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J Biol Chem, Vol. 273, Issue 48, 32182-32186, November 27, 1998
From the Division of Hematology/Oncology, Department of Medicine,
Beth Israel Deaconess Medical Center and Harvard Medical School,
Massachusetts, Boston 02115
Transformation is accompanied by the
down-regulation of the high molecular weight isoforms of non-muscle
tropomyosin. Several lines of evidence suggest that tropomyosin
down-regulation may be essential for ras-induced
tumorigenicity. It is unclear which of the many signaling pathways
downstream of Ras are involved in tropomyosin down-regulation. Here we
demonstrate that Raf activation induces tropomyosin down-regulation
comparable to that induced by Ras. Expression of the effector-domain
mutant Ras-G12V,Y40C, which is unable to bind Raf, induced only modest
down-modulation of tropomyosin. Treatment with the MEK-specific
inhibitor PD98059 had little effect on tropomyosin levels in
ras- or raf-transformed cells. In contrast, a
mutant form of MEK-1, MEK-1-S218A,S222A, restored tropomyosin levels in
ras-transformed NIH3T3 cells almost to the levels observed
in non-transformed cells. MEK-1-S218A,S222A does not inhibit MEK
phosphorylation and is a poor inhibitor of ERK phosphorylation. These
data suggest that this mutant form of MEK-1 interferes with a yet
uncharacterized pathway controlled by Raf. We conclude that the
ras-induced down-modulation of tropomyosin is predominantly
Raf-mediated, but MEK-independent, and that a novel pathway exists
downstream of Raf which may play an important role in regulation of the cytoskeleton.
Malignant transformation results in the down-modulation of the
non-muscle high molecular weight tropomyosin isoforms
TM-1,1 TM-2, and TM-3. These
actin-binding cytoskeletal proteins are virtually undetectable in
transformed cell lines generated by the induced expression of oncogenes
such as ras, src, raf, mos, fms, fes, or erbB
(1-5), or by infection with tumor inducing viruses such as SV40, Rous
sarcoma virus, adenovirus type 5, or MC29 (4, 6). Tumorigenic cell
lines produced by the exposure of SV40-immortalized prostate cells to
x-rays also have markedly lower levels of the isoforms TM-1 and TM-3
compared with those of the normal parental line (7). It is unclear how
tropomyosin is down-regulated in tumor cells. Hendricks and Weintraub
(4) and Cooper et al. (3) have shown that down-modulation of
tropomyosin occurs both at the transcriptional as well as at the
post-transcriptional level. TM down-modulation observed in tumor cells
is not an artifact of prolonged cell culture or deliberate
transformation, since it has also been observed in freshly isolated
human prostate, breast, ovarian, and squamous cell carcinoma tissues
(8-12). These data suggest that TM down-modulation is consistently
associated with tumorigenesis regardless of the specific genetic
alterations responsible for the transformed phenotype.
Several lines of evidence suggest that TM down-modulation is essential
for transformation. TM-1 down-modulation resulting from the expression
of a TM-1 antisense construct is sufficient to induce anchorage
independence in immortal hamster embryo cells (13) and the
reintroduction of TM-1 or TM-2 into ras-transformed cells
reduces or eliminates anchorage-independent growth and tumorigenicity (5, 14-16). Analyses of multiple clones from rodent melanoma and lung
carcinoma cell lines have demonstrated a strong inverse correlation
between metastatic capacity and TM levels (17, 18). Collectively, these
results suggest that the expression of high molecular weight
tropomyosin plays an important role in maintaining the non-transformed phenotype.
Oncogenic Ras mutants activate many different downstream signaling
pathways including those controlled by the activity of Rac, Rho, Cdc42,
Ral, phosphatidylinositide 3-kinase and Raf (see Joneson and Bar-Sagi
and Katz and McCormick, for reviews (19, 20)). To identify which
pathway(s) downstream of Ras may be involved in TM down-modulation, we
analyzed the effects of activated forms of Raf, Rac, Rho, Lbc, and Ral,
as well as the Ras effector-domain mutant Ras-G12V,Y40C, on TM levels.
We also investigated the effects of MEK and Raf inhibition in
ras- and raf-transformed cells using the MEK
inhibitor PD98059 and a mutant form of MEK-1, MEK-S218A,S222A. The
results of our studies implicate Raf in ras-induced TM
down-modulation but suggest that the critical downstream effectors may
be MEK-independent. Our data provide evidence for the existence of Raf
substrates in addition to MEK-1 and MEK-2 which may control the levels
of actin-binding proteins including the high molecular weight TMs and
regulate the phenotype of transformed cells.
Cell Lines and Tissue Culture--
NIH3T3 and
ras-transformed NIH3T3 cell lines were generous gifts of L. Feig, Tufts University (16). All cell lines were grown in Dulbecco's
modified Eagle's medium (Mediatech, Herndon, VA) containing 10% calf
serum and 50 µg/ml gentamicin (Life Technologies, Inc., Gaithersburg, MD).
Monoclonal Antibodies--
TM311, a monoclonal antibody which
recognizes a common epitope in the first exon of TM-1, TM-2, and TM-3
(21) was purchased from Sigma. Anti-Raf (clone C-12) and anti-ERK-1
(clone K-23) were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA) and anti-MEK-1 (clone 25) was purchased from Transduction
Laboratories (Lexington, KY). Anti-phospho-MEK and anti-phospho-ERK
antibodies were purchased from New England Biolabs (Beverley, MA).
Anti-H-Ras (clone LA069) was purchased from Quality Biotech (Camden,
NJ). Secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG) conjugated to horseradish peroxidase were purchased from Bio-Rad.
Treatment with PD98059--
A total of 200,000 cells per well
were plated in 6-well plates in Dulbecco's modified Eagle's medium
with 10% serum. The following day, the medium was replaced by fresh
Dulbecco's modified Eagle's medium with 5% serum including the
indicated concentration of PD98059 (New England Biolabs) in
Me2SO or an equal volume of Me2SO (0.1% v/v).
Cells were incubated for the times indicated.
Expression Vectors and Stable Transfections--
The expression
vectors ral-G23V in pJ4
Fibroblasts grown to 50% confluency in a 6-well plate in medium
without serum were stably transfected with the above expression vectors
(0.5-2 µg of DNA) using 10 µl of LipofectAMINE (Life Technologies, Inc.). Expression vectors were co-transfected with pSVneo
in a molar ratio of 10:1, except for rho-Q63L in
pZipneo and MEK-1-S218A,S222A in pAUCT, which
contain the neoR gene, as indicated. Clones were
selected using cloning cylinders and grown in medium with 10% serum
and 500 µg/ml G418 (Mediatech). Transfections with
MEK-1-S218A,S222A in pAUCTneo were performed in
the presence of 1 µg/ml tetracycline and cells were kept in
tetracycline-containing medium unless otherwise indicated.
Whole Cell Extracts and Western Blotting--
Nearly confluent
monolayers of fibroblast cultures in 6-well plates were washed twice
with cold phosphate-buffered saline. Cells were lysed with 250 µl of
lysis buffer (1% SDS in 62.5 mM Tris, pH 6.8) containing
protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride) and phosphatase inhibitors (1 mM EGTA, 1 mM Na3VO4,
10 mM sodium fluoride, 10 mM
ERK In-gel Kinase Assay and MEK and ERK-Phosphorylation
Assays--
The ERK in-gel kinase assay was performed as described
previously (16). In vivo Raf and MEK activities were
determined by assaying the levels of phosphorylation of their
substrates MEK-1, and ERK-1 and ERK-2, respectively, using the
anti-phospho-MEK and anti-phospho-ERK antibodies from New England
Biolabs. ERK phosphorylation levels were normalized to the total ERK
levels as determined by Western blotting using an anti-ERK-1 antibody from Santa Cruz Biotechnology.
Role of Raf in ras-mediated Down-modulation of Tropomyosin--
We
previously demonstrated that v-Ki-ras-transformed NIH3T3
cells have substantially reduced levels of the high molecular weight
isoforms of tropomyosin (TM-1, TM-2, and TM-3; Ref. 16). Ras is a
molecular switch controlling many different signaling pathways involved
in tumorigenesis. In order to determine which signaling pathway(s)
downstream of Ras might be involved in TM down-regulation, NIH3T3 cell
lines stably expressing the activated forms of Raf, Rac, Rho, Ral, or
Lbc were generated and the levels of high molecular weight isoforms of
tropomyosin were determined by Western blotting. Cell lines expressing
the activated forms of Rac, Rho, Ral, or Lbc did not have reduced TM
levels relative to non-transformed NIH3T3 cells (not shown). In
contrast, cell lines transfected with constitutively active forms of
Raf (v-raf and raf-20A) had markedly decreased
TM-1 and TM-2 levels and a complete disappearance of TM-3, identical to
the changes induced by v-Ki-ras (Fig.
1). These results indicate that Raf
activity is sufficient to down-regulate TM expression.
To determine if Raf activity is essential for ras-induced TM
down-modulation, we tested the effects of the Ras effector-domain mutant, Ras-G12V,Y40C, which is unable to bind Raf, in non-transformed NIH3T3 cells. NIH3T3 cell lines were stably transfected with vector only or with vectors containing H-ras-G12V,Y40C or
H-ras-G12V. Expression of the transfected genes was
validated by Western blot analysis (Fig.
2A) and examination of
cellular morphology. NIH3T3 cells transfected with vector alone were
flat and grew as a monolayer. The H-ras-G12V-transfected
cells showed a spindle-shaped morphology and grew in multiple layers.
The H-ras-G12V,Y40C-transfected cells showed an intermediate
phenotype (not shown). To confirm that the Ras-G12V,Y40C mutant was
indeed unable to interact with Raf, we determined the levels of
ERK-phosphorylation in these cell lines. To exclude the influence of
growth factors, the cells were serum-starved for 24 h. Fig.
2B shows that the levels of ERK-phosphorylation in the
ras-G12V,Y40C-transfected cells (normalized to total ERK protein levels) were comparable to those in normal NIH3T3 cells transfected with vector only. In contrast, the levels of ERK-1 and
ERK-2 phosphorylation were increased 4.0- and 2.5-fold, respectively, in ras-G12V-transformed cells relative to normal NIH3T3
cells. Stable expression of H-ras-G12V induced a 98%
decrease in the combined levels of TM-2 and TM-3 as determined by
densitometry and a 90% reduction in those of TM-1 (Fig.
2C). Conversely, stable expression of
H-ras-G12V,Y40C induced only a 40% decline in the TM
levels. This difference in TM down-regulation was not due to differences in transgene expression, since the levels of
H-Ras-G12V,Y40C present in the transfectants used in this study were
actually higher than those of H-Ras-G12V (Fig. 2A). This
result suggests that Raf activity is necessary for the complete
down-regulation of tropomyosin induced by the ras oncogene,
but that Raf-independent pathways may contribute to TM
down-regulation.
Role of MEK in ras- and raf-mediated Down-modulation of
Tropomyosin--
In addition to the Ras-G12V,Y40C study, we tested the
effects of PD98059, a well characterized inhibitor of Raf-mediated MEK activation (31), on TM levels in ras- and
raf-transformed NIH3T3 cells. Ras-transformed
cells were treated with various concentrations of PD98059 in
Me2SO or with Me2SO alone for 5, 24, and
48 h and ERK-1 and ERK-2 activities were assayed using the in-gel
kinase assay. ERK activities in ras-transformed cells were
almost completely inhibited when the cells were treated with the MEK
inhibitor PD98059 for 24 or 48 h (Fig.
3A). Treatment with PD98059
for 24 h at concentrations of 25, 50, and 100 µM
decreased ERK-1 phosphorylation (normalized) by 83, 94, and 97%,
respectively, and ERK-2 phosphorylation by 80, 90, and 98%,
respectively. Treatment at the same concentrations of PD98059 for
48 h decreased the level of ERK-1 phosphorylation by 78, 65, and
55%, and that of ERK-2 by 87, 62, and 58%. The drug had only a modest
effect on TM-2 levels and almost no effect on TM-1 levels (Fig.
3B). In fact, the TM-1 levels actually decreased below
baseline level when cells were incubated with PD98059 at a
concentration of 100 µM (Fig. 3B).
Furthermore, PD98059 failed to restore TM-3 expression. The results
shown in Fig. 3 have been corroborated in a subsequent study in which
the duration of exposure to PD98059, and suppression of ERK activity,
were extended to 78 h. In this study, TM levels remained depressed
to the same extent as those observed at 48 h of exposure (not
shown).
Results similar to those reported above were obtained with
raf-transformed cells. In these studies, both Raf-20A, a
constitutively active form of Raf-1 with 313 amino acids deleted from
the NH2 terminus and containing only the kinase domain, and
v-Raf, which contains all effector domains of Raf-1, were used. Both
forms of Raf preferentially phosphorylate ERK-2. Levels of
phosphorylated ERK-2 in raf-transformed cells were
comparable to those in v-Ki-ras-transformed cells, whereas levels of phosphorylated ERK-1 in
raf-transformed cells were approximately 70% lower than
those in ras-transformed cells (Fig.
4A). Treatment of
raf-20A-transformed cells with the MEK inhibitor at 50 µM for 48 h decreased the normalized levels of
phosphorylated ERK-1 and ERK-2 by 97 and 90%, respectively. Treatment
of v-raf-transformed cells with the inhibitor resulted in
virtually undetectable ERK-1 phosphorylation and a more than 95%
decrease of ERK-2 phosphorylation (Fig. 4A). As with the
ras-transformed cells, MEK inhibition in
raf-transformed cells resulted only in a minor increase
in TM-2 levels and no change in those of TM-1 or TM-3 (Fig.
4B).
MEK-1-S218A,S222A Inhibits ras-induced Down-modulation of
Tropomyosin--
Expression of the MEK-1 mutants MEK-1-S217A and
MEK-1-S221A in ras-transformed NIH3T3 cells induces
reversion to a non-transformed phenotype with flattening of the cells
and restitution of actin cables (32). This observation suggests that
expression of mutants of MEK-1 in which the serine phosphorylation
sites of Raf have been mutated may affect cytoskeletal organization,
possibly by restoring the expression of various actin-binding proteins
known to be down-modulated in transformed cells. To test this
hypothesis, we evaluated the effects of the MEK-1 mutant
MEK-1-S218A,S222A on tropomyosin expression in
v-Ki-ras-transformed NIH3T3 cells. In this study,
v-Ki-ras-transformed cells were stably transfected with
tetracycline-regulable MEK-1-S218A,S222A. In the absence of
tetracycline, the level of MEK-1-S218A,S222A increased 9-fold over
background within 48 h (Fig.
5A). At this point, the levels of phosphorylated ERK-1 and ERK-2 (normalized) were decreased by 60 and
63%, respectively, compared with those of cells grown in the presence
of tetracycline (Fig. 5B), indicating that MEK-1-S218A,S222A was functionally active. Addition of tetracycline to cell cultures of
untransfected v-Ki-ras-transformed cells had no effect on
ERK phosphorylation levels (not shown). The degree of MEK inhibition induced by MEK-1-S218A,S222A (Fig. 5B) was much less than
that induced by PD98059 (Fig. 3A). In addition,
MEK-S218A,S222A expression did not inhibit MEK-1 phosphorylation in
v-Ki-ras-transformed cells (Fig. 5C), suggesting
that MEK-S218A,S222A does not interfere with Raf activity in these
cells. Despite the lack of Raf inhibition and the incomplete
down-regulation of ERK phosphorylation, the levels of all three high
molecular weight isoforms of TM in MEK-S218A,S222A expressing cells
were up-regulated to levels comparable to those observed in
non-transformed NIH3T3 cells (Fig. 5D). Addition of tetracycline to cell cultures of untransfected
v-Ki-ras-transformed cells had no effect on TM levels (Fig.
5D). The restorative effect of MEK-S218A,S222A on TM levels,
its modest inhibitory effect on ERK phosphorylation, and the minimal
effect of PD98059 on TM expression all support the contention that the
ability of MEK-S218A,S222A to normalize TM levels in
ras-transformed NIH3T3 fibroblasts cannot be attributed to
the disruption of the Raf/MEK/ERK pathway. Rather, the data suggest
that the MEK-S218A,S222A mutant affects the cytoskeleton by interfering
with another, yet to be characterized, signaling cascade controlled by
Raf.
The aggressive behavior of ras-transformed cells is a
result of its ability to activate multiple signaling pathways capable of cooperating in the promotion of anchorage-independent growth, motility, invasiveness, and metastasis. Of the various signaling molecules downstream of Ras, the serine/threonine kinase Raf is perhaps
the most vital to the transforming activity of the GTPase. Constitutively active raf mutants are able to directly
transform NIH3T3 cells (33, 34) and inhibition of Raf suppresses
ras-induced transformation of NIH3T3 cells (32). Active
forms of Rac, Ral, and Rho on the other hand, are either
non-transforming, weakly transforming, or functional only when
introduced in combinations (26, 35, 36). In the studies reported
herein, we assessed the ability of active mutants of raf, rac,
rho, ral, and lbc to down-modulate the high molecular
weight TM isoforms and observed that raf was the only one
capable of inducing TM down-modulation when introduced as a single
oncogene. This parallel between the ability of an oncogene to
down-regulate TM and to transform immortalized fibroblasts not only
singles out Raf as the downstream Ras target most essential for this
cytoskeletal modification but also reinforces the notion that TM
down-modulation is a critical step in the acquisition of the malignant phenotype.
The fact that Raf activity induces the down-modulation of TM does not
necessarily indicate that it plays an essential role in
ras-mediated cytoskeletal modifications. To determine if Raf activity was necessary for ras-induced TM down-modulation,
we assessed the effects of the effector-domain mutant Ras-G12V,Y40C, which is unable to interact with Raf. The expression of this Ras mutant
does indeed reduce TM levels, indicating that signaling pathways other
than those activated through Raf may contribute to the cytoskeletal
modifications observed in ras-transformants. However, this reduction is only modest and does not approach
the nearly complete down-modulation induced by Ras-G12V or oncogenic Raf. These data suggest that Raf activation is essential for the nearly
complete disappearance of the high molecular weight TMs observed in
ras-transformed cells.
So far, the only well characterized signaling pathway downstream of Raf
is the MEK/ERK pathway. Since Raf activity appears to be essential for
the ras-induced TM down-modulation, it was expected that
inhibition of MEK in ras- and raf-transformed
cells would lead to TM restoration. Surprisingly, the MEK inhibitor PD98059 had only a modest effect on ras- or
raf-induced down-regulation of TM levels. In the presence of
PD98059, only the TM-2 isoform was partially up-regulated while TM-1
and TM-3 isoforms were virtually unaffected. These data indicate that
MEK activity is not necessary for complete ras- or
raf-induced down-regulation of TM.
The inability of PD98059 to restore TM levels contrasts sharply
with the effects of MEK-1-S218A,S222A, which increased the levels of
all three high molecular weight TM isoforms almost to those observed in
non-transformed NIH3T3 cells. The different effects of
MEK-1-S218A,S222A and PD98059 on TM down-modulation are not due to
differences in their respective potency. In fact, the MEK inhibitor
suppresses ERK phosphorylation far better than does
MEK-1-S218A,S222A.
The mutant MEK-1-S218A,S222A used in our studies lacks the serine
phosphorylation sites at positions 218 and 222, thereby preventing it
from being activated by Raf. In a yeast two-hybrid screening, this
mutant was found to bind tightly to all Raf isoforms (38).
Furthermore, of 29 clones isolated, Raf proteins were the only ones
identified as interacting with this mutant MEK. The related triple
mutant MEK-1-K97A,S218A,S222A, which has an additional defect in ATP
binding, co-immunoprecipitates with Raf and ERK (39). These MEK mutants
and others have been shown to block chemotaxis toward fibronectin and
to suppress the transformed phenotype of ras-transformed
cells (30, 32). Because of these properties, these MEK mutants are
regarded as dominant negative forms of MEK-1. However, despite this
designation, few of these mutants have actually been shown to inhibit
the Raf/MEK/ERK pathway. In fact, numerous studies, including those in
the present report, have shown that MEK-1 mutants in which one or more
of the amino acids at positions Lys-97, Ser-218, and Ser-222 have been
mutated to alanine are very poor inhibitors of MEK and/or ERK
activation (32, 39, 40). Our results suggest that MEK-1-S218A,S222A interferes with a yet uncharacterized pathway distinct from the MEK/ERK
cascade leading to the down-regulation of tropomyosin.
Although Raf isoforms were the only MEK partners identified in earlier
two-hybrid experiments (38), MEK has recently been shown to bind to the
adaptor protein Grb10 and to the carboxyl-terminal region of kinase
suppressor of Ras (41-43). Thus, the ability of MEK-1-S218A,S222A to
normalize TM levels in ras-transformed cells may be due to
an interaction between the mutant form of MEK-1 and one of these
proteins or others yet to be identified.
MEK is not the only downstream effector of Raf. In some cells, Raf
activates the p70 S6 kinase (44) and NF- Our results on the regulation of TM are reminiscent of those reported
by Ramocki et al. (29) on the regulation of MyoD in transfected fibroblasts. Although MyoD expression has recently been
shown to be ERK-dependent in C2C12 myoblasts (46), Ramocki et al. (29) reported that the ras- and
raf-CAAX-mediated down-regulation of MyoD transcriptional
activity in C3H10T1/2 fibroblasts co-transfected with MyoD and a
muscle-specific luciferase reporter gene was not reversed by PD98059.
These results and ours emphasize the notion that not all Raf-induced
phenomena are dependent on the activity of MEK. Ramocki et
al. (29) also observed that the Ras-G12V,Y40C effector domain
partially inhibited MyoD transcriptional activity, a finding comparable
to our observation that the Ras-G12V,Y40C mutant has a modest
down-modulating effect on TM levels. Collectively, our data and those
of Ramocki et al. (29) indicate that Raf activation is the
dominant means by which Ras reduces TM levels and suppresses MyoD
transcriptional activity in transformed fibroblasts. Our results
support the notion that MEK-independent signaling pathways exist
downstream of Ras and Raf that regulate cellular transformation and differentiation.
We thank M. White (University of Southwestern
Texas), L. Feig (Tufts University), B. Schaffhausen (Tufts University),
D. Toksoz (Tufts University), D. Roth (Beth Israel Deaconess Medical
Center), C. Der (University of North Carolina), and M. Symons (Onyx
Pharmaceuticals) for providing expression vectors. We thank R. Erikson (Harvard University) for helpful discussions.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
TM, tropomyosin; MEK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase; ERK, extracellular signal-regulated kinase.
Ras- and Raf-induced Down-modulation of
Non-muscle Tropomyosin Are MEK-independent*
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(22, 23), v-raf in
pBR322 (24), and raf-20A in pJ4 (25) were generous gifts of L. Feig, Tufts University. pBR322 was obtained from D. Roth, Beth
Israel Deaconess Medical Center. Rho-Q63L in
pZipneo (26) was provided by C. Der, University of North
Carolina. pSR
and flag-tagged onco-lbc (a rho-specific
GEF) (27) in pSR were obtained from D. Toksoz, Tufts University.
H-Ras-G12V and the effector-domain mutant
H-Ras-G12V,Y40C in pSR (28, 29) were generous gifts of M. White, University of Southwestern Texas. c-Myc-tagged
Rac-1-Q61L (26) in pRC/CMVneo was
obtained from B. Schaffhausen, Tufts University. The dominant-negative form of MEK-1, MEK-1-S218A,S222A, in pAUCTneo
(30), was a generous gift of M. Symons, Onyx Pharmaceuticals and
contains a neomycine resistance gene (neoR), the
tetracycline transactivator under control of a cytomegalovirus promoter, and MEK-1-S218A,S222A under the control of a
minimal cytomegalovirus promoter fused to the tetracycline operon.
-glycerophosphate, and 1 mM sodium pyrophosphate). Samples were boiled to reduce the viscosity and protein concentrations were determined using the bicinchoninic acid protein assay from Pierce
(Rockford, IL). Protein samples were mixed 4 to 1 (v/v) with 5 × loading buffer (6% SDS, 62.5 mM Tris, pH 6.8, 25%
-mercaptoethanol, 50% glycerol, and 0.02% bromphenol blue). Equal
amounts of protein per lane were electrophoresed on an
SDS-polyacrylamide gel and subsequently analyzed by Western blotting.
Equal loading of the samples was assessed by staining the blots with a
0.1% Ponceau S, 5% acetic acid solution (Sigma). The membranes were
probed with antibodies directed against the relevant proteins and
proteins were visualized by chemiluminescence as described previously
(16). Densitometric analysis was performed using the Bio-Rad Imaging Densitometer GS-700 in combination with Molecular Analyst Software.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (49K):
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Fig. 1.
A, expression levels of Raf-20A and
v-Raf in stably transfected cells. NIH3T3 cells were stably transfected
with either vector alone (lanes labeled pBR322 and
pJ4 ) or with pBR322 containing v-raf (lanes
labeled v-Raf) or pJ4 containing raf-20A
(lanes labeled Raf-20A). Numbers indicate
separate clones stably transfected with the respective oncogenes. P11
represents a pool of clones stably expressing v-Raf. B,
expression levels of high molecular weight TM isoforms in
raf-transfected cells (see also A).
v-Ki-ras indicates a NIH3T3 cell line stably expressing
v-Ki-ras (16). NIH3T3 indicates the parental cell
line.
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Fig. 2.
A, H-Ras levels in
H-ras-G12V,Y40C- and H-ras-G12V-transfected
cells. NIH3T3 cells were stably transfected with either vector alone
(lane labeled pSR ) or with pSR containing
H-ras-G12V,Y40C (lanes labeled
V12,C40) or H-ras-G12V (lanes labeled
V12). Numbers indicate separate clones stably
transfected with the respective oncogenes. B,
phosphorylation levels of ERK-1 and ERK-2 in Ras-G12V,Y40C- and
Ras-G12V-transfected cells. Upper panel, levels of ERK
phosphorylation in NIH3T3 cells stably transfected with
H-ras-G12V,Y40C or H-ras-G12V (see legend to Fig.
2A). Lower panel, levels of total ERK-1 and ERK-2 in the
same samples as in the upper panel. C, levels of tropomyosin
expression in Ras-G12V,Y40C- and Ras-G12V-transfected cells (see
A).
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Fig. 3.
A, in-gel ERK kinase activities of
ras-transformed cells treated with the MEK inhibitor
PD98059. V-Ki-ras-transformed cells were treated for 24 or 48 h
with the indicated concentrations of PD98059 in Me2SO. As a
control, cells were treated with Me2SO only. B,
TM levels in PD98059-treated ras-transformed cells (see
A).
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Fig. 4.
A, phosphorylation levels of ERK-1 and
ERK-2 in raf-transformed cells treated with 50 µM PD98059 in Me2SO or Me2SO only
for 48 h. Upper panel, levels of phosphorylated ERK-1
and ERK-2 in raf-20A- and
v-raf-transformed cells in the presence (+) or absence ( 
)
of PD98059. Numbers indicate the various clones. P11
represents a pool of clones stably expressing v-Raf.
v-Ki-ras indicates v-Ki-ras-transformed cells
grown under the same conditions as the raf-transformed
cells. Lower panel, levels of total ERK-1 and ERK-2 in the
same samples as in the upper panel. B, TM levels in
raf-transformed cells treated with 50 µM
PD98059 (+) or Me2SO () for 48 h. (see
A).
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Fig. 5.
A, levels of MEK-1-S218A,S222A under the
control of tetracycline. Ras-transformed cells were stably
transfected with MEK-1-S218A,S222A
(v-Ki-ras/MEK-1AA) under the control of a
tetracycline-repressible promoter. Cells were grown in medium with (+)
or without tetracycline ( ) for 48 h and whole cell extracts of
these cells were analyzed by Western blotting using an anti-MEK-1
antibody identifying both endogenous MEK-1 and the transfected
MEK-1-S218A,S222A. As control, non-transformed NIH3T3 (NIH3T3) and
non-transfected v-Ki-ras-transformed NIH3T3
(v-Ki-ras) cells were used. In the presence of tetracycline,
MEK levels in the stable transfectants are similar to those in
non-transfected ras-transformed cells, while in the absence
of tetracycline, MEK-1-S218A,S222A expression is increased 9-fold over
background. B, phosphorylation levels of ERK-1 and ERK-2 in
ras-transformed cells transfected with
tetracycline-regulable MEK-1-S218A,S222A. Upper panel,
levels of ERK phosphorylation in ras-transformed cells
stably transfected with tetracycline-regulable MEK-1-S218A,S222A
(v-Ki-Ras/MEK-1AA) grown in the presence (+) or absence
() of tetracycline. The cell extracts used in this experiment were
the same as those used in A. Lower panel, levels
of total ERK-1 and ERK-2 in the same samples as in the upper
panel. C, phosphorylation levels of MEK-1 in
ras-transformed cells transfected with
tetracycline-regulable MEK-1-S218A,S222A (v-Ki-Ras/MEK-1AA)
grown in the presence (+) or absence () of tetracycline. Since the
high expression of the MEK-1-S218A,S222A prevents normalization to
total MEK levels, loading of the total amount of protein in each lane
was determined by Ponceau S staining and no significant differences in
loading were observed (not shown). D, left panel,
TM levels in ras-transformed cells stably transfected with
tetracycline-regulable MEK-S218A,S222A (v-Ki-Ras/MEK-1AA)
grown in the presence (+) or absence () of tetracycline.
Non-transformed NIH3T3 (NIH3T3) and
ras-transformed NIH3T3 (v-Ki-Ras) were used as
controls. The cell extracts used for this experiment were the same as
those used in A. Right panel, as a control for the effects
of tetracycline on TM expression, ras-transformed cells
(v-Ki-ras) were grown in the presence (+) or absence () of
tetracycline.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B (45). Whether these
factors play a role in ras-induced down-regulation of TM remains to be determined. The activation of NF-B by raf
is probably mediated by JNK (45). Since the expression of a
constitutively active Rac, which activates JNK, does not down-modulate
TM (not shown), it seems unlikely that NF-B activation would be
responsible for the TM down-modulation observed in
ras-transformed cells. We observed that rapamycin, an
inhibitor of p70 S6 kinase, had no effect on TM levels in
ras-transformed cells (not shown). These negative data
suggest that the non-MEK Raf target responsible for TM down-regulation
may be novel.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Div. of
Hematology/Oncology, Dept. of Medicine, Beth Israel Deaconess Medical Center, RE 302, 41 Avenue Louis Pasteur, Boston, MA 02115. Tel.: 617-667-0430; Fax: 617-975-5398; E-mail:
jmier{at}bidmc.harvard.edu.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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