Role of Tyrosine Kinase Activity of Epidermal Growth Factor
Receptor in the Lysophosphatidic Acid-stimulated Mitogen-activated
Protein Kinase Pathway*
Jess M.
Cunnick
§¶,
Jay F.
Dorsey
§,
Todd
Standley
,
James
Turkson
,
Alan J.
Kraker
,
David W.
Fry
,
Richard
Jove
, and
Jie
Wu
§
§§
From the
Molecular Oncology Program, H. Lee Moffitt
Cancer Center and Research Institute, § Department of
Medical Microbiology and Immunology,
Department of Biochemistry
and Molecular Biology, University of South Florida College of Medicine,
Tampa, Florida 33612 and 
Parke-Davis
Pharmaceutical Research, Ann Arbor, Michigan 48105
 |
ABSTRACT |
Recent evidence indicates that the epidermal
growth factor (EGF) receptor mediates a branch of lysophosphatidic acid
(LPA)-induced signal transduction pathways that activate
mitogen-activated protein (MAP) kinase. However, it is unclear whether
the intrinsic tyrosine kinase activity of EGF receptor is involved. We
previously showed that reactive oxygen species (ROS) were involved in
the LPA-stimulated MAP kinase pathway. Here, we identify tyrosine
phosphorylation of EGF receptor as an LPA signaling step that requires
ROS. To evaluate the role of the tyrosine kinase activity of EGF
receptor in the LPA-stimulated MAP kinase pathway, we examined the
effects of an EGF receptor-specific tyrosine kinase inhibitor,
PD158780. PD158780 potently inhibited the LPA-stimulated MAP kinase
kinase 1/2 (MKK1/2) activation and EGF receptor tyrosine
phosphorylation in HeLa cells, while it had no detectable effect on
c-Src kinase activity. PD158780 also inhibited LPA-induced MKK1/2
activation and DNA synthesis in NIH 3T3 cells. Furthermore, we compared
LPA-stimulated MKK1/2 and MAP kinase activation, transcriptional
activity of the c-fos promoter, and DNA synthesis in B82L
cells, which lack endogenous EGF receptor, and B82L cells expressing
kinase-defective or wild-type human EGF receptor. Results obtained from
analysis of these cell lines suggest that the EGF receptor tyrosine
kinase contributes to the LPA-stimulated MAP kinase activation,
c-fos transcription, and mitogenesis.
 |
INTRODUCTION |
Lysophosphatidic acid
(LPA)1 is a bioactive
phospholipid present in serum. LPA concentrations in serum are normally
in the range of 2-20 µM (1), but higher concentrations
have been reported (2). LPA induces cellular responses by binding to a
specific cell surface receptor(s) that is coupled to Gi,
Gq, and G12/13 subfamilies of heterotrimeric
G-proteins (1, 3). LPA rapidly activates the mitogen-activated protein
(MAP) kinase cascade, consisting of Ras, Raf-1, MAP kinase kinase (MKK
or MEK) 1 and MKK 2 (MKK1/2), and MAP kinases (also called ERKs)
(4-9). It has been observed that LPA, as well as other agonists of G
protein-coupled receptors, induces tyrosine phosphorylation of several
signaling proteins in diverse cell types (6, 10-15). Activation of
protein tyrosine kinases, such as Src and PYK2, by agonists of G
protein-coupled receptors have been reported in certain cell types (15,
16), but not others (17). The LPA-stimulated MAP kinase pathway is sensitive to certain protein tyrosine kinase inhibitors such as genistein (6).2 These
observations suggest that regulation of protein tyrosine phosphorylation is an important signaling mechanism of LPA and other
agonists of G protein-coupled receptors.
Recently, it was reported that LPA and some other agonists of G
protein-coupled receptors stimulate tyrosine phosphorylation of the
epidermal growth factor (EGF) receptor (14, 15). Expression of a
truncated human EGF receptor lacking the cytoplasmic domain in Rat1
cells abrogated LPA-stimulated MAP kinase activation, suggesting that
the EGF receptor mediates at least a branch of the LPA-stimulated MAP
kinase activation pathway (14). However, it is unclear whether
intrinsic tyrosine kinase activity of EGF receptor is involved in G
protein-coupled receptor-stimulated tyrosine phosphorylation of the EGF
receptor, MAP kinase activation, and downstream cellular
responses such as gene transcription and DNA synthesis.
EGF stimulates tyrosine phosphorylation of its receptor by inducing
homodimerization of EGF receptor and heterodimerization of EGF receptor
with ErbB-2 or ErbB-4, and activation of these receptor tyrosine
kinases (18-21). However, an increase in EGF receptor tyrosine
phosphorylation can also occur without activation of the receptor
tyrosine kinase. First, EGF receptor is basally active in the absence
of EGF. The basal phosphorylating activity of EGF receptor is balanced
by dephosphorylating activity (22). Thus, suppression of the
dephosphorylating activity, such as incubation of cells with a protein
tyrosine phosphatase inhibitor, will increase tyrosine phosphorylation
of EGF receptor.2 Second, other cellular kinases such as
c-Src or putative Src-activated tyrosine kinases may also cause
tyrosine phosphorylation of EGF receptor (15, 23). By using transient
expression of the Src-inactivating kinase, CSK, or a dominant negative
c-Src mutant, it was reported that c-Src mediates G
-
and LPA-induced tyrosine phosphorylation of the EGF receptor in COS
cells (15). However, similar experiments indicated that c-Src is not
involved in LPA signaling in Rat1 cells (3, 17).
The main objective of this study is to evaluate whether the intrinsic
EGF receptor tyrosine kinase activity contributes to the LPA-induced
tyrosine phosphorylation of the EGF receptor and MAP kinase pathway. We
previously found that reactive oxygen species (ROS) are involved in the
LPA-stimulated MAP kinase activation pathway (25). We report here that
LPA-induced tyrosine phosphorylation of the EGF receptor requires ROS.
Importantly, using a newly developed specific inhibitor of EGF receptor
tyrosine kinase (24) and B82L cells that lack EGF receptor, we have
found that the intrinsic tyrosine kinase activity of the EGF receptor
is involved in the LPA-stimulated MAP kinase pathway.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Lysophosphatidic acid (1-oleoyl) and enolase were
from Sigma. Anti-phosphotyrosine antibody PY20 was from Transduction
Laboratories. Anti-EGF receptor antibody LA22 and anti-Src antibody
GD11 were from UBI. Polyclonal antibody to EGF receptor was from Santa
Cruz Biotech. Heregulin and anti-ErbB-2 antibody were from NeoMarkers. Anti-Active MAPK antibody and the luciferase assay system were from
Promega. Anti-MAP kinase antibody TR12 was kindly provided by Dr.
Michael J. Weber (University of Virginia). Enhanced chemiluminescent SuperSignalTM substrate kit was from Pierce. Ray-tide
was from Calbiochem. Monoclonal antibody against amino acid residues
2-17 of Src (N2-17) was from the National Cancer Institute
Repository. [methyl-3H]Thymidine (85.5 Ci/mmol) was from NEN Life Science Products. PD158780
(4-[(3-bromophenyl)amino]-6-(methylamino)pyrido[3,4-d]pyrimidine) is a specific inhibitor of the EGF receptor tyrosine kinase recently developed at Parke-Davis Pharmaceutical Research (25).
Cell Culture--
HeLa cells and NIH 3T3 cells were grown and
serum-starved as described previously (24). B82L cell lines were grown
as described (26).
Immunoprecipitation and Immunoblotting--
For immunoblotting
analyses of EGF receptor or ErbB-2 following immunoprecipitation, cells
were lysed in (1 ml/10-cm plate) lysis buffer (50 mM
Tris·HCl, pH 7.3, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet
P-40, 0.5 mM Na3VO4, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 100 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml each of aprotinin, leupeptin, and pepstatin). Precleared supernatants were incubated with
anti-EGF receptor antibody LA22. Immunoprecipitates were collected with
protein G-agarose. Immunoprecipitated proteins were separated on 7.5%
SDS-polyacrylamide gels and transferred to nitrocellulose filters (in
some experiments, Immobilon-P filters were used). For immunoblotting
analyses using anti-EGF receptor and anti-ErbB-2 antibodies, blots were
probed with antibodies in 10 mM Tris·HCl, pH 7.5, 100 mM NaCl, 0.05% Tween 20 (TBST) containing 5% dry milk.
For immunoblotting analyses using anti-phosphotyrosine antibody (PY20),
filters were blocked with ovalbumin in TBST. Blots were developed by
the enhanced chemiluminescent method following incubation with
horseradish peroxidase-conjugated secondary antibodies.
Kinase Assays--
Total MKK1/2 activity was determined using a
kinase defective p42MAPK mutant (KR) as substrate as
described previously (24). Total MKK1/2 activity is used as a measure
of activation of the MAP kinase pathway (24, 27). Activation of MAP
kinase, p42MAPK and p44MAPK, was assayed by
immunoblotting using the anti-Active MAPK antibody (Promega), which
only reacts with the activated dual threonine and
tyrosine-phosphorylated p42MAPK and p44MAPK.
For Src kinase activity assays, c-Src was immunoprecipitated from cell
lysates using the anti-Src antibody N2-17 (in some experiments, monoclonal antibody GD11 was used). Src immune complexes were mixed
with Src-kinase buffer (25 mM Hepes, 50 mM
NaCl, 0.01% Brij-35, 10 mM MgCl2, 25 µM [
-32P]ATP (1,000 cpm/pmol))
containing 75 µM Raytide in a total volume of 30 µl.
After incubating at 30 °C for 12 min, the reaction was terminated by
addition of 50 µl of 10% phosphoric acid. Following a brief
centrifugation, 60 µl of each supernatant was spotted onto P81
filters. The filters were washed 5 times with 2% phosphoric acid and
radioactivity remaining on the filters was determined. In experiments
using enolase as Src substrate, enolase (5 µg/reaction) was denatured
with 25 µM acetic acid at 30 °C for 5 min and added to
1/10 of the total kinase reaction volume. Phosphorylation of acid-denatured enolase was analyzed by a PhosphorImager following SDS-polyacrylamide gel electrophoresis.
Transcriptional Activity of c-fos Promoter--
Plasmid pLucSRE
(28) contains a luciferase reporter gene controlled by two tandem
copies of the c-fos promoter. To determine transcriptional
activity of the c-fos promoter, cells were seeded in 35-mm
plates in triplicate 20-24 h prior to transfection. A mixture of
pLucSRE (1.5 µg) and the
-galactosidase expression vector
pCMV
gal (0.075 µg) was used to transfect each plate of cells using
the Superfect transfection reagent (Qiagen) according to the
supplier's instruction. Twenty-two h after transfection, cells were
serum-starved for 24 h and then stimulated as indicated in the
legend of Fig. 8. Luciferase activity was determined using the
Luciferase Assay System from Promega according to supplier's protocol
and a Barthold luminometer.
-Galactosidase activity was determined
by a colorimetric assay as described (28). Luciferase activity was
normalized to
-galactosidase activity as an internal control for
transfection efficiency.
DNA Synthesis--
DNA synthesis was determined by
[3H]thymidine incorporation assay essentially as
described (29). Statistical analyses were performed using the
nonparametric Wilcoxon Rank Sum test as well as two sample t
test (assuming unequal variances). Differences in means were
considered statistically significant when both tests indicated p < 0.05.
 |
RESULTS |
Stimulation of Tyrosine Phosphorylation of EGF Receptor by
LPA--
To determine whether LPA stimulates tyrosine phosphorylation
of EGF receptor in HeLa cells, confluent, serum-starved cells were
treated with LPA or EGF and lysed in detergents. EGF receptor was
immunoprecipitated from cell lysates and analyzed with an antibody
against phosphotyrosine. Fig.
1A shows that LPA induced tyrosine phosphorylation of EGF receptor in HeLa cells. Slightly higher
levels of EGF receptor tyrosine phosphorylation were consistently detected in cells treated with LPA for 2.5 min than for 4 min, suggesting a transient nature of the LPA-stimulated tyrosine
phosphorylation of EGF receptor. Quantification of the EGF receptor
bands by a densitometer indicated that a 2.5-min stimulation of HeLa
cells with 10 µM LPA (a near-saturation concentration for
HeLa cells) increased tyrosine phosphorylation of the EGF receptor
approximately 3-fold (3.1 ± 0.5 in four experiments), which is
similar to that induced by 0.17 nM EGF in HeLa cells. Other
experiments showed that 10 µM LPA and 0.17-0.34
nM EGF activate MKK 1 and MKK 2 (MKK1/2) to a similar
extent in HeLa cells.2

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Fig. 1.
LPA induces tyrosine phosphorylation of the
EGF receptor. A, serum-starved HeLa cells were left
untreated, or treated with LPA or EGF as indicated. The EGF receptor
was immunoprecipitated from cell lysates, and analyzed by
immunoblotting with the anti-phosphotyrosine antibody PY20.
B, serum-starved HeLa cells were treated with LPA (40 µM, 2.5 min), H2O2 (5 mM, 5 min), heregulin (50 nM, 4 min), or EGF
(3.4 nM, 3 min). Cell lysates were incubated with
anti-phosphotyrosine antibody PY20 to immunoprecipitate
tyrosine-phosphorylated proteins. Immunoprecipitated proteins were
probed with an anti-ErbB-2 antibody.
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To assess whether LPA also induces tyrosine phosphorylation of other
growth factor receptors, we analyzed tyrosine phosphorylation of ErbB-2
in HeLa cells. While both heregulin and EGF markedly induced tyrosine
phosphorylation of ErbB-2 in HeLa cells, LPA did not appear to affect
ErbB-2 in HeLa cells (Fig. 1B). In other experiments,2 we found no detectable tyrosine
phosphorylation of the PDGF receptor in LPA-stimulated NIH 3T3 and Rat1
cells (HeLa cells have no PDGF receptor), while control experiments
showed that LPA-stimulated tyrosine phosphorylation of EGF receptor in
NIH 3T3 cells. Thus, LPA appears to preferentially induce tyrosine
phosphorylation of EGF receptor.
Role of ROS in LPA-induced Tyrosine Phosphorylation of the EGF
Receptor--
We reported previously that LPA rapidly increased
intracellular levels of ROS and that ROS were involved in
LPA-stimulated MKK1/2 activation (24). Similarly, LPA-stimulated Raf-1
activation is also mediated by ROS.2 Other evidence
suggests that ROS modulate the MAP kinase pathway at signaling steps
upstream of the Ras protein (30). To assess whether ROS have a role in
LPA-induced tyrosine phosphorylation of the EGF receptor, we analyzed
the effect of the antioxidant N-acetylcysteine (NAC) on
LPA-induced tyrosine phosphorylation of the EGF receptor. Our previous
studies showed that NAC attenuated the LPA-stimulated MKK1/2 activity
in HeLa cells (24). As illustrated in Fig.
2A, NAC effectively blocked
the LPA-induced tyrosine phosphorylation of the EGF receptor in HeLa
cells. Consistent with these results, direct exposure of HeLa cells to
H2O2 induced tyrosine phosphorylation of EGF
receptor (Fig. 2A). The increase in tyrosine phosphorylation of EGF receptor resulted in increased binding affinity of EGF receptor
to the signaling adaptor protein Grb2, as demonstrated by binding to a
GST-Grb2 fusion protein in vitro (Fig. 2C),
confirming a previous observation (14).

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Fig. 2.
Inhibition of LPA-induced tyrosine
phosphorylation of the EGF receptor by antioxidants. A and
B, serum-starved HeLa cells were pretreated with NAC (30 mM, 90 min) or left untreated, followed by stimulation with
LPA (10 µM, 2.5 min), EGF (0.34 nM, 3 min),
or H2O2 (2 mM, 3 min). EGF receptor
was immunoprecipitated from cell lysates with anti-EGF receptor
antibody LA22 and analyzed by immunoblotting with the
anti-phosphotyrosine antibody PY20 (A). After removing the
antibody from the filter, the filter was reprobed with a polyclonal
anti-EGF receptor antibody (B). (C), HeLa cells
were treated with LPA, NAC + LPA, EGF, or left untreated as above, and
cell lysates were incubated with agarose-conjugated GST-GRB2 fusion
protein. GRB2-associated EGF receptor was then analyzed by
immunoblotting with an anti-EGF receptor antibody. D, HeLa
cells were pretreated with aminotriazole (ATZ, 50 mM, 90 min), L-histidine (His, 50 mM, 60 min), dimethyl sulfoxide (4%, 20 min) or left
untreated, followed by stimulation for 2.5 min with LPA (10 µM) or EGF (0.34 nM). Tyrosine
phosphorylation of the EGF receptor was analyzed as described in
panel A.
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To verify that the inhibitory effect of NAC was attributable to its ROS
scavenger/antioxidant activity, we determined whether other
antioxidants could similarly block the LPA-induced tyrosine phosphorylation of EGF receptor. L-Histidine and dimethyl
sulfoxide are hydroxyl radical scavengers (31) that inhibit the
LPA-induced MKK1/2 activation in HeLa and NIH 3T3 cells (24). Fig.
2D shows that both L-histidine and dimethyl
sulfoxide attenuated the LPA-induced tyrosine phosphorylation of EGF
receptor. By contrast, the catalase inhibitor aminotriazole, which
augments the LPA stimulated MKK1/2 activity (24), slightly enhanced the
LPA-induced tyrosine phosphorylation of EGF receptor in HeLa cells.
These data indicate that ROS are required for LPA-stimulated tyrosine
phosphorylation of the EGF receptor.
ROS Negatively Regulate c-Src Activity--
A protein tyrosine
kinase that could potentially contribute to tyrosine phosphorylation of
the EGF receptor is c-Src. It was observed previously that LPA
marginally activated c-Src kinase in neuroblastoma cells, but not in
fibroblasts (32). In addition, a recent report suggests that c-Src
mediates G
- and LPA-induced tyrosine phosphorylation
of the EGF receptor (15). Despite many attempts, we have not been able
to detect activation of c-Src tyrosine kinase activity by LPA in HeLa
cells (for an example, see Fig.
3B) or NIH 3T3
cells.2 Because ROS positively regulate tyrosine
phosphorylation of EGF receptor and the MAP kinase pathway in HeLa
cells, we examined whether H2O2 could activate
c-Src kinase in HeLa cells. Serum-starved HeLa cells were treated with
H2O2 or LPA and c-Src was immunoprecipitated. c-Src kinase activity was determined in the immune complexes using either acid-denatured enolase (Fig. 3A) or Raytide (Fig.
3B) as substrate. As shown in Fig. 3, basal activity of
c-Src kinase was detectable in serum-starved HeLa cells.
H2O2 treatment reduced c-Src kinase activity in
a concentration-dependent manner. These data indicate that
ROS negatively regulate c-Src kinase activity, which is opposite to the
effects of ROS on LPA-stimulated tyrosine phosphorylation of EGF
receptor and the MAP kinase pathway (Ref. 24, Fig. 2).

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Fig. 3.
H2O2 decreases c-Src
kinase activity in HeLa cells. A, serum-starved HeLa cells
were treated for 3 min with the indicated concentrations of
H2O2 and c-Src kinase activity was determined
in immune complexes using acid-denatured enolase as substrate.
B, serum-starved HeLa cells were treated for 3 min with LPA
(10 µM), H2O2 (2 mM)
or mock-treated. c-Src was immunoprecipitated from cell lysates and Src
kinase activity was determined using Raytide as substrate.
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Inhibition of LPA-stimulated MKK1/2 Activation and Tyrosine
Phosphorylation of the EGF Receptor by PD158780--
The compound
PD158780 is a newly developed, specific inhibitor of the EGF receptor
tyrosine kinase (25). PD158780 is more potent than another previously
reported specific inhibitor of the EGF receptor tyrosine kinase,
PD153035 (33). In human epidermoid carcinoma cells and in mouse
fibroblasts, PD158780 inhibits EGF-stimulated autophosphorylation of
its receptor with an IC50 of approximately 13 nM (34). To evaluate whether the intrinsic EGF receptor
tyrosine kinase activity is required for the LPA-stimulated MAP kinase pathway, we determined whether inhibition of the EGF receptor tyrosine
kinase activity by PD158780 affected LPA-stimulated MKK1/2 activation.
Serum-starved HeLa cells were preincubated with 0-100 nM
PD158780 followed by LPA stimulation, and MKK1/2 activity was assayed.
Fig. 4A shows that PD158780
inhibited LPA-stimulated MKK1/2 activity in a
concentration-dependent manner in HeLa cells. The
IC50 value for the effect of PD158780 on LPA-stimulated
MKK1/2 activation is approximately 20 nM, which is very
similar to that for inhibition of EGF receptor autophosphorylation.
Fig. 4B illustrates the inhibitory effects of 50 nM PD158780 on LPA- and EGF-stimulated MKK1/2 activity in
HeLa. In other experiments, we examined whether PD158780 affected
PDGF-stimulated MKK1/2 activity in NIH 3T3 cells. Although control
experiments showed that PD158780 (50 nM) inhibited LPA- and
EGF-stimulated MKK1/2 activation (Fig. 4, C and
D), preincubation of NIH 3T3 cells with PD158780 (50 nM) had little effect on the PDGF-stimulated MKK1/2
activity in NIH 3T3 cells (Fig. 4D).

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Fig. 4.
Inhibition of LPA-stimulated MKK1/2
activation by PD158780. A, confluent, serum-starved HeLa
cells were incubated for 120 min with the indicated concentrations of
PD158780 or mocked-treated, and then stimulated with LPA (10 µM, 4 min). B, HeLa cells were preincubated
with 50 nM PD158780 or mock-treated, and then stimulated
for 4 min with LPA (10 µM), EGF (0.34 nM), or
an equal volume of 1% fatty acid-free bovine serum albumin
(N). C and D, confluent, serum-starved
NIH 3T3 cells were pretreated with 50 nM PD158780 followed
by stimulation with LPA (10 µM), EGF (3.4 nM), or PDGF-BB (1.25 nM). Total MKK1/2
activity was determined by phosphorylation of a kinase-defective
p42MAPK mutant (KR). Quantification of KR
phosphorylation was accomplished by a PhosphorImager after
SDS-polyacrylamide gel electrophoresis.
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To confirm that the inhibitory effect of PD158780 on LPA-stimulated
MKK1/2 activation is due to suppression of LPA-induced tyrosine
phosphorylation of the EGF receptor, we examined LPA-induced tyrosine
phosphorylation of the EGF receptor in HeLa cells with or without
PD158780 pretreatment. Confluent, serum-starved HeLa cells were
pretreated with PD158780 (50 nM) or mock-treated, followed by LPA stimulation. The EGF receptor was immunoprecipitated and analyzed by immunoblotting with an anti-phosphotyrosine antibody. As
shown in Fig. 5, preincubation of HeLa
cells with PD158780 (50 nM) completely blocked LPA-induced
tyrosine phosphorylation of the EGF receptor.

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Fig. 5.
PD158780 blocks LPA-induced tyrosine
phosphorylation of the EGF receptor. Confluent, serum-starved HeLa
cells were incubated with PD158780 (50 nM, 10 min) or
mocked-treated, and then stimulated with LPA (40 µM, 2.5 min) or bovine serum albumin as control (N). EGF receptor
was immunoprecipitated from cell lysates, separated on 7.5%
SDS-polyacrylamide gels, transferred to an Immobilon-P filter, and
analyzed with the anti-phosphotyrosine antibody PY20 (A).
After removing the PY20 antibody from the filter, the filter was
reprobed with a polyclonal anti-EGF receptor antibody
(B).
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To assess the possibility that PD158780 may nonspecifically inhibit
c-Src kinase activity, we immunoprecipitated c-Src from serum-starved
HeLa cells and determined c-Src kinase activity in the presence or
absence of PD158780 (50 nM) in the kinase reaction. Alternatively, serum-starved HeLa cells were treated with PD158780 (50 nM) and the c-Src kinase activity was subsequently
determined following immunoprecipitation. Even though our assay is able
to detect not only the basal kinase activity of c-Src but also
decreases in c-Src kinase activity below the basal level (Fig. 3), no
inhibitory effect of PD158780 on c-Src kinase activity was detected
either in vitro or in vivo (Fig.
6). These data indicate that the
inhibitory effect of PD158780 on LPA-stimulated MKK1/2 activation is
not attributed to the nonspecific effect of PD158780 on c-Src kinase activity.

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Fig. 6.
PD158780 has no inhibitory effect on c-Src
kinase activity in vitro and in vivo. For
determining the in vitro effect of PD158780 on c-Src kinase
activity, c-Src was immunoprecipitated from confluent, serum-starved
HeLa cells, and then c-Src kinase activity was assayed using Raytide as
substrate in the presence or absence of 50 nM PD158780. For
the in vivo effect of PD158780, serum-starved cells were
incubated with 50 nM PD158780 for 120 min and c-Src kinase
activity was assayed after immunoprecipitating c-Src from cell
lysates.
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Comparison of LPA-stimulated MKK1/2 and MAP Kinase Activation in
Mouse B82L Fibroblasts and B82L Cell Lines Expressing the Wild-type and
Kinase-defective Human EGF Receptor--
B82L is a mouse L fibroblast
cell line that has no detectable EGF receptor mRNA and protein
(Refs. 35 and 36; Fig. 7A). To
further evaluate the role of intrinsic kinase activity of EGF receptor
in the LPA-stimulated MAP kinase pathway, we compared the
LPA-stimulated MKK1/2 activity in parental B82L cells, and transfected
B82L cell lines expressing the wild-type human EGF receptor
(B82L/hER+) or the kinase-defective human EGF receptor
(B82L/hER
) (27). Immunoblotting analysis confirmed that
EGF receptor was expressed in B82L/hER+ and
B82L/hER
cells (Fig. 7A). The level of EGF
receptor expression in B82L/hER
cells appears to be
higher than that in B82L/hER+ cells (Fig.
7A).

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Fig. 7.
LPA-stimulated MKK1/2 and MAP kinase
activation in B82L cell lines. Serum-starved B82L,
B82L/hER+, and B82L/hER cells were stimulated
with LPA (50 µM, 3 min), EGF (1.7 nM, 3 min),
or left untreated. A, portions of cell lysates (10 µg)
were analyzed by immunoblotting for EGF receptor using a polyclonal
anti-EGF receptor antibody that reacts with both mouse and human EGF
receptor. B, the activity of MKK1/2 was determined using a
kinase-defective p42MAPK mutant (KR).
C, quantification of KR phosphorylation in two experiments
with a PhosphorImager. The averages and errors are shown.
Phosphorylation of KR in serum-starved B82L cells was set as 1 unit.
D and E, activation of MAP kinase was analyzed by
immunoblotting of cell lysates (10 µg) using an anti-Active MAP
kinase antibody that only reacts with the activated, dual threonine and
tyrosine phosphorylated forms of p42MAPK and
p44MAPK (D), and a duplicate filter (control)
was probed with antibody TR12 that reacts with total
p42MAPK and p44MAPK proteins
(E).
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LPA weakly activated MKK1/2 in B82L cells (2.9 ± 0.01-fold),
indicating that one or more EGF receptor-independent pathways exists
for MKK1/2 activation in this cell line (Fig. 7, B and C). This observation is consistent with the notion that
multiple signaling pathways mediate LPA signal transduction, including the LPA-stimulated MAP kinase pathway, and that EGF receptor mediates one of these pathways. While expression of kinase-defective human EGF
receptor increased LPA-stimulated MKK1/2 activation by 96% (5.7 ± 0.13-fold activation), expression of wild-type human EGF receptor
increased LPA-stimulated MKK1/2 activation by 196% (8.6 ± 0.31-fold activation) (Fig. 7, B and C). These
observations suggest that EGF receptor is involved in one of the LPA
signal transduction pathways that activate the MAP kinase cascade, and that another cellular kinase activity as well as the intrinsic kinase
activity of EGF receptor both contribute to the function of EGF
receptor in these cells.
To confirm that similar changes in MAP kinase activation also occur in
B82L, B82L/hER+, B82L/hER
cells, LPA- and
EGF-stimulated p42MAPK/p44MAPK activation was
analyzed. As shown in Fig. 7D, LPA- and EGF-stimulated activation of p42MAPK/p44MAPK in these cells
correlated well with that of MKK1/2. Additional experiments were also
performed to analyze tyrosine phosphorylation of Shc proteins. Shc has
been implicated in mediating signal transduction of receptor tyrosine
kinases (37). The 52- and 46-kDa Shc proteins were detected in B82L and
the two B82L-derived cell lines. The 52-kDa Shc is basally
phosphorylated on tyrosine in B82L cells. While LPA did not increase
tyrosine phosphorylation of Shc in B82L or B82L/hER
cells, it stimulated tyrosine phosphorylation of the 52-kDa Shc in
B82L/hER+ cells.2 However, a much higher level
of 52-kDa Shc tyrosine phosphorylation was observed in EGF-stimulated
B82L/hER
cells (most likely due to heterodimerization of
EGF receptor with ErbB-2) than that detected in LPA-stimulated
B82L/hER
cells and B82L/hER+
cells.2 Therefore, the levels of Shc tyrosine
phosphorylation did not appear to correlate well with activation of the
MAP kinase pathway (Fig. 7) or transcriptional activity of the
c-fos promoter described below (Fig.
8).

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Fig. 8.
LPA- and EGF-induced c-fos
promoter transcriptional activity in B82L cell lines and in HeLa cells.
A, B82L cell lines were co-transfected with pLucSRE and
pCMV gal. Serum-starved transfected cells in triplicates were
stimulated with LPA (50 µM, 3 h) or EGF (10 nM, 6 h) or left untreated. Luciferase activity was
then determined in cell lysates and normalized for transfection
efficiency with -galactosidase activity. Similar results were
obtained in repeated experiments. B, HeLa cells were
transfected with pLucSRE and pCMV gal. Serum-starved transfected
cells were pretreated with PD158780 (50 nM, 1 h) or
mock-treated. Following stimulation with LPA (10 µM,
8 h) or EGF (0.85 nM, 8 h), luciferase activity
was measured and normalized to -galactosidase activity. The data
represent the mean and standard deviations of a triplicate and a
duplicate experiment. The normalized luciferase activities in
non-stimulated B82L (A) and HeLa cells (B) were
arbitrarily set as 1 unit.
|
|
c-fos Promoter Activation and DNA Synthesis--
LPA is known to
induce transcription of the c-fos gene (38, 39) and have
mitogenic activity in some cells (1, 2). Previous studies in NIH 3T3
cells suggest that induction of c-fos transcription by LPA
is mediated primarily via the serum response element (SRE), which is
controlled by both the MAP kinase-regulated ternary complex factor as
well as the MAP kinase-independent, Rho-mediated serum response factor
(39). To further assess the role of EGF receptor in LPA signaling, we
compared transcriptional activity of the c-fos promoter in
B82L, B82L/hER+, and B82L/hER
cells. pLucSRE,
which contains a luciferase reporter gene controlled by the
c-fos promoter, was transfected into B82L,
B82L/hER+, and B82L/hER
cells. Luciferase
activity in the cell lysates were measured after stimulation of cells
with LPA or EGF. As shown in Fig. 8A, low, but detectable,
LPA-stimulated luciferase activity was observed in B82L cells. On the
other hand, EGF did not activate the c-fos promoter in B82L
cells. While expression of the kinase-defective EGF receptor increased
c-fos promoter activity in LPA-stimulated B82L/hER
cells to 2.2 ± 0.07-fold of that observed
in LPA-stimulated B82L cells, expression of the wild-type EGF receptor
further increased c-fos promoter activity in LPA-stimulated
B82L/hER+ to 6.6 ± 0.26-fold of that detected in
LPA-stimulated B82L cells (Fig. 8A). Increases in
c-fos promoter activity were also observed in EGF-stimulated
B82L/hER
cells and B82L/hER+ cells (Fig.
8A). These changes in c-fos promoter activity
were similar to the changes observed above in MKK1/2 and MAP kinase activation in these three cell lines, suggesting that EGF receptor contributes to the LPA-induced c-fos transcriptional
activity.
c-fos promoter activity was also analyzed in HeLa cells.
Fig. 8B shows that the EGF receptor inhibitor PD158780
reduced the LPA-stimulated c-fos promoter activity by 62%
and the EGF-stimulated c-fos promoter activity by 54%.
Thus, inhibition of the EGF receptor tyrosine kinase activity decreased
the LPA- and EGF-stimulated c-fos promoter activity,
indicating that the intrinsic EGF receptor tyrosine kinase contributes
to the LPA-induced c-fos transcription in HeLa cells.
Analysis of DNA synthesis by the [3H]thymidine
incorporation assay in three independent triplicate experiments
indicated that LPA did not induce DNA synthesis in B82L cells (Table
I). DNA synthesis in LPA-treated
B82L/hER
and B82L/hER+ cells was 1.16 ± 0.27-fold and 1.49 ± 0.24-fold of the controls, respectively
(Table I). While the 16% increase in DNA synthesis in LPA-stimulated
B82L/hER
cells is not statistically significant
(p > 0.05 in both the Wilcoxon Rank Sum test and the
t test), the 49% increase in DNA synthesis induced by LPA
in B82L/hER+ cells is statistically significant
(p < 0.001 in both tests). Thus, the DNA synthesis
analysis indicates that EGF receptor tyrosine kinase activity
contributes to the LPA-induced mitogenic response.
View this table:
[in this window]
[in a new window]
|
Table I
LPA-stimulated DNA synthesis in B82L cell lines
Cells were grown to 50% confluence and serum-starved for 24 h,
and then stimulated with LPA (50 µM), EGF (10 nM), or mock-treated with BSA. Six h (experiments I and II)
or 18 h (experiment III) after stimulation,
[methyl-3H]thymidine (2.5 µCi/ml) was added to
the cells. Twenty-four h after stimulation, cells were processed to
determine [3H]thymidine incorporation into DNA. Each
experiment was performed in triplicate. The lower levels of
[3H]thymidine incorporation into DNA in transfected cells is
probably due to overexpression of dihydrofolate reductase in these
cells, which is predicted to increase de novo synthesis of dTMP.
|
|
Surprisingly, EGF, which clearly activates MAP kinase and the
c-fos promoter in B82L/hER+ cells to higher
extents than that induced by LPA, did not stimulate DNA synthesis in
B82L/hER+ cells. In other experiments, we stimulated
serum-starved B82L/hER+ cells with various concentrations
of EGF (0.05-20 nM) and detected no EGF-stimulated DNA
synthesis at all concentrations tested. In addition, we also detected
no EGF-stimulated DNA synthesis in B82L/hER+ cells in the
presence of insulin. This observation suggests that while the EGF
receptor-mediated MAP kinase-c-fos pathway is necessary for
the LPA-induced DNA synthesis in B82L cell lines, activation of the MAP
kinase-c-fos pathway alone is insufficient to induce DNA
synthesis in these cells. Signaling pathways besides MAP kinase and
c-fos transcription must also be activated for induction of DNA synthesis in B82L cell lines. Our data suggest that LPA is capable
of activating these additional signaling pathways.
To further evaluate the role of EGF receptor tyrosine kinase activity
in the LPA-stimulated mitogenic response, we analyzed LPA- and
EGF-stimulated DNA synthesis in NIH 3T3 cells. Fig.
9 shows that LPA (50 µM)
and EGF (10 nM) induced DNA synthesis in NIH 3T3 cells
approximately 16- and 24-fold, respectively. Addition of 50 nM PD158780 to the medium inhibited the EGF-stimulated DNA synthesis by 85% and the LPA-stimulated DNA synthesis by 63%. Fig. 4
shows that this concentration of PD158780 does not affect PDGF receptor
kinase in NIH 3T3 cells. A recent study by one of us (34) also
indicated that PD158780 was very specific for the EGF receptor tyrosine
kinase at nanomolar concentrations and did not affect PDGF- and basic
fibroblast growth factor-stimulated DNA synthesis at nanomolar
concentrations. Thus, the result shown in Fig. 9 confirms that EGF
receptor tyrosine kinase activity is involved in the mitogenic response
induced by LPA.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
Inhibition of LPA-induced DNA synthesis by
PD158780. Serum-starved NIH 3T3 cells were treated with PD158780
(50 nM, 1 h) or mock-treated, and then stimulated with
LPA (50 µM) or EGF (10 nM). Eighteen h after
stimulation, [methyl-3H]thymidine (3 µCi/ml)
was added and incubation continued for 6 h. After which, cells
were processed for determination of [3H]thymidine
incorporation into DNA (29). Data shown are the mean and standard
deviations of two independent experiments performed in
triplicate.
|
|
 |
DISCUSSION |
We previously reported that LPA rapidly raises intracellular
concentrations of ROS in HeLa cells and that ROS participate in the
LPA-stimulated MAP kinase pathway (24). Evidence presented in Fig. 2
indicates that induction of tyrosine phosphorylation of EGF receptor by
LPA requires ROS. Involvement of ROS in EGF-elicited tyrosine
phosphorylation of its receptor and signaling pathways was also
reported recently by other investigators (40). Although the mechanisms
by which ROS mediate tyrosine phosphorylation of EGF receptor and other
growth factor receptors remains to be delineated, a possible mechanism
is by inhibiting a specific protein tyrosine phosphatase activity
rather than directly increasing the EGF receptor kinase activity (22).
Protein tyrosine phosphatases contain a catalytic cysteine residue and
are sensitive to oxidative inhibition (41-44). Inhibition of protein
tyrosine phosphatase activity would shift the balance of tyrosine
phosphorylation and dephosphorylation of EGF receptor toward the
phosphorylation direction. Because of differences in sensitivity to
oxidative inhibition and in cellular location, inhibition of a specific
protein tyrosine phosphatase activity may be achieved under
physiological conditions in which only low levels of ROS are produced.
This protein tyrosine phosphatase inhibition hypothesis predicts that
basal phosphorylation of EGF receptor occurs in the absence of EGF, and
that inhibition of protein tyrosine phosphatase activity will increase
tyrosine phosphorylation of EGF receptor. Indeed, basal phosphorylation
of the EGF receptor in serum-starved cells was detectable in HeLa cells
(Figs. 1, 2, and 5). Moreover, treatment of HeLa cells with the protein tyrosine phosphatase inhibitor Na3VO4 resulted
in increased tyrosine phosphorylation of EGF receptor.2
Tyrosine phosphorylation has been recognized as an important signaling
mechanism of LPA and other G protein-coupled receptors (6, 10-15).
Recent evidence indicates that tyrosine phosphorylation of EGF receptor
mediates a branch of the LPA signaling pathways that leads to MAP
kinase activation (14, 15). While conflicting evidence has been
reported regarding the role of c-Src in signal transduction by LPA and
G protein-coupled receptors (14, 17), the role of intrinsic kinase
activity of EGF has not been critically evaluated. In the present
investigation, experiments were carried out to assess the involvement
of intrinsic kinase activity of EGF receptor in LPA-stimulated tyrosine
phosphorylation of EGF receptor and the MAP kinase activation pathway.
Our results show that in HeLa cells: (i) while LPA-stimulated MAP
kinase pathway and tyrosine phosphorylation of EGF receptor are
positively regulated by ROS, ROS negatively regulate c-Src kinase
activity; (ii) PD158780, a specific inhibitor for the EGF receptor
tyrosine kinase, is very potent in inhibiting LPA- and EGF-induced
MKK1/2 activation. PD158780 also inhibits LPA-induced c-fos
promoter activity in HeLa cells. Furthermore, PD158780 inhibits
LPA-induced tyrosine phosphorylation of the EGF receptor, whereas it
has no inhibitory effects on c-Src tyrosine kinase both in
vitro and in vivo. These observations suggest that the
intrinsic EGF receptor tyrosine kinase activity contributes to the
LPA-stimulated MAP kinase pathway in HeLa cells and that c-Src is
probably not involved. Other evidence has recently been obtained by
Boyer et al. (45) that indicates c-Src is not involved in
the EGF-stimulated MAP kinase pathway in NBT-II carcinoma cells.
Although our experiments suggest that c-Src is probably not involved in
the LPA-stimulated MKK1/2 activation in HeLa cells, we do not exclude
the possibility that other members of the Src family of protein kinases
may be involved. Many protein tyrosine kinases are positively regulated
by tyrosine phosphorylation. In contrast, c-Src is activated primarily
by dephosphorylation of a tyrosine residue at its C-terminal regulatory
region. This is consistent with our observation that exposure of cells
to H2O2, which inhibits protein tyrosine
phosphatase activities, decreases c-Src kinase activity (Fig. 3). On
the other hand, some other members of the Src family kinases, such as
Lck in T cells, are activated predominantly by autophosphorylation in
the kinase domain (46, 47). It is possible that one of these Src family
kinases or other cellular tyrosine kinases also contribute to
LPA-induced tyrosine phosphorylation of the EGF receptor in HeLa cells.
Furthermore, our data in no way argue against the possibility that
c-Src and EGF receptor may cooperatively regulate certain signal
transduction pathways that mediate cell transformation and
tumorigenesis (48).
The role of EGF receptor in LPA-stimulated MAP kinase activation was
further evaluated in mouse fibroblasts B82L, B82L/hER
,
and B82L/hER+ cells. LPA weakly activates MKK1/2 and MAP
kinases in B82L cells, suggesting that at least one alternate, EGF
receptor-independent signaling mechanism exists in this cell line. This
finding is in agreement with the notion that more than one signaling
pathway mediates LPA-stimulated MAP kinase activation. Expression of a kinase-defective human EGF receptor in B82L cells increases the level
of MAP kinase activation in LPA-stimulated cells, implying that another
cellular kinase activity contributes to the function of EGF receptor in
mediating the LPA signaling pathway in B82L cell lines. Because
cross-phosphorylation of the EGF receptor by other ErbB family members
requires EGF-induced heterodimerization, the cellular kinase that
contributes to the function of the kinase-defective EGF receptor in LPA
signaling may be a non-receptor tyrosine kinase. Importantly,
expression of the wild-type human EGF receptor in B82L cells further
increases LPA-stimulated MKK1/2 and MAP kinase activation to a level
higher than that of B82L cells expressing the kinase-defective EGF
receptor. These observations indicate that intrinsic tyrosine kinase
activity of EGF receptor as well as another cellular kinase both
contribute to the role of EGF receptor in the LPA-stimulated MAP kinase
pathway in B82L cell lines.
To further assess the role of EGF receptor in LPA signaling, we
analyzed the LPA-stimulated c-fos promoter activity and DNA synthesis. LPA weakly induces c-fos promoter activity in
B82L cells, which may be attributed primarily to Rho-mediated serum response factor activity that is independent of MAP kinase (39). Higher
levels of c-fos promoter activity were detected in
LPA-treated B82L/hER
and B82L/hER+ cells,
corresponding to higher levels of MKK1/2 and MAP kinase activation in
these cells. Thus, these data again suggest that EGF receptor
contributes to LPA signaling and that both the intrinsic EGF receptor
kinase as well as another cellular kinase are involved. At the same
time, LPA-stimulated c-fos promoter activity was inhibited by PD158780 in HeLa cells (Fig. 8B). This observation is
consistent with the model that EGF receptor kinase has a role in LPA
signaling.
Further support for a role of EGF receptor tyrosine kinase in LPA
signaling was obtained from DNA synthesis analysis. LPA has no
mitogenic activity in B82L cells that lack EGF receptor. While the
lower levels of increase in MAP kinase activation and c-fos
promoter activity in B82L/hER
cells did not result in a
statistically significant increase in LPA-induced DNA synthesis in
B82L/hER
cells, expression of the wild-type EGF receptor
in B82L/hER+ cells restores the mitogenic activity of LPA
in this fibroblast line. Furthermore, the LPA-stimulated DNA synthesis
is inhibited by a specific inhibitor of EGF receptor tyrosine kinase.
Therefore, the role of EGF receptor tyrosine kinase in mediating LPA
signaling is biologically relevant.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Gordon Gill and Michael Weber
for the B82L cell lines, Dr. Michael Weber for the TR12 antibody, Dr.
Alan Cantor of the Moffitt Biostatistics Core for statistical analyses,
and Drs. Doug Cress, Nancy Olashaw, Ed Seto, and Hua Yu for critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by Junior Faculty Research
Award JFRA-647 (to J. W.) from the American Cancer Society,
American Cancer Society, Florida Division, Grant F96USF-2 (to J. W.), University of South Florida Research and Creative Scholarship
Grant (to J. W.), and National Institutes of Health Grant CA55652 (to
R. J.).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 Training Grant T32 DA 07245 from the National
Institutes of Health.
§§
To whom correspondence should be addressed: Molecular Oncology
Program, MDC 44, H. Lee Moffitt Cancer Center and Research Institute,
12902 Magnolia Dr., Tampa, FL 33612. Tel.: 813-979-6713; Fax:
813-979-6700; E-mail: wu{at}moffitt.usf.edu.
1
The abbreviations used are: LPA,
lysophosphatidic acid; MAP kinase, mitogen-activated protein kinase;
MKK1/2, MAP kinase kinase 1 and MAP kinase kinase 2; ROS, reactive
oxygen species; EGF, epidermal growth factor; SRE, serum response
element; PDGF, platelet-derived growth factor; NAC,
N-acetylcysteine.
2
J. M. Cunnick, J. F. Dorsey, Q. Chen,
and J. Wu, unpublished data.
 |
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