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J Biol Chem, Vol. 274, Issue 38, 27177-27184, September 17, 1999
From the Department of Microbiology, Health Sciences Center,
University of Virginia, Charlottesville, Virginia 22908
Epidermal growth factor stimulates migration of a
number of cell types, yet the signaling pathways that regulate
epidermal growth factor-stimulated migration are poorly defined. In
this report, we employ a transient transfection migration assay
to assess the role of components of the Ras-mitogen-activated protein (MAP) kinase signaling pathway in epidermal growth factor-stimulated chemotaxis of rat embryo fibroblasts. Expression of dominant negative Ras blocks epidermal growth factor-mediated chemotaxis, while constitutively active Ras has no effect on chemokinesis or chemotaxis. PD98059 and U0126, inhibitors of MAP kinase kinase (MEK) activity, decreased epidermal growth factor-stimulated migration, while kinase-defective MEK1, an inhibitor of MAP kinase activation, enhanced
migration. To understand the paradoxical effects of these molecules on
epidermal growth factor-induced migration, we examined the role of
c-Raf on migration. Expression of either wild type c-Raf or the
catalytic domain of c-Raf effectively inhibited epidermal growth
factor-stimulated cell migration. We suggest that, whereas Ras activity
is necessary to promote epidermal growth factor-stimulated migration,
sustained activation of c-Raf may be important in down-regulating migratory signaling pathways triggered by epidermal growth factor receptor activation. Further, activation of c-Raf upon inhibition of
the MEK-MAP kinase pathway may contribute to the inhibition of cell
migration observed with pharmacological MEK inhibitors.
Regulated cell migration is physiologically important for
embryonic development, wound healing, and immunological responses associated with inflammation. Moreover, unregulated migration is a
central feature of some pathological states, most notably tumor
metastasis. While it is clear that regulating cell migration is
important for homeostasis of an organism, the signal transduction events that regulate motility are poorly defined.
Cells can be stimulated to move toward soluble factors (chemotaxis)
including growth factors, such as epidermal growth factor (EGF),1 or substrate-bound
components (haptotaxis), typically ECM. EGF binding to its cell surface
receptor elicits signal transduction pathways that culminate in
migration (1-3). It has been reported that EGF-stimulated migration
requires receptor tyrosine kinase activity and autophosphorylation
induced upon EGF binding (2-4). Phosphotyrosine residues on the
activated EGFR bind Src homology 2 domains of PLC Binding of Grb2 and/or Shc to the activated EGFR stimulates the Ras The role of MAP kinase in mediating steps necessary for migration is
somewhat less clear. Haptotactic migration of FG pancreatic carcinoma
cells or Rat1 fibroblasts to collagen or fibronectin, respectively, can
be blocked by the MEK-specific inhibitor, PD98059 (17, 18). Such
migration can be stimulated by ectopically expressed constitutively
active MEK, suggesting a regulatory role for MAP kinase in haptotaxis
(17, 18). PDGF-stimulated chemotaxis of Rat1 fibroblasts was unaffected
by PD98059 treatment, implying that MAP kinase is not necessary for
PDGF-mediated migration (18). Recent reports have indicated that
EGF-stimulated MAP kinase activation is not sufficient to promote
migration of mouse NR6 cells expressing the EGFR (3). However,
treatment of these cells with PD98059 inhibits EGF-stimulated
migration, suggesting that MAP kinase is necessary for migration to
occur (19).
In this study, we employed a transient transfection migration assay to
assess the role of components of the Ras-Raf-MEK-MAP kinase signaling
pathway in EGF-stimulated REF52 cell migration. As reported previously,
treatment of cells with PD98059 inhibited EGF-stimulated cell
migration. However, additional experiments showed that overexpression
of catalytically inactive MEK stimulated EGF-induced migration, whereas
the overexpression of activated MEK failed to stimulate cell migration.
Catalytically inactive MEK binds to and titrates c-Raf away from
endogenous substrates while the pharmacological MEK inhibitors increase
c-Raf activity. These observations prompted examination of the role of
c-Raf in the regulation of EGF induced cell migration. We show that
overexpression of either wild type c-Raf or activated c-Raf
consistently inhibited EGF-induced cell migration of REF52 cells.
We conclude that, whereas Ras activity is necessary to promote
EGF-stimulated migration, the sustained activation of c-Raf in REF52
cells may be important in down-regulating the migratory signaling
pathways triggered by EGF receptor activation.
Reagents--
REF52 cells were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Life
Technologies, Inc.), 10 µg/ml penicillin (Life Technologies, Inc.),
and 0.25 µg/ml streptomycin (Life Technologies, Inc.). EGF (Sigma)
was reconstituted in DMEM containing 0.1% fatty acid-free BSA (Sigma). PDGF-BB (Amersham Pharmacia Biotech) was reconstituted in 4 mM HCl, 0.1% BSA. PD98059 (Biomol) and U0126 (Promega),
MEK inhibitors, were reconstituted in Me2SO and used at a
final concentration of 50 µM. U73122 (Biomol), a PLC
inhibitor, and the inactive congener U73433 (Biomol) were reconstituted
in chloroform and stored at Plasmids--
All constructs in this study utilized a
cytomegalovirus promoter to drive expression of a
peptide-epitope-tagged protein. HA-tagged A17Ras, N17Ras, and L61Ras
(20, 21) were kind gifts of R. Mattingly and I. Macara (University of
Virginia, Charlottesville, VA); HA-tagged V12Ras, cloned into pDCR, was
supplied by M. Wigler (Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY); FLAG-tagged wild type c-Raf, activated c-Raf (22W) (22),
and the empty vector pC were a gift of R. Jove (University of South
Florida, Tampa, FL). The empty vector for A17Ras, N17Ras, and L61Ras
constructs, pKH, was generated by digesting A17Ras with
BamHI and EcoRI, followed by religation to remove
A17Ras coding sequence. Wild type MEK1, activated MEK1 (S218/222D), and
kinase-defective MEK1 (K97A) subcloned into pCMVHA have been described
previously (23). FLAG-tagged ERK2 was constructed by digesting murine
erk2 in pBluescript with HindIII and
XbaI and subcloning into the same sites in pcDNA3-FLAG. Extraneous pBluescript sequence between the HindIII site and
the erk2 start site was removed using the Transformer
mutagenesis kit (CLONTECH) and an oligonucleotide
spanning the FLAG coding sequence and the erk2 start
site.2 The GFP expression
plasmid, pEGFP-C1 (CLONTECH), was used as a
transfection control.
Transfection Assays--
For migration assays, cells
(0.5-1.0 × 106/100-mm dish) were transfected with 10 µg of DNA in a 4:1 ratio of expression plasmid to GFP (which
identifies positively transfected cells) using 60 µl of Superfect
(Qiagen). Between 24 and 48 h after the addition of DNA, the
transfection efficiency was determined by calculating the ratio of
GFP-positive cells to total cells (counted in four random
(magnification, ×320) fields using a Ziess Axiovert 135TV inverted
fluorescence microscope). Migration of transfected cells was assessed
using the Boyden chamber assay described below.
For activity assays, 0.5-1.0 × 106 cells, seeded on
100-mm dishes, were transfected with 9.9 µg of mutant MEK1 DNA and
0.1 µg of FLAG-ERK2 or 9.9 µg of mutant c-Raf DNA and 0.1 µg of
wild type MEK1. Twenty-four to 48 h later, the cells were
stimulated, harvested, and used for protein kinase assays described
previously (23).
Immunofluorescence--
Co-expression of individual constructs
together with GFP was confirmed by transfecting 2.5-5 × 104 cells plated on coverslips in 35-mm dishes with 2 µg
of total DNA in a 4:1 ratio of test construct to GFP using 10 µl of
Superfect (Qiagen). Twenty-four to 48 h later, the cells were
fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton
X-100. Cells transfected with individual test constructs and GFP were stained with monoclonal antibodies to the peptide tag expressed on each
ectopically expressed protein (12CA5 for HA-tagged Ras and MEK, M2
(Sigma) for FLAG-tagged c-Raf) at a final concentration of 1.5-5
µg/ml. Texas red-conjugated goat anti-mouse IgG was used as a
secondary antibody at a final concentration of 1.5-2 µg/ml. The
number of cells expressing both green (GFP) and red (test protein)
fluorescence was determined by counting four random 400× magnification
fields using a Leitz DMR fluorescence microscope. Co-expression
frequency of each test construct and GFP was always greater than
80%.
Cell Migration and Adhesion Assays--
Cell migration was
assessed using modified Boyden chambers (tissue culture-treated, 6.5 mm
diameter, 10 µm thickness, 8 µm pores; BioCoat, Becton Dickinson).
For growth factor-mediated chemotaxis, the indicated concentrations of
EGF or 6.7 ng/ml PDGF-BB were placed in the lower chamber in DMEM in
the absence of serum (DMEM/0). To assess fibronectin stimulated
haptotaxis, the underside of the membrane was coated with 1.5 µg/ml
fibronectin suspended in PBS in the lower chamber and PBS was placed in
the upper chamber. After incubating the chambers overnight at 4 °C,
the fibronectin solutions were removed and DMEM/0 was added to each
chamber. For adhesion assays, 2.5 × 104 cells were
plated on either uncoated 24-well plates or plates coated with 1.5 µg/ml fibronectin overnight at 4 °C and allowed to adhere for
6 h at 37 °C. The cells were washed twice in PBS, fixed with
4% paraformaldehyde at room temperature for 20 min, and washed twice
with PBS. For inhibitor studies, cells were pretreated with each
inhibitor for 15 min (16 h for herbimycin) prior to harvest. The
inhibitor remained in both chambers of each well for the duration of
the migration assay.
Migration assays were performed by suspending 1 × 105
REF52 cells in DMEM/0 in the upper chamber and allowing the cells to migrate for 6 h at 37 °C. Nonmigrating cells on the upper side of the membrane were removed with a cotton swab. Cells that had migrated to the underside of the membrane were washed twice in PBS,
fixed with 4% paraformaldehyde at room temperature for 20 min, and
washed twice with PBS. Migration or adhesion was assessed by counting
four 320× fields of cells using a Ziess Axiovert 135TV inverted
fluorescence microscope. Nontransfected cells were stained with crystal
violet prior to counting to aid in visualization. For experiments using
transfected cells, the number of fluorescent and total cells that
migrated (observed by phase microscopy without counter staining) were
determined and used to calculate the percentage of fluorescent cells
that migrated. This percentage was normalized to the transfection
efficiency for each construct to obtain the "relative migratory
index." The data presented represent the mean ± standard
deviation. Each experimental group was analyzed using single-factor
analysis of variance. If the global F test for differences among any one of the groups was significant at the 5% level, we proceeded to investigate which transfected group(s) differed from vector controls using Student's t tests assuming unequal
variance. Statistical significance was defined as p < 0.05.
EGF Stimulates Migration of Fibroblasts--
EGF has been reported
to stimulate migration of many normal and tumorigenic cells (1),
including NR6 fibroblasts engineered to express exogenous EGFR (2, 3).
To identify signals that regulate EGF-stimulated migration of the
normal rat embryo fibroblasts, REF52, we determined the optimal
concentration of EGF required to stimulate migration of these cells.
Cells were allowed to migrate for 6 h at 37 °C in response to
increasing concentrations of EGF placed in the lower well of a Boyden
chamber (Fig. 1). The maximal, 2-fold
increase in migration was induced with 3-10 ng/ml EGF (Fig. 1).
Higher, mitogenic concentrations of EGF (25 ng/ml; Fig. 1) failed to
stimulate migration significantly above background levels, consistent
with reports of PDGF-stimulated migration (24).
EGF-stimulated NR6 migration requires EGFR tyrosine phosphorylation and
kinase activity (2) as well as PLC Overexpression of Dominant Acting Ras Affects EGF-stimulated
Migration--
Because EGF-stimulated migration requires receptor
activation and tyrosine phosphorylation (Ref. 2 and Table I) as well as
receptor-mediated PLC Differential Effects of MEK1 Inhibitors on EGF-stimulated
Migration--
To identify Ras effectors that may mediate chemotaxis
in response to EGF, we examined molecular components of the
Ras-Raf-MEK-MAP kinase pathway. Initially, we tested the effects of
PD98059, an inhibitor of MEK activation (25), on EGF-stimulated
migration of REF52 cells. Cells pretreated for 15 min with 50 µM PD98059 or Me2SO were stimulated to
migrate to 10 ng/ml EGF in the continued presence or absence of
inhibitor. PD98059 decreased EGF-stimulated cell migration to levels
comparable with chemokinesis (Table I). U0126, an inhibitor of MEK
activation and MEK catalytic activity (26), inhibited EGF-stimulated
migration as well as chemokinesis (Table I; data not shown). These data
are consistent with a role for MEK in EGF-stimulated migration.
To further explore the requirement for MEK1 activation in migration,
expression constructs encoding kinase-defective MEK1, activated MEK1 or
the empty vector control (pCHA) were co-transfected into REF52 cells
with GFP-expressing plasmids and EGF-stimulated migration of
transfected cells was assessed (Fig. 3).
Surprisingly, kinase-defective MEK1 expression enhanced EGF-stimulated
migration 2-fold while having no effect on chemokinesis (Fig. 3,
A and B). In contrast, activated MEK1 had no
significant effect on chemokinesis or chemotaxis (Fig. 3, A
and B), and neither of the mutant MEK1 constructs affected
adhesion to fibronectin (Fig. 3C). The enzymatic activities
of kinase-defective and activated MEK1 constructs were confirmed by
measuring the activity of co-expressed FLAG-ERK2 (Fig. 3D).
As anticipated, kinase-defective MEK1 inhibited EGF-stimulated ERK2
activity approximately 50% while activated MEK1 increased ERK2
activity approximately 4-5-fold in unstimulated cells. These data
indicate that MEK-dependent activation of MAP kinase is not sufficient for EGF-stimulated migration of REF52 cells. Since kinase-defective MEK titrates c-Raf away from endogenous substrates, the paradoxical effects of the pharmacological MEK inhibitors and
kinase-defective MEK on cell migration indicated the possible involvement of c-Raf in both the regulation of MEK/MAP kinase activation and cell migration.
c-Raf Expression Inhibits EGF-stimulated Migration--
Recently,
it has been shown that PD98059, in addition to blocking ERK activity,
increases c-Raf activity in Swiss 3T3 cells treated with a number of
stimuli (27). Thus, we tested the effects of PD98059 and a second MEK
inhibitor, U0126, on c-Raf activity in REF52 cells. Cells were
pretreated with 50 µM PD98059, 50 µM U0126,
or Me2SO for 15 min and stimulated with 10 ng/ml EGF or vehicle for 1 h in the continued presence or absence of either MEK
inhibitor. To quantitate c-Raf activity, immunoprecipitated, endogenous
c-Raf was tested for its ability to phosphorylate kinase-defective MEK
in vitro (Fig. 4). PD98059 had
no effect on basal c-Raf activity in unstimulated cells; U0126
marginally increased basal c-Raf activity (Fig. 4A).
However, PD98059 or U0126 treatment enhanced EGF-stimulated c-Raf
activity approximately 2-3- or 5-6-fold, respectively, relative to
the level observed from EGF treatment alone (Fig. 4A).
EGF-stimulated MAP kinase phosphorylation was decreased by PD98059
treatment and completely blocked by U0126 treatment (Fig.
4B). Basal MAP kinase phosphorylation was blocked by both
PD98059 and U0126, as evidenced by the failure to detect binding of a
phospho-ERK antibody and as the lack of mobility shift of ERK2 on ERK2
immunoblots (Fig. 4B). Thus, these results indicate that
PD98059 or U0126 inhibition of MEK can lead to an increase in c-Raf
activity presumably by blocking MAP kinase-dependent negative feedback inhibition of c-Raf (28-30).
To determine if increased c-Raf activity was sufficient to inhibit
EGF-induced cell migration, expression constructs encoding wild type
c-Raf, the catalytic domain of c-Raf (22W), or the empty vector control
(pC) were transfected together with GFP-expressing plasmids into REF52
cells and the effects on cell motility were assessed (Fig.
5). Expression of individual c-Raf
constructs had no significant effect on chemokinesis (Fig.
5A). However, both wild type c-Raf and 22W inhibited
EGF-stimulated migration approximately 30-50% (Fig. 5B).
The ability of 22W or wild type c-Raf to inhibit migration was not due
to an effect on adhesion since cells expressing these constructs
adhered to fibronectin as well as cells expressing the negative vector
controls (Fig. 5C).
The activity of each c-Raf construct was assessed by co-transfecting
empty vector, wild type c-Raf, or truncated c-Raf together with
HA-tagged wild type MEK1 into REF52 cells. Immunoprecipitated MEK1
activity was assayed by virtue of its ability to phosphorylate ERK2
in vitro (Fig. 5D). As shown in Fig.
5D, wild type c-Raf and 22W increased c-Raf activity
approximately 4-6-fold in unstimulated cells, relative to the empty
vector control. EGF treatment increases c-Raf activity approximately
7-10-fold in cells expressing wild type c-Raf or 22W compared with
EGF-stimulated cells expressing the empty vector control (Fig.
5D). Taken together, these data indicate that increased
c-Raf activity correlates with the inhibition of EGF-stimulated
migration. These results also suggest the possibility that treatment of
cells with PD98059 or U0126 may indirectly lead to the activation of
c-Raf and inhibition of migration through an alternative
c-Raf-dependent pathway.
In this report, we provide evidence that EGF-stimulated migration
of REF52 cells is differentially regulated by Ras and its downstream
effector, c-Raf. Blocking Ras activity by transient overexpression of
dominant negative Ras constructs (A17Ras or N17Ras) in a population of
REF52 cells substantially inhibits chemokinesis and EGF-stimulated
chemotaxis while failing to significantly inhibit haptotactic migration
to fibronectin. However, transient expression of activated variants of
Ha-Ras (V12Ras or L61Ras) fail to stimulate chemokinesis, chemotaxis,
or haptotaxis. These results confirm previously reported
studies carried out in other cell types, showing that Ras is necessary
but not sufficient to promote cell migration to many growth factors
(12-14). We also present the first evidence for c-Raf being a
regulator of the cell migration signaling pathway. Treatment of REF52
cells with the MEK inhibitor PD98059 or U0126 inhibited migration of
REF52 cells toward EGF. Treatment of REF52 cells with PD98059 or U0126 also resulted in a detectable enhancement of c-Raf kinase activity in
response to EGF. Expression of wild type c-Raf or activated c-Raf,
however, significantly inhibited EGF-induced REF52 cell migration.
These studies provide evidence that the activation state of c-Raf may
be an important sensor in regulating the cell migration signaling pathway.
Several downstream effectors of Ras, including MAP kinase, have been
suggested as potential regulators of cell motility including chemotaxis
and haptotaxis (3, 14, 17, 18). Inhibition of MAP kinase activation
with the MEK-specific inhibitor, PD98059 (25, 27), is reported to
significantly reduce fibronectin-stimulated migration of Rat1 cells
(18), collagen-stimulated migration of FG carcinoma cells (17), and
EGF-stimulated migration of NR6 or human diploid fibroblast cell lines
(19). Indeed, we find that both PD98059 and the MEK inhibitor, U0126,
inhibit EGF-stimulated migration of REF52 cells (Table I), consistent
with MAP kinase playing a role in the regulation of EGF-stimulated
migration. Interestingly, U0126 appears to be a more effective
inhibitor of cell migration (Table I), inhibiting both EGF-induced
migration and chemokinesis. This may reflect the fact that U0126 is a
significantly more potent inhibitor of MAP kinase (Ref. 26 and Fig.
4B) and would suggest that even chemokinesis requires some
basal level of MAP kinase activity. Alternatively, U0126 may be acting
on another pathway important for chemokinesis. Treatment of cells with
PD98059 or U0126 also led to an elevation of c-Raf activity upon EGF
stimulation. The stimulation of c-Raf activity in REF 52 cells is
similar to previously reported results of c-Raf activation by PD98059
in Swiss 3T3 cells treated with a number of growth factors and hormones
(27). Our observations with the U0126 inhibitor provide additional
evidence for the role for MAP kinase or other downstream kinases in the
feedback regulation of c-Raf.
Initially, we observed that overexpression of kinase-defective MEK1,
which inhibits MAP kinase activation, enhanced EGF-stimulated migration
of REF52 cells (Fig. 3). These data, coupled with the observation that
activated MEK constructs, which effectively activated MAP kinase,
failed to enhance chemokinesis or EGF-induced migration, suggest that
state of c-Raf activation may be important in regulating the migration
response to EGF. Indeed, overexpression of wild type c-Raf or the
catalytic domain of c-Raf resulted in the inhibition of EGF-stimulated
cell migration without effecting chemokinesis or the adhesion to
fibronectin. In the absence of EGF, cells expressing the activated
c-Raf constructs exhibited MAP kinase levels comparable to
EGF-stimulated control cells (Fig. 5). Therefore, elevation of MAP
kinase activity alone is insufficient to stimulate cell migration in
the absence of EGF. In addition, high constitutive levels of MAP kinase
activity achieved upon stimulation of c-Raf-expressing cells with EGF
did not result in increased cell migration. Thus, the activation of MAP
kinase alone is insufficient to drive cell migration, leading us to
suggest that the activation state of c-Raf could be an important
regulator of the cellular migratory response.
The mechanism(s) by which c-Raf might negatively regulate cell
migration is presently unknown. The ability of c-Raf overexpression to
inhibit EGF-stimulated migration suggests that c-Raf may down-regulate EGF-dependent signals that promote migration, including
signals to or from Ras and PLC An alternative possibility is that c-Raf targets cellular components of
the migration machinery, perhaps components of the adhesion-signaling
pathway, in response to EGF-stimulated signaling events. Expression of
an activated form of c-Raf, Raf-BXB, has been reported to suppress
integrin activation, suggesting that c-Raf may inhibit engagement of
activated integrins with ECM proteins (40). In addition, cells
overexpressing active c-Raf appeared to spread more poorly compared
with controls (40). The c-Raf-dependent inhibition of
integrin binding of ECM proteins may well correlate with the inability
of the cell to reorganize existing focal adhesions, resulting in an
inhibition of cell migration. Indeed, EGF-stimulated migration of NR6
cells overexpressing the EGFR has been correlated with the loss of
focal adhesions following EGF treatment (19). Treatment of these cells
with PD98059 both inhibits migration and reduces the extent of focal
adhesion loss in response to EGF, leading the authors to suggest that
MAP kinase regulates EGF-initiated migration by promoting focal
adhesion disassembly and remodeling. However, since PD98059 also
enhances growth factor stimulated c-Raf activity (27, Fig. 4), PD98059
could inhibit focal adhesion disassembly by enhancing c-Raf activity.
While we found no alteration in the ability of cells overexpressing
c-Raf to adhere to fibronectin (Fig. 5), more subtle changes in
adhesion complexes or adhesion-based signaling may be compromised by
c-Raf overexpression.
We are grateful to Drs. R. Mattingly, I. Macara, M. Wigler, and R. Jove for plasmid constructs used in this
study and Eric Bissonette and Dr. Gina Petroni of the University of
Virginia Cancer Center Biostatistics Core for assistance with the
statistical analysis. We thank Drs. J. Taylor, K. Martin, and W.-C.
Xiong for helpful suggestions.
*
This work was supported in part by National Institutes of
Health Grants CA29243 and CA40042 (to J. T. P.) and National
Institutes of Health Grants CA39076 and GM47332 (to M. J. W.).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.
§
Supported by National Research Service Award 5F32 GM18672-02.
¶
To whom correspondence should be addressed: Dept. of
Microbiology, Box 441, Health Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5395; Fax: 804-982-1071; E-mail: jtp@virginia.edu.
2
S. Eblen, unpublished data.
The abbreviations used are:
EGF, epidermal
growth factor;
ECM, extracellular matrix;
EGFR, EGF receptor;
PLC
c-Raf-mediated Inhibition of Epidermal Growth
Factor-stimulated Cell Migration*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Grb2, and Shc
in vivo (5-8). Cells treated with the pharmacological PLC
inhibitor, U73122, or expressing a mutant EGFR incapable of activating
PLC fail to migrate to EGF indicating that PLC activity is
necessary for migration in response to EGF (3).
Raf MEK ERK1/ERK2 (MAP kinase) signal transduction pathway (4).
Several groups have investigated the role of this pathway in cell
motility. In particular, Ras activity appears to be indispensable for
cell motility in a number of systems (9-16). Indeed, overexpression of
dominant interfering Ras proteins inhibits chemokinesis,
PDGF-stimulated migration (13), and wound repair (15). However,
expression of dominant negative Ras is reported to have no effect on
haptotactic migration toward fibronectin (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Aliquots were dried under a
nitrogen stream, resuspended in DMEM containing 1% fatty acid-free
BSA, and used immediately at a final concentration of 1 µM. Wortmannin (Sigma), herbimycin (Calbiochem), and
genistein (Life Technologies, Inc.) were reconstituted in
Me2SO and used at final concentrations of 100 nM, 875 nM, and 100 µg/ml, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Migration of REF52 cells to EGF. REF52
cells were stimulated to migrate to 1, 3, 10, and 25 ng/ml EGF. The
data represent the mean relative migration ± standard deviation
for three independent experiments.
activity (3). We assessed the
requirement for these activities, as well as PI3K activity, in
EGF-stimulated migration of REF52 using pharmacological inhibitors.
Treatment of REF52 cells with the tyrosine kinase inhibitors genistein
or herbimycin completely inhibited chemokinesis and EGF-mediated
chemotaxis (data not shown), thereby supporting the previous report of
a requirement for tyrosine phosphorylation (2). Treatment of REF52
cells with 1 µM U73122, a PLC inhibitor, blocked
EGF-stimulated migration (Table I) while
having no effect on chemokinesis (data not shown). The inactive
congener U73433, had no effect on cell migration. Finally, cells
treated with PI3K inhibitors wortmannin or LY294002, failed to inhibit
EGF-stimulated migration (Table I) at concentrations that blocked
PDGF-stimulated migration (data not shown). These experiments support
the previously reported requirement for PLC activation in
EGF-stimulated cell migration and interestingly fail to provide
evidence for a significant role of PI3K.
EGF-mediated chemotaxis of REF52 cells in the presence of metabolic
inhibitors
activation (Ref. 3 and Table I), we sought to
determine which components of the EGF receptor signaling pathway
regulated migration. Previous reports have implicated Ras as a
regulator of cell migration (9-16). Thus, we employed a transient
transfection migration assay (see "Experimental Procedures") to
assess the effects of mutant Ras expression on EGF-stimulated migration. REF52 cells were transfected with mutant Ras constructs; the
transfected cells were harvested 24-48 h later, and cell migration to
10 ng/ml EGF was measured as described under "Experimental Procedures." Both EGF-mediated chemotaxis and chemokinesis of REF52
cells expressing dominant negative Ras (A17Ras or N17Ras) were reduced
compared with cells expressing an empty vector control (pKH; Fig.
2, A and B). In
contrast, expression of constitutively active Ras had no effect on
chemokinesis or EGF-stimulated motility (Fig. 2, A and
B). Overexpression of A17Ras or N17Ras had no significant effect on fibronectin-stimulated migration (Fig. 2C),
indicating that dominant negative Ras likely blocks migration by
affecting EGFR-regulated signaling rather than direct inhibition of
components of the migration machinery. Dominant negative Ras expression
also had no effect on cell adhesion (Fig. 2D). Western
analysis indicated that Ras expression in REF52 cells was equivalent
among the mutant Ras constructs (data not shown). In parallel
experiments, expression of N17Ras and V12Ras in REF52 cells effectively
blocked PDGF-stimulated migration in agreement with previous reports
(data not shown; Ref. 13). Together, these data show that a
Ras-dependent signal is required for EGF-mediated
migration.
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Fig. 2.
EGF-stimulated migration requires functional
Ras. REF52 cells were transiently transfected with activated Ras
(L61Ras and V12Ras) or dominant negative Ras (A17Ras or N17Ras)
expression constructs or empty vector negative controls (pKH for
A17Ras, N17Ras, and L61Ras; pDCR for V12Ras) together with
GFP-expressing plasmids. Transfected cells were stimulated to migrate
toward vehicle (A), 10 ng/ml EGF (B), or 1.5 µg/ml fibronectin (C) or allowed to adhere to 1.5 µg/ml
fibronectin (D). The relative migratory index or relative
adhesion (see "Experimental Procedures") was determined 6 h
later. The data represent the mean ± standard deviation for five
to nine independent experiments. *, p values < 0.05 determined as described under "Experimental Procedures." EGF and
fibronectin stimulated 2.0 ± 0.9- and 2.5 ± 0.5-fold
increases in migration relative to basal chemokinesis,
respectively.
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Fig. 3.
Kinase-defective MEK1 expression enhances
EGF-stimulated migration. REF52 cells, transiently transfected
with kinase-defective MEK1 (KD MEK1), activated MEK1
(Act MEK1) expression constructs, or pCHA, the empty vector
control, were stimulated to migrate to vehicle (A) or 10 ng/ml EGF (B), or allowed to adhere to 1.5 µg/ml
fibronectin (C). The data represent the mean relative
migratory index or mean relative adhesion (see "Experimental
Procedures") ± standard deviation for six to nine independent
experiments. *, p values < 0.05 determined as
described under "Experimental Procedures." EGF stimulated 3.0 ± 1.2-fold increase in migration above mock-stimulated migration.
D, the kinase activities of each construct were assessed
in vitro. REF52 cells were co-transfected with FLAG-tagged
wild type ERK2 and HA-tagged kinase-defective MEK1, activated MEK1, or
pCHA. Following a 10-min mock or EGF (10 ng/ml) stimulation,
ERK2-mediated, in vitro phosphorylation of myelin basic
protein (MBP) was assessed. The numbers under
each pair of lanes indicate the -fold increase in ERK2 activity
relative to unstimulated pCHA. Levels of transfected mutant MEK1
expression were determined by Western analysis of whole cell lysates
using a HA-specific antibody (MEK1:IB). Relative
ERK2 levels in each immunoprecipitate were determined by blotting with
a FLAG-specific monoclonal antibody
(ERK2:IB).
View larger version (36K):
[in a new window]
Fig. 4.
PD98059 or U0126 stimulates c-Raf
activity. A, endogenous c-Raf was immunoprecipitated
from REF52 cells following vehicle or 10 ng/ml EGF stimulation for
1 h with or without PD98059 or U0126 treatment. c-Raf activity was
determined by measuring the relative level of MEK1 phosphorylation
following incubation of immunoprecipitated c-Raf with kinase-defective
MEK1 substrate (KD MEK1) in vitro as
described under "Experimental Procedures." Levels of c-Raf in each
immunoprecipitate were determined by Western analysis using a
c-Raf-specific monoclonal antibody. B, ERK phosphorylation,
detected by Western analysis of whole cell lysates using a
phosphospecific ERK monoclonal antibody (phospho-ERK), was
used as a measure of ERK activation. Serum-depleted REF52 cells were
pretreated with 50 µM PD98059 or 50 µM
U0126 for 15 min, followed by stimulation with vehicle or 10 ng/ml EGF
for 1 h in the continuous presence of 50 µM PD98059
or 50 µM U0126. Western analysis of ERK2
(ERK2:IB) using monoclonal antibody B3B9
confirmed equivalent levels of ERK in each lane. The data shown are
representative of at least three independent experiments.
View larger version (19K):
[in a new window]
Fig. 5.
c-Raf expression inhibits EGF-stimulated
migration. REF52 cells expressing pC, empty vector control, wild
type c-Raf (Wt c-Raf), or the catalytic domain of c-Raf
(22W) were stimulated to migrate to vehicle (A)
or 10 ng/ml EGF (B) or allowed to adhere to 1.5 µg/ml
fibronectin (C). The data represent the mean relative
migratory index or mean relative adhesion (see "Experimental
Procedures") ± standard deviation for seven to nine independent
experiments. *, p values < 0.05 determined as
described under "Experimental Procedures." EGF stimulated 3.5 ± 1.5-fold increase in migration. D, the activity of each
c-Raf construct was determined by its ability to activate co-expressed
epitope-tagged MEK1. Immunoprecipitated MEK1 activity was measured as
the ability to phosphorylate ERK2 in vitro
(ERK:P). The -fold increase in MEK1 activity of each
construct ± EGF stimulation relative to unstimulated pC is
indicated. Relative levels of immunoprecipitated MEK1 were detected
using an HA-specific antibody (MEK:IB).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Indeed, negative feedback mechanisms have been described that lead to Ras desensitization in response to
growth factor stimulation (32-39). In some cases, uncoupling Ras
activation from growth factor receptor signaling involves MEK- or MAP
kinase-dependent phosphorylation of SOS, and correlates with the dissociation of SOS from the growth factor receptor complex either by release from Grb2 (32-35) or from Shc as a Grb2/SOS complex (36-38). While c-Raf overexpression results in increased MEK activity (Fig. 5), it is unlikely that MEK-dependent desensitization
of Ras, mediated by SOS phosphorylation, regulates EGF-stimulated migration since active MEK overexpression has no effect on
EGF-stimulated migration (Fig. 3). In addition, hormone-induced
activation of 
Raf1:ER (an estrogen-regulated version of activated
c-Raf) has been reported to increase MEK, MAP kinase activity and SOS
phosphorylation in NIH3T3 cells, yet cells containing activated c-Raf
retain the ability of EGF to stimulate Ras activity (39). However,
prolonged increases in c-Raf activity did result in decreased EGFR
tyrosine phosphorylation (39), suggesting that proteins that interact directly with the EGFR, including PLC, may not be activated to the
same level in cells overexpressing active c-Raf. Therefore, increased
c-Raf activity may indirectly alter PLC-mediated migratory events
following EGF stimulation.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by Cancer Center Training Grant NCI T32 CA09109.
ABBREVIATIONS
, phospholipase C;
MAP kinase, mitogen-activated protein kinase;
MEK, MAP kinase kinase;
ERK, extracellular signal-regulated kinase;
PDGF, platelet-derived growth factor;
REF, rat embryo fibroblasts;
BSA, bovine serum albumin;
PBS, phosphate-buffered serum;
PI3K, phosphoinositide 3-kinase;
GFP, green fluorescent protein;
DMEM, Dulbecco's modified Eagle's medium;
HA, hemagglutinin.
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RESULTS
DISCUSSION
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