From the Cork Cancer Research Center and Departments
of § Medicine and ¶ Microbiology, National University
of Ireland, Cork, Ireland and the
Center for Experimental
Therapeutics, Department of Pharmacology, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6160
Received for publication, December 1, 2000, and in revised form, January 22, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we examined the mitogen-activated
protein kinase (MAPK) cascade in micrometastatic cell lines generated
from rib bone marrow (RBM) of patients undergoing resection of
esophagogastric malignancies. The molecular mechanism(s) involved in
esophagogastric MAPK activation have not previously been investigated.
Constitutive activation of both ERK1 and -2 isoforms was evident in
each of the five RBM cell lines. Elk-1, a transcription factor
activated by the ERK1/2 pathway was also found to be constitutively
activated. Cell lines generated from metastases of involved lymph nodes
(OC2) and ascites (OC1) of patients with esophageal cancer do not
display, however, hyperphosphorylation of ERK1/2. Constitutive RBM
ERK1/2 activation is protein kinase C and phosphatidylinositol
3-kinase dependent. Surprisingly, constitutive ERK1/2 activation is
MEK-independent. Pharmacological inhibition of MEK with two specific
inhibitors, PD 98059 and U0126, were both ineffective in blocking ERK
activation. Similarly, the use of a dominant negative MEK mutant was
without effect. Interestingly, experiments overexpressing two different dominant negative Pak1 mutants significantly reduced RBM ERK1/2 activation, albeit not to the same extent for all cell lines. We also
examined the role of three different phosphatases, PAC1, MKP-1, and -2. While RBM ERK1/2 activation was found to be PAC1- and
MKP-2-independent, surprisingly, MKP-1 was down-regulated in all five
RBM cell lines. In conclusion, we provide evidence for the first time
for a MEK-independent constitutive ERK1/2 activation pathway in
esophagogastric RBM cell lines. These findings have important
implications for drug treatment strategies which currently target MEK
in other forms of cancer.
Esophageal cancer is an aggressive tumor which responds poorly to
treatment and has a poor prognosis (1, 2). Approximately half of
patients diagnosed with localized esophageal cancer die of metastatic
disease within the first 2 years following tumor resection. We have
succeeded in developing a number of esophagogastric cell lines from the
rib bone marrow of patients (3). We have previously shown that these
cells are viable, proliferate, and grow independently in tissue culture
and form malignant tumors in athymic nude mice (4). These metastatic
cells are representative of the disseminated progenitors of secondary
tumors and are the appropriate targets for treatment.
The precise molecular events leading to the acquisition of the
metastatic phenotype remain largely unknown. Members of the Ras
superfamily of small GTP-binding proteins have been implicated in tumor
progression and are found to be activated in 20 to 30% of tumors (5).
In its active GTP-bound state, Ras activates the serine threonine
kinase Raf (6). Raf upon activation in turn phosphorylates the dual
specific kinase MEK1 (also
known as MAP kinase kinase or MAPKK) which in turn phosphorylates the
MAP kinases ERK1 and -2 (for extracellular regulated kinases 1 and 2).
Although MEK is the only known kinase directly downstream of Raf, there
are several lines of evidence suggesting that Raf can also activate
other effectors (7, 8). In vitro MEK1 is phosphorylated by
ERKs, Cdk2, and the p21-activated kinase Pak1 (9, 10). The Pak kinases
Pak1, -2 and -3, are a family of protein kinases that are regulated by
GTP-bound Rac and Cdc42 and are candidates for effectors that mediate
both actin and JNK signaling (for review, see Ref. 11).
The MAPKs are activated by the reversible dual threonine and tyosine
phosphorylation of a conserved T-X-Y motif (12). The reversible nature of MAPK phosphorylation suggests that phosphatases play a key role in regulating MAPK activity (for review, see Ref. 13).
MAPK phosphatase-1 and -2 (MKP-1, -2) as well as PAC1, a phosphatase
which shares a highly homologous C-terminal catalytic domain with MKP-1
(14), are known to inactivate ERKs (15) and possibly
JNKs/stress-activated protein kinases (12, 16). While PAC1 is
expressed predominantly in hematopoietic cells, MKP-1 and -2 are more
widely expressed and are induced by growth factors and genotoxic
and environmental stresses (17, 18).
Constitutive activation of ERKs in human malignancies has been
previously documented (19-22), albeit the detailed mechanism(s) underlying such activation have not been well characterized. In this
study, we undertook to characterize the MAPK signaling pathway in
micrometastatic cell lines generated from rib bone marrow of patients
undergoing resection of esophagogastric malignancies, which until now
had remained unexplored. Using complementary genetic and
pharmacological approaches, we now demonstrate that in all five
esophagogastric rib bone marrow (RBM) cell lines examined, both ERK1
and -2 isoforms are activated in a MEK- and Raf-1-independent manner.
In contrast, however, ERK1/2 activation was found to be PKC-, PI3K- and
Pak1-dependent. Moreover, we did not detect PAC1 expression, but found that MKP-1 but not MKP-2 was down-regulated. In
addition, the activation of ERK1/2 correlated with increased activation
of the downstream transcription factor Elk-1.
Reagents
EDTA, EGTA, leupeptin, Nonidet P-40, soybean trypsin inhibitor,
aprotinin, Cells
Generation of the esophagogastric metastatic rib bone marrow
cell lines (lines 1-5) used throughout this study have previously been
described in detail by our group, and were obtained from patients
undergoing resection of esophagogastric cancers (4). Briefly, RBM1 and
-5 cell lines are cells cultured from two different patients with
adenocarcinoma of the gastroesophageal junction. RBM2 and -4 cell lines
are cells cultured from two different patients with adenocarcinoma of
the esophagus and finally, the RBM3 cell line are cells cultured from a
patient with squamous carcinoma of the esophagus. OC1 and OC2 are
squamous cell lines established from malignant ascites and a lymph
node, respectively, of two patients with esophageal cancer. Finally,
OC3 are adenocarcinoma cells established from a metastatic lymph node
of a Barrett's esophageal lesion. We used normal human bone marrow
stromal (HBMS) cells throughout this study as control. CACO-2, HT-29,
and SW480 cells were also employed for comparative purposes in some
experiments throughout this study.
Cell Culture
RBM(1-5), OC1, -2, and -3, CACO-2, HT-29, and SW480 were
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine,
100 µg/ml streptomycin, and 100 units/ml penicillin. Cells were
washed twice with DMEM, and 1 × 105 cells/well were
plated into 6-well tissue culture plates. After 48 h cells were
cultured in medium supplemented with antibiotics and 0.5% FCS for all
experiments performed unless otherwise stated. HBMS cells, obtained
from the ATCC (Rockville, MD), were cultured in DMEM supplemented with
10% FCS, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, and 1 mM sodium pyruvate as previously
described (23). After 48 h cells were cultured in 6-well plates
(1 × 105/well) in DMEM supplemented with antibiotics
and 0.5% FCS, for all experiments carried out unless otherwise stated.
Plasmids
cDNA expression plasmids utilizing the cytomegalovirus
promoter to express Myc-tagged Pak1, Pak1R299,
Pak1L83,L86, and Pak1L83,L86,R299 were the
generous gift of Dr. J. Field (University of Pennsylvania School of
Medicine). The Pak constructs have been previously described in detail
(24-26). Briefly, Pak1 is wild type Pak, Pak1L83,L86 is a
hyperactive Pak, Pak1R299 lacks kinase activity and
Pak1L83,L86,R299 is a mutant that lacks kinase activity and
also fails to bind either Rac or Cdc42 (26). The dominant-negative form
of MEK1 was a generous gift of Drs. O. A. Coso and J. S. Gutkind (National Institutes of Health).
Immunoblot Analysis
RBM(1-5) cell lines, OC1, -2, and -3, CACO-2, HT-29, SW480, and
HBMS cells were cultured in DMEM supplemented with antibiotics and
0.5% FCS. Cells were lysed in ice-cold lysis buffer as previously described (27). Supernatants were used for immunoblotting with specific
antibodies for the phosphorylated or total p42/p44 MAPK, JNK1/2, or
p38, using the experimental conditions described by the manufacturer
(New England Biolabs). Similarly, membranes were incubated with
antibodies for Raf-1 (C-12), and MKP-1 (M-18) and -2 (S-18) (Santa Cruz
Biotechnology Inc., Santa Cruz, CA). Chemiluminescent substrates were
used to reveal positive bands that were visualized after exposure to
Hyperfilm ECL (Amersham Pharmacia Biotech). All immunoblots were
performed in triplicate.
In Vitro MAPK Assay
ERK, MEK, and Raf kinase assays were performed as previously
described (28) with minor modifications. Briefly, cells were lysed in
100 µl of modified RIPA buffer containing 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 10 mM sodium
flouride, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM
pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 25 mM Tris-HCl, pH 7.4, 1% Triton X-100, and 0.5% Nonidet
P-40. Supernatants were incubated with mild agitation for 60 min at
4 °C with 5 µg of anti-ERK-1(C-16) (Santa Cruz Biotechnology, Inc.), anti-MEK-1 (Transduction Laboratories), or anti-Raf-1 (Santa Cruz Biotechnology, Inc.) antibodies preabsorbed to protein A-Sepharose beads (Amersham Pharmacia Biotech). Immunoprecipitates were washed three times with cold phosphate-buffered saline containing 1% Nonidet
P-40 and 2 mM Na3VO4, once with 100 mM Tris, pH 7.5, 0.5 M LiCl, and once with MAP
kinase reaction buffer (12.5 mM MOPS, pH 7.5, 12.5 mM Semi-quantitative RT-PCR
Total RNA from RBM(1-5), OC1, -2, and -3, CACO-2, HT-29,
SW480, and HBMS cells was isolated using the acid
guanidium:phenol:chloroform method (29). Total RNA was digested with
RNase-free DNase I (RQ1; Promega, Madison, WI) and subsequently
re-extracted by the acid guanidium:phenol:chloroform method. PCR was
performed in a final volume of 50 µl containing 2 µl of cDNA,
15 pmol of each primer, 1.5 mM MgCl2, 200 µM each dNTP, and 1 unit of Taq DNA polymerase, all in 1 × supplied Taq buffer (Promega).
Sequences of primers used were: Transient Transfection of Cells
To investigate constitutive Elk-1 transcriptional activation, we
employed the PathDetect trans-reporting system as previously described but with minor modifications (27). Plasmid DNAs were transiently transfected into RBM(1-5), OC1, -2, and -3 using
LipofectAMINETM 2000 Reagent (Life Technologies) according
to the manufacturer's instructions. For all experiments, 1 µg of
Renilla luciferase vector pRL-SV40 (Promega Corp.) was included which
provides constitutive luciferase expression in transfected cells and
serves as an internal control to normalize transfection efficiency.
Cells were co-transfected with luciferase reporter (pFR-Luc) and
Renilla plasmids, along with the GAL-4-c-Elk-1 expression plasmid or
pFC-dbd plasmid as a negative control. Transfections were carried out
using a total of 5 µg of DNA (3 µg of GAL-4-c-Elk-1 plus 1 µg of
pFR-Luc plus 1 µg of Renilla or 3 µg of pFC-dbd plus 1 µg of
pFR-Luc plus 1 µg of Renilla). Forty-eight hours following
transfection, cells were washed twice in phosphate-buffered saline and
refed with DMEM supplemented with antibiotics and 0.5% FCS for a
further 48 h. Both firefly- and Renilla-luciferase activities were
measured using a commercial Dual LuicferaseTM Reporter
Assay Kit (Promega) following the manufacturer's instructions.
Stable Transfection of Cells
To establish stable cell lines expressing Pak1,
Pak1R299, Pak1L83,L86,R299,
Pak1L83,L86, and DN MEK, each construct was co-transfected
with pCDNA3 into RBM(1-5) cell lines, OC1, -2, and -3. Transfections were carried out using LipofectAMINETM 2000 Reagent and a total of 10 µg of DNA (5 µg of each test plasmid plus
5 µg of pCDNA3) according to the manufacturer's instructions. Following 48-72 h after the addition of DNA, cells were washed twice
in phosphate-buffered saline, and split 1:3 into 100-mm diameter
dishes. The transfected cells were selected in growth medium containing
600 µg/ml Geneticin (G418; Life Technologies Inc., Grand Island, NY).
After 4-8 weeks, individual cell colonies were transferred for clone
expansion and maintained in culture medium supplemented with 600 µg/ml G418. Protein expression levels were determined by Western blot
analysis of G418-selected cell foci using anti-Myc tag mAb 9E10
(Calbiochem) or an anti-MEK antibody (New England Biolabs) for Pak1 and
MEK transfections, respectively. Blots were visualized with the
procedure outlined in the enhanced chemiluminescence kit (Amersham
Pharmacia Biotech).
Cell Proliferation Assays
MTT Assay--
RBM(1-5) cell lines were seeded onto 96-well
plates at 5 × 103 cells per well in growth medium and
grown overnight prior to the initiation of any experimental treatments.
Following the indicated treatments, MTT (5 µg/ml) was added to each
well of a 96-well plate and the reduction of MTT was assayed to
calculate cell numbers as previously described (30).
[3H]Thymidine Incorporation--
Cells were seeded
sparsely in 6-well plates, and grown in DMEM supplemented with 10% FCS
and antibiotics for 24 h. The cells were then starved by culturing
in the same medium without serum for 48 h. The medium was then
removed, and cells were refed with medium containing 10% FCS and 0.5 µCi/well [3H]thymidine (Amersham Pharmacia Biotech).
The cells were incubated for 48 h after which
[3H]thymidine incorporation was performed as previously
described (31).
Cytotoxicity Assay--
For experiments using various kinase
inhibitors, cytotoxicity was measured by the MTT cell viability assay
described above.
Statistical Analysis
Results are expressed as mean ± S.E. Statistical
comparisons were made by using analysis of variance with subsequent
application of Student's t test, as appropriate.
Constitutive Activation of ERK1/2 in RBM(1-5)--
To determine
whether ERKs are activated in esophagogastric RBM we evaluated
the phosphorylation of ERK1/2 using phosphospecific antibodies as an
index of ERK1/2 activation (27). All five of the RBM cell lines when
cultured in 0.5% FCS, had substantially higher levels of
phosphorylated ERK1/2 when compared with HBMS cells, HT-29, CACO-2,
SW480, and OC1, -2, and -3 (Fig.
1B). ERK1/2 was defined as
constitutively activated in RBM(1-5) as cells grown in the presence of
serum (S) or treated with PMA for 5 min did not demonstrate a further
increase in phosphorylation (Fig. 1B). Results are shown for
RBM1 but similar results were observed for RBM(2-5) (data not shown).
Since previous reports have linked constitutive ERK activation with ERK
hyperexpression (20, 22), we examined ERK expression by immunoblot
analysis. We failed to see any significant difference in ERK protein
expression when RBM(1-5) cell lines were compared with HBMS cells,
HT-29, CACO-2, SW480 and OC1, -2, and -3 (Fig. 1A).
To verify that the observed phosphorylated ERK1/2 in RBM(1-5) was
active and capable of in vitro phosphorylation of cellular proteins, we used immunoprecipitated ERK from equal volumes of protein
extracted from RBM(1-5). HBMS cells and cells grown in the presence of
10% FCS were employed for comparison purposes in in vitro
kinase assays with myelin basic protein as a substrate. A
representative experiment is shown in Fig. 1C which
corroborates Western blot data analysis (Fig. 1B), again
demonstrating a marked increase in the activation state of ERK in RBM
cells compared with normal HBMS cells. No further increase in ERK
activity was observed when RBM1 cells were grown in the presence of
10% FCS (Fig. 1C). Similar results were obtained for
RBM(2-5) (data not shown).
To address the question of whether other members of the MAPK cascade
could also be constitutively activated, we examined the phosphorylation
of both JNK and p38. We failed to see either JNK or p38 phosphorylation
in each of the five cell lines examined (Fig. 1, D and
E). Treatment of the RBM1 cell line with UV for 5 min
is shown as a positive control (Fig. 1, D and
E).
PKC, and PI 3-Kinase but Not MEK Are Involved in RBM(1-5) ERK1/2
Activation--
To address the mechanism(s) of constitutive ERK1/2
activation in RBM(1-5), cells were pretreated for 1 h with GF
109203X (5 µM), a PKC inhibitor (32). GF 109203X (5 µM) abolished ERK1/2 activation while the PKA inhibitor
H-89 (10 µM) was without effect (Fig.
2A). Likewise, staurosporine
(100 nM), a nonspecific serine threonine kinase inhibitor,
also abolished RBM(1-5) ERK1/2 constitutive activation (Fig.
2A). Results are shown for RBM1 but similar results were
observed for RBM(2-5) (data not shown). Since MEK is the upstream
activator of ERKs (33, 34), we addressed the role of MEK1/2 in the
observed constitutive activation of ERK-1 and -2 in RBM(1-5). Cells
were incubated with PD 98059 (20 µM) or U0126 (20 µM), both inhibitors of MEK (35, 36). The compound U0126
has been identified as a selective inhibitor of MEK-1 and MEK-2, both
in vitro and in vivo (36). Surprisingly, we
observed that both inhibitors failed to alter the phosphorylation of
ERK1/2 in RBM(1-5) (Fig. 2A). Again, results are shown for
RBM1 but similar results were observed for RBM(2-5) (data not shown).
To verify that both inhibitors, however, could abolish ERK
phosphorylation, HBMS cells were pretreated with PD 98059 (20 µM) and U0126 (20 µM) for 1 h. ERK1/2
phosphorylation was completely abolished in the presence of both
inhibitors when serum-starved HBMS cells were cultured in DMEM plus
10% FCS (S) for 24 h or treated with PMA (100 nM) for
5 min (Fig. 2B). Therefore, both inhibitors are fully
capable of "shutting down" activation of the MAPK pathway. To
investigate the role that PI3K may have in RBM(1-5) constitutive ERK1/2 activation, cells were treated with two PI3K inhibitors LY294002
(20 µM) and wortmannin (100 nM) (37). Both
inhibitors abolished the phosphorylation of ERK1/2 in RBM(1-5) (Fig.
2A). Similarly, ERK1/2 kinase activity could be altered
pharmacologically. GF 109203X (5 µM), as well as LY294002
(20 µM) and wortmannin (100 nM) abolished
ERK1/2 kinase activity (Fig. 2C), while PD 98059 (20 µM), U0126 (20 µM), and H89 (10 µM) were without effect (Fig. 2C).
Constitutive ERK1/2 Activation is Raf- and MEK-independent in
RBM(1-5)--
Since both PD98059 and U0126, two specific inhibitors
of MEK (35, 36), failed to alter the activation of ERK1/2 in RBM(1-5) (Fig. 2, A and C), we turned our attention to
MEK. We examined the expression, phosphorylation, and activation of MEK
in RBM(1-5). The phosphospecific MEK1/2 antibodies (New England
Biolabs) only recognize the phosphorylated forms of MEK1/2 but do not
distinguish between the two isoforms. As expected, serum-starved HBMS
cells, as well as OC1, did not exhibit MEK1/2 phosphorylation (Fig.
3C). Similar results were
found for OC2, -3, CACO-2, HT-29, and SW480 (data not shown). However,
MEK1/2 was not found to be phosphorylated in serum-starved RBM(1-5)
(Fig. 3B), despite the existence of ERK activation. When
RBM1, HBMS, and OC1 cells were cultured in DMEM supplemented with 10%
FCS (S) or treated with PMA (100 nM) for 5 min, MEK1/2 was
phosphorylated (Fig. 3, B and C). Results are
shown for RBM1, but similar observations were noted for RBM(2-5) (data
not shown). Since a previous report has linked MEK hyperexpression with
ERK constitutive activation (21), we performed immunoblot analysis to
determine the expression level of MEK protein in RBM(1-5) versus HBMS, OC1, -2, and -3, using antibodies that
recognize total MEK1/2. Unlike previous reports (21), we failed to see any significant differences in MEK1/2 expression for all cells analyzed
(Fig. 3A). To reconfirm the absence of MEK activation in
RBM(1-5) we also performed a MEK kinase assay using kinase inactive
GST-ERK-1 (K71A, Upstate Biotechnology Inc.) as substrate (28).
Consistent with our immunoblot results, we failed to observe an
increase in MEK kinase activity (Fig. 3, D and
E). Results are shown for RBM1, but were similar for
RBM(2-5) (data not shown). Cells cultured in the presence of serum (S)
or treatment with PMA (100 nM) for 5 min, however, showed a
marked increase in MEK activity, as assessed by GST-ERK1
phosphorylation (Fig. 3, D and E). Thus,
RBM(1-5) have a functional MEK as well as ERK.
Since Raf lies directly upstream of MEK, we also investigated whether
Raf is activated or not in RBM cell lines. For each of the five cell
lines examined we failed to see either Raf phosphorylation (Fig.
3F) or activation (Fig. 3G). However, cells
cultured in the presence of serum or treatment with PMA (100 nM) for 5 min showed a marked increase in Raf-1
phosphorylation (Fig. 3F) as well as Raf-1 activity as
assessed by GST-MEK-1 phosphorylation (Fig. 3G) as
previously described (38). Thus, RBM(1-5) have a functional Raf-1 and
MEK as well as ERK.
Dominant Negative MEK Does Not Disrupt ERK1/2 Constitutive
Activation in RBM(1-5)--
To further confirm that ERK activation is
MEK-independent in RBM(1-5), cells were stably transfected with a DN
MEK mutant. In agreement with our results using the two specific MEK
inhibitors, we failed to see any change in ERK1/2 phosphorylation in
RBM(1-5) when cells overexpressed DN MEK versus
nontransfected cells (Fig. 4,
A and B). The DN MEK plasmid utilized was not
HA-tagged. We attempted to verify the expression of the mutant by
Western blot analysis but failed to see a significant difference in the
expression levels between transfected and nontransfected cells (data
not shown). However, the DN MEK mutant was capable of inhibiting ERK activation by PMA (100 nM, 5 min) in OC1, -2, and -3 cells
(Fig. 4C). To further confirm that cells were transfected,
we analyzed the level of MEK activation by Western blot analysis. In
all five RBM cell lines examined (Fig. 4D) as well as OC1,
-2, and -3 cells (Fig. 4E), we observed a significant
reduction in MEK1/2 phosphorylation to basal levels in cells that
stably expressed the DN MEK mutant versus the nontransfected
cells when cells were stimulated with PMA (100 nM, 5 min).
Pak1 Contributes to the Constitutive Activation of ERK1/2 in
RBM(1-5)--
Since both MEK inhibitors or DN MEK failed to alter the
activation of ERK1/2 in RBM(1-5), we sought to identify other kinases which may lie upstream in the MAPK signaling pathway of these cells.
Since we observed that the two PI3K inhibitors LY294002 and wortmannin
both abolished ERK1/2 activation in RBM(1-5) (Fig. 2, A and
C), we turned our attention to Pak1, a kinase which has previously been shown to be activated by PI3K (25). Pak has been shown
to activate JNK (39), as well as ERK (40). We developed stable
RBM(1-5) cell lines that expressed the various Pak mutants, confirmed
by Western blot analysis of the Myc-tagged plasmids (Fig.
5A). Pak1 is wild type Pak,
Pak1R299 lacks kinase activity,
Pak1L83,L86,R299 is a mutant that lacks kinase activity and
also fails to bind either Rac or Cdc42, and Pak1L83,L86 is
a hyperactive Pak. The two kinase-deficient Pak mutants are both
dominant negative forms of Pak. Pak1 is seen as a 65-kDa band.
Therefore, we tested the effects of the different Pak1 mutants versus empty vector on the constitutive activation of ERK1/2
in RBM(1-5). We observed that wild type Pak1 did not affect ERK1/2 activation (Fig. 5B). When we tested the two kinase mutants
Pak1R299 and Pak1L83,L86,R299, however, we
consistently found that both reduced ERK1/2 phosphorylation in all of
the RBM cell lines, albeit not to the same extent. We observed that the
two Pak1 mutants continuously had less of an effect on RBM4 and -5 ERK
activation in comparison to RBM1-3 (Fig. 5, C and
D). A reduction in phosphorylated ERK in cells
overexpressing the dominant negative Pak mutants has previously been
described by Tang et al. (26). Interestingly, cells
overexpressing the hyperactive Pak1L83,L86 showed a further
increase in ERK1/2 phosphorylation (Fig. 5, E and
F). This increase in ERK1/2 activation was MEK-independent but PI3K dependent (Fig. 5, E and F). Again,
results are shown for RBM1 but were similar for RBM(2-5) (data not
shown). We also examined whether cells expressing the different Pak1
mutants could alter the levels of ERK and MEK protein expression. In
all five cell lines expressing the four different Pak1 mutants, we
failed to see any changes in protein expression (data not shown). Since Pak has been shown to activate JNK (39), we also examined whether the
hyperactive Pak1 mutant, Pak1L83,L86, which brought about a
further increase in ERK activation, could increase JNK activation in
RBM(1-5). We failed, however, to see up-regulation of JNK in
RBM(1-5) (Fig. 5G)
RBM(1-5) Demonstrate Constitutive Transcriptional Activation of
Elk-1--
To further evaluate the level of activation in RBM(1-5),
we investigated whether the observed upstream constitutive activation of ERK1/2 may influence gene expression. RBM(1-5) transiently transfected with GAL-c-Elk-1 demonstrated an elevated level of Elk-1
activation when compared with HBMS, OC1, -2, and -3 (Fig. 6A). To investigate the
upsteam kinases involved in the Elk-1 activation, cells were treated
with the PKC inhibitor GF 109203X (5 µM). GF 109203X
brought about a significant reduction in the activation of Elk-1 (Fig.
6B). Similarly, the PI3K inhibitors LY294002 (20 µM) and wortmannin (100 nM), reduced the
levels of Elk-1 activation (Fig. 6B). In contrast, however,
both PD 98059 (20 µM) and U0126 (20 µM),
failed to abrogate the levels of Elk-1 activation, thereby implying a
MEK-independent Elk-1 up-regulation in RBM(1-5) (Fig.
6B).
Since we observed that the two kinase-deficient Pak1 mutants,
Pak1L83,L86,R299 and Pak1R299 were capable of
reducing the levels of ERK1/2 activation in RBM(1-5) (Fig. 5,
C and D), we decided to investigate if they could
also alter the levels of constitutive Elk-1 activation. Stable cell lines overexpressing each of the Pak1 mutants were transiently transfected with GAL-c-Elk-1 plus pFR-Luc plus Renilla as described above. Stable cell lines overexpressing wild type Pak1 (Pak1) did not
display altered levels of Elk-1 activation in all five RBM cell lines
examined (Fig. 6C). Both kinase-deficient Pak mutants, Pak1R299 and Pak1L83,L86,R299, however, were
efficacious in significantly reducing the levels of Elk-1 activation in
RBM1 and -2 but not in RBM3, -4, and -5 (Fig. 6C). The
hyperactive mutant, Pak1L83,L86, significantly increased
the level of Elk-1 activation in RBM2 and -3 but not in RBM1, -4, and
-5. Thus, while Pak1 seems to play a definitive role in ERK1/2
activation in RBM(1-5), its role in Elk-1 activation is more complex.
Pak1 appears to influence Elk-1 activation in RBM1, -2 and -3, but is
ineffective in RBM4 and -5, suggesting that there may be other kinases
operating in these two cell lines lying outside of the Pak pathway (39,
40) which influence the level of activation of Elk-1. These results are
consistent with those for ERK activation where
Pak1L83,L86,R299 and Pak1R299 were less
effective in RBM4 and -5 ERK down-regulation, thereby indicating that
these two cell lines may be differentially regulated compared with
RBM1-3.
Constitutive Activation of ERK1/2 in RBM(1-5) Is
MKP-1-dependent But MKP-2 and PAC1-independent--
The
constitutive activation of ERKs suggested the possibility of an
abnormal dephosphorylation mechanism of ERKs. PAC1, a member of the ERK
phosphatase family is induced in response to ERK activation (14, 41).
Therefore, to examine whether abnormal ERK down-regulation mechanisms
were present, we examined PAC1 gene expression by RT-PCR because
PTPases, including PAC1, are principally up-regulated at the
transcription level in response to ERK activation (42). PAC1 gene
expression was not observed in RBM(1-5) or OC2 cells, but was present
in OC1, -3, HT-29, CACO-2, SW480, and HBMS cells showing no ERK
activation, and could be up-regulated in the presence of serum (Fig.
7A). We then examined the role
of two other protein-tyrosine phosphatases MKP-1 and -2 using
antibodies which are specific for both. Like PAC1, both MKP-1 and -2 are reported to be up-regulated upon ERK activation (16, 43).
Interestingly, in our study, we found that the levels of MKP-1
expression in RBM(1-5) showing constitutive ERK1/2 activation was
significantly lower than that in unstimulated HBMS, CACO-2, HT-29,
SW480, OC1, -2, and -3 cells showing no ERK activation (Fig.
7B). No changes in MKP-2 expression were observed (Fig. 7C).
RBM(1-5) Cell Proliferation is PKC- and PI
3-Kinase-dependent But MEK-independent--
The MAPK
pathway is essential in cellular growth and differentiation (44). Thus,
we examined the role of the constitutive activation of ERK1/2 on
RBM(1-5) cell growth. To investigate the role of MEK in RBM cell
proliferation, cells were treated with PD 98059 (20 µM).
This MEK inhibitor failed to significantly reduce the cell number
(using the MTT assay) or DNA synthesis (using [3H]thymidine incorporation) (Fig.
8). PD 98059 was effective, however, in
significantly reducing HT-29 cell growth (data not shown). We also
investigated the role of PKC, and PI3K in RBM(1-5) cell proliferation.
PKC inhibition with GF 109203X (5 µM) or pretreatment with PMA (100 nM) for 48 h which down-regulates PKC
(27) significantly reduced cell growth (Fig. 8). Similar results were
observed when cells were treated with the PI3K inhibitors, LY294002 (20 µM) and wortmannin (100 nM). Wortmannin is
unstable in solution and PI3K levels remain suppressed for only 9-12 h
after drug addition (45), thus cells were washed and pretreated a
second time with wortmannin (100 nM) for another 12 h.
Thus, ERK1/2 constitutive activation in RBM(1-5) correlates with
cellular proliferation.
The present results have identified for the first time that the
mitogen-activated protein kinases ERK1 and -2 as well as the transcription factor Elk-1 are constitutively activated in
esophagogastric RBM cell lines. To our surprise, the observed
activation of ERKs was not accompanied by MEK activation. Until now,
the activated status of ERK has been shown to be predominantly
accompanied by MEK activation (33, 34). One previous report has
documented the importance in Swiss 3T3 fibroblasts of MEK activation
for the initiation of MAPK activation by PDGF but also emphasize that prolonged MAPK activation by PDGF is MEK-independent (46). Other reports documenting constitutive ERK activation have shown it to be
synonymous with hyperexpression of either MEK or ERK (21, 22, 34). In
contrast to all of these reports, however, we have failed to make any
correlation between constitutive ERK activation and ERK or MEK
hyperexpression or indeed with MEK activation. It may be possible that
esophagogastric RBM cells may display elevated levels of receptor
tyrosine kinases, or intermediate molecules which may explain the
constitutive activation of ERKs observed in these cells. These
questions are currently being addressed in our laboratory. Indeed, it
has recently been shown that growth factor receptor tyrosine kinases
are overexpressed in esophageal squamous cell carcinomas relative to
normal tissue (47).
Although the specific role of ERKs in esophagogastric RBM cells is not
known, it is reasonable to speculate that chronic activation of the ERK
signaling cascade may influence the local metastatic environment to
favor progression of esophagogastric metastatic cancer. Indeed,
constitutive ERK activation has previously been shown to confer a
tumorigenic and metastatic potential (48) as well as regulate cell
motility and matrix metalloproteinase expression (49). Our data
demonstrate that selective inhibition of the upstream kinase MEK does
not affect esophagogastric RBM cell growth. This is in contrast to
recent studies whereby inhibition of MEK attenuated the in
vivo invasiveness of head and neck squamous cell carcinoma (50)
and suppressed the growth of colon tumors in vivo (51).
Pharmacological inhibition of PI3K and PKC, however, both proved
efficacious in significantly reducing esophagogastric RBM cell growth
as well as ablating ERK activation (52). Our studies revealed that
while PI3K seems to be necessary for ERK activation and esophagogastric
metastatic cell growth, it does not require Raf activation as Raf was
found not to be activated in these cell lines. Furthermore, upon
examination of the K-ras gene in RBM cells, we did not
detect the existence of point
mutations.2 This
is in agreement with a previous report demonstrating the absence of
K-ras gene mutations in a series of cell lines established from esophageal cancer (2).
The involvement of PI3K lead us to investigate the role of the
serine-threonine kinase Pak (p65Pak) a known effector of
PI3K (25). The kinase-deficient Paks (Pak1R299 and
Pak1L83,L86,R299) both significantly reduced the
phosphorylation levels of ERK1 and -2. Interestingly, we observed that
hyperactive Pak (Pak1L83,L86) could further enhance the
activation state of ERKs in esophagogastric RBM cells. This activation
by Pak was found to be sensitive to PI3K inhibition but not MEK
inhibition. Although Pak is not usually associated with ERK activation,
a recent report has shown that Pak can cooperate with Raf to activate
ERK in a cross-cascade activation (26). It remains to be investigated
whether Pak is interacting with Raf within RBM(1-5). However, the
absence of MEK involvement in Pak1 signaling in RBM(1-5) ERK
activation reveals a novel signaling pathway in these cells. Although
Pak-induced JNK activation has also been previously demonstrated (26),
our study shows neither JNK nor p38 were involved in the signaling cascade in RBM(1-5).
Abundant data indicate that constitutive activation of the ERK cascade
increases the expression of PAC1, a member of the MAPK phosphatase
family (14, 42) providing a pivotal role for this phosphatase in
feedback inactivation of the stimulated ERK signaling pathway. PAC1
exhibits stringent substrate specificity for ERK and constitutive PAC1
expression inactivates ERK. While PAC1 expression is associated
predominantly with hematopoeitic cells (14), we have now shown that it
is also present in CACO-2, HT-29, SW480, OC1, and -3 cells and can be
up-regulated in the presence of serum. We failed, however, to detect
PAC1 gene expression in RBM cell lines, thus ruling out the possibility
of a role for this phosphatase in the observed ERK constitutive
activation. Two other protein phosphatases, MKP-1 and MKP-2 (17, 18),
may further explain the constitutive activation of ERKs in RBM(1-5).
Previous reports have shown that both MKP-1 and -2 expression is
up-regulated upon ERK activation (16, 43). Moreover, up-regulation of
normal MKP-1 mRNA and protein has been implicated in clinical
specimens of a group of early stage carcinomas and in various stages of breast and prostate carcinoma (54, 55). MKP-1 may also act as a tumor
suppressor (54) as overexpressed MKP-1 has been shown to down-regulate
ras-dependent mitogenic signals (53). In
contrast to these reports, however, we have observed a significant
decrease in MKP-1 expression in RBM(1-5) while MKP-2 expression
remained unaltered. Although the significance of MKP-1 down-regulation and its mechanistic basis require additional investigation, we may
speculate that an abnormal down-regulation mechanism of ERKs may exist
in RBM(1-5) involving MKP-1. In conclusion, although the
complexity of the signaling cascade which converges on ERK seems
daunting, delineation of this pathway may uncover new cellular targets
for the pharmacological manipulation of esophagogastric metastatic RBM cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, sodium orthovanadate, magnesium chloride, sodium flouride, and myelin basic protein were purchased from
Sigma. Cell culture medium, fetal calf serum (FCS) and antibiotics were
purchased from Life Technologies, Inc. (Dublin, Ireland). Phorbol
12-myristate 13-acetate (PMA), GF 109203X, LY294002, wortmannin, H-89
(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfinamide), the two MEK inhibitors,
2-(2'-amino-3'-methoxyphenyl)-oxanapthalen-4-one (PD98059) and
1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthiobutadiene) (U0126) and
the anti-Myc tag monoclonal antibody 9E10 were purchased from
Calbiochem (Nottingham, United Kingdom).
[32P]Orthophosphate (6000-7000 Ci/mmol),
-[32P]ATP (3000 Ci/mmol), and protein A-Sepharose were
purchased from Amersham Pharmacia Biotech (Dublin, Ireland). Phosphorus
p42/p44 MAPK, JNK, and p38 MAPK antibody kits were purchased from New England Biolabs (Beverly, MA). Rabbit polyclonal antibodies anti-Raf-1 (C-12), anti-MKP-1 (M-18) and -2 (S-18) were obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). The PathDetectTM
in vivo signal transduction pathway
trans-reporting system was purchased from Stratagene (La
Jolla, CA). The Dual LuciferaseTM Reporter Assay Kit was
purchased from Promega (Madison, WI).
-glycerophosphate, 7.5 mM
MgCl2, 0.5 mM EGTA, 0.5 mM NaF, and
0.5 mM vanadate) and resuspended in 30 µl of reaction buffer (kinase reaction buffer with 20 µM ATP, 1 µCi of
[
-32P]ATP, and 3.3 mM dithiothreitol and
0.5 µg of myelin basic protein, 0.3 µg of kinase inactive
GST-ERK1(K71A) (Upstate Biotechnology Inc.) or 0.1 µg of
kinase-inactive GST-MEK-1(K97A) (Upstate Biotechnology, Inc.).
Incubations were carried out for 30 min at 30 °C. Reactions were
terminated by the addition of 10 µl of Laemmli sample buffer. Samples
were run on a 10 or 7.5% SDS-PAGE gel, and visualized by
autoradiography. The MAPK, MAPKK, and Raf-1 activity was defined as the
amount of radioactivity incorporated into myelin basic protein,
GST-ERK1, or GST-MEK-1, respectively, under these conditions. Alternatively, the reaction was terminated by spotting 30 µl of reaction mixture onto 1-inch squares of phosphocellulose paper (P81;
Whatman International Ltd., Maidstone, United Kingdom). Free
-32P was removed by three washes in 180 mM
phosphoric acid as previously described (26). Incorporated
radioactivity was determined by liquid scintillation counting. All
assays were performed in triplicate.
-actin (primer positions correspond
to nucleotides 541-571 (sense primer) and nucleotides 1171-1201
(antisense primer) on human
-actin mRNA, sense:
5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3';
-actin, antisense:
5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'; PAC-1 (20), sense:
5'-TTGCCCTACCTGTTCCTGGG-3'; PAC-1, antisense:
5'-GTCTCAAACTGCAGCAGCTG-3'. The PCR conditions were as follows:
94 °C, 15 s; 64 °C, 20 s; 72 °C, 1 min, following an
80 °C, 3-min step prior to the addition of Taq (hot
start), with 23 cycles for
-actin and 33 cycles for PAC-1. 10 µl
of each PCR was resolved on ethidium bromide-stained 3:1
NuSieve:Agarose gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
Constitutive activation of p44/p42 MAPK in
esophagogastric micrometastatic rib bone marrow cell lines.
Immunoblot analysis using anti-MAPK antibodies that react with total
p44/p42 MAPK (A) and phospho-anti-MAPK, anti-JNK, and
anti-p38 antibodies that react only with the phosphorylated forms of
p44/p42 MAPK, JNK, and p38 (B, D and E) was
carried out as described under "Materials and Methods." Kinase
activity as described under "Materials and Methods" was monitored
either by autoradiography or by liquid scintillation counting
(C). HBMS cells, HT-29, CACO-2, SW480, OC1, -2, and -3 and
RBM(1-5) were grown in DMEM supplemented with antibiotics and 0.5%
FCS (A-E). RBM1 was cultured in 10% FCS (S) for 24 h
or stimulated with PMA (100 nM) for 5 min (A and
B). RBM1 cells were treated with UV for 5 min as positive
control for JNK and p38 phosphorylation (D and
E). Twenty µg of protein was loaded per lane
(A-E). The figure is representative of three
separate blots. C, results are expressed as mean ± S.E. from three independent experiments.
View larger version (27K):
[in a new window]
Fig. 2.
Effect of different inhibitors on
constitutive p44/p42 MAPK activation in esophagogastric micrometastatic
rib bone marrow cell lines. Immunoblot analysis (A and
B) and immunoprecipitation (C) were carried out
as described under "Materials and Methods." HBMS cells, OC1, -2, and -3 and RBM1 were grown in DMEM supplemented with antibiotics and
0.5% FCS (A and C). HBMS cells were cultured
with and without 10% FCS (S) or stimulated with PMA (100 nM) for 5 min (B). Cells were preincubated with
GF 109203X (GF) (5 µM), PD 98059 (PD) (20 µM), U0126 (20 µM),
LY294002 (LY)(20 µM), wortmannin (W) (100 nM), H-89 (10 µM), and staurosporine
(St) (100 nM) for 1 h prior to analysis.
Twenty µg of protein was loaded per lane (A-C).
The figure is representative of four separate blots. C,
results are expressed as mean ± S.E. from four independent
experiments.
View larger version (33K):
[in a new window]
Fig. 3.
Absence of MEK1/2 and Raf-1 activation in
esophagogastric micrometastatic rib bone marrow cell lines.
Immunblot analysis as described under "Materials and Methods" was
performed using anti-MEK1/2 antibodies that react with total MEK1/2
(A), phospho-anti-MEK1/2 antibodies that react only with the
phosphorylated forms of MEK1/2 (B and C), and
Raf-1 (C-12) antibody that reacts with both phosphorylated and
non-phosphorylated Raf-1 (F and G). Aliquots of
cell extracts were loaded on a 7.5% SDS-PAGE for F and
G and all other gels were 10%. Twenty µg of protein was
loaded per lane (A-D, F, and G) and
are representative of three different blots. Kinase activity as
described under "Materials and Methods" was monitored either by
autoradiography (D) or by liquid scintillation counting
(E). E, results are expressed as mean ± S.E. from three independent experiments.
View larger version (49K):
[in a new window]
Fig. 4.
Dominant negative MEK does not disrupt
p44/p42 MAPK constitutive activation. Immunoblot analysis as
described under "Materials and Methods" was performed using
phospho-anti-MAPK and phospho-anti-MEK1/2 antibodies that react only
with the phosphorylated forms of p44/p42 MAPK (A-C) and
MEK1/2, respectively (D and E). A
represents non-transfected RBM(1-5) cell lines. B
represents stably transfected RBM(1-5) cell lines with a DN MEK
mutant. C represents stably transfected OC1, -2, and -3 cell
lines with a DN MEK mutant, in the presence and absence of PMA (100 nM, 5 min). D and E represent stably
transfected and nontransfected RBM(1-5) and OC1, -2, and -3 cells,
respectively, in the presence and absence of PMA (100 nM, 5 min). All cells were cultured in DMEM supplemented with antibiotics and
0.5% FCS. Twenty µg of protein was loaded per lane
(A-E). The figure is representative of three
separate blots.
View larger version (50K):
[in a new window]
Fig. 5.
Involvement of Pak1 in RBM(1-5) p44/p42 MAPK
activation. A, Western blot showing stable expression
of Pak1 mutants. Twenty µg of extracts was probed with antibody 9E10,
which recognizes the Myc tag on the Pak1 constructs. Pak1 is seen as a
65-kDa band. B-F, Western blot analysis of ERK1/2 with
phospho-specific ERK antibodies. G, Western blot analysis of
JNK with a phospho-specific JNK antibody. All cells were cultured in
DMEM supplemented with antibiotics and 0.5% FCS. E and
F, cells were treated with PD 98059 (PD) (20 µM), wortmannin (W) (100 nM), or
LY294002 (LY) (20 µM) for 1 h prior to
analysis. Similar results were obtained in five independent
experiments.
View larger version (17K):
[in a new window]
Fig. 6.
Constitutive transcriptional activation of
Elk-1 in transiently transfected RBM(1-5) RBM(1-5), HBMS cells, OC1,
-2, and -3, (A and B), and stable
cell lines RBM(1-5) expressing the different Pak1 constructs
(C) were transiently co-transfected with GAL4-c-Elk-1
together with pFR-Luc reporter and Renilla plasmids. A
and B, cells were preincubated with GF 109203X
(GF) (5 µM), PD 98059 (PD) (20 µM), LY294002 (LY) (20 µM), or
wortmannin (W) (100 nM) for 12 h prior to
analysis. Luciferase activity was determined on cell extracts as
described under "Materials and Methods." The results shown are
mean ± S.E. of six independent experiments. C,
significant (*, p < 0.05) changes from nontransfected
cells.
View larger version (63K):
[in a new window]
Fig. 7.
MKP-1 but not MKP-2 or PAC1 is down-regulated
in RBM(1-5). CACO-2, SW480, HT-29, OC1, -2, -3, HBMS cells, and
RBM(1-5) were subjected to RT-PCR analysis as described under
"Materials and Methods" (A). PCR products derived from 1 µg of total RNA were applied to each lane. For immunoblot analysis,
aliquots of extracts containing 40 µg of protein were loaded on a
10% SDS-PAGE and analyzed using anti-MKP-1 (M-18) (B), and
-2 (S-18) (C) antibodies. Similar results were obtained in
three independent experiments.
View larger version (15K):
[in a new window]
Fig. 8.
Inhibition or down-regulation of PKC as well
as inhibition of PI3K but not MEK1/2 inhibits growth of esophageal
micrometastatic RBM cell lines. Cell growth was determined either
by reduction of MTT or by [3H]thymidine incorporation.
Cells were preincubated with GF 109203X (GF) (5 µM), PD 98059 (PD) (20 µM),
LY294002 (LY)(20 µM), or wortmannin
(W) (100 nM) for 24 h prior to analysis.
PKC down-regulation was achieved by treating the cells with PMA (100 nM) for 48 h. The results shown are mean ± S.E.
of six independent experiments. Significant (*, p < 0.05) changes from untreated cells are shown in C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We acknowledge Dr. J. Fields for critical analysis of this manuscript and for providing the Pak1 mutants.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Irish Cancer Society and a Higher Education Authority Grant.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.
** To whom correspondence and reprints should be addressed: Cork Cancer Research Center, Mercy Hospital, Cork, Ireland. Tel.: 21-4271971 (ext. 5397); Fax: 21-4345300; E-mail: geraldc@iol.ie.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M010847200
2 O. P. Barry and S. Aarons, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular-regulated kinase; RBM, rib bone marrow cells; HBMS, human bone marrow stromal cells; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun NH2-terminal kinase; Pak, p21-activated kinase; MKP-1, MAPK phosphatase-1; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfinamide; PD98059, 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one; U0126, (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthoio]butadiene); FCS, fetal calf serum; DN, dominant negative; GST, glutathione S-transferase; RT-PCR, reverse transcriptase-polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Montesano, R., Hollstein, M., and Hainaut, P. (1996) Int. J. Cancer 69, 225-235[CrossRef][Medline] [Order article via Infotrieve] |
2. | Nishira, T., Hashimoto, Y., Katayama, M., Mori, S., and Kuroki, T. (1993) J. Cancer Res. Clin. Oncol. 119, 441-449[Medline] [Order article via Infotrieve] |
3. | O'Sullivan, G. C., Collins, J. K., O'Brien, F., Crowley, B., Murphy, K., Lee, G., and Shanahan, F. (1995) Gastroenterology 109, 1535-1540[Medline] [Order article via Infotrieve] |
4. | O'Sullivan, G. C., Sheehan, D., Clarke, A., Stuart, R., Kelly, J., Kiely, M. D., Walsh, T., Collins, K., and Shanahan, F. (1999) Gastroenterology 116, 543-548[Medline] [Order article via Infotrieve] |
5. | Lowry, D. R., and Willumsen, B. R. (1993) Annu. Rev. Biochem. 62, 851-891[CrossRef][Medline] [Order article via Infotrieve] |
6. | Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214[Medline] [Order article via Infotrieve] |
7. |
Lenormand, P.,
McMahon, M.,
and Pouyssegur, J.
(1996)
J. Biol. Chem.
271,
15762-15768 |
8. |
Finco, T. S.,
Westwick, J. K.,
Norris, J. L.,
Beg, A. A.,
Der, C. J.,
and Baldwin, A. S., Jr.
(1997)
J. Biol. Chem.
272,
24113-24116 |
9. |
Frost, J. A.,
Steen, H.,
Shapiro, P.,
Lewis, T.,
Ahn, N.,
Shaw, P.,
and Cobb, M. H.
(1997)
EMBO J.
16,
6426-6438 |
10. | Rossomando, A. J., Dent, P., Sturgill, T. W., and Marshak, D. R. (1994) Mol. Cell. Biol. 14, 1594-1602[Abstract] |
11. | Manser, E., and Lim, L. (1999) Prog. Mol. Subcell. Biol. 22, 115-133[Medline] [Order article via Infotrieve] |
12. |
Raingeaud, J.,
Gupta, S.,
Rogers, J. S.,
Dickens, M.,
Han, J.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
J. Biol. Chem.
270,
7420-7426 |
13. | Guan, K.-L., and Dixon, J. E. (1993) Semin. Cell Biol. 4, 389-396[CrossRef][Medline] [Order article via Infotrieve] |
14. | Rohan, P. J., Davis, P., Moskaluk, C. A., Kearns, M., Krutzsch, H., Siebenlist, U., and Kelly, K. (1993) Science 259, 1763-1766[Medline] [Order article via Infotrieve] |
15. | Bokemeyer, D., Sorokin, A., and Dunn, M. J. (1997) J. Am. Soc. Nephrol. 8, 40-50[Abstract] |
16. |
Chu, Y.,
Solski, P. A.,
Khosravi-Far, R.,
Der, C. J.,
and Kelly, K.
(1996)
J. Biol. Chem.
271,
6497-6501 |
17. |
Liu, Y.,
Gorospe, M.,
Yang, C.,
and Holbrook, N. J.
(1995)
J. Biol. Chem.
270,
8377-8380 |
18. | Keyse, S. M., and Emslie, E. A. (1992) Nature 359, 644-647[CrossRef][Medline] [Order article via Infotrieve] |
19. | Towatari, M., Lida, H., Tanimoto, M., Iwata, H., Hamaguchi, M., and Saito, H. (1997) Leukemia 11, 479-484[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Kim, S. C.,
Hahn, J.-S.,
Min, Y. H.,
Yoo, N.-C.,
Ko, Y.-W.,
and Lee, W.-J.
(1999)
Blood
93,
3893-3899 |
21. | Oka, H., Chatani, Y., Hoshino, R., Ogawa, O., Kakehi, Y., Terachi, T., Okada, Y., Kawaichi, M., Kohno, M., and Yoshida, O. (1995) Cancer Res. 55, 4182-4187[Abstract] |
22. |
Sivaraman, V. S.,
Wang, H.-Y.,
Nuovo, G. J.,
and Malbon, C. C.
(1997)
J. Clin. Invest.
99,
1478-1483 |
23. |
Roecklein, B. A.,
and Torok-Storb, B.
(1995)
Blood
85,
997-1005 |
24. | Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M., and Chernoff, J. (1997) Curr. Biol. 7, 202-210[Medline] [Order article via Infotrieve] |
25. |
Tang, Y., Yu, J.,
and Field, J.
(1999)
Mol. Cell. Biol.
19,
1881-1891 |
26. |
Tang, Y.,
Marwaha, S.,
Rutkowski, J. L.,
Tennekoon, G. I.,
Phillips, P. C.,
and Field, J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5139-5144 |
27. |
Barry, O. P.,
Kazanietz, M. G.,
Pratico, D.,
and FitzGerald, G. A.
(1999)
J. Biol. Chem.
274,
7545-7556 |
28. |
Ellinger-Ziegelbauer, H.,
Brown, K.,
Kelly, K.,
and Siebenlist, U.
(1997)
J. Biol. Chem.
272,
2668-2674 |
29. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Barry, O. P.,
Savani, R. C.,
Pratico, D.,
and FitzGerald, G. A.
(1998)
J. Clin. Invest.
102,
136-144 |
31. | Blume-Jensen, P., Claesson-Welsh, L., Siegbahn, A., Zsebo, K. M., Westermark, B., and Heldin, C. H. (1991) EMBO J. 10, 4121-4128[Abstract] |
32. |
Toullec, D.,
Pianetti, P.,
Coste, H.,
Bellevergue, P.,
Grand-Perret, T.,
Ajakane, D.,
and Kirilovsky, J.
(1991)
J. Biol. Chem.
266,
15771-15781 |
33. | Crews, C. M., Alessandrini, A., and Erikson, R. L. (1992) Science 258, 478-481[Medline] [Order article via Infotrieve] |
34. |
Zheng, C.-F.,
and Guan, K.-L.
(1993)
J. Biol. Chem.
268,
11435-11439 |
35. |
Alessi, D. R.,
Cuenda, A.,
Cohen, P.,
Dudley, D. T.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
27489-27494 |
36. |
Favata, M. F.,
Horiuchi, K. Y.,
Manos, E. J.,
Daulerio, A. J.,
Stradley, D. A.,
Feeser, W. S.,
Van Dyk, D. E.,
Pitts, W. J.,
Earl, R. A.,
Hobbs, F.,
Copeland, R. A.,
Magolda, R. L.,
Scherle, P. A.,
and Trzaskos, J. M.
(1998)
J. Biol. Chem.
273,
18623-18632 |
37. | Wymann, M. P., Bulgarelli-Leva, G., Zvelebil, M. J., Pirola, L., Vanhaesebroeck, B., Waterfield, M. D., and Panayotou, G. (1996) Mol. Cell. Biol. 16, 1722-1733[Abstract] |
38. |
Zhang, P.,
Wang, Y.-Z.,
Kagan, E.,
and Bonner, J. C.
(2000)
J. Biol. Chem.
275,
22479-22486 |
39. | Tang, Y., Chen, Z., Ambrose, D., Liu, J., Gibbs, J. B., Chernoff, J., and Field, J. (1997) Mol. Cell. Biol. 17, 4454-4464[Abstract] |
40. | Khosravi-Far, R., Chrzanowska-Wodnicka, M., Solski, P. A., Eva, A., Burridge, K., and Der, C. J. (1994) Mol. Cell. Biol. 14, 6848-6857[Abstract] |
41. | Hunter, T. (1995) Cell 80, 225-236[Medline] [Order article via Infotrieve] |
42. |
Becker, S.,
Quay, J.,
and Soukup, J.
(1991)
J. Immunol.
147,
4307-4312 |
43. |
Franklin, C. C.,
and Kraft, A. S.
(1997)
J. Biol. Chem.
272,
16917-16923 |
44. |
Finlay, G. A.,
Thannickal, V. J.,
Fanburg, B. L.,
and Paulson, K. E.
(2000)
J. Biol. Chem.
275,
27650-27656 |
45. |
Laird, A. D.,
Taylor, S. J.,
Oberst, M.,
and Shalloway, D.
(1995)
J. Biol. Chem.
270,
26742-26745 |
46. | Grammer, T. C., and Blenis, J. (1997) Oncogene 14, 1635-1642[CrossRef][Medline] [Order article via Infotrieve] |
47. | Nemoto, T., Ohashi, K., Akashi, T., Johnson, J. D., and Hirokawa, K. (1997) Pathobiology 65, 195-203[Medline] [Order article via Infotrieve] |
48. |
Welch, D. R.,
Sakamaki, T.,
Pioquinto, R.,
Leonard, T. O.,
Goldberg, S. F.,
Hon, Q.,
Erikson, R. L.,
Rieber, M.,
Rieber, M. S.,
Hicks, D. J.,
Bonventre, J. V.,
and Alessandrini, A.
(2000)
Cancer Res.
60,
1552-1556 |
49. |
McCawley, L. J.,
Li, S.,
Wattenberg, E. V.,
and Hudson, L. G.
(1999)
J. Biol. Chem.
274,
4347-4353 |
50. | Simon, C., Hicks, M. J., Nemechek, A. J., Mehta, R., O'Malley, B. W., Jr., Goepfert, H., Flaitz, and Boyd, D. (1999) Br. J. Cancer 80, 1412-1419[CrossRef][Medline] [Order article via Infotrieve] |
51. | Sebolt-Leopold, J. S., Dudley, D. T., Herrera, R., Van Becelaere, K., Wiland, A., Gowan, R. C., Tecle, H., Barrett, S. D., Bridges, A., Pryzbranowski, S., Leopold, W. R., and Saltiel, A. R. (1999) Nature Med. 5, 810-816[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Hu, L.,
Zaloudek, C.,
Mills, G. B.,
Gray, J.,
and Jaffe, R. B.
(2000)
Clin. Cancer Res.
6,
880-886 |
53. | Sun, H., Tonks, N. K., and Bar-Sagi, D. (1994) Science 266, 285-288[Medline] [Order article via Infotrieve] |
54. | Loda, M., Capodieci, P., Mishra, R., Yao, H., Corless, C., Grigioni, W., Wang, Y., Magi-Galluzzi, C., and Stork, P. J. (1996) Am. J. Pathol. 149, 1553-1564[Abstract] |
55. | Magi-Galluzzi, C., Mishra, R., Fiorentino, M., Montironi, R., Yao, H., Capodieci, P., Wishnow, K., Kaplan, I., Stork, P. J., and Loda, M. (1997) Lab. Invest. 76, 37-51[Medline] [Order article via Infotrieve] |