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INTRODUCTION |
Cells use a limited number of intracellular signaling pathways for
a variety of cellular responses. One of these pathways is the classical
mitogen-activated protein kinase
(MAPK)1 cascade that is
involved in the regulation of cell proliferation, cell differentiation,
and embryonic early development. In response to extracellular stimuli
MAP kinase kinase (MAPKK, also known as MEK), a direct activator for
MAPK/extracellular signal-regulated kinase, becomes activated and then
activates MAPK in the cytoplasm (1-3). The activated MAPK is
translocated into the nucleus (4-6) where it phosphorylates several
nuclear targets such as Elk-1 (7, 8). This signaling event is thought
to stimulate the induction of an immediate early gene fos
(9), which may, with some other transcription factors, activate many
late genes required for the S phase entry of quiescent fibroblastic
cells. Several experiments revealed a requirement of MAPK for
serum-induced cell growth (10, 11). Thus, the MAPK cascade is important
for the initiation of cell proliferation.
Ras is an upstream regulator of the MAPK cascade. Oncogenic Ras induces
malignant transformation of fibroblastic cells, and as earlier cell
responses, it causes both the S phase entry and the morphological
changes (12, 13). A dominant-negative form of MAPKK blocks oncogenic
Ras-induced cell transformation (14), and overexpression of MAPK
phosphatase, MKP1/CL100, inhibits Ras-induced S phase entry of
quiescent cells (15). These results suggest that the activation of the
MAPK cascade is essential for the oncogenic Ras-initiated cell
proliferation and transformation. Thus, the classical MAPK cascade has
been thought to function primarily as a mediator of mitogenic signals
through gene expression. On the other hand, little has been known about
its role in the morphological changes induced by Ras.
Constitutively active mutants of MAPKK are successfully generated by
substituting acidic amino acids for two serine residues in the
activation phosphorylation sites (16-20). These mutants are shown to
be able to induce malignant cell transformation of fibroblasts (14,
20), differentiation of PC12 cells (14), oocyte maturation (21, 22),
and mesoderm induction in Xenopus (23-25). However, there
have been no reports demonstrating the ability of constitutively active
MAPKK to induce the S phase entry of quiescent cells or early
morphological changes.
We previously showed that a leucine-rich nuclear export signal (NES)
(residues 33-44) in the N-terminal region of MAPKK directs cytoplasmic
localization of MAPKK (26). Disruption of the NES by substituting
alanines for essential leucines dramatically enhanced the ability of
constitutively active MAPKK to induce malignant cell transformation
(27), suggesting a potential role for the NES of MAPKK to suppress
abnormal cellular responses. Moreover, it has been shown that MAPK
binds to the N-terminal region of MAPKK (28). Through this binding,
MAPK appears to be retained to the cytoplasm (28). Extracellular
stimuli may induce dissociation of this MAPKK·MAPK complex, and MAPK
is then translocated to the nucleus (28). Thus, MAPKK may function as a
cytoplasmic anchor for MAPK.
In this study, we show evidence that the MAPK cascade is involved in
the control of cell morphology and is sufficient for initiation of cell
proliferation. We then demonstrate dominant-negative effects of the
N-terminal region of MAPKK on the MAPK cascade signaling.
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EXPERIMENTAL PROCEDURES |
DNA Constructions
pSR
HA-MAPKK and pSR
HA-LA-SDSE MAPKK
were produced as described (27). pSR
HA-SASA MAPKK was generated from
an EcoRI fragment of Xenopus MAPKK cDNA (29)
subcloned into M13mp18 by the methods of Kunkel et
al. (30) using a mutagenic primer
5'-GGGCAACTCATAGACGCCATGGCAAATGCCTTTGTTGGGACAAGATCC-3' as described
(27). The open reading frame of SASA MAPKK was amplified by polymerase
chain reaction with a 5' primer, 5'-ACTCAGATCTAACATGCCTAAAAAGAAG-3', and a 3' primer, 5'-GCCAAGATCTCTCACACTCCGGCGGCAT-3', which produce BglII sites at both ends of SASA MAPKK. The BglII
fragment of SASA MAPKK was cloned into pSR
HA1, yielding
pSR
HA-SASA MAPKK. An N-terminal region (residues 1-60) of wild-type
MAPKK and that of LA MAPKK (26) were amplified by polymerase chain
reaction with a 5' primer, 5'-CCGGGGATCCATGCCTAAAAAGAAGCCTAC-3',
and a 3' primer, 5'-GGCCGAATTCAACTTTCTGCTTCTGGGTGA-3', which
produce a BamHI site at the 5'-terminal and an
EcoRI site at the 3'-terminal. Each
BamHI-EcoRI fragment was cloned into
pSR
HA1, yielding pSR
HA-MAPKK-(1-60) and
pSR
HA-LA-MAPKK-(1-60), respectively. pSR
-RasV12 was
kindly given by Dr. S. Hattori. The BglII fragment of
LA-SDSE MAPKK was cloned into pET-16b (Novagen) to produce
pET16b-LA-SDSE MAPKK (His-LA-SDSE MAPKK). The open reading frame of
Xenopus MAPK was cloned into pcDL-SR
457 to obtain
pSR
-MAPK.
Preparation of Recombinant Proteins--
Histidine-tagged
Xenopus MAPKK (His-MAPKK) and histidine-tagged
Xenopus LA-SDSE MAPKK (His-LA-SDSE MAPKK) were expressed in Escherichia coli strain BL21 (DE3) pLysS pT-Trx by
incubating with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside for 5 h at 25 °C and purified as described (17). His-MAPKK (2 mg/ml) and His-LA-SDSE MAPKK (2 mg/ml) were dialyzed against the injection buffer
(20 mM K-Hepes, pH 7.4, 120 mM KCl) before injection.
Cells--
Swiss 3T3 cells and MDCK cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% FCS and
antibiotics (100 units/ml penicillin and 0.2 mg/ml kanamycin). For
microinjection, cells were plated on glass coverslips or CELLocate
coverslips (Eppendorf, Inc.), and grown as above to subconfluence. For
Fos induction and BrdUrd incorporation assays, Swiss 3T3 cells were plated as above, grown to confluence, and then left overnight in
serum-free medium to ensure that all the cells had entered the
quiescent state. PD098059 (final 50 µM, from New England
Biolabs), when used, was added to the culture media 30 min prior to injection.
Microinjection--
Microinjection was performed with an IM-188
apparatus (Narishige) as described previously (26). An anti-Ras mAb
Y13-259 (10 mg/ml) and control rat IgG (10 mg/ml) were dialyzed against the injection buffer (see "Preparation of Recombinant Proteins") before injection. For BrdUrd incorporation assay, BrdUrd (0.5 mg/ml)
and insulin (1 µg/ml) were added to the culture media 1 h after
injection, and then cells were cultured for 30 h.
Cell Staining--
Cells were fixed and permeabilized as
described previously (26). To detect BrdUrd-positive cells, cells were
incubated for 40 min at 37 °C with anti-BrdUrd mAb (5 µg/ml,
Becton Dickinson), DNase I (0.5 mg/ml), and 0.1% bovine serum albumin
followed by an RITC-conjugated goat anti-mouse antibody (Cappel) and
DAPI (0.1 mg/ml). Hemagglutinin (HA)-tagged MAPKK proteins were
detected with anti-HA mAb (12CA5). Focal adhesions were visualized with anti-vinculin mAb (VIN-11-5). Anti-Ras mAb (Y13-259) was a kind gift
from Dr. S. Hattori. Injected Ras antibody and control rat IgG were
detected with RITC-conjugated anti-rat antibody (Cappel). Anti-Fos
antibody was purchased from Upstate Biotechnology Inc. An
anti-Xenopus MAPK antibody was produced in rabbit by using bacterially expressed recombinant His-tagged MAPK as an antigen. Fluorescence and phase contrast images were observed in an Axiophot (Zeiss) with a 63× plan-Neofl. (1.25 NA) objective.
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RESULTS |
Activation of MAPKK Induces Early Morphological Changes Independent
of Cellular Ras Function--
Although the cell transformation by
constitutively active MAPKK is often accompanied by morphological
changes of the cell, the effect of the MAPKK activation on early (
24
h) morphological changes has been poorly understood. To examine the
effect of activation of MAPKK on cell morphology in fibroblasts, we
injected the nuclei of subconfluent Swiss 3T3 cells with a mammalian
expression vector encoding an NES-disrupted, constitutively active
MAPKK, LA-SDSE MAPKK in which leucines in NES are replaced by alanines
and two serines in the activation phosphorylation site by aspartic acid and glutamic acid, respectively (27). In agreement with our previous
report (27), morphological changes with several protrusions and
processes (Fig. 1A,
LA-SDSE, MAPKK) and disruption of stress fibers
(data not shown) were induced in cells expressing LA-SDSE MAPKK within
18 h. Signs of the morphological changes were first seen at ~8 h
after injection. Rounding or spindle-shaped morphologies with
refractile appearances were seen under phase contrast microscope (Fig.
1A, LA-SDSE, phase). We observed
disruption of focal adhesions by LA-SDSE expression, as determined by
vinculin staining (Fig. 1A, LA-SDSE,
vinculin). Normal, flattened cell morphologies with normal
focal adhesions were seen in cells expressing wild-type (WT) MAPKK
(Fig. 1A, WT). We then expressed active MAPKK in
an epithelial cell line MDCK which is polarized and has well developed cell-cell contacts including tight junctions. An expression plasmid encoding WT MAPKK or LA-SDSE MAPKK was injected into the nuclei of a
few neighboring cells in a single colony consisting of about 100 cells.
Although no apparent changes in cell morphology were induced by
expression of WT MAPKK, a normal cobblestone-like cell shape was broken
in cells expressing LA-SDSE MAPKK; the cells became rounded and shrunk
(Fig. 1B). Phase contrast images revealed the appearance of
thin gaps between cells expressing active MAPKK (Fig. 1B,
LA-SDSE, phase), suggesting a reduced cell-cell
attachment among neighboring cells. In addition, accumulation of actin
filaments in the cell periphery was observed in LA-SDSE
MAPKK-expressing cells (data not shown). Thus, expression of
constitutively active MAPKK induces similar morphological changes in
both fibroblastic and epithelial cells.

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Fig. 1.
Morphological changes of fibroblastic and
epithelial cells induced by LA-SDSE MAPKK. A,
morphological changes and disruption of focal adhesions in Swiss 3T3
cells expressing LA-SDSE MAPKK. Swiss 3T3 cells were grown on the
coverslips. pSR HA-MAPKK (WT, 350 µg/ml) or
pSR HA-LA-SDSE MAPKK (LA-SDSE, 350 µg/ml) was injected
into the nuclei of cells. After 18 h, the cells were stained with
antibody to HA (12CA5) or antibody to vinculin, followed by RITC- or
fluorescein isothiocyanate-conjugated secondary antibodies. The
rhodamine and phase contrast images of one representative field for
each sample are shown (upper four panels). Vinculin-staining
images of the plasmid-injected cells (lower two panels) were
3 times as magnified as upper four panels. B,
effect of LA-SDSE MAPKK expression on MDCK cells. pSR HA-MAPKK
(WT, 350 µg/ml) or pSR HA-LA-SDSE MAPKK
(LA-SDSE, 350 µg/ml) was injected into the nuclei of MDCK
cells in a single colony. After 18 h, cells were stained with
12CA5 followed by an fluorescein isothiocyanate-conjugated secondary
antibody. Fluorescein and phase images of each representative field are
shown. Cytoplasmic localization of WT MAPKK is not so clear in MDCK
cells in this image probably because nuclei are in lower part of cells.
Staining intensity for LA-SDSE MAPKK is a little bit stronger than that
for WT MAPKK because of the more rounded cell morphologies
(LA-SDSE, MAPKK). Arrowheads indicate
gaps between cells (LA-SDSE, phase).
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The altered morphology of constitutively active MAPKK-expressing cells
might not be a direct consequence of the activation of the MAPK cascade
but rather result from some autocrine mechanism that leads to the
activation of Ras which might induce morphological responses
independent of the MAPK cascade (14). To address this, we investigated
the effect of a neutralizing antibody against Ras, Y13-259, on the
morphological changes induced by LA-SDSE MAPKK. The monoclonal antibody
Y13-259 has been shown to block cellular H-, K-, and N-Ras function
(31, 32), and in agreement with a previous report with NIH 3T3 cells
(31), injection of Y13-259 into the cytoplasm blocked serum-induced S
phase entry of quiescent Swiss 3T3 cells (Fig.
2A, Y13-259).
Blocking efficiency was quite high when the entry of S phase was
determined by incorporation of bromodeoxyuridine (BrdUrd) into the
nucleus; only 6 out of 100 injected cells incorporated BrdUrd in
the first experiment and 8 out of 100 in the second experiment. Control
rat IgG did not block BrdUrd incorporation at all (Fig. 2A,
cont. IgG). These results confirm that Y13-259 is a strong
and specific neutralizing antibody. Y13-259 was pre-injected into the
cytoplasm of Swiss 3T3 cells inside the marked area of coverslips, and
after 30 min the plasmid DNA encoding LA-SDSE MAPKK was injected into
the nucleus of these cells. As shown in Fig. 2B,
morphological changes, typical of those induced by LA-SDSE MAPKK,
occurred even in the Y13-259-injected cells. The cells became rounded
and shrunk, appeared refractile, and had some protrusions and
processes. The antibody-injected cells were indistinguishable from
uninjected cells. These results suggest that constitutively active
MAPKK-induced morphological changes do not require Ras function.
Consistent with this, expression of a dominant-negative form of Ras did
not inhibit LA-SDSE MAPKK-induced morphological changes (data not
shown). Therefore, it is suggested that morphological changes are
primarily downstream events from MAPKK, although some effect through
possible autocrine mechanism cannot be ruled out because cells
surrounding the active MAPKK-expressing cells sometimes showed slight
morphological changes.

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Fig. 2.
Effect of anti-Ras neutralizing antibody on
MAPKK-induced morphological changes. A, Y13-259 blocks
serum-induced S phase entry of quiescent Swiss 3T3 cells. A
neutralizing antibody against Ras (Y13-259) (10 mg/ml) or
control rat IgG (10 mg/ml) was injected into the cytoplasm of
serum-starved confluent Swiss 3T3 cells. 1 h after injection,
cells were stimulated with 20% FCS, cultured for 30 h in the
presence of BrdUrd, and then stained with antibody to BrdUrd followed
by an RITC-labeled secondary antibody. Injected IgG was visualized by
staining with an RITC-labeled goat anti-rat antibody.
Arrowheads indicate IgG-injected cells. B,
Y13-259 does not block MAPKK-induced morphological changes. Y13-259 (10 mg/ml) was preinjected into the cytoplasm of several cells inside the
marked area of coverslips. 30 min after injection, pSR HA-LA-SDSE
MAPKK (200 µg/ml) was injected into the nuclei of the cells in the
same area. After 18 h, cells were stained with antibody to HA tag
(MAPKK). Injected Ras antibody was visualized with an
RITC-labeled goat anti-rat antibody (Ras antibody). In phase
contrast images, black arrowheads indicate the cells
injected with both the antibody and the MAPKK plasmid, whereas
white arrowheads indicate the cells injected with only
MAPKK. Surrounding uninjected cells also showed slight signs of
morphological changes. At least 50 cells were injected with Y13-259,
and no cells showed signs of reverted morphologies. Experiments were
repeated 3 times and gave similar results.
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Then we tested whether oncogenic Ras-induced early morphological
changes are mediated through a function of MAPKK. When an expression
vector encoding RasV12 was injected into the nuclei of
Swiss 3T3 cells, the cells became rounded, shrunk, and refractile, and
numerous pinocytotic vacuoles appeared (33) (Fig.
3A,
). An inhibitor of MAPKK
activation, PD098059 (34), significantly inhibited these phenotypes
(Fig. 3A, PD098059), whereas Me2SO (a
solvent for PD098059) had no effect (Fig. 3A,
DMSO). To confirm further a requirement of MAPKK for induction of these phenotypes by Ras, WT MAPKK, or SASA MAPKK (with an
HA tag), a dominant-negative form of MAPKK in which two activating
serines are replaced by alanines (35), was co-expressed with oncogenic
Ras. Expression of SASA MAPKK, but not that of WT MAPKK, completely
blocked the RasV12-induced phenotypes (Fig. 3B).
The staining intensity for HA tag confirmed the same level of
expression of WT and SASA MAPKK. These results, together with the
observation that inhibition of Ras did not interfere with the ability
of active MAPKK to induce morphological changes (Fig. 2B),
suggest a linkage between the classical MAPK cascade and the control of
cell morphology.

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Fig. 3.
MAPKK function is required for oncogenic
Ras-induced early morphological changes. A, an
inhibitor of MAPKK activation inhibits oncogenic Ras-induced
morphological changes. PD098059 (final 50 µM) or control
Me2SO (DMSO) was pre-added to the culture media.
After 30 min incubation, pSR -RasV12 (200 µg/ml) was
injected. After 18 h, cells were stained for Ras expression with
antibody to Ras (Y13-259), and expression levels were judged to be the
same among each sample by staining intensity. Phase contrast images of
representative fields were shown. Arrowheads indicate the
injected cells. B, expression of a dominant-negative form of
MAPKK blocks oncogenic Ras-induced morphological changes. pSR HA-SASA
MAPKK (+ SASA KK) or pSR HA MAPKK (+ WT KK)
(300 µg/ml for each) was co-injected with pSR -RasV12
(200 µg/ml). After 18 h, cells were stained for Ras
(Ras) and MAPKK (MAPKK) expression, and phase
contrast images are also shown. At least 50 cells were injected, and
over 90% of the cells co-injected with SASA MAPKK showed normal,
flattened morphology. Cytoplasmic localization of WT MAPKK is often
unclear in cells with rounded morphologies (+ WT KK,
MAPKK). Experiments were repeated 3 times with similar
results.
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Activation of MAPKK Is Sufficient for Initiation of Cell
Proliferation--
Although previous studies suggested the importance
of the MAPK cascade in the initiation of cell proliferation, it has not been determined whether activation of the MAPK cascade alone is sufficient for induction of an immediate early gene fos and
the S phase entry of quiescent cells. To test this, we first injected the MAPKK plasmids into the nuclei of quiescent Swiss 3T3 cells, and
induction of endogenous Fos was examined. Expression of LA-SDSE MAPKK,
but not that of WT MAPKK, induced endogenous Fos, as revealed by the
appearance of bright nuclear staining with anti-Fos antibody (Fig.
4A). This level of Fos
induction was roughly the same as the level of Fos that was induced by
stimulation with 20% fetal calf serum, as judged by the staining
intensity (data not shown). Then, to examine the S phase entry, we
produced bacterially expressed WT and LA-SDSE MAPKK proteins, and we
injected each of them into the cytoplasm of all the quiescent Swiss 3T3
cells inside the specified circle on the marked coverslips. After
injection, cells were incubated for 30 h in the presence of
insulin (36) and BrdUrd. Whereas none of WT MAPKK-injected cells
incorporated BrdUrd, approximately 40% of LA-SDSE MAPKK-injected cells
incorporated BrdUrd (Fig. 4B, BrdUrd). Therefore,
activation of the MAPK cascade may be sufficient for the S phase entry
of quiescent cells, as well as the induction of Fos.

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Fig. 4.
Activation of MAPKK is sufficient for the S
phase entry of quiescent cells. A, LA-SDSE MAPKK
induces endogenous Fos expression in quiescent Swiss 3T3 cells.
pSR HA-LA-SDSE MAPKK (LA-SDSE) or pSR HA MAPKK
(WT) (350 µg/ml for each) was injected into the nuclei of
serum-starved confluent Swiss 3T3 cells. After 5 h, cells were
fixed and stained simultaneously with antibody to HA tag
(MAPKK) and antibody to Fos (Fos). Nuclei of all
cells were visualized with DAPI (DAPI). At least 40 cells
were injected, and Fos induction was observed in all the cells
expressing LA-SDSE MAPKK. Experiments were repeated twice with the same
results. B, injection of LA-SDSE MAPKK protein induces the S
phase entry of quiescent Swiss 3T3 cells. Bacterially expressed
His-LA-SDSE MAPKK (LA-SDSE) or His-MAPKK (WT) (2 mg/ml for each) was injected into the cytoplasm of serum-starved
confluent Swiss 3T3 cells. Then BrdUrd (0.5 mg/ml) and insulin (1 µg/ml) were added to the media and incubated for 30 h. All the
cells inside the specified circle of each marked coverslip were
injected. Cells were stained for BrdUrd incorporation into the nuclei
(BrdU). Cell nuclei were also visualized with DAPI. The
purity of each protein is more than 90%. The activity of recombinant
His-LA-SDSE MAPKK protein to phosphorylate kinase-negative
Xenopus MAPK was about 45 times as high as that of WT MAPKK
protein (data not shown). More than 10 circles were injected for one
experiment. Experiments were repeated 3 times with similar
results.
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Expression of the N-terminal Region of MAPKK Causes
Dominant-negative Effects--
In our previous report we proposed a
potential role of NES of MAPKK to prevent malignant cell transformation
(27). To test whether the NES-containing region has regulatory roles in
the serum-induced G0/S transition and the Ras-induced
morphological changes, we produced expression vectors encoding an
HA-tagged N-terminal fragment of WT or NES-disrupted (LA) MAPKK (1-60
amino acids) (Fig. 5A). This
region contains both the MAPK-binding site (residues 1-32) and the NES
sequence (residues 33-44) (28). We expressed each of these fragments
together with MAPK in Swiss 3T3 cells and determined their
intracellular localization. The WT fragment was localized in the
cytoplasm, whereas the LA fragment was present in both the nucleus and
the cytoplasm. MAPK was localized in the cytoplasm when co-expressed
with the WT fragment, whereas it was present in both the nucleus and
the cytoplasm when expressed with the LA fragment (Fig.
5B).

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Fig. 5.
Characterization of expression vectors
encoding the N-terminal region of MAPKK. A, schematic
representation of HA-tagged N-terminal fragments of MAPKK. 1-60-amino
acid region of Xenopus MAPKK containing the NES sequence
(NES, 33-44) and the MAPK binding site
(MAPK binding, 1-32) was cloned into pSR HA
(WT). Two critical leucines in the NES sequence,
Leu33 and Leu37 (LL) were replaced
by alanines (AA) to disrupt the NES activity in LA.
B, cytoplasmic retention of MAPK by expression of the N-terminal
fragment of MAPKK. pSR HA-MAPKK-(1-60) (WT) or
pSR HA-LA-MAPKK-(1-60) (LA) (350 µg/ml for each) was
injected together with pSR -MAPK (150 µg/ml) into the nuclei of
Swiss 3T3 cells. After 5 h, cells were fixed and stained
simultaneously with antibody to HA tag (MAPKK fragment) and
antibody to Xenopus MAPK (MAPK).
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Then we expressed these fragments in quiescent Swiss 3T3 cells, and we
examined the effect on the serum-induced S phase entry of cells.
Expression of an empty vector did not affect 20% fetal calf
serum-induced BrdUrd incorporation into the nucleus (Fig. 6A). Expression of the WT
fragment, however, blocked the serum-induced S phase entry, whereas
expression of the LA fragment did not significantly inhibit the BrdUrd
incorporation (Fig. 6A). Fig.
7B represents a quantification
of the result; 78% of vector-injected cells entered the S phase under
the conditions, whereas none of the WT fragment-expressing cells
entered the S phase, and 54% of the LA fragment-expressing cells
incorporated BrdUrd.

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Fig. 6.
Expression of the N-terminal fragment of
MAPKK blocks the serum-induced S phase entry of quiescent Swiss 3T3
cells. A, pSR HA-MAPKK-(1-60) (WT) or
pSR HA-LA-MAPKK-(1-60) (LA) (350 µg/ml for each) was
injected into the nuclei of serum-starved confluent Swiss 3T3 cells.
After 5 h, cells were stimulated with 20% FCS (serum
+) and incubated for 30 h in the presence of BrdUrd. Cells
were fixed and stained simultaneously with antibody to HA tag
(injected) and antibody to BrdUrd (BrdU). As a
control, pSR HA (vector, 350 µg/ml) was injected and
stained as well in the absence (serum ) or presence
(serum +) of serum stimulation. Arrowheads in the
panel BrdU indicate positions of nuclei of the
injected cells. B, quantification of the results in
A. Percentages of BrdUrd-positive cells in the injected
cells are shown. At least 100 cells were injected for each
sample.
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Fig. 7.
Expression of the N-terminal fragment of
MAPKK blocks oncogenic Ras-induced early morphological changes.
pSR -RasV12 (200 µg/ml) was co-injected with
pSR HA-MAPKK-(1-60) (+ WT) or pSR HA-LA-MAPKK-(1-60)
(+ LA) (300 µg/ml for each) into the nuclei of Swiss 3T3
cells. After 18 h, cells were fixed and stained as in Fig.
3B. At least 100 cells were injected, and a representative
field for each sample is shown. Experiments are repeated 3 times with
similar results.
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Finally, we examined the effect of these fragments on the oncogenic
Ras-induced morphological changes. Each of these fragments was
co-expressed with RasV12 in subconfluent Swiss 3T3 cells.
Expression of the WT fragment inhibited the Ras-induced morphological
changes; the cells were flattened and had few pinocytotic vacuoles and
few protrusions (Fig. 7, + WT). In contrast, expression of
the LA fragment did not inhibit the Ras-induced phenotypes; the cells
were refractile and had many pinocytotic vacuoles and protrusions (Fig.
7, + LA). The results depicted in Fig. 6 and Fig. 7 reveal
dominant-negative effects of the N-terminal region of MAPKK on the MAPK signaling.
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DISCUSSION |
Although it was previously shown that the MAPK pathway is involved
in a variety of cellular responses, the relationship between the MAPK
cascade and the cell morphology remained undefined. In addition, it was
also unclear whether the MAPK pathway was actually able to induce the S
phase entry of quiescent cells. By using a constitutively active mutant
of MAPKK (LA-SDSE MAPKK) whose kinase activity is the highest in the
recombinant MAPKK mutants but still lower than that of normally
activated, dually phosphorylated MAPKK (27), we have here shown a link
between the MAPK cascade and the control of cell morphology, and we
have also shown the ability of the MAPK cascade to induce the
expression of Fos and the S phase entry.
A number of experiments suggested that Ras induces morphological
changes and gene expression through different effector proteins (37,
38). The Raf/MAPK cascade, one of the downstream components of Ras, was
thought to function to transmit mitogenic signals to the nucleus to
induce gene expression and not to participate in the regulation of cell
morphology or actin reorganization (14, 38). The present results
demonstrate that active MAPKK is able to induce dramatic changes in
cell morphology which are accompanied by disruption of both actin
stress fibers and focal adhesions and that inhibition of Ras function
does not suppress the MAPKK-induced morphological changes. Moreover, we
observed that inhibition of MAPKK or expression of the N-terminal
portion of MAPKK suppresses oncogenic Ras-induced morphological
changes. These results therefore suggest that the
Ras-dependent morphological changes are at least partly
mediated by the MAPKK/MAPK cascade. Downstream events or target
molecules of MAPK which are involved in morphological changes remain to
be elucidated. It has been reported that activation of Raf suppresses
integrin activation probably through MAPK activation (39). It is
possible that the MAPK cascade affects cytoskeletal network and cell
adhesion machinery through phosphorylation of cytoplasmic target proteins.
There were many reports showing that the MAPK pathway stimulates
transcriptional activation from the exogenously transfected serum
response element derived from the fos gene. However,
c-fos gene transcription in vivo seems to require
some chromatin modification such as histone H4 acetylation (40), and
thus does not occur readily. Our observation that active MAPKK induces
expression of endogenous Fos protein suggests that activation of the
MAPK pathway may result in some change in chromatin structure as well as in activation of transcription factors. We have further found that
active MAPKK induces the S phase entry of G0-arrested
cells. This is the first direct evidence that activation of the MAPK pathway alone is sufficient to induce initiation of cell proliferation and thus reinforces the essential role of the MAPK pathway for growth control.
Finally we have shown dominant-negative effects of the N-terminal
fragment of MAPKK on the MAPK signaling. One of possible molecular
mechanisms for these effects is that the fragment with the normal NES
retains endogenous MAPK in the cytoplasm through the MAPK-binding site
and thus suppresses nuclear import of MAPK in response to upstream
stimuli. The other is that the fragment inhibits the complex formation
between MAPK and MAPKK. Recently it has been reported that a bacterial
toxin which causes anthrax of animals inhibits the MAPK signaling by
cleaving an N-terminal region of MAPKK (1-7 amino acids of human
MAPKK1) (41). Since this region may contribute to the MAPK binding
(28), the cleavage may prevent the complex formation between MAPKK and
MAPK. Thus, it is likely that the complex formation between MAPKK and
MAPK contributes to an increase in efficiency of the MAPKK-induced MAPK
activation in normal signal transmission.
In conclusion, our results here suggest that MAPKK is a key molecule in
the Ras pathway, controlling both cell morphology and cell
proliferation, and demonstrate an important regulatory role of the
N-terminal region of MAPKK for the MAPK signaling. The results might
also explain an underlying mechanism through which constitutive
activation of the MAPK cascade alone can lead to cell transformation
with typical morphological alterations.