(Received for publication, September 29, 1994; and in revised form, December 9, 1994)
From the
The proposal that epidermal growth factor (EGF) activates
phospholipase D (PLD) by a mechanism(s) not involving
phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P)
hydrolysis was examined in Swiss 3T3 fibroblasts. EGF, basic fibroblast
growth factor (bFGF), bombesin, and platelet-derived growth factor
(PDGF) activated PLD as measured by transphosphatidylation of butanol
to phosphatidylbutanol. The increase in inositol phosphates induced by
bFGF, EGF, or bombesin was significantly enhanced by Ro-31-8220,
an inhibitor of protein kinase C (PKC), suggesting that
PtdIns(4,5)P
-hydrolyzing phospholipase is coupled to the
receptors for these agonists but that the response is down-regulated by
PKC. Activation of PLD by EGF was inhibited dose dependently by the PKC
inhibitors bis-indolylmaleimide and Ro-31-8220, which also
inhibited the effects of bFGF, bombesin, and PDGF. Down-regulation of
PKC by prolonged treatment with 4
-phorbol 12-myristate 13-acetate
also abolished EGF- and PDGF-stimulated phosphatidylbutanol formation.
EGF and bombesin induced biphasic translocations of PKC
and
to the membrane that were detectable at 15 s. In the presence of
Ro-31-8220, translocation of PKC
became evident, and
membrane association of the
- and
-isozymes was enhanced
and/or sustained in response to the two agonists. The inhibitor also
enhanced EGF-stimulated [
H]diacylglycerol
formation in cells preincubated with
[
H]arachidonic acid, which labeled predominantly
phosphatidylinositol, but inhibited
[
H]diacyl-glycerol production in cells
preincubated with [
H]myristic acid, which labeled
mainly phosphatidylcholine. These data support the conclusion that EGF
can stimulate diacylglycerol formation from PtdIns(4,5)P
and that PKC performs the dual role of down-regulating this
response as well as mediating phosphatidylcholine hydrolysis. In
summary, all of the results of the study indicate that PLD activation
by EGF is downstream of PtdIns(4,5)P
-hydrolyzing
phospholipase and is dependent upon subsequent PKC activation.
Epidermal growth factor (EGF) ()stimulates a number
of cellular responses following binding to its specific cell surface
receptor and activation of the intrinsic tyrosine kinase(1) .
Much evidence has been gathered on the EGF receptor signaling system
using the A431 cells. PI-PLC-
1 is one of the proteins that is
activated by EGF through interaction of the enzyme with
autophosphorylation sites on the cytoplasmic tail of the receptor (2, 3, 4) . The activated PLC catalyzes the
hydrolysis of PtdIns(4,5)P
to generate Ins(1,4,5)P
and 1,2-diacylglycerol (DAG), which elevates cytosolic
Ca
and activates protein kinase C (PKC),
respectively.
Recent evidence shows that many agonists, including EGF, also stimulate DAG production through the hydrolysis of PtdCho and that phospholipase D (PLD) is the major enzyme involved(5) . PLD initially produces PtdOH, which may have second messenger roles, but can also be rapidly converted by PtdOH phosphohydrolase to DAG(6) . Most agonists induce a biphasic production of DAG, with PI-PLC being responsible for the initial rapid increase and PtdCho-hydrolyzing phospholipase D (PC-PLD) for the second sustained increase.
Although agonist-induced activation of PLD is a widely occurring phenomenon, the regulatory mechanisms involved are not well defined. Evidence for the involvement of PKC in the regulation of PLD has been obtained in many different cell systems with various agonists. Addition of the phorbol ester, PMA, increases PLD activity in many cells, and treatment of cells with PKC inhibitors or down-regulation of the enzyme abolishes PLD activation by PMA and most agonists(5, 7) . Overexpression of PKCs in fibroblasts (8, 9, 10) also enhances the PLD response. Similarly, when cells are depleted of PKC by antisense methods, PLD activation is inhibited(11) .
Based on the evidence that PKC
plays a major role in the regulation of PLD, it has been proposed that
DAG produced by initial PtdIns(4,5)P breakdown activates
PKCs, which then directly or indirectly activate
PLD(12, 13, 14, 15, 16) .
However, evidence for this sequential mechanism is limited, and there
have been several reports presenting contrary findings. For example,
certain agonists in some cell types do not induce detectable increases
in InsPs, while they elicit a sustained DAG increase originating from
PtdCho(17, 18, 19, 20, 21) .
There have also been contradictory results concerning the effect of EGF
on PtdIns(4,5)P
metabolism. This growth factor induces
PtdIns(4,5)P
hydrolysis in hepatocytes and A431 cells (22) but produces no detectable increase in InsPs in fibroblast
cell lines, although it stimulates
mitogenesis(18, 23, 24, 25) . The
apparent activation of PLD in the absence of PtdIns(4,5)P
hydrolysis suggests the existence of a PKC-independent pathway (19) .
In the present report, we have studied the mechanisms of activation of PC-PLD by EGF in Swiss 3T3 cells. In contrast to the report of Cook and Wakelam(19) , we observed a small but reproducible increase in InsP formation in EGF-stimulated cells, which was enhanced by a PKC inhibitor. In addition, we provide evidence for the involvement of PKC in the activation of PLD by EGF.
Figure 1:
Agonist-stimulated PLD activity.
Subconfluent, quiescent cells were labeled with 1 µci/ml
[H]myristic acid overnight, and the cells were
preincubated and stimulated with various agonists including EGF (100
nM), bFGF (100 µg/ml), bombesin (Bomb) (100
nM), and PDGF (50 µg/ml) for 15 min in the presence of
0.3% 1-butanol. [
H]PtdBut accumulation was
measured as described under ``Experimental Procedures,'' and
net cpm values over the background were calculated and plotted as means
± S.E. of four determinations in a representative experiment. In
this and subsequent figures, C refers to control cells
incubated without agonist.
A key issue in the present study was to obtain data for or
against the hypothesis that PLD activation requires PKC activation and
DAG production by PI-PLC as prerequisites. As shown in Fig. 2,
PDGF and bombesin were the most potent agonists for PtdIns(4,5)P hydrolysis in Swiss 3T3 cells, whereas bFGF and EGF produced
small but reproducible increases, i.e. they were always
observed and ranged between 11 and 47% (EGF) and 8 and 45% (bFGF) with
averages of 23% (n = 7) (EGF) and 27% (n = 5) (bFGF) (see also Fig. 3).
Figure 2:
Agonist-stimulated PI-PLC activity.
Subconfluent, quiescent cells were labeled with 0.5 µCi/ml
myo-[H]inositol overnight, and the cells were
preincubated and stimulated with each agonist (100 nM EGF, 100
µg/ml bFGF, 100 nM bombesin (Bomb), and 50
µg/ml PDGF) for 15 min in the presence of 20 mM LiCl.
Accumulation of
H-labeled InsPs was determined as
described under ``Experimental Procedures.'' Each column
represents the mean ± S.E. for a representative experiment
performed in triplicate.
Figure 3:
Effect of a PKC inhibitor,
Ro-31-8220, on growth factorstimulated inositol phosphate
accumulation in Swiss 3T3 fibroblasts. Cells, prelabeled with
myo-[H]inositol, were preincubated with 10
µM Ro-31-8220 for 1 h followed by incubation with
each agonist (100 µg/ml bFGF, 100 nM EGF, 100 nM bombesin (Bomb), and 50 µg/ml PDGF) for 15 min in the
presence of 20 mM LiCl. InsPs were fractionated on a
AG 1-X8 anion-exchange column. Data presented as the quotient of
InsPs divided by Ins plus InsPs represent means for a typical
experiment performed in duplicate.
Figure 4:
Effect of PKC inhibitors on
[H]PtdBut accumulation in EGF-treated Swiss 3T3
cells. Cells prelabeled with [
H]myristic acid
were preincubated with increasing concentrations of PKC inhibitors
(bIM, Ro-31-8220, and chelerythrine) for 1 h prior to incubation
with 100 nM EGF. Control cells received the vehicle (0.1%
Me
SO) instead of the PKC inhibitors. The generation of
PtdBut in response to a 15-min stimulation with EGF was determined.
Results, given as percent maximal EGF response on
[
H]PtdBut formation are the means ± S.E.
of four determinations in a representative
experiment.
Because
different PKC isoforms could be involved in the regulation of PLD, we
examined the in vitro effects of the two most potent
inhibitors, bIM and Ro-31-8220, on the PKC isoforms (,
,
, and
), which exist in Swiss 3T3 cells. Using
recombinant enzymes (29) , MBP phosphorylation activity was
measured in the presence of increasing concentrations of the
inhibitors. The two compounds showed similar inhibitory patterns on the
PKC isoforms, although Ro-31-8220 was slightly more potent (Fig. 5, A and B). Both inhibitors were more
potent on PKC
and -
than on PKC
and were almost
ineffective on PKC
. The concentrations that were effective in
vitro were much lower than those required to affect PLD activation
in the intact cells (cf.Fig. 4and Fig. 5).
Figure 5:
Effect of PKC inhibitors, bIM and
Ro-31-8220, on phosphorylating activity of various PKC isozymes.
Partially purified recombinant PKC isoforms were incubated with
increasing concentrations of bIM (A) or Ro-31-8220 (B) in the PKC assay mixture described under
``Experimental Procedures.'' The incorporation of
[P] into substrate (MBP) was measured for 15 min
at 30 °C as described, and the percent inhibition of
phosphorylation was calculated. Data represent means for a typical
experiment performed in duplicate.
Receptor tyrosine kinase activity is necessary for the activation of
PLD by EGF and PDGF(19) . ()Because bIM and
Ro-31-8220 are competitive inhibitors with respect to ATP, it was
possible that they attenuated PLD activation by inhibiting EGF receptor
tyrosine kinase. We therefore examined the tyrosine phosphorylation of
proteins in EGF-stimulated cells in the absence or in the presence of
the inhibitors. Western blotting with anti-phosphotyrosine antibody (Fig. 6) demonstrated that EGF stimulated the phosphorylation of
the EGF receptor (178 kDa) and other cellular proteins (115, 85, and 45
kDa). The inhibitors did not inhibit the tyrosine phosphorylation of
any of those proteins. In fact, bIM and, to a greater extent,
Ro-31-8220 enhanced the tyrosine phosphorylation of the EGF
receptor (Fig. 6B). This is in accord with the effects
of PKC on the receptor, as described earlier.
Figure 6:
Effect of PKC inhibitors on tyrosine
phosphorylation stimulated by EGF. Subconfluent and quiescent cells
were preincubated with PKC inhibitors (A, 5 µM bIM and B, 10 µM Ro-31-8220) for 1 h
prior to incubation with 100 nM EGF. Control cells received
the vehicle (0.1% MeSO) instead of the PKC inhibitors.
Tyrosine phosphorylation in response to a 5-min stimulation with EGF
was determined by Western analysis against anti-phosphotyrosine
antibodies (PY-20).
PKC, -
,
-
, and -
have been reported to be present in Swiss 3T3 cells
by Olivier and Parker (35) who observed that treatment of cells
with 500 nM PMA for 48 h almost completely down-regulated the
,
, and
isozymes, although the rate of loss of each
isoform was not uniform. In contrast, the treatment had no effect on
the cellular level of PKC
. We tested the effect of PKC
down-regulation on EGF-stimulated PtdBut formation. As shown in Fig. 7, the treatment itself significantly enhanced the
radioactivity in PtdBut, suggesting that the basal activity of PLD was
increased. However, the ability of EGF and PDGF to promote PtdBut
accumulation in the PKC down-regulated cells was completely or almost
completely abolished. These results strongly support our conclusion
that PKC is critically involved in PLD activation by EGF.
Figure 7:
Effect of PKC down-regulation on
EGF-stimulated [H]PtdBut accumulation. Swiss 3T3
cells were pretreated for 48 h with 500 nM PMA. Following a
15-min stimulation with 100 nM EGF in the PtdBut, formation
was measured as described under ``Experimental Procedures.''
The results were normalized on the basis of the cpm in the total lipid
extract from each dish and are means ± S.E. of four
determinations in a representative experiment. Control cells received
vehicle (0.1% Me
SO) instead of
PMA.
Figure 8:
Effect
of PKC inhibitors, Ro-31-8220 and bIM, on
[H]PtdBut accumulation stimulated by various
growth factors in Swiss 3T3 cells. Cells, prelabeled with
[
H]myristic acid, were preincubated with 1
µM bIM (A) or 10 µM Ro-31-8220 (B) for 1 h. Control cells received the vehicle (0.1%
Me
SO) instead of the PKC inhibitors. The generation of
PtdBut in response to a 15-min stimulation with each agonist (100
µg/ml bFGF, 100 nM bombesin, and 50 µg/ml PDGF) was
determined. Results given as percent control radioactivity in PtdBut
obtained from each agonist-stimulated cells are means ± S.E. of
four determinations in a representative
experiment.
Figure 9:
EGF-stimulated PtdOH and DAG production in
Swiss 3T3 cells prelabeled with [H]myristic acid
or [
H]arachidonic acid. PtdOH and DAG production
was measured in Swiss 3T3 cells stimulated by 100 nM EGF in
the absence or presence of 10 µM Ro-31-8220 after
prelabeling with different fatty acid precursors. A,
[
H]myristic acid; B,
[
H]arachidonic acid. At the indicated times, the
incubations were terminated, lipids were extracted, and the amount of
PtdOH and DAG was measured as described under ``Experimental
Procedures.'' Control cells were incubated with medium only for
each time in the presence or absence of PKC inhibitor. Results are
given as percent control value of DAG and PtdOH at each time point and
are means ± S.E. of four determinations in a representative
experiment.
When PtdOH and DAG were examined in the
[H]arachidonic acid-labeled cells, the
radioactivity in DAG was higher at 1 min than at 15 min (Fig. 9B), consistent with the activation of PI-PLC.
The DAG species generated from PtdIns(4,5)P
by PI-PLC
usually accumulate transiently because of the down-regulation of the
response and because the DAG is metabolized by DAG kinase and other
enzymes. Thus, the increase in [
H]PtdOH level at
15 min in Fig. 9B is probably due to the formation of
PtdOH from DAG derived from PtdIns(4,5)P
. Interestingly, in
the presence of Ro-31-8220, the increases in
[
H]DAG at 1 min and in
[
H]PtdOH at 15 min in the cells labeled with
arachidonic acid were significantly enhanced (Fig. 9B).
This is consistent with the effect of the PKC inhibitor to counteract
the down-regulation of the activation of PI-PLC by EGF, as observed for
InsPs production (Fig. 3). (
)
Figure 10:
Bombesin-induced PKC translocation. The
quiescent cells preincubated with either vehicle (0.1%
MeSO) or 10 µM Ro-31-8220 for 1 h were
treated with 100 nM bombesin for the indicated times. Cell
homogenates were fractionated into cytosolic (C) and
particulate (M) fractions. An equal amount of proteins was
analyzed by 10% SDS-polyacrylamide gel electrophoresis and Western
blotting as described under ``Experimental
Procedures.''
Both bombesin and EGF
induced a biphasic translocation of PKC ( Fig. 10and Fig. 11) and also caused a rapid translocation of PKC
. The
membrane association of these isozymes was enhanced and/or prolonged by
Ro-31-8220, which also promoted significant association of both
isozymes in the absence of the agonists ( Fig. 10and 11). (
)PKC
was not translocated by these agonists at any
time (data not shown) in agreement with observations by other
investigators(20, 35, 41) .
Figure 11: EGF-induced PKC translocation. Cells were treated with 100 nM EGF for the indicated times in the absence or presence of 10 µM Ro-31-8220. Cytosolic (C) and particulate (M) fractions were prepared, and an equal amount of protein for each fraction was analyzed by 10% SDS-polyacrylamide gel electrophoresis and Western blotting as described under ``Experimental Procedures.''
It is becoming clear that PtdCho hydrolysis by PLD is a
widespread cellular response to many
agonists(5, 6, 7) . Through this pathway,
many known or potential signaling molecules such as PtdOH, lysoPtdOH,
DAG, monoacylglycerol, and arachidonic acid and its metabolites can be
produced. Most G-protein-coupled and receptor tyrosine kinase-coupled
agonists have been shown to stimulate PtdCho hydrolysis by PLD by a
process that appears to be downstream of the initial Ins(1,4,5)P and DAG formation by PI-PLC and to involve PKC
activation(5, 6, 12, 14, 15) .
In A431 cells, EGF has been shown to stimulate the hydrolysis of
PtdIns(4,5)P via the activation of PLC
due to the
tyrosine kinase activity of the EGF receptor(3) . Although Cook
and Wakelam (19) have suggested that EGF does not induce the
hydrolysis of PtdIns(4,5)P
in Swiss 3T3 fibroblasts, the
present report provides evidence indicating that PI-PLC is also coupled
to the EGF receptor in these cells. We consistently observed a small
but reproducible increase in InsPs in EGF-stimulated cells, and this
was enhanced by the PKC inhibitor Ro-31-8220 (Fig. 3). The
enhancing effect of the inhibitor is probably due to its prevention of
down-modulation of the EGF receptor by PKC. Evidence for such
down-regulation has been provided by Wahl and Carpenter (42) and is in accordance with the findings of Davis (30) and others(22, 31) . The inability of
Cook and Wakelam (19) to observe an increase in InsPs
with EGF in Swiss 3T3 cells may relate to their use of cells that had
grown to confluency for 48 h. We have observed that such cells show
diminished responses to EGF.
In additional support of the involvement of PI-PLC in the activation of PC-PLD in Swiss 3T3 cells is the observation that the relative efficacy of EGF, PDGF, and bombesin in stimulating PtdBut formation (Fig. 1) is the same as for activating PI-PLC (Fig. 2). Similar observations have been made in hepatocytes stimulated by various agonists(43) .
Because of our evidence that EGF is coupled to the PI-PLC signaling system in Swiss 3T3 cells, we expected that the activation of PLD by EGF would be PKC dependent. In contrast to the report of Cook and Wakelam(19) , three potent, selective PKC inhibitors (bIM, Ro-31-8220, and chelerythrine) dose-dependently antagonized EGF-induced PtdBut formation (Fig. 4). The potency of the inhibitors on PLD activation by EGF was similar to that for inhibition of other PKC-mediated cellular responses(32, 33, 34) . The concentrations required to inhibit PLD were much higher than those required to inhibit PKC isozymes in vitro (Fig. 5), reflecting limitations and differences in the permeability of intact cells to the inhibitors. To our knowledge, Fig. 5presents the first data on the effects of bIM and Ro-31-8220 on individual PKC isozymes.
Fig. 8illustrates that 10 µM Ro-31-8220 was unable to completely abolish the stimulation of PLD by four different agonists. Although this could merely be due to an inability to achieve full inhibition of PKC isozymes at this non-toxic dose, it is also possible that PKC-independent mechanisms of PLD activation could exist. However, the fact that down-regulation of PKC eliminated the ability of EGF or PDGF to stimulate PLD (Fig. 7) argues against this possibility. In these experiments, PKC depletion produced a substantial increase in basal PLD activity. The basis for this is unknown, but it could imply a role for PKC in the regulation of the synthesis or degradation of the enzyme. The basal level of PLD activity observed in Fig. 7and Fig. 8is probably due to the existence of multiple PLD isozymes, as has been speculated(44, 45, 46, 47) . Such PKC-independent isozymes could be regulated by other mechanisms, e.g. those involving small G-proteins such as ARF and Rho(48, 49, 50) .
In our studies of the
regulation of PLD by PKC, we did not employ the PKC inhibitors,
staurosporine and sphingosine, since these have been shown to stimulate
PLD (51, 52) via the activation of pertussis
toxin-sensitive G-proteins or other mechanisms. This probably accounts
for the fact that these inhibitors usually do not produce complete
inhibition of the activation of PLD(6) . ()Another
issue is that PKC is often involved not only in the stimulation of PLD
by an agonist but also in the down-regulation of its receptor (22, 31, 42, 43, 53, 54, 55, 56, 57) .
Thus, in the presence of a PKC inhibitor, downregulation of the
receptor could be blocked, resulting in enhancement of the signal for
PtdBut accumulation. As a result of these positive and negative
effects, PKC inhibitors could thus have variable effects on PLD
activity.
In any consideration of the role of PKC in the regulation
of PLD, another point should be recognized. This is that the DAG
derived from PtdIns(4,5)P breakdown may be considered to
act as a ``trigger'' for PLD activation via PKC, i.e. continuing production of DAG from this source may not be required.
This is because, once activated, PLD can produce DAG from PtdCho via
PtdOH phosphohydrolase, and this DAG can then sustain PKC activation
and hence PLD activity. This would explain why PLD activation can
continue despite down-regulation of the PI-PLC response.
The
conclusion that the EGF receptor, like the receptor for bombesin, is
linked to PI-PLC in Swiss 3T3 cells is further supported by the PKC
translocation studies of Fig. 10and Fig. 11. These show
a movement of PKC to the membrane in EGF-stimulated cells in the
presence of Ro-31-8220. Because we have shown that the inhibitor
increases InsPs production (Fig. 3) and
[
H]DAG production in response to EGF in
[
H]arachidonic acid-labeled cells (Fig. 9B), it is likely that the translocation reflects
DAG formation from PtdIns(4,5)P
in the membrane. In IIC9
fibroblasts, it has been shown that PKC
translocation requires
concurrent rises in DAG and Ca
, i.e. PtdIns(4,5)P
hydrolysis(20) .
There have
been some efforts to define the role of the various PKC isoforms in the
regulation of PLD. In membranes prepared from CCL39 fibroblasts, only
PKC and -
were activators of PLD(58) , but whole
cells were not examined. In other studies, PKC
and -
were
implicated in Madin-Darby canine kidney cells (11) and in
messangial cells(59) . PKC
1 overexpression in rat
fibroblasts has been observed to enhance agonist-stimulated PLD
activity(8, 9) , and overexpression of PKC
enhances it in Swiss 3T3 cells(10) . In contrast, transfection
of PKC
constructs into Cos-1 cells resulted in partial suppression
of the PLD response to PMA(60) . Except for the latter
observation, these findings implicate PKC-
, -
, and -
in
the stimulation of PLD. Since PKC
is
Ca
-independent, it may be the isozyme principally
involved in the ``feedback'' activation of PLD that occurs
when DAG is derived mainly from PtdCho after the initial phase of
agonist stimulation.
Although there is evidence that PLD can be activated by a phosphorylation-independent mechanism in membranes from CCL-39 cells (61) , the stimulation has been shown to require ATP-dependent phosphorylation in many cell types(62, 63) . Since we observed that Ro-31-8220 blocked PLD activation but promoted membrane association of PKCs, it seems that translocation of PKC protein per se does not activate PLD, i.e. it also requires the catalytic activity of the kinase.
Clearly, more work needs to be
done to more rigorously define the regulation of PC-PLD by G-protein-
and tyrosine kinase-linked agonists. Most important is the need to
purify and characterize the PC-PLD isozymes involved and to demonstrate
if they are direct substrates for PKC isozymes and if their
phosphorylation is correlated with activation. ()