 |
INTRODUCTION |
Cholecystokinin
(CCK)1 and related peptides
are potent growth factors in the gastrointestinal tract (1, 2). CCK
stimulates normal growth of the pancreas (3-6) and might be involved
in growth of human pancreatic cancer (7, 8). Exogenous administration of CCK can lead to pancreatic hyperplasia, dysplasia, and malignancies (9) and accelerates the growth of malignant pancreatic tissue (10-12).
In several cancer cell lines, CCK promotes growth (13). Furthermore,
endogenous hypercholecystokininemia promotes carcinogenesis in the
hamster (14). CCK binds to and activates receptors (CCKA and CCKB) belonging to the seven-transmembrane-spanning
family of G protein-coupled receptors (GPCRs) (15, 16). A major
signaling cascade stimulated by both CCKA and
CCKB receptors is pertussis toxin-insensitive activation of
phospholipase C-
and subsequent activation of protein kinase C (PKC)
(2, 16).
Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) plays
a key role in mediating proproliferative effects of both receptor
tyrosine kinases (RTKs) such as the epidermal growth factor (EGF)
receptor (EGFR) and GPCR (17). Depending on receptor and cell type,
GPCR-induced ERK1/2 activation may involve stimulation of nonreceptor
tyrosine kinases of the Src family and Pyk-2, receptor tyrosine kinases
(most notably the EGFR), and phosphatidylinositol 3-kinase, leading to
activation of Ras (17-24). Moreover, Gq-coupled receptors
can activate ERK1/2 by a PKC-dependent Ras-independent pathway involving direct activation of Raf-1 (17, 25, 26).
CCK stimulates ERK1/2 by a mechanism depending on activation of
phospholipase C and phorbol ester-sensitive PKCs (27, 28). In agreement
with a PKC-dependent, Ras-independent mechanism of CCK-induced ERK1/2 activation, expression of dominant-negative Ras did
not inhibit CCK-induced ERK1/2 activation in primary pancreatic acini
(29). However, down-modulation of PKC by long term treatment with
phorbol ester only partially inhibits CCK-induced ERK1/2 activation
(28, 30), indicating that additional mechanisms are involved in
CCK-induced ERK1/2 activation. CCK has been shown to induce tyrosine
phosphorylation of Shc and Pyk-2 as well as complex formation of Grb2
with Shc and Pyk-2 in native rat pancreatic acini (27, 31), events
occurring in Ras-dependent ERK1/2 activation (17, 19). In
CCKB-transfected Chinese hamster ovary cells, the
CCKB receptor agonist gastrin activates ERK1/2 by a Shc-
and Src-dependent mechanism (32). In CCKB
receptor-transfected gastric epithelial cells, gastrin induces EGFR
tyrosine phosphorylation (33). Phosphatidylinositol 3-kinase may
represent an additional signaling intermediate in gastrin-induced
ERK1/2 activation (34, 35). Thus, a number of possible signaling
intermediates of the Ras-dependent pathway have been
assigned to CCK receptor-induced ERK1/2 activation, but it is unclear
whether these signaling pathways operate within one cell type and
whether cross-talk exists to mediate CCK-induced ERK1/2 activation.
In the present study, we sought to delineate the contribution of the
EGFR, Src family tyrosine kinases, and PKC as well as their cross-talk
in CCK-induced activation of the ERK1/2 pathway in the pancreatic
acinar carcinoma cell line AR42J. Our data show that different signals
emanating from the CCK receptor cooperate to mediate stimulation of
ERK1/2; activation of Yes and the EGFR mediate Shc-Grb2 recruitment and
Ras activation, and activation of PKC, most likely PKC
, potentiates
ERK1/2 activation at the Ras/Raf level.
 |
EXPERIMENTAL PROCEDURES |
Materials--
CCK octapeptide, human recombinant EGF,
[Glu52]diphtheria toxin (CRM197), and pertussis toxin
were obtained from Sigma. pUSEamp expression vector containing dominant
negative Ras (N17Ras), agarose-conjugated Ras-binding domain, and
glutathione S-transferase-MEK1 were from Upstate
Biotechnology, Inc. (Lake Placid, NY). Dulbecco's modified Eagle's
medium (DMEM), fetal calf serum, LipofectAMINE 2000, and penicillin/streptomycin were from Invitrogen. The EGFR-specific tyrosine kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), the PKC inhibitors bisindoylmaleimide (GF109203X),
Gö6976 and rottlerin, the Src family kinase inhibitor
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1), 1,2-bis(2-aminophenoxy)ethene
N,N,N',N'-tetraacetic acid acetoxymethyl ester, 12-O-tetradecanoylphorbol-13-acetate
(TPA), all dissolved in dimethyl sulfoxide, hepatocyte growth factor (HGF), and gastrin were obtained from Calbiochem. The ERK1/2 activity assay was from Cell Signaling (Beverly, MA). pEGFP-C1 was from Clontech (Palo Alto, CA). The CCKA
receptor antagonist L364,717 and the CCKB receptor
antagonist L365,260 were kind gifts of ML Laboratories PLC (Liverpool, UK).
Antibodies--
Monoclonal anti-
-actin, affinity-purified
horseradish peroxidase-conjugated anti-mouse, anti-rabbit, and
anti-sheep IgG were obtained from Sigma. Sheep anti-EGFR used for
immunoblotting and polyclonal anti-Shc IgG used for
immunoprecipitation, anti-pan-Ras IgG, and agarose-conjugated
Ras-binding domain were from Upstate Biotechnology. The antibody
against Tyr(P)418-Src was from
BIOSOURCE (Camarillo, CA). The monoclonal
anti-Grb2, anti-Fyn, anti-Yes, anti-PKC
, anti-PKC
, and
anti-PKC
as well as monoclonal anti-Shc IgGs were from Transduction
Laboratories (Lexington, KY). The anti-c-Src IgG (clone 327) was
obtained from Calbiochem. The antibody raised against dually
phosphorylated activated ERK1/2 was from Cell Signaling. Monoclonal
anti-phosphotyrosine, polyclonal goat anti-EGFR and anti-Raf-1 used for
immunoprecipitation, rabbit anti-Raf-1 IgG used for reprobing of the
immunoblots, and horseradish peroxidase-conjugated anti-goat IgG were
from Santa Cruz. Neutralizing and anti-HB-EGF was from R & D Systems
(Minneapolis, MN).
Cell Culture and Transfection--
AR42J cells were cultured in
DMEM containing 10% fetal calf serum and antibiotics (36). For
transient transfection, cells were cultured in 8-cm2 dishes
and transfected with 3 µg of Csk (C-terminal
Src kinase) in pSG5 or pUSEamp containing
dominant negative Ras (N17Ras) using LipofectAMINE 2000 according to
the instructions of the manufacturer. The efficiency of transfection
was monitored by transfecting the cells with a plasmid coding for the
green fluorescent protein (pEGFR-C1). Pertussis toxin treatment was
carried out by incubating the cells with 200 ng/ml pertussis toxin for
12 h prior to the experiments (28).
Immunoprecipitation and Immunoblotting--
AR42J cells were
incubated with appropriate agents at 37 °C. At specified times, the
incubation was stopped by the addition of lysis buffer (50 mM Hepes, pH 7.0, 100 mM NaCl, 0.2 mM MgSO4, 0.5 mM
Na3VO4, 0.4 mM phenylmethylsulfonyl
fluoride, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml
aprotinin). The extracts were clarified by centrifugation and incubated
sequentially (2 h for each incubation at 4 °C) with antibodies as
indicated and protein A/G-Sepharose (Amersham Biosciences) with gentle
agitation. Immunoprecipitates were washed three times with lysis
buffer, boiled for 3 min in 4× Laemmli sample buffer, separated on
SDS-polyacrylamide gels under reducing conditions, and
electrotransferred to nitrocellulose membranes (16, 28).
Antigen-antibody complexes were visualized using horseradish
peroxidase-conjugated IgGs and the enhanced chemiluminescence system.
Where appropriate, a 50-µl aliquot of clarified whole-cell lysate was
mixed with an equal volume of 4× Laemmli sample buffer and resolved by
SDS-PAGE for confirmation of identity of proteins after
immunoprecipitation. For reprobing, blots were incubated in stripping
buffer (62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol) at 50 °C for 30 min, washed
extensively with phosphate-buffered saline, reblocked as described
(28), and reprobed with appropriate antibodies. To determine Src family
kinase activity, cell lysates were either subjected to
immunoprecipitation with anti-Src, anti-Yes, or anti-Fyn or directly
analyzed by immunoblotting with an antibody recognizing Tyr(P)418-Src. Tyr418 is an
autophosphorylation site and thus reflects activation of Src family
kinases (37). Blots were stripped and reprobed with appropriate
antibodies to verify the amount of immunoprecipitated protein.
Ras Activation Assay--
Ras activation was determined by
affinity precipitation of activated Ras from cell lysates using
agarose-conjugated Ras-binding domain. Cells were stimulated, washed
once with phosphate-buffered saline, and lysed in a buffer (Ras lysis
buffer) containing 25 mM Hepes, pH 7.5, 150 mM
NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, and 8 µl of agarose-conjugated
Ras-binding domain. After a 30-min incubation at 4 °C, immune
complexes were washed three times with Ras lysis buffer, followed by
analysis of the immune complexes by anti-Ras immunoblotting.
Raf-1 Assay--
The Raf-1 assay was carried out similarly as
described previously (28) with some modifications. Cells were
stimulated and lysed, and Raf-1 was immunoprecipitated with anti-Raf-1
antibody as described above in the immunoprecipitation and
immunoblotting protocol. The immunoprecipitates were washed twice in
lysis buffer and once in assay buffer (20 mM MOPS, pH 7.2, 25 mM 2-glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM
dithiothreitol). The immune complexes were incubated with inactive
fusion proteins glutathione S-transferase-MEK1 (0.4 µg) in
a buffer containing 15 mM MOPS, pH 7.2, 20 mM
2-glycerol phosphate, 5 mM EGTA, 1 mM
Na3VO4, 1 mM dithiothreitol, 150 µM ATP, and 25 µM MgCl2 in a
final volume of 45 µl for 30 min at 30 °C with gentle agitation.
Reactions were stopped by the addition of 15 µl of 4× sample buffer,
followed by boiling of the samples and SDS-PAGE. Phosphorylation of
MEK1 by immunoprecipitated Raf-1 was determined by immunoblotting with an antibody specific for the phosphorylated form of MEK.
PKC Translocation Assay--
The PKC translocation assay was
carried out as described recently (38). Cells were incubated for 2 days
in serum-free DMEM and stimulated as indicated. Thereafter, cells were
washed and scraped into 1 ml of homogenization buffer (20 mM Tris/HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 10 µg/ml
leupeptin, 10 µg/ml aprotinin), lysed by sonication, and centrifuged
for 1 h at 100,000 × g at 4 °C. Supernatants
were used as a source of cytosolic protein. Pellets were resonicated in
1 ml of homogenization buffer supplemented with 1% Triton X-100 and
centrifuged for 1 h at 100,000 × g, yielding the
solubilized particulate fractions. Protein concentration was
determined, and the fractions were analyzed by immunoblotting.
Reproducibility of Results--
Results are representative of at
least three experiments on different occasions giving similar results.
 |
RESULTS |
Inhibition of EGFR Signaling Reduces CCK- and Gastrin-induced
ERK1/2 Activation in AR42J Cells--
The pancreatic
cell line AR42J is a well established model for CCK-induced signaling
and expresses both CCKA and CCKB receptors, both of which are activated by CCK and stimulate ERK1/2 by a
PKC-dependent mechanism (27, 28, 30, 39). CCK activated
ERK1/2 with a maximum after 3 min as determined by immunoblotting of
cell lysates with an antibody recognizing specifically the dually
phosphorylated activated form of ERK1/2 (Fig.
1A). To determine whether the
EGFR is involved in CCK-induced activation of ERK1/2, we tested the effect of AG1478, a well established inhibitor of the EGFR tyrosine kinase, on CCK-induced ERK1/2 activation. As shown in Fig. 1, B and C, AG1478 strongly inhibited CCK-induced
ERK1/2 phosphorylation and activation. AG1478 had no effect on ERK1/2
activation in response to HGF, which activates the c-Met/HGF receptor
tyrosine kinase, showing the specificity of this compound for the EGFR
tyrosine kinase (Fig. 1B). AG1478 had only a small if any
effect on ERK1/2 phosphorylation in response to TPA (Fig.
1D), an activator of PKC. These data indicate that maximal
ERK1/2 activation in response to CCK requires EGFR tyrosine kinase,
whereas TPA-induced ERK1/2 activation is mainly EGFR-independent in
AR42J cells.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
The EGFR-specific tyrosine kinase inhibitor
AG1478 inhibits CCK-induced ERK1/2 activation. AR42J cells were
incubated with CCK (10 nM), EGF (10 nM), HGF
(10 nM), or TPA (100 nM) for 3 min or the
indicated time in the absence or presence of AG1478 (250 nM) or vehicle (Me2SO). A and
C-E, ERK1/2 activation was determined by immunoblotting of
cellular lysates with an antibody that specifically recognizes the
dually phosphorylated activated form of ERK1 and ERK2. Before reprobing
with anti-ERK1/2, filters were stripped. B, following cell
lysis, ERK1/2 was immunoprecipitated, and its activity was assayed with
recombinant Elk-1 as a substrate by anti-phospho-Elk-1 immunoblotting.
Con, control.
|
|
To differentiate which CCK receptor subtype mediates EGFR tyrosine
kinase-dependent ERK1/2 activation, we investigated the effect of AG1478 on ERK1/2 phosphorylation in response to gastrin, which activates only the CCKB receptor. As shown in Fig.
1E, AG1478 strongly inhibited gastrin-induced ERK1/2
phosphorylation, indicating that ERK1/2 activation in response to
CCKB receptor stimulation is
EGFR-dependent.
CCK Induces Tyrosine Phosphorylation of the EGFR and Shc as Well as
Complex Formation of the EGFR with Grb2 and Shc--
Certain GPCRs
have been reported to induce tyrosine phosphorylation and activation of
the EGFR (24, 40). To investigate if CCK induces tyrosine
phosphorylation of the EGFR, cells were stimulated with CCK or EGF
followed by immunoprecipitation of the EGFR and analysis of the
immunoprecipitates by anti-phosphotyrosine immunoblotting. As shown in
Fig. 2, incubation of the cells with CCK
caused rapid tyrosine phosphorylation of the EGFR. CCK-induced tyrosine
phosphorylation of the EGFR was, however, considerably smaller than the
effect of EGF. The EGFR-specific tyrosine kinase inhibitor AG1478
abolished CCK-induced EGFR tyrosine phosphorylation, indicating that
the EGFR tyrosine kinase domain mediates CCK-induced EGFR tyrosine
phosphorylation.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
CCK causes tyrosine phosphorylation of the
EGFR and Shc and complex formation of Shc with both EGFR and Grb2.
Cells were exposed to CCK (10 nM) or EGF (20 nM) in the absence or presence of AG1478 or vehicle
(Me2SO) for 3 min or the indicated time periods. Following
cell lysis, immunoprecipitation (IP) was carried out with
anti-EGFR or anti-Shc, and the immunoprecipitates were analyzed by
anti-phosphotyrosine, anti-EGFR, and anti-Grb2 immunoblotting
(IB). Following stripping, blots were reprobed with
anti-EGFR (A-C) or anti-Shc (B).
|
|
EGFR activation involves complex formation of the EGFR with the adaptor
proteins Grb2 and Shc and tyrosine phosphorylation of Src homology 2 domain-containing substrates such as Shc (17). Determination of Grb2
immunoreactivity in EGFR immunoprecipitates showed that CCK increased
complex formation between the EGFR and Grb2 (Fig. 2B).
Analysis of Shc immunoprecipitates by anti-phosphotyrosine, anti-Grb2,
and anti-EGFR immunoblotting revealed that CCK induced tyrosine
phosphorylation of p47Shc and p52Shc as well as
complex formation of Shc with Grb2 and the EGFR (Fig. 2C).
Taken together, these data demonstrate that CCK induces tyrosine phosphorylation of the EGFR and Shc as well as formation of a complex
between Shc, Grb2, and the EGFR, events closely related to EGFR activation.
AG1478 abolished CCK-induced complex formation with Grb2 and the
tyrosine-phosphorylated EGFR (Fig. 2B), indicating the
involvement of the EGFR tyrosine kinase activation in this process. In
contrast, AG1478 had no effect on tyrosine phosphorylation of Shc and
its complex formation with Grb2 in response to HGF (data not shown), which activates the c-Met/HGF receptor tyrosine kinase, thus confirming that AG1478 specifically antagonized the effect of the EGFR tyrosine kinase in our system.
The addition of GF109203X, a chemical inhibitor of PKC, or
loading of the cells with the intracellular calcium chelator
1,2-bis(2-aminophenoxy)ethene N,N,N',N'-tetraacetic acid
acetoxymethyl ester did not prevent CCK-induced tyrosine
phosphorylation of the EGFR (data not shown), indicating that an
increase in intracellular calcium or activation of PKC is not essential
for CCK-induced EGFR transactivation. Moreover, since cleavage of
pro-HB-EGF by metalloproteinases has been shown to mediate EGFR
transactivation by Gi- and Gq-coupled receptors
in several different cell lines (41), we investigated the effect of
neutralizing anti-HB-EGF antibody and of the HB-EGF inhibitor
[Glu52]diphtheria toxin (CRM197) on CCK-induced tyrosine
phosphorylation of the EGFR and ERK1/2 activation. Anti-HB-EGF or
CRM197 had no effect on CCK responses (data not shown), indicating that
HB-EGF is not involved in CCK-induced EGFR and ERK1/2 activation in
AR42J cells.
Ras Is Involved in CCK-induced ERK1/2
Activation--
Whereas there is agreement concerning the
requirement of Ras in EGF- and Gi-coupled receptor-induced
ERK1/2 activation, Gq-coupled receptor-induced ERK1/2
activation has been reported to be mediated by both
Ras-dependent or -independent pathways (17, 23, 42-44). Adenoviral expression of dominant negative Ras was found to have no
effect on CCK-induced activation of ERK1/2 in cultured primary pancreatic acinar cells (29), and CCK appears to have no significant effect on the amount of activated GTP-bound Ras in freshly prepared pancreatic acinar cells (39, 45), suggesting that Ras may not be
involved in CCK-induced ERK1/2 activation in native pancreatic acinar
cells. If CCK-induced activation of ERK1/2 is mediated by EGFR
transactivation in pancreatic AR42J cells, CCK-induced ERK1/2
activation should depend on Ras activation. Involvement of Ras in
CCK-induced ERK1/2 activation was determined in AR42J cells transiently
transfected with dominant-negative Ras (N17Ras) or empty vector. As
shown in Fig. 3A, N17Ras
inhibited CCK-induced ERK1/2 phosphorylation.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
CCK induces activation of Ras, which is
essential for CCK-induced ERK1/2 activation. A, AR42J
cells were transiently transfected with N17Ras or empty vector. After
24 h, cells were serum-starved for 5 h and were then exposed
to CCK (10 nM) for 3 min at 37 °C. Following lysis,
cellular proteins were analyzed by anti-phospho-ERK1/2 immunoblotting
(IB). The blots were stripped and reprobed with anti-ERK2.
B, cells were stimulated with CCK (10 nM), TPA
(10 nM), or EGF (10 nM) in the presence or
absence of AG1478 (250 nM) for 3 min. Following lysis,
equal amounts of cellular extracts were incubated with Ras binding
domain (RBD) peptide coupled to agarose. Affinity
precipitates were analyzed for GTP-bound activated Ras by anti-pan-Ras
immunoblotting. The asterisk represents a significant
difference (p < 0.05) according to analysis of
variance; n.s., not significant; Con,
control.
|
|
The findings that dominant-negative Ras and AG1478 inhibited
CCK-induced ERK1/2 activation suggest involvement of both the EGFR and
Ras in CCK-induced ERK1/2 activation. Therefore, we studied the effect
of CCK, EGF, and AG1478 on Ras activity. Both CCK and EGF increased the
amount of GTP-bound Ras in an AG1478-sensitive fashion (Fig.
3B), indicating that CCK induces Ras activation through an
EGFR-dependent mechanism. TPA also caused activation of Ras
(Fig. 3B). However, TPA-induced Ras activation was less affected by AG1478 than the CCK response, indicating that phorbol ester-induced Ras activation occurs mainly through an EGFR-independent mechanism.
Src Family Tyrosine Kinases Are Involved in CCK-induced Ras and
ERK1/2 Activation--
In the majority of studies,
GPCR-induced ERK1/2 activation depends on activation of Src family
tyrosine kinases (17). To study the role of Src family tyrosine kinases
in CCK-induced ERK1/2 activation, we first investigated the effect of
PP1, a specific inhibitor of Src family kinases, on CCK-induced
activation of ERK1/2 and Src family tyrosine kinases. Because there is
a correlation between autophosphorylation of Tyr418 and
activation of Src kinase (37), Src kinase activation can be detected by
immunoblotting with an antibody recognizing the autophosphorylated form
of Src (Tyr(P)418-Src). As shown in Fig.
4A, CCK caused an increase in
the proportion of autophosphorylated Src family kinase; this was
inhibited by PP1. The inhibitory effect of PP1 on CCK-induced Src
family kinase autophosphorylation closely correlated with its effect on
CCK-induced ERK1/2 phosphorylation, providing evidence that PP1 indeed
inhibited CCK-induced ERK1/2 activation by interfering with activation
of Src family kinases. The involvement of Src family tyrosine kinase in
CCK-induced ERK activation was confirmed by experiments in which Csk,
which phosphorylates and thereby blocks activation of Src family
tyrosine kinases (46, 47), was transfected into the cells, followed by
detection of CCK-induced ERK1/2 activation. As shown in Fig.
4B, expression of Csk reduced CCK-induced ERK1/2 phosphorylation significantly.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 4.
Role of Src family tyrosine kinases in
CCK-induced ERK1/2 activation, tyrosine phosphorylation of the EGFR and
Shc, and recruitment of Shc and Grb2 to the EGFR. A,
cells were exposed to CCK (10 nM) in the presence or
absence of the indicated concentration of PP1 for 3 min. Cell lysates
were analyzed by anti-phospho-418-Src, anti-phospho-ERK1/2, and
anti- -actin immunoblotting. B, Csk- or mock-transfected
cells were stimulated with CCK for 3 min followed by lysis of the cells
and analysis of the ERK phosphorylation by anti-ERK1/2. The blot was
stripped and reprobed with anti-ERK2. C, cells were
stimulated with CCK or EGF (10 nM) in the presence or
absence of PP1 (10 µM) for 3 min. Following cell lysis,
equal amounts of cellular protein extracts were incubated with Ras
binding domain (RBD) peptide coupled to agarose. Affinity
precipitates were analyzed for GTP-bound activated Ras by anti-pan-Ras
immunoblotting. D, pertussis toxin
(PTx)-pretreated and -untreated cells were exposed to CCK
(10 nM) for the indicated time. Following cell lysis,
autophosphorylation of Src family tyrosine kinases was detected by
immunoblotting with anti-Tyr(P)418-Src. E and
F, immunoprecipitation was carried out with anti-EGFR
(E) or anti-Shc (F), and immunoprecipitates were
analyzed by anti-phosphotyrosine, anti-EGFR, anti-Grb2, and anti-Shc
immunoblotting. Blots were stripped and reprobed with anti-EGFR
(E) or anti-Shc (F). Con,
control.
|
|
PP1 also reduced EGF-induced ERK1/2 phosphorylation (Fig.
4A), but this effect was clearly smaller than its effect on
CCK-induced ERK1/2 phosphorylation. PP1 inhibited CCK-induced Ras
activation, whereas the response to EGF remained unchanged (Fig.
4C). Together, these results demonstrate that CCK-induced,
but not EGF-induced, ERK1/2 activation depends on stimulation of Src
family tyrosine kinases, which are involved in CCK-induced ERK1/2
activation upstream of Ras activation.
Since CCK receptor activation can lead to activation of pertussis
toxin-sensitive G proteins (48-51) and Gi-coupled
receptors can activate Src by G
, which then induces tyrosine
phosphorylation of Shc, we studied the effect of pertussis toxin, which
inactivates Gi/o-type G proteins, on CCK-induced
autophosphorylation of Src family kinase. As shown in Fig.
4D, treatment of the cells with pertussis toxin had no
effect on CCK-induced autophosphorylation of Src family kinase.
Furthermore, pertussis toxin treatment did not alter CCK-induced
tyrosine phosphorylation of Shc (data not shown). These data suggest
that Gi/o proteins do not participate in CCK-induced
tyrosine phosphorylation of Shc.
Recent studies provided heterogeneous data concerning the involvement
of Src family kinases in GPCR-induced EGFR transactivation (42, 43,
52-57). Src family kinases might be involved in GPCR-induced assembly
of the Ras activation complex either by mediating tyrosine phosphorylation of the EGFR (53, 55, 57) and/or Shc (20, 58). To
investigate whether activation of Src family tyrosine kinases is
involved in CCK-induced EGFR tyrosine phosphorylation and/or
recruitment of the docking proteins Shc and Grb2 to the EGFR, cells
were incubated with CCK or EGF in the presence or absence of PP1
followed by immunoprecipitation of the EGFR or Shc and analysis of the
immunoprecipitates by anti-phosphotyrosine, anti-Shc, anti-Grb2, and
anti-EGFR immunoblotting. As shown in Fig. 4E, PP1 had no
effect on CCK-induced tyrosine phosphorylation of the EGFR but
completely inhibited CCK-induced tyrosine phosphorylation of Shc (Fig.
4F). These data indicate that CCK-induced EGFR tyrosine phosphorylation is Src family kinase-independent, whereas CCK-induced recruitment of Shc and Grb2 to the EGFR and tyrosine phosphorylation of
Shc require Src family kinase activity. PP1 had no effect on EGF-induced tyrosine phosphorylation of Shc (Fig. 4F).
To investigate which Src family tyrosine kinases are activated in
response to CCK and could therefore participate in CCK-induced ERK1/2
activation, cells were stimulated with CCK followed by immunoprecipitation of Src, Fyn, and Yes and analysis of the
immunoprecipitates for Tyr(P)418-Src immunoreactivity. As
shown in Fig. 5, A-C, CCK
caused rapid autophosphorylation of Src, Fyn, and Yes, indicating that
CCK induces activation of these Src kinases. Since EGF can also induce activation of Src family tyrosine kinases (59), we investigated the
effect of AG1478 on CCK-induced Src family Tyr418
phosphorylation. As shown in Fig. 5D, AG1478 had no
significant effect on CCK-induced Src family tyrosine kinase
autophosphorylation, showing that CCK-induced Src family kinase
activation does not require activation of the EGFR.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 5.
CCK induces activation of Src, Fyn, and
Yes. Cells were exposed to CCK (10 nM) in the presence
or absence of AG1478 (250 nM), CCK-A, or CCK-B receptor
blocker for the indicated time or 3 min (E). Following cell
lysis, immunoprecipitation was carried out with anti-Src
(A), anti-Fyn (B), or anti-Yes
(C and E), and immunoprecipitates were
analyzed by anti-Tyr(P)418-Src. Blots were stripped and
reprobed with anti-Src (A), anti-Fyn (B), and
anti-Yes (C). Cell lysates were analyzed by
anti-Tyr(P)418-Src (D).
|
|
It has recently been shown that gastrin induces complex formation
between Src and Shc in CCKB/gastrin receptor-expressing Chinese hamster ovary cells (32). Similarly, lysophosphatidic acid,
which activates a Gi-coupled receptor, causes complex
formation of Shc with Src but not with Fyn or Yes (20), whereas
angiotensin II induces association of Shc with Fyn in cardiac myocytes
(21) or with Src in vascular smooth muscle cells (57), indicating that
GPCR-induced signaling toward Shc can involve several different members
of the Src family tyrosine kinases depending on receptor and cell type.
To examine which Src family kinases might be involved in CCK-induced
tyrosine phosphorylation of Shc and complex formation between the EGFR,
Grb2, and Shc, EGFR and Shc immunoprecipitates from CCK-pretreated and
untreated cells were examined for the presence of Src, Fyn, and Yes
immunoreactivity. As illustrated in Fig.
6, CCK led to an increase in Yes
immunoreactivity in both EGFR and Shc immunoprecipitates, indicating
that Yes could be involved in tyrosine phosphorylation of Shc and its
complex formation with the EGFR and Grb2. Src immunoreactivity was
detected in both EGFR and Shc immunoprecipitates, but there was no
clear increase in Src immunoreactivity in EGFR and Shc
immunoprecipitates upon treatment of the cells with CCK (data not
shown). Fyn immunoreactivity was undetectable in Shc or EGFR
immunoprecipitates. These data point to a critical role of Yes in
CCK-stimulated tyrosine phosphorylation of Shc and its complex
formation with Grb2.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
CCK increases Yes immunoreactivity in EGFR
and Shc immunoprecipitates. Cells were exposed to CCK (10 nM) for the indicated time. Following cell lysis,
immunoprecipitation was carried out with anti-EGFR (A) or
anti-Shc (B), and immunoprecipitates were analyzed by
anti-Yes. Blots were stripped and reprobed with anti-EGFR
(A) or anti-Shc (B).
|
|
Because Yes appears to play a critical role in CCK-induced signal
transduction, we examined which CCK receptor subtype mediates CCK-induced tyrosine phosphorylation of Yes using the CCK receptor subtype-specific inhibitors. As shown in Fig. 5E,
CCK-induced tyrosine phosphorylation of Yes was inhibited by
CCKB, but not by CCKA receptor antagonist,
indicating that the CCKB receptor mediates CCK-induced Yes activation.
PKC Augments CCK-induced ERK1/2 Signaling at the Ras/Raf
Level--
Fig. 7 compares the effects
of CCK with that of EGF with respect to induction of tyrosine
phosphorylation of Shc, Shc-Grb2 complex formation, and activation of
Ras, Raf, and ERK1/2. CCK was a much weaker stimulator of the ERK
cascade than EGF up to the level of Ras, whereas at the level of Raf-1,
the effects of CCK and EGF were almost equal. This indicates signal
amplification between Ras and Raf-1. Activation of PKC appears to
mediate this signal amplification, because CCK-induced Ras, Raf-1, and
ERK1/2 activation were inhibited by down-modulation of PKC by TPA,
whereas more proximal responses (activation of Shc/Grb2) were not
influenced by this treatment.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of prolonged TPA treatment on CCK- or
EGF-induced tyrosine phosphorylation of Shc and Shc-Grb2 complex
formation. A, TPA-pretreated and control cells were exposed
to CCK (10 nM) or EGF (3 nM) for 3 min. Cell
lysates were immunoprecipitated with anti-Shc followed by
anti-phosphotyrosine and anti-Grb2 immunoblotting, followed by
stripping and reprobing of the blot with anti-Shc. The same lysates
were used for immunoprecipitation with anti-Raf-1. The Raf-1 immune
complexes were incubated with inactive fusion proteins glutathione
S-transferase-MEK1. Phosphorylation of MEK1 by
immunoprecipitated Raf-1 was determined by immunoblotting with an
antibody specific for the phosphorylated form of MEK. The blot was
reprobed with anti-Raf-1. Aliquots of the lysates were analyzed by
anti-phospho-ERK1/2 immunoblotting, followed by reprobing of the blot
with anti-ERK2. Furthermore, equal amounts of cellular extracts were
incubated with Ras binding domain (RBD) peptide coupled to
agarose. Affinity precipitates were analyzed for GTP-bound activated
Ras by anti-pan-Ras immunoblotting. B, cells preincubated
with 10 nM TPA or vehicle (dimethyl sulfoxide) for 18 h
were analyzed by anti-PKC , -PKC , and -PKC immunoblotting.
Con, control.
|
|
Since PKC has been proposed as a modulator of Ras-Raf activation and
activation of PKC is clearly involved in CCK-induced ERK1/2 activation
(25, 26, 60), we studied the effect of prolonged treatment of the cells
with TPA, which leads to down-modulation of diacylglycerol-sensitive
classical and novel PKC isoenzymes, on CCK-induced tyrosine
phosphorylation of Shc, Shc-Grb2 complex formation, and activation of
Ras, Raf, and ERK1/2. As shown in Fig. 7, TPA long term treatment had
no effect on CCK- or EGF-induced tyrosine phosphorylation of Shc or its
complex formation with Grb2 (Fig. 7), although CCK-induced ERK1/2
phosphorylation tested in the same cell lysates was clearly inhibited
(Fig 7). A similar result was obtained when PKC activation was
inhibited by GF109203X (data not shown). Prolonged TPA treatment
inhibited CCK-induced Ras and Raf-1 activation without influencing the
effect of EGF (Fig. 7), suggesting a role of PKC in CCK-induced
activation of Ras and Raf-1.
Immunoblotting analysis revealed expression of three major phorbol
ester- or diacylglycerol-responsive PKC isoforms (i.e. PKC
, -
, and -
) in AR42J cells and revealed that prolonged
treatment of the cells with TPA led to down-modulation of these PKC
isoenzymes (Fig. 7, lower panel). To provide
evidence which PKC isoform might be involved in CCK-induced ERK1/2
activation, we studied the effect of CCK on PKC activation as detected
by translocation of PKC from the cytosol to the membrane compartment.
As illustrated in Fig. 8A, CCK
caused rapid translocation of PKC
, -
, and -
from the cytosol
to the membrane fraction, indicating that CCK causes activation of
these PKC isoenzymes.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 8.
CCK induces activation of
PKC and its complex formation with Raf-1.
A, cells were incubated for 2 days in serum-free DMEM and
stimulated as indicated with CCK (10 nM) or TPA (10 nM). Thereafter, cells were washed, scraped, homogenized,
and separated into cytosol and membrane fractions as described under
"Experimental Procedures." The fractions were analyzed by
immunoblotting with anti-PKC , - , and - . B, cells
were exposed to CCK in the absence or presence of Gö6976 (10 µM) or rottlerin (10 µM) for 3 min. Cell
lysates were analyzed by anti-phospho-ERK1/2, followed by
stripping and reprobing of the blot with anti-ERK2. C, cells
were stimulated with CCK for the indicated time. Cell lysates were
immunoprecipitated with anti-Raf-1. The immunoprecipitates were
analyzed by anti-PKC immunoblotting.
|
|
To examine which PKC isoenzyme mediates CCK-induced activation of Raf-1
and subsequently ERK1/2, we studied the effect of Gö6976, an
inhibitor of calcium-dependent PKC isoenzymes, and rottlerin, an inhibitor of PKC
, on CCK-induced ERK1/2
phosphorylation. As illustrated in Fig. 8B, Gö6976 and
rottlerin had no effect on CCK-induced ERK1/2 activation, suggesting
that conventional PKCs and PKC
are unlikely to be involved in
CCK-induced ERK1/2 activation.
 |
DISCUSSION |
The mechanisms by which GPCRs activate ERK1/2 are
characterized by considerable heterogeneity. Depending on receptor and
cell type, GPCRs have been shown to mediate Ras-independent ERK1/2 activation via stimulation of PKC or Ras-dependent ERK1/2
activation via activation of receptor and nonreceptor tyrosine protein
kinases (17). In the present study, we provide evidence that
activations of the EGFR, Yes, and PKC
are involved in CCK-induced
ERK1/2 activation and cooperate to accomplish CCK-induced ERK1/2 activation.
We found that CCK caused activation of the EGFR, which is essential for
full activation of ERK1/2 in response to CCK. The evidence for this is
based on the findings that CCK caused tyrosine phosphorylation of the
EGFR and Shc, Shc-Grb2 complex formation, recruitment of these docking
proteins to the EGFR as well as Ras, and ERK1/2 activation through an
EGFR tyrosine kinase-dependent pathway. CCK-induced
AG1478-sensitive ERK1/2 phosphorylation was mimicked by the
CCKB receptor agonist gastrin, indicating that CCKB receptor activation mediates
EGFR-dependent ERK1/2 activation. This hypothesis is
supported by a recent study showing that gastrin induces EGFR tyrosine
phosphorylation in CCKB/gastrin receptor-transfected gastric epithelial cells (33).
The mechanism of CCK receptor-induced EGFR tyrosine phosphorylation is
unclear. The inhibitory effect of AG1478 suggests that CCK-induced EGFR
tyrosine phosphorylation is due to EGFR autophosphorylation. It has
recently been shown that cleavage of pro-HB-EGF by metalloproteinases mediates EGFR transactivation in response to Gi- and
Gq-coupled receptors in several cell types (41). Moreover,
in CCKB receptor-transfected gastric epithelial cells,
gastrin induces release of HB-EGF into the medium, which causes EGFR
tyrosine phosphorylation (33). Thus, it is possible that CCK also
induces EGFR tyrosine phosphorylation by activation of
metalloproteinase and proteolytic processing of EGFR ligand precursors
in pancreatic AR42J cells. However, in the present study, neither the
HB-EGF inhibitor [Glu52]diphtheria toxin (CRM197) nor
neutralizing anti-HB-EGF IgG had any effect on CCK-induced ERK1/2
activation, suggesting that HB-EGF may not be involved in CCK-induced
EGFR and ERK1/2 activation in AR42J cells. However, it is possible that
proteolytic cleavage of an EGFR ligand precursor different from HB-EGF
mediates the effects of CCK on EGFR tyrosine phosphorylation. Src
family kinases have been implicated in GPCR-induced EGFR tyrosine
phosphorylation, and GPCR can induce association of Src with the EGFR
(53-55, 57). In other studies, GPCR-induced EGFR tyrosine
phosphorylation was found to be Src-independent (52, 56). In the
present study, inhibition of Src kinases had no significant effect on
CCK-induced EGFR tyrosine phosphorylation, indicating that Src family
kinases may not be involved in CCK-induced EGFR tyrosine
phosphorylation. However, because CCK induced activation of Yes and its
recruitment to the EGFR, Yes may modulate signaling from the
CCK-stimulated EGFR.
Whereas there is consent for the requirement of Ras in EGF- and
Gi-mediated ERK1/2 activation, Gq-mediated
ERK1/2 activation can occur through Ras-dependent or
-independent pathways depending on receptor and cell type (17, 23, 25).
Adenoviral expression of dominant-negative Ras in primary rat
pancreatic acini was found to have no effect on CCK-induced activation
of ERK1/2 (29), and CCK has no significant effect on the amount of
activated GTP-bound Ras, although it appears to enhance GTP turnover on
Ras (39, 45), suggesting that Ras may not be involved in CCK-induced ERK1/2 activation in these cells. The present study shows that CCK
caused activation of Ras and that CCK-induced ERK1/2 activation was
abolished by dominant negative Ras in pancreatic AR42J cells, thus
providing strong evidence for the notion that CCK stimulates ERK1/2
through a Ras-depending pathway in these cells.
Depending on receptor and cell type, GPCR-induced ERK1/2 activation
depends completely or partially on EGFR or platelet-derived growth
factor receptor activation or is independent from RTK transactivation (17). Even in cases where GPCR-induced ERK1/2 activation is completely
dependent on RTK transactivation, the strong stimulatory effect of
Gq-coupled receptors on ERK1/2 often contrasts with rather
small effects on RTK tyrosine phosphorylation (56, 57, 61-63). In the
present study, CCK activated ERK1/2 and Raf-1 to a similar extent as
EGF, and activation of the EGFR and Ras was required for full
activation of ERK1/2 by CCK. However, compared with EGF, CCK caused
only small increases in tyrosine phosphorylation of the EGFR and Shc,
Shc-Grb2 complex formation, and Ras activation. Thus, in CCK-induced
activation of the ERK cascade, signal amplification occurs between Ras
and Raf. This is concluded from our finding that down-modulation
of phorbol ester-sensitive PKCs inhibited CCK-induced activation of
Ras, Raf, and ERK1/2, but not tyrosine phosphorylation of Shc and its
complex formation with Grb2. Recent studies have shown that activation
of PKC may modulate Ras-dependent ERK activation by fine
tuning Ras-Raf activation (25, 26, 60). The finding that phorbol
ester-induced Ras and ERK1/2 activation was much less sensitive to EGFR
tyrosine kinase inhibition compared with the effects of CCK is well in
agreement with the assumption that PKC-activating phorbol esters act at
the level of Ras-Raf in AR42J cells.
CCK induces activation of PKC
as well as of PKC
and -
(64)
(present study). The present study reveals that inhibition of
conventional PKCs did not prevent CCK-induced ERK1/2 activation, indicating that PKC
is not involved. The findings that PKC
complexed with Raf-1, that this complex formation was increased by CCK
stimulation, and the lack of effect of the PKC
inhibitor rottlerin
on CCK-induced ERK1/2 activation suggest involvement of PKC
in
CCK-induced ERK1/2 activation. However, the specificity of rottlerin
has recently been challenged (65). Taking into account that PKC
forms a complex with Raf-1 in fibroblasts (66-68), the data of the
present study suggest that CCK-induced activation of Raf-1 and
subsequent stimulation of ERK1/2 by CCK is mediated by activation of
PKC
, which then potentiates CCK stimulation of the ERK cascade at
the level of Ras/Raf.
The PKC inhibitor GF109203X has recently been shown to inhibit
CCK-induced tyrosine phosphorylation of Shc in native pancreatic acini
(27), whereas TPA mimicked it (30), suggesting involvement of PKC in
CCK-induced tyrosine phosphorylation of Shc. We also observed that TPA
induces tyrosine phosphorylation of Shc and Shc-Grb2 complex formation
(data not shown). However, GF109203X or PKC down-modulation by
prolonged TPA treatment had no effect on CCK-induced tyrosine
phosphorylation of Shc or its complex formation with Grb2, suggesting
that activation of PKC is not involved in CCK-induced tyrosine
phosphorylation of Shc in AR42J cells.
Src family tyrosine kinases can be activated by various extracellular
signals, including GPCRs and RTKs (59, 69). The present study shows
that CCK caused increase in tyrosine phosphorylation of three different
Src family tyrosine kinases (i.e. Src, Fyn, and Yes on
tyrosine 418), reflecting their activation. The mechanisms by which
GPCRs activate Src family kinases are still incompletely understood.
The
2-adrenergic receptor activates Src by promoting its
interaction with the adaptor protein arrestin (70). A number of studies
indicate pertussis toxin-sensitive activation of Src, Fyn, Yes, and Lyn
in various cell types (22, 71-73). It has previously been shown that
-subunits of Gs and Gi, but not
G
q, can activate Src directly (74). Our finding that
pertussis toxin had no effect on CCK-induced autophosphorylation of Src
family tyrosine kinases argues against an involvement of Gi
proteins in CCK-induced Src activation despite the ability of CCK
receptors to activate pertussis toxin-sensitive G proteins (48-51). A
recent study has shown that the Gq-coupled angiotensin II
receptor activates Src directly without the contribution of G proteins
(75). However, whether CCK receptors utilize similar mechanisms to
activate Src family kinases remains to be established.
There is strong evidence that Src family tyrosine kinases can be
involved in Gq-coupled receptor-induced ERK1/2 activation, but this is not universal (42, 52, 53, 55-57, 76, 77). These tyrosine
kinases might be involved in GPCR-induced assembly of the Ras
activation complex by mediating tyrosine phosphorylation of the EGFR
(53, 55, 57) and/or Shc (20, 59). GPCR stimulation has been reported to
induce complex formation of Src or Fyn with Shc and of Src with the
EGFR (20, 21, 32, 57). The present study shows that GPCR activation can
induce complex formation of Yes with both the EGFR and Shc. In
particular, our data indicate that CCK-induced ERK1/2 activation
requires a concerted action of both the EGFR and Yes that converge at
the level of Shc-Grb2 complex formation. This assumption is based on
the findings that complex formation between Shc and Grb2 as well as
their association with the EGFR and the downstream events including Ras
and ERK1/2 activation were sensitive to Src family kinase inhibition in
addition to their dependence on the EGFR tyrosine kinase and that Yes
was activated and recruited to both Shc and the EGFR in response to CCK. CCK also induced activation of Src and Fyn, but it did not cause
robust increase in complex formation between Src and the EGFR or Shc,
and Fyn immunoreactivity was undetectable in EGFR or Shc
immunoprecipitates. Thus, Src and Fyn probably have roles in CCK
signaling other than inducing tyrosine phosphorylation of Shc. Our
finding that CCKB, but not CCKA receptor
antagonist inhibited CCK-induced tyrosine 418 phosphorylation of Yes
suggests that the CCKB receptor mediates CCK-induced
activation of Yes.
In contrast to CCK, EGF-induced activation of the ERK1/2 pathway
appears to be less dependent on Src family kinase activation than the
CCK-induced response, because inhibition of Src family kinase had no
effect on EGF-induced tyrosine phosphorylation of Shc, its complex
formation with Grb2, and Ras activation. PP1 significantly reduced
EGF-induced activation of ERK1/2, but the inhibitory effect of PP1 on
EGF-induced ERK1/2 activation was clearly smaller than its effect on
CCK-induced ERK1/2 activation. In agreement with our data, a recent
study shows that expression of a kinase-inactive mutant of Src does not
alter EGF-induced tyrosine phosphorylation of Shc, Shc-Grb2 complex
formation, and ERK1/2 activation in epithelial cells (78).
In conclusion, the present study demonstrates that three different
signals emanating from the Gq-coupled CCK receptor mediate ERK1/2 activation, including transactivation of the EGFR and activation of Yes, which are recruited to and essential for formation of the EGFR-Grb2-Shc complex, which in turn mediates CCK-induced Ras
activation. Activation of PKC, most likely PKC
, provides a third
signal essential for full CCK-induced ERK1/2 activation at the Ras/Raf level.