(Received for publication, April 26, 1995; and in revised form, July 3, 1995)
From the
NIH 3T3 cells stably transfected with the gene encoding
phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) from Bacillus cereus display a chronic elevation of intracellular
diacylglycerol levels and a transformed phenotype. We have used such
PC-PLC-transformed cells to evaluate the roles of the cytoplasmic
serine/threonine kinases Raf-1, protein kinase C (
PKC) and
protein kinase A (PKA) in oncogenesis and mitogenic signal transduction
elicited by phosphatidylcholine hydrolysis. We demonstrate here that
stable expression of dominant negative mutants of both
PKC and
Raf-1 lead to reversion of PC-PLC-transformed cells. Interestingly,
expression of kinase defective
PKC also reverted NIH 3T3 cells
transformed by the v-Ha-ras oncogene. Activation of PKA in
response to elevation of cAMP levels also lead to reversion of
PC-PLC-induced transformation, implicating PKA as a negative regulator
acting downstream of PC-PLC. On the other hand, inhibition or depletion
of phorbol ester responsive PKCs attenuated but did not block the
ability of PC-PLC-transformed cells to induce DNA synthesis in the
absence of growth factors. These results clearly implicate both Raf-1
and
PKC as necessary downstream components for transduction of the
mitogenic/oncogenic signal generated by PLC-mediated hydrolysis of
phosphatidylcholine and suggest, together with other recent evidence, a
bifurcation in the signaling pathway downstream of PC-PLC.
Recently, evidence for a crucial role of phospholipase
C-mediated hydrolysis of phosphatidylcholine (PC) ()in
mitogenic signaling in different mammalian cells and in the maturation
of Xenopus oocytes has
accumulated(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
In fact, exogenous addition of a phosphatidylcholine-hydrolyzing
phospholipase C (PC-PLC) from Bacillus cereus is able to mimic
both a significant portion of the mitogenic response to PDGF in Swiss
3T3 fibroblasts and the constitutive activation of protein kinase C
(PKC) in v-ras- or v-src-transformed NIH 3T3
cells(5, 12) . Furthermore, constitutive expression of
the gene (plc) encoding B. cereus PC-PLC leads to a
chronic elevated level of intracellular DAG and oncogenic
transformation of NIH 3T3 cells (13) . Both in Xenopus oocytes and in murine fibroblasts, a variety of experimental
approaches have revealed that PLC-mediated hydrolysis of PC, elicited
either by the endogenous activity or by the exogenous addition of the
bacterial enzyme, is located downstream of
Ras(3, 8, 12, 14, 15) . It
has also been shown that PC-PLC may be involved in coupling Ras to
activation of Raf(16) . However, the mechanism whereby PC-PLC
transduces mitogenic signals conveyed by p21 Ras remains to be
elucidated. But, since PC-PLC generates the second messenger
diacylglycerol (DAG) capable of activating PKC isozymes (17) possible downstream targets may include one or more
specific PKC isozymes (12, 18, 19) or perhaps
other hitherto undetected DAG-regulated kinases. A direct activation of
Raf-1 by PC-derived DAG may also be possible since the enzyme contains
a cysteine finger in the regulatory domain similar to the DAG binding
motifs of PKC and DAG kinase(20) . The role of the atypical
PKC subtype in mitogenic signal transduction is particularly
interesting since it resembles Raf-1 both in terms of structural
organization, insensitivity to Ca
and phorbol esters,
and a ubiquitous expression pattern(21, 22) .
Furthermore, a requirement for the
PKC isotype in the Ras-mediated
maturation pathway in Xenopus oocytes and for serum-activated
DNA synthesis in mouse fibroblasts has been
documented(18, 19) , suggesting an important role for
PKC in mitogenic signaling. Also,
PKC, but not Raf-1, is
required for stimulation of the stromelysin promoter via a
PDGF/Ras/PC-PLC-responsive element(23) .
In the study
presented here, we show that dominant negative mutants of both Raf-1
and PKC revert the transformed phenotype of cells stably
transfected with the PC-PLC gene(13) . Consistent with a
downstream location of PC-PLC relative to Ras, we also found that
v-Ha-ras-transformed cells acquired a normal, nontransformed
phenotype following transfection with a dominant negative mutant of
PKC. As recently demonstrated for both v-ras- and
v-raf-transformed fibroblasts(24, 25) , PKA
activation blocked both transformation, and the mitogenic signal
elicited by chronic PLC-catalyzed hydrolysis of PC. Taken together,
these results clearly show that PC-derived DAG acts upstream of Raf-1
and establish
PKC as a novel downstream mediator of Ras/PC-PLC
signaling.
Figure 2:
Immunostaining of cells expressing B.
cereus PC-PLC with an affinity-purified antibody. Note the
transformed morphology of the cells expressing PC-PLC alone versus the flat, normal morphology of the cells expressing dominant
negative Raf-1 (M1-dnRaf) or dominant negative PKC (P18-dn
PKC) mutants, even though they still express
PC-PLC. Vector control denotes NIH 3T3 cells stably transfected with
the empty expression vector. Magnification,
400
.
Figure 3:
NIH 3T3 cells expressing PC-PLC form
colonies in soft agar, while stable transfection of these cells with
plasmids expressing dominant negative mutants of either Raf-1 (M1-dnRaf) or PKC (P18-dn
PKC) completely
abolished their ability to anchorage-independent
growth.
Figure 1:
Expression of a kinase defective mutant
of PKC (dn
PKC) and the N-terminal regulatory domain of Raf-1
(dnRaf) in transfected cell lines. A, immunoblot analysis of
PKC overexpression. Cellular proteins (5 µg) were resolved by
SDS-polyacrylamide gel electrophoresis, electrophoretically transferred
onto an Immobilon P membrane, and incubated with a polyclonal
anti-
PKC antibody. The molecular mass of
PKC was estimated to
65 kDa. Equal protein loading in each well was verified by reprobing
the blot with an anti-
-actin antibody (not shown). Note that the P18-dn
PKC-1 cell line was included as a negative control
since it showed a transformed phenotype indistinguishable from the
parental P18 cell line. P18 indicates P18 cells stably
transfected with empty expression vector. B, immunoblot
analysis of dnRaf expression in M1. The cells were incubated for 24 h
in the presence or absence of 1 µM Cd
prior to extraction of cellular proteins. Seventy µg of
protein was loaded into each well. The molecular mass of dnRaf was
estimated to 32 kDa. M1 denotes M1 cells stably transfected
with the empty expression plasmid. Cd
was used to
increase the expression of dnRaf from the human metallothionein IIa
promoter.
Figure 4:
Reversion of the transformed phenotype of
P18 cells by stable expression of a dominant negative mutant of
PKC correlates both with the loss of ability to induce DNA
synthesis in the absence of growth factors and the failure to
transactivate reporter plasmids containing binding sites for the
transcription factors NF-
B or AP-1 following serum starvation. A, serum-starved NIH 3T3 cells expressing PC-PLC induced DNA
synthesis in the absence of added mitogens while transfection of plc clones with either dominant negative Raf-1 (M1-dnRaf) or dominant negative
PKC (P18-dn
PKC) made the cells quiescent.
The magnitude of the mitogenic response is expressed relative to the
response to 10% serum, which was set to 100%. The data are expressed as
means with standard errors of from three to more than 10 other
independent experiments performed in triplicate. M1-vectorcontrol denotes a clone isolated from the M1 cell line
stably transfected with the empty expression vector pMT-hyg. P18-vectorcontrol is the parental P18 cell
line stably transfected with the pRc/CMV expression plasmid and is
representative of 13 different isolated clones. B, following
serum deprivation, PC-PLC-transformed cells (P18) display
constitutive activation of the transcription factors NF-
B and
AP-1, while stable expression of a dominant negative mutant of
PKC
completely blocks this growth factor-independent activation. The CAT
activities determined for the parental NIH 3T3 cells were set to 1.0.
The data are expressed as the mean ± S.E. for one experiment
performed in triplicate and are representative of two other independent
experiments with similar results.
Figure 5:
Reversion of v-ras-transformed
cells by stable expression of a dominant negative mutant of PKC. A, overexpression of dn
PKC in two cell lines established
following transfection of v-ras-transformed NIH 3T3 cells with
pRcCMV
was visualized by immunoblotting
with a polyclonal anti-
PKC antibody. B, the v-ras cells expressing dn
PKC contain activated Ras as evidenced by
the amount of Ras-bound GTP in serum-starved cells. The
PC-PLC-transformed cell lines P18 and P12 do not contain elevated
levels of Ras-GTP. The combined data for both cell lines are shown (PC-PLC), and v-ras-dn
PKC represents the combined data for two independently isolated
clones. The data are expressed as the mean with standard error of five
to 10 independent experiments. The molar ratio of GTP to GDP was
calculated as described under ``Materials and Methods.'' C, the mitogen-independent induction of DNA synthesis of the
parental v-ras-transformed cells is abolished following stable
expression of dn
PKC. D, v-ras cells stably
expressing dn
PKC do not form colonies in soft
agar.
Figure 6:
Loss
of growth factor-independent activation of the transcription factors
NF-B and AP-1 in v-ras cells following stable expression
of dn
PKC. The experiments were performed as described in the
legend to Fig. 3B. The data represent the means with
standard errors of three independent transfections per reporter plasmid
and are representative of two other experiments showing similar
results.
Figure 7:
The
mitogenic signal elicited by PC-PLC is not dependent on classical,
TPA-responsive PKC subtypes but is blocked following treatment with PKA
activating agents. A, neither depletion of TPA-responsive PKC
subtypes or inhibition of PKC by GF 109203X blocked the mitogenic
signal elicited by v-ras or PC-PLC (P18 and P12) expression.
To down-regulate PKC TPA was added to the culture medium during serum
starvation (24 h) to a final concentration of 500 ng/ml (LongtermTPA). The PKC inhibitor GF 109203X (500
nM) was added to quiescent cultures 18 h prior to cell
harvesting and determination of [H]thymidine
incorporation. Short term stimulation with 100 ng/ml TPA (ShorttermTPA) was performed for 1 h following serum
starvation. Addition of GF 109203X totally abolished the mitogenic
response resulting from short term stimulation with TPA (data not
shown). Openbars indicate the induction of DNA
synthesis observed in the absence of growth factor addition. B, increasing the intracellular cAMP level either indirectly
by forskolin treatment or directly by addition of 8-bromo-cAMP (8-Br-cAMP) inhibited DNA synthesis induced by both PC-PLC and
v-ras, while 8-bromo-cGMP (8-Br-cGMP) was without
effect. Additions of 10 ng/ml PDGF (BBhomodimer),
0.5 mM 8-bromo-cAMP, 0.5 mM 8-bromo-cGMP, or 0.1
mM forskolin are indicated (+). The results in A and B are expressed as means with standard errors of
three independent experiments performed in triplicate. The mitogenic
response to 10 ng/ml PDGF (BB) was used to set the 100%
level.
A number of recent reports have firmly established that PC
hydrolysis is critically involved in growth factor-mediated mitogenic
signal
transduction(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16) .
Hydrolysis of PC can be catalyzed either by PC-PLC or
PC-PLD(2, 6) . However, PC-PLC action seems to be
responsible for the sustained increase in cellular DAG levels observed
in fibroblasts upon growth factor stimulation or following
transformation by v-ras or v-src oncogenes(5, 10, 14, 15) .
Importantly, PC-PLC has been shown to act downstream of Ras and
upstream of the serine/threonine kinase
Raf-1(3, 14, 15, 16) . Consistent
with a critical role of PC-PLC in mitogenic signal transduction, we
have recently shown that constitutive expression of the gene (plc) encoding B. cereus PC-PLC leads to
transformation of NIH 3T3 cells and that the transformed phenotype is
completely dependent on plc expression, resulting in a chronic
increase of the cellular DAG mass(13) . In the present report,
we extend upon these findings and show that there seems to be a direct
relationship between the expression level of the bacterial PC-PLC, the
resulting DAG levels, and the extent of oncogenic transformation
achieved as evaluated by parameters such as cell morphology, growth
pattern, cell doubling times, size of soft agar colonies, and induction
of DNA synthesis in the absence of growth factor addition. We also show
that like v-ras-transformed cells, plc-transformed
cells possess activated AP-1 and NF-B transcription factors
following serum starvation. Thus, transformation by plc closely mimicks the behavior of v-Ha-ras-transformed NIH
3T3 cells, perhaps indicating that constitutive expression of PC-PLC
leads to activation of most, if not all, of the downstream targets of
Ras. In keeping with this notion, we found that we could revert the
transformed phenotype of plc expressing cells by coexpression
of a dominant negative mutant of Raf-1. This result corroborates the
findings of Cai et al.(16) who, by employing colony
formation assays with cotransfection of PC-PLC and dominant negative
Raf-1 mutants, recently demonstrated that PC-PLC is unable to bypass
the block to proliferation caused by dominant negative Raf mutants.
They also found that treatment with D609, a specific inhibitor of
PC-PLC enzyme activity(45) , completely inhibited the
activation of Raf-1. In addition, we demonstrate for the first time
that
PKC is required for transformation and growth
factor-independent DNA synthesis in both v-ras- and plc-transformed fibroblasts. Although our present results
permit the conclusion that both Raf-1 and
PKC are required for
transduction of the signal elicited by PLC-catalyzed hydrolysis of PC,
this study does not address the mechanism(s) of activation of these
kinases. The mechanism(s) of activation of Raf is not completely
understood. However, it seems clear that the N-terminal regulatory
domain of Raf binds to the effector loop of GTP-bound Ras and that Ras
may serve to ferry the Raf kinase to the plasma membrane where it is
subsequently
activated(46, 47, 48, 49, 50, 51, 52) .
At the membrane, Raf is part of a complex including GTP-bound Ras,
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase-1, and mitogen-activated protein kinase. Following
phosphorylation, mitogen-activated protein kinase is released from this
complex(47, 53) . GTP-bound Ras is not by itself able
to activate the kinase activity of Raf(34, 52) ,
suggesting that an additional signal is needed. Recent results pinpoint
membrane-associated second messengers as likely candidates for the
second signal needed for full activation of
Raf(16, 46, 48) . Thus, this signal could be
provided by DAG generated by PC hydrolysis interacting with the
cysteine finger in the regulatory CR1 domain of raf-1 p74(16, 20) . Alternatively, PC-derived DAG may
activate a kinase(s) distinct from phorbol ester-sensitive PKCs, which
then can activate Raf by direct
phosphorylation(55, 56) . Whatever the mechanism, our
present results and those of Cai et al.(14, 16) together strongly implicate a crucial
role for PC-derived DAG in this mitogenic signaling pathway and place
this step downstream of Ras but upstream of Raf. Additional support for
this notion is provided by our demonstration that activation of PKA by
treatment with forskolin and/or 8-bromo-cAMP reverted the transformed
phenotype of plc-transformed cells as evidenced by
morphological reversion, loss of ability to form colonies in soft agar
and lack of induction of DNA synthesis in the absence of growth
factors. Since Raf-1 has been shown to be the target of the inhibitory
action of PKA(24, 44) , our results with forskolin and
8-bromo-cAMP confirm the downstream location of Raf-1 relative to
PC-PLC. Furthermore, we show that plc-transformed cells do not
contain elevated levels of GTP-bound Ras. Together with the previous
finding that expression of PC-PLC is able to relieve the block to
proliferation imposed by expression of the N-17 dominant negative
mutant of Ras(14) , this strongly supports the conclusion that
PC-PLC acts downstream of Ras.
As for Raf-1, the mechanism(s) of
activation of the atypical subtype of PKC is not completely
understood. However, it is clear that the enzyme is not activated by
phorbol esters or short-chain DAGs(22, 36) . Recently,
it was reported that in vitro
PKC can be activated by
phosphatidylinositol 3,4,5-trisphosphate, a product of PI
3-kinase(57) . If this mechanism is active in vivo, a
direct link between PI 3-kinase activation following binding to
activated tyrosine kinase receptors and stimulation of
PKC can be
envisioned. Interestingly, Ras may contribute to the activation of PI
3-kinase by directly interacting with the catalytic p110
subunit(58, 59) . This would place both activation of
PI 3-kinase and
PKC downstream of Ras. There is also evidence that
PKC is activated in vivo by treatment of NIH 3T3 cells
with sphingomyelinase C capable of generating the lipid second
messenger ceramide and that ceramide can activate
PKC in
vitro(60) . Thus,
PKC may be regulated by different
lipid mediators. Different lines of evidence suggest that PC-PLC is
acting upstream of
PKC(19, 26, 60) .
Considering the fact that
PKC is activated by ceramide, the
downstream location relative to PC-PLC is completely consistent with
the prevalent model for tumor necrosis factor
signaling where an
acidic sphingomyelinase C is activated by DAG generated by a
membrane-bound PC-PLC(45) . As previously shown for Raf-1, it
has recently been demonstrated that
PKC interacts with Ras both in vitro and in vivo and that the in vivo interaction is dependent on GTP-bound active Ras and takes place
between the N-terminal regulatory domain of
PKC and the effector
domain of Ras(61) . Thus, analogous to Raf, the
PKC-Ras
interaction may serve to bring
PKC to the membrane where its
kinase activity is induced by a lipid mediator(s). This activation is
probably direct since, contrary to Raf-1(56) , there is no
evidence for a role of phosphorylation/dephosphorylation in the
activation of the kinase activity of
PKC. Thus, the same role for
PC-derived DAG proposed for Raf-1 activation above may be applicable to
PKC, which is similar to Raf-1 in its structural organization.
Hitherto, experiments directly addressing binding and activation by
physiological relevant PC-derived DAG species have not been performed
for these kinases. Alternatively, as outlined above, PC-derived DAG may
activate a sphingomyelinase C, which in turn produces ceramide that
will directly activate
PKC.
In view of the proven direct
interaction between Ras and PKC, our results with expression of
the kinase-defective mutant of
PKC in v-ras cells could
be explained as simply due to a blockade of the ability of Ras to
interact productively with both
PKC, Raf-1, and PI 3-kinase as
well as other presently unknown downstream targets that may be
dependent on binding to the effector domain of activated Ras. However,
this model fails to explain why dominant negative mutants of both
PKC and Raf is able to revert plc-transformed cells since
PC-PLC has been convincingly shown, by different experimental
approaches, to act downstream of Ras (3, 14-16, this work).
Colony formation assays have revealed that cotransfection of activated
PKC is not able to relieve the block to proliferation of NIH 3T3
cells imposed by a dominant negative Raf-1 mutant. Likewise, an
activated form of Raf-1 did not abolish the growth inhibitory action of
a dominant negative
PKC mutant(61) . Furthermore,
expression of dominant negative
PKC in NIH 3T3 cells did not
inhibit PDGF-stimulated Raf-1 phosphorylation of mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase-1. (
)Also, a kinase defective mutant of
PKC, but not a
similar mutant of Raf-1, is able to block the activation of the
stromelysin promoter mediated through a novel palindromic PDGF, Ras and
PC-PLC responsive element(23) . Together with our present
results, these findings suggest a bifurcation of the mitogenic
signaling pathway downstream of PC-PLC with Raf-1 and
PKC located
on separate branches. Thus, more than one signaling pathway need to be
activated in order to bring about mitogenesis or oncogenic
transformation.