©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ras Mediates the Activation of Phospholipase D by v-Src (*)

(Received for publication, November 11, 1994; and in revised form, January 12, 1995)

Hong Jiang (1) Zhimin Lu (1) Jing-Qing Luo (1) Alan Wolfman (2) David A. Foster (1)(§)

From the  (1)Institute for Biomolecular Structure and Function and the Department of Biological Sciences, The Hunter College of The City University of New York, New York, New York 10021 and (2)The Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We demonstrated previously that v-Src activates a phospholipase D (PLD) activity (Song, J., Pfeffer, L. M., and Foster, D. A.(1991) Mol. Cell. Biol. 11, 4903-4908) and that this activation is dependent upon a G protein(s) (Jiang, H., Alexandropoulos, K., Song, J., and Foster, D. A.(1994) Mol. Cell. Biol. 14, 3676-3682). An in vitro PLD assay was developed to study G protein involvement in v-Src-induced PLD activity. Maximal PLD activity in membranes isolated from v-Src-transformed cells was dependent upon both GTP and cytosol. In this report, we present three lines of evidence demonstrating that v-Src-induced PLD activity is mediated by Ras. First, a neutralizing Ras monoclonal antibody (Y13-259) inhibits PLD activity in membranes isolated from v-Src-transformed Balb/c 3T3 cells. Second, immobilized Ras protein depleted cytosol of the ability to stimulate PLD activity. This effect was dependent upon preloading immobilized Ras with GTP. Last, expression of a dominant negative Ras mutant in v-Src-transformed cells reduced PLD activity to the level observed in the nontransformed parental cells. These data establish a novel role for Ras in the regulation of PLD activity.


INTRODUCTION

There is a strong correlation between the activation of phospholipase D (PLD) (^1)and mitogenesis (Boarder, 1994; Foster, 1993). Protein-tyrosine kinase activity is also widely implicated in mitogenic signaling (Fantl et al., 1993) and commonly leads to an elevation of PLD activity. v-Src (Song et al., 1991), v-Fps (Jiang et al., 1994b), and the receptors for both platelet-derived growth factor (Plevin et al., 1991) and epidermal growth factor (Kaszkin et al., 1992; Song et al., 1994) have all been shown to activate PLD activity. The primary metabolite of PLD is phosphatidic acid, which is a biologically active phospholipid (see Foster(1993) for review). Phosphatidic acid can be converted into a variety of lipid second messengers such as diglyceride and lysophosphatidic acid. Diglyceride activates protein kinase C (Nishizuka, 1992), and lysophosphatidic acid is mitogenic (van Corven et al., 1990). Thus, the activation of PLD can generate a variety of intracellular second messengers that may play an important role in the generation of intracellular signals that lead to cell division. We demonstrated previously that v-Src activates a PLD activity that can be distinguished from the PLD activity induced by phorbol esters that activate protein kinase C (Song and Foster, 1993) and that this PLD activity is dependent upon a G protein(s) (Jiang et al., 1994a). In this report, we present data implicating the monomeric G protein Ras in the activation of PLD by v-Src.


EXPERIMENTAL PROCEDURES

Cells and Cell Culture Conditions

Normal and v-Src-transformed Balb/c 3T3 and NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum (HyClone). Cell cultures were made quiescent by growing to confluence and then replacing with fresh media containing 0.5% newborn calf serum for 1 day.

Materials

GTPS and GMP-PNP were purchased from Boehringer Mannheim; [^3H]myristate (NET-830) and [^3H]arachidonate (NET-2982) were obtained from DuPont NEN. Precoated Silica Gel 60A thin layer chromatography plates were from Scientific Products.

Assay of Membrane PLD Activity

Cell cultures were grown to confluence, at which time the media were replaced with fresh media containing 0.5% newborn calf serum for 1 day. This treatment reduced background PLD activity presumably because of serum stimulation. Cells in 150-mm culture dishes were then prelabeled overnight for 14-16 h in 20 ml of Dulbecco's modified Eagle's media containing 0.5% newborn calf serum. Isotopes were included as follows: 20 µCi of [^3H]myristate (40 Ci/mmol) or 15 µCi of [^3H]arachidonate (240 Ci/mmol). PLD activity in isolated membranes was determined using conditions established by Olson et al.(1991) and Conricode et al.(1992) with modifications. Prelabeled cells were washed twice with cold isotonic phosphate-buffered saline, suspended in hypotonic buffer (25 mM Hepes, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride), allowed to swell for 10 min, and then broken by Dounce homogenation (30 strokes with type B pestle). The disrupted cells were centrifuged at 500 times g for 5 min to clear nuclei and unbroken cells, and the supernatant was then centrifuged at 44,000 rpm for 45 min in an SW 50 rotor. The supernatant was saved as the cytosolic fraction (approximately 1.5 mg/ml protein); the membrane fraction was recovered from the pellet by resuspending in hypotonic buffer and adjusted to a final protein concentration of 4 mg/ml. The resuspended membranes were put on ice for 30 min and then passed through a 25-gauge needle to break up membrane fragments. The 100 µg of membrane protein and 150 µg of cytosolic protein (if included) were then recombined by dilution with assay buffer (25 mM Hepes, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 5 mM MgCl(2), 100 mM KCl, 10 mM NaCl, 0.16 mM CaCl(2), 1 mM dithiothreitol, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium vanadate, 1 mM ATP, 3 mM creatine phosphate, 4 units/ml creatine phosphate kinase) to 500 µl. The PLD assay is then initiated by addition of ethanol to a final concentration of 1.0%. The reaction was terminated by the addition of organic solvent. PLD activity was determined by the transphosphatidylation of labeled membrane phospholipids to phosphatidylethanol (PEt) as described previously (Jiang et al., 1994a).

Transfection and G418 Selection

NIH 3T3 cells were plated at a density of 10^5 cells/100-mm dish 18 h prior to transfection. Transfections were performed by using Lipofectamine reagent (Life Technologies, Inc.) according to the vendor's instructions. Transfected cultures were selected in 400 µg/ml G418 for 10-14 days at 37 °C. At that time G418-resistant colonies were pooled and expanded for further analysis.


RESULTS AND DISCUSSION

To examine the mechanism of PLD activation by v-Src, we developed an in vitro PLD assay to examine PLD activity in isolated membranes where greater than 90% of the increased PLD activity in v-Src-transformed cells fractionated. In vitro PLD activity was optimized in membranes isolated from v-Src-transformed cells that had been prelabeled with [^3H]myristate. [^3H]Myristate is incorporated almost exclusively into phosphatidylcholine, the substrate for the PLD activated by v-Src (Song et al., 1991; Song and Foster, 1993). Maximal PLD activity, as determined by the transphosphatidylation of phosphatidylcholine to PEt in the presence of exogenously provided ethanol, was dependent upon cytosol and the nonhydrolyzable GTP analog GTPS. Cytosol and GTPS had a much smaller effect on PLD activity in membranes from Balb/c 3T3 cells. The effect of cytosol and GTPS on PLD activity in the v-Src-transformed cells appeared to be synergistic in that the increase in PLD activity in the presence of both GTPS and cytosol was greater than the sum of individual effects of either GTPS or cytosol alone (Table 1). The synergistic effect of cytosol and GTPS and the increased PLD activity in membranes from v-Src-transformed cells were not observed when the cells were prelabeled with [^3H]arachidonate, which is incorporated into phopspholipids (including phosphatidylcholine) not utilized by the PLD activated by v-Src (Song and Foster, 1993). When cells were pretreated with the protein-tyrosine kinase inhibitor genistein, the effect of cytosol and GTPS was reduced to that observed in the parental Balb/c 3T3 cells (Table 1). Thus, the pattern of PLD activity in membranes from v-Src-transformed cells can be clearly distinguished from that in the parental Balb/c 3T3 cells and from that observed in membranes from v-Src-transformed cells treated with genistein or prelabeled with [^3H]arachidonate. The differences observed in the in vitro PLD activity in the membranes isolated from the v-Src-transformed and parental Balb/c 3T3 cells were identical to the differences observed previously in intact cells (Song et al., 1991; Song and Foster, 1993; Jiang et al., 1994a) and strongly suggest that the increased PLD activity in the membranes from the v-Src-transformed cells is caused by v-Src.



The monomeric G protein Ras has been implicated in v-Src-initiated intracellular signals and transformation (Smith et al., 1986; Nori et al., 1991; DeClue et al., 1991; Qureshi et al., 1992). To determine whether Ras contributes to the G protein requirement for v-Src-induced PLD activity, we examined the effect of the neutralizing Ras monoclonal antibody Y13-259 (Furth et al., 1982; Smith et al., 1986) on PLD activity in membranes from v-Src-transformed cells. Y13-259 antibody was incubated with the membrane fraction (where Ras localizes) for 45 min prior to addition of cytosol and GTPS. As shown in Fig. 1a, pretreatment with Y13-259 reduced PLD activity to about half that observed in the untreated cells. This effect could be competed away with an excess of exogenously Ras protein. A non-neutralizing Ras antibody (Y13-238) had little or no effect on the PLD activity in membranes isolated from the v-Src-transformed cells (Fig. 1a). Interestingly, the effect of Y13-259 on PLD activity was dependent upon the presence of the cytosolic fraction as shown by the lack of effect of Y13-259 on the PLD activity membranes treated only with GTPS. Y13-259 had little or no effect upon the PLD activity in membranes from v-Src-transformed cells in the absence of cytosol (Fig. 1a). Y13-259 also had no effect upon the PLD activity observed in membranes isolated from Balb/c 3T3 cells or in membranes isolated from v-Src-transformed cells that had been prelabeled with [^3H]arachidonate (Fig. 1b). Additionally, if the v-Src-transformed cells were treated with the protein-tyrosine kinase inhibitor genistein, the effect of Y13-259 was lost (Fig. 1b). Thus, the effect of the neutralizing Ras antibody is likely specific for v-Src-induced PLD activity. These data demonstrate a functional requirement for Ras for the elevated PLD activity in membranes from v-Src-transformed cells.


Figure 1: A neutralizing Ras antibody inhibits PLD activity in membranes from v-Src-transformed cells. a, membranes from v-Src-transformed cells, prelabeled with [^3H]myristate, were prepared, and PLD activity was determined in the presence of both cytosol and GTPS as described in Table 1. The effect of Ras monoclonal antibodies Y13-259 (neutralizing) and Y13-238 (non-neutralizing) on PLD activity was examined by adding increasing amounts of antibody to the reaction mix as shown. Where indicated, purified Ras protein (12 µg) was included to compete with Y13-259. Membranes were incubated with antibodies for 45 min at 22 °C prior to the addition of cytosol, GTPS, and ethanol at 37 °C. The effect of Y13-259 in the absence of cytosol is also presented (b). Ras monoclonal antibodies Y13-259 and Y13-238 were added to membranes isolated from Balb/c 3T3 cells, v-Src-transformed cells prelabeled with [^3H]arachidonate, and v-Src-transformed cells treated with genistein, and PLD activity was determined as in a. The data are presented as in Table 1as the cpm/mg of membrane protein and represent the average of duplicate determinations ± the range from a representative experiment that was repeated at least three times.



As shown in Fig. 1a, the effect of Y13-259 was dependent upon the inclusion of the cytosolic fraction, suggesting a cytosolic downstream Ras effector molecule. We therefore examined whether immobilized Ras could deplete the cytosolic fraction of a factor(s) required for v-Src-induced PLD activity in a GTP-dependent manner. Upon separation of cytosolic and membrane fractions, the cytosolic fraction was incubated with immobilized Ras proteins loaded with either GTP (nonhydrolyzable GMP-PNP) or GDP. The immobilized Ras proteins were spun out, the cytosolic fraction was added back to the membranes, and PLD activity was determined. As shown in Fig. 2, there was a GTP-dependent depletion of the stimulatory effect of the cytosolic fraction on PLD activity in membranes isolated from v-Src-transformed cells. These data implicate a cytosolic factor as a GTP-dependent target of Ras function in the v-Src-induced activation of PLD. Although the identities of putative cytosolic factor(s) binding to Ras remain to be determined, the data demonstrate a GTP dependence for Ras function in v-Src-induced PLD activity.


Figure 2: Ras depletes the cytosol of its stimulatory potential in a GTP-dependent manner. Membranes from v-Src-transformed cells, prelabeled with [^3H]myristate, were prepared, and PLD activity was determined in the presence and absence of cytosol (10 µM GTPS was included in all samples). The cytosolic fractions were either untreated or pretreated with immobilized bovine serum albumin (BSA), Ras GMP-PNP, or Ras GDP. After a 1-h incubation at 4 °C, the immobilized bovine serum albumin and Ras proteins were spun out, the cytosolic fractions were added back to the membranes, and PLD activity was determined as in Table 1. Immobilized Ras and bovine serum albumin were prepared as described previously (DiBattiste et al., 1993). The data represent the average of duplicate determinations ± the range from a representative experiment that was repeated at least three times.



The data presented in Fig. 1and Fig. 2strongly implicate Ras in v-Src-induced PLD activity in a cell-free system. To test for Ras involvement in the activation of PLD activity by v-Src in intact cells, we transfected a dominant negative Ras mutant (Feig and Cooper, 1988) into NIH 3T3 cells transformed by v-Src. NIH 3T3 cells were used instead of Balb/c 3T3 cells because of a higher transfection efficiency. As a control for the effects of the Ras mutant, we used a dominant negative Raf-1 mutant (Kolch et al., 1991) that was shown previously to block v-Src-induced transformation without inhibiting v-Src-induced PLD activity in Balb/c 3T3 cells (Qureshi et al., 1993). Plasmids expressing the dominant negative Ras and Raf-1 mutants also expressed the selectable G418 resistance gene. G418-resistant colonies were selected and pooled to avoid clonal variation. Overexpression of Ras and Raf-1 proteins was confirmed by Western blot analysis (data not shown). The G418-selected v-Src-transformed cells expressing the Ras and Raf-1 mutants had a reduced ability to form colonies in soft agar. There was a greater than 80% reduction in colony number for the Ras mutant and greater than 70% reduction for the Raf-1 mutant (data not shown). Additionally, colonies that did form in soft agar were smaller than those observed for the v-Src-transformed cells. These data are consistent with previous results showing that both Raf-1 and Ras are required for v-Src-induced transformation (Smith et al., 1986; Qureshi et al., 1993). The inhibitory effect of the Ras and Raf mutants on v-Src-induced transformation suggested that in addition to being expressed, the dominant negative mutants were also functional. In v-Src-transformed NIH 3T3 cells prelabeled with [^3H]myristate, expression of the dominant negative Ras mutant reduced PLD activity to the level of PLD activity observed in the parental NIH 3T3 cells (Fig. 3). When the cells were prelabeled with [^3H]arachidonate instead of [^3H]myristate, there was no observable difference in the PLD activity between the v-Src-transformed cells and the v-Src-transformed cells expressing the dominant negative Ras mutant (Fig. 3). As demonstrated previously in Balb/c 3T3 cells (Qureshi et al., 1993), expression of the dominant negative Raf-1 mutant did not inhibit PLD activity in v-Src-transformed NIH 3T3 cells (Fig. 3). The inability of the Raf-1 mutant to inhibit PLD activity suggests that the elevated levels of PLD activity in cells expressing v-Src is not caused by secondary effects of transformation since the transformed phenotype is inhibited in these cells. The data presented in Fig. 3provide evidence in intact cells that v-Src-induced PLD activity is mediated by Ras.


Figure 3: A dominant negative Ras mutant blocks PLD activity in v-Src-transformed cells. NIH 3T3 cells, v-Src-transformed NIH 3T3 cells, and v-Src-transformed NIH 3T3 cells stably transfected with plasmids expressing dominant negative mutants of Ras (pZIP M17) and Raf-1 (p301) were prelabeled with either [^3H]myristate (Myr) or [^3H]arachidonate (Ara) as shown. The PLD activity in these cells was then determined by determining the level of PEt as a percentage of the total cpm incorporated per culture dish in the presence of exogenously supplied ethanol (1.0%). The data are the average of duplicate determinations ± the range from a representative experiment repeated three times. v-Src-transformed NIH 3T3 cells (provided by R. Jove, University of Michigan) were transfected with plasmids expressing dominant negative mutants for Ras (Feig and Cooper, 1988; Cai et al., 1990) and Raf-1 (Kolch et al., 1991) as described previously (Qureshi et al., 1993). pZIP M17 contains the ras gene with Asn-17 substituted for Ser-17 in pZIPneoSV(X) (Cai et al., 1990). p301 contains a mutated raf-1 gene, with the lysine in the ATP-binding site changed to tryptophan, cloned into pMNC (Kolch et al., 1991). The plasmids expressing the dominant negative mutants also expressed the G418 resistance gene. The control NIH 3T3 and v-Src-transformed cells were stably transfected with the parental vector for pZIP M17, pZIPneoSV(X), which expressed the G418 resistance gene, and these cells were maintained under G418 selection in the same way as the cells expressing the dominant negative ras gene. G418-resistant colonies were selected and pooled to avoid clonal variants. The transformed phenotype of cells expressing the dominant negative mutants was examined by testing for the ability to form colonies in soft agar as described previously (Qureshi et al., 1993).



The monomeric G proteins Rho (Bowman et al., 1993; Malcolm et al., 1994) and ARF (ADP-ribosylation factor) (Brown et al., 1993; Cockroft et al., 1994) have recently been reported to be regulators of PLD activity. ARF and Rho have been implicated in the regulation membrane traffic and cytoskeletal assembly (Ridley and Hall, 1992; Kahn, 1993). Thus, the PLD activated by mitogenic stimuli like v-Src may be distinct from the PLD activated by non-mitogenic stimuli. Consistent with this, C3 exoenzyme of Clostridium botulinum, which inhibits Rho family G proteins (Ridley and Hall, 1992), had no effect upon v-Src-induced PLD activity. (^2)What role Ras may play in the activation of PLD by v-Src is not yet clear. Attempts to activate PLD activity directly with purified Ras in cell membranes and to isolate PLD activity with immobilized Ras proteins in detergent lysates of v-Src-transformed cells were not successful,^2 suggesting that PLD is not a direct target of Ras. However, it was recently reported that PLD activity is elevated in v-Ras-transformed cells (Carnero et al., 1994), suggesting that an activated Ras may increase PLD activity in intact cells. Several recent reports have demonstrated a physical interaction with potential Ras effector molecules including Raf-1 (Moodie et al., 1993; Vojtek et al., 1993; Warne et al., 1992; Zhang et al., 1993), phosphatidylinositol 3-kinase (Rodriguez-Viciana et al., 1994), and Ral guanine nucleotide-releasing factor (Kikuchi et al., 1994; Hofer et al., 1994). As demonstrated in Fig. 2, Ras-GTP binds to a soluble factor that is required for the cytosol to activate PLD in membranes from v-Src-transformed cells. Since the dominant negative Raf-1 mutant does not prevent PLD activation, this factor is not likely to be Raf-1. We have determined that phosphatidylinositol 3-kinase localizes with the membrane fraction in v-Src-transformed cells(^3); thus, the cytosolic factor is not likely to be phosphatidylinositol 3-kinase either. A possible role for Ral guanine nucleotide-releasing factor or another yet to be identified downstream target of Ras in v-Src-induced PLD activity remains to be determined; however, data presented here establish that Ras is a component in the signaling machinery activated by v-Src that results in PLD activation.


FOOTNOTES

*
This investigation was supported by National Institutes of Health Grant CA46677, Council for Tobacco Research Grant 3075, and PSC-CUNY Research Award 664259 (to D. A. F.); a Research Centers in Minority Institutions (RCMI) award from the Division of Research Resources, National Institutes of Health (RR 03037) (to Hunter College); and National Institutes of Health Grant GM49652 (to A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Inst. for Biomolecular Structure and Function and Dept. of Biological Sciences, Hunter College of City University of New York, 695 Park Ave., New York, NY 10021. Tel.: 212-772-4075; Fax: 212-772-5227; foster{at}genectr.hunter.cuny.edu.

(^1)
The abbreviations used are: PLD, phospholipase D; PEt, phosphatidylethanol; GTPS, guanosine 5`-3-O-(thio)triphosphate; GMP-PNP; 5`-guanyl-beta,-imidodiphosphate.

(^2)
H. Jiang, Z. Lu, J.-Q. Luo, A. Wolfman, and D. A. Foster, unpublished results.

(^3)
H. Jiang, Z. Lu, J.-Q. Luo, A. Wolfman, and D. A. Foster, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Joan Brugge and Rich Jove for the v-Src-transformed Balb/c 3T3 and NIH 3T3 cells, respectively; Geoffrey Cooper and Larry Feig for the dominant negative Ras mutant; and Ulf Rapp for the dominant negative Raf-1 mutant. We thank Akira Kikuchi and Rusty Williams for communicating results prior to publication. We thank Larry Feig, Marcello Curto, and Sergey Bychenok for comments on the manuscript.


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