(Received for publication, November 11, 1994; and in revised form, January 12, 1995)
From the
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.
There is a strong correlation between the activation of
phospholipase D (PLD) ()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.
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 [H]myristate.
[
H]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
GTP
S. Cytosol and GTP
S had a much smaller effect on PLD
activity in membranes from Balb/c 3T3 cells. The effect of cytosol and
GTP
S 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 GTP
S and cytosol was greater than the sum of individual
effects of either GTP
S or cytosol alone (Table 1). The
synergistic effect of cytosol and GTP
S and the increased PLD
activity in membranes from v-Src-transformed cells were not observed
when the cells were prelabeled with
[
H]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 GTP
S 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 [
H]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
GTP
S. 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 [
H]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
[H]myristate, were prepared, and PLD activity was
determined in the presence of both cytosol and GTP
S 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,
GTP
S, 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
[
H]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
[H]myristate, were prepared, and PLD activity was
determined in the presence and absence of cytosol (10 µM GTP
S 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
[H]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 [
H]arachidonate instead of
[
H]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
[H]myristate (Myr) or
[
H]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. ()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,
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(
); 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.