any factors present in serum. Polymine concentrations were maximal on day 5. By day 9, they returned to initial levels and remained at these fairly high values until day 21. Since we have previously shown (Jumarie and Malo, 1994, In Vitro Cell. Dev. Biol., 30A:753–760) that triiodothyronine (T₃) stimulates differentiation but not proliferation of Caco-2 cells maintained in TS-defined medium, we investigated if it induces differentiation by a polyamine-dependent mechanism. Short- and long-term measurements revealed similar ODC activity and polyamine levels whether T₃ was present or not in the culture medium. These results clearly demonstrate that polyamine synthesis is more likely to be associated with Caco-2 cell proliferation, and that the T₃ effect on Caco-2 cell differentiation does not involve polyamine biosynthesis. Moreover, our data show that ODC activity is not solely regulated by intracellular polyamine concentration. © 1995 Wiley-Liss Inc.     

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

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 p42^MAPK-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.