From the Division of Molecular Oncology, Institute
for Genetic Medicine, and Graduate School of Science, Hokkaido
University, Sapporo 060-0185 and the § Department of
Oncogene Research, Research Institute for Microbial Diseases, Osaka
University, Suita 565-0871, Japan
Received for publication, August 9, 2002, and in revised form, November 1, 2002
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ABSTRACT |
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Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from
gastritis to cancer. The CagA protein is the product of the
cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine
phosphorylation. The phosphorylated CagA binds and activates SHP-2
phosphatase and thereby induces a growth factor-like morphological
change termed the "hummingbird phenotype." In this work, we
demonstrate that CagA is also capable of interacting with C-terminal
Src kinase (Csk). As is the case with SHP-2, Csk selectively binds
tyrosine-phosphorylated CagA via its SH2 domain. Upon complex
formation, CagA stimulates Csk, which in turn inactivates the Src
family of protein-tyrosine kinases. Because Src family kinases
are responsible for CagA phosphorylation, an essential prerequisite of
CagA·SHP-2 complex formation and subsequent induction of the
hummingbird phenotype, our results indicate that CagA-Csk
interaction down-regulates CagA·SHP-2 signaling by both
competitively inhibiting CagA·SHP-2 complex formation and reducing
levels of CagA phosphorylation. We further demonstrate that
CagA·SHP-2 signaling eventually induces apoptosis in AGS cells. Our
results thus indicate that CagA-Csk interaction prevents excess cell
damage caused by deregulated activation of SHP-2. Attenuation of CagA
activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.
Helicobacter pylori is a micro-aerophilic spiral-shaped
bacterium (1) and is estimated to infect about half of the world's population. It colonizes the human stomach and persists for several decades, causing chronic gastritis and peptic ulcer diseases (2). Recent epidemiological studies have further indicated that H. pylori infection is associated with the development of gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue
lymphoma (3-6).
The H. pylori cagA gene encodes the 120- to 145-kDa CagA
protein, and it is a marker for the presence of the cag
pathogenicity island (7-9). More recently, the biological
heterogeneity of H. pylori has been recognized, and H. pylori strains harboring the cagA gene have been
suspected to have a specific responsibility in promoting the
atrophic-metaplastic mucosal lesions that represent the most recognized
pathway in multistep intestinal-type gastric carcinogenesis. Thus they
are considered to be more virulent than cagA-negative
strains (5, 10, 11). Consistently, molecular epidemiological studies
have suggested that cagA-positive H. pylori infection significantly increases the risk of gastric carcinoma (5, 12,
13).
In vitro infection of gastric epithelial cells with
cagA-positive, but not cagA-negative, H. pylori induces a unique morphological change termed the
hummingbird phenotype, which is characterized by strong elongation of
the cell (14-16). During the infection, the CagA protein, which is
produced within H. pylori, is translocated from the
bacteria into the attached epithelial cells via the bacterial type IV
injection apparatus. The translocated CagA protein then localizes at
the inner surface of the plasma membrane and undergoes tyrosine
phosphorylation by the Src family of protein-tyrosine kinases such as
c-Src, Lyn, Fyn, and Yes (17-23). In vivo tyrosine phosphorylation sites of CagA are characterized by Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, which vary in number among different H. pylori strains (24, 25).
We as well as others have demonstrated that CagA is the essential and
sufficient H. pylori factor in the induction of the hummingbird phenotype (15, 16, 22). Tyrosine phosphorylation of CagA is
a prerequisite for the morphological changes. We have further shown
that CagA binds and stimulates an
SH2 1 domain-containing
protein-tyrosine phosphatase, SHP-2, in a tyrosine phosphorylation-dependent manner (16). Thus, CagA induces
the hummingbird phenotype by recruiting and activating SHP-2 at the plasma membrane. Considering the critical roles of SHP-2 in
transmission of mitogenic signals as well as in regulation of cell
motility (26-28), perturbation of SHP2 by CagA is thought to be
substantially involved in pathological processes that are associated
with cagA-positive H. pylori infection.
Although it is known that the majority of the CagA proteins in host
cells form complexes with SHP-2, we found in this work that CagA is
also capable of binding C-terminal Src kinase (Csk) (29-33). Upon
binding with Csk, CagA stimulates its kinase activity and thereby
inactivates Src family kinases. Given that Src family kinases are
responsible for CagA phosphorylation (22, 23) and that this CagA
phosphorylation is an essential prerequisite for CagA·SHP-2 complex
formation and subsequent induction of the hummingbird phenotype (16),
our findings indicate that CagA-Csk interaction down-regulates
CagA·SHP-2 signaling that perturbs cellular functions.
Antibodies--
Anti-Csk polyclonal antibody C-20 (Santa Cruz
Biotechnology), hemagglutinin (HA)-epitope-specific monoclonal antibody
12CA5 and anti-Myc monoclonal antibody 9E10 were used as primary
antibodies for immunoblotting and immunoprecipitation. Anti-HA
polyclonal antibody Y-11 (Santa Cruz Biotechnology),
anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology),
anti-c-Src polyclonal antibody N-16 (Santa Cruz Biotechnology),
Anti-Phospho-Src (Tyr-416) polyclonal antibody (Cell Signaling),
and anti-nonphospho-Src (Tyr-416) 7G9 monoclonal antibody (Cell
Signaling) were used as primary antibodies for immunoblotting. Normal
rabbit IgG was purchased from Santa Cruz Biotechnology.
Construction of DNA--
Mammalian expression vectors for
HA-tagged wild-type CagA (WT CagA-HA) isolated from NCTC11637 H. pylori and its phosphorylation-resistant mutant (PR CagA-HA) have
been described previously (16). EPIYA mutants of WT CagA-HA, abCCC and
ABccc, were generated from HA-tagged wild-type CagA by substituting
tyrosine residues that constitute EPIYA motifs with alanine residues
using a Chameleon site-directed mutagenesis kit (Stratagene), and the
DNA fragments encoding these mutants were cloned into a pSP65SR Cell Culture and Transfection--
AGS human gastric epithelial
cells and monkey COS-7 cells were, respectively, cultured in RPMI 1640 medium and Dulbecco's modified Eagle medium supplemented with 10%
fetal bovine serum. Expression vectors were transiently transfected
into the cells by using LipofectAMINE 2000 reagent (Invitrogen) as
previously described (16). Cells were harvested at 36 h after
transfection. The morphology of the AGS cells was examined at 17 h
post-transfection.
Immunoprecipitation and Immunoblotting--
Cells were lysed in
lysis buffer as described previously (16). Cell lysates were treated
with the appropriate antibody, and then immune complexes were trapped
on protein A- or protein G-Sepharose beads. Total cell lysates and
immunoprecipitates were subjected to SDS-PAGE. Proteins transferred to
polyvinylidene difluoride membranes (Millipore) were soaked in
solutions of primary antibodies and then visualized using Western blot
chemiluminescence reagent (PerkinElmer Life Sciences). Quantitation of
chemiluminescence on the immunoblotted membrane was performed by using
a luminescence image analyzer (LAS1000, FUJIFILM).
In Vitro Kinase Assay--
Csk kinase activity was measured with
the use of poly(Glu,Tyr) 4:1 (Sigma) as a substrate (34). Cells
were lysed in lysis buffer mB (50 mM HEPES-NaOH, pH 8.0, 100 mM NaCl, 5 mM EDTA, and 1% Brij-35)
containing 2 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, and 10 µg/ml aprotinin. Cell lysates were
treated with anti-Myc monoclonal antibody, and the immune complex was
then trapped on protein G-Sepharose beads. After wash for three times
with lysis buffer mB and two times with wash buffer (50 mM
HEPES-NaOH, pH 8.0, 100 mM NaCl, 3 mM
MnCl2, and 0.1 mM
Na3VO4), immunoprecipitates were incubated in
25 µl of kinase assay buffer (50 mM HEPES-NaOH, pH 8.0, 100 mM NaCl, 10 mM MgCl2, and 0.1 mM Na3VO4) containing 20 µM ATP and 2 µg of poly(Glu,Tyr) 4:1 at 30 °C for
indicated time. The reaction was terminated by the addition of SDS gel
loading buffer. The reaction mixtures were then subjected to SDS-PAGE
followed by immunoblotting. The amount of phosphorylated tyrosine
residues in poly(Glu,Tyr) 4:1 were quantified using the luminescence
image analyzer.
Colony Suppression Assay--
AGS cells (1.5 × 106) were transfected with 1.5 µg of the
puromycin-resistant gene (pBabe-puro) and 28.5 µg of expression
plasmid by using LipofectAMINE 2000 reagent. At 16 h after the
transfection, cells were collected, split into twelve 100-mm plates,
and selected in RPMI 1640 medium/10% fetal bovine serum containing 0.4 µg/ml puromycin for 10 days. After the drug selection, the cells were stained with May-Giemsa solution. Densities of colonies were
quantitated by using the LAS1000.
Flow Cytometric Analysis--
AGS cells (1.5 × 106) were transfected with expression vector or control
empty vector. At 36 h after the transfection, cells were
collected, treated with Cy3-conjugated Annexin V (Bio Vision), and
subjected to flow cytometric analysis using FACSCalibur (BD Biosciences).
Physical Interaction between CagA and Csk--
The cagA
gene was isolated from the genome of H. pylori standard
strain NCTC11637 and, after addition of a C-terminal hemagglutinin (HA)
tag, was cloned into the mammalian expression vector pSP65SR
To determine which EPIYA motifs were involved in Csk binding in COS-7
cells, we employed two EPIYA mutants of CagA, abCCC and ABccc (Fig.
1A). In the abCCC mutant, the former two EPIYA motifs were
converted into phosphorylation-resistant EPIAA sequences. Similarly,
the latter three EPIYA motifs, which were generated by duplication of
an EPIYA-containing 34-amino acid sequence three times, were replaced
by EPIAA to make the ABccc mutant. Co-expression studies in
COS-7 cells revealed that both abCCC and ABccc mutants were capable of
binding Csk, indicating that multiple EPIYA motifs can independently
bind Csk once they are tyrosine-phosphorylated (Fig. 1B,
lanes 5-8).
To confirm the CagA-Csk interaction in gastric epithelial cells, CagA
was transiently expressed in the AGS human gastric carcinoma cell line.
From the cell lysates prepared, Csk was immunoprecipitated using
anti-Csk, and the anti-Csk immunoprecipitates were immunoblotted with
anti-HA, which specifically detects the HA-tagged CagA. As expected,
endogenous Csk was capable of specifically binding wild-type, but not
the phosphorylation-resistant, CagA in AGS cells (Fig. 1C).
Hence, CagA formed a complex with endogenous Csk in gastric epithelial
cells, and the complex formation was strictly dependent on tyrosine
phosphorylation of CagA.
Involvement of the Csk SH2 Domain in CagA·Csk Complex
Formation--
The above-described observations suggested that
CagA-Csk interaction involved the phosphotyrosine-containing EPIYA
motifs of CagA. Like SHP-2, Csk possesses an SH2 domain, which
specifically binds a phosphotyrosine-containing peptide (35-38). To
determine the role of the Csk SH2 domain in CagA binding, we generated
a Csk mutant, CskS109C, in which serine 109 was replaced by cysteine to
destroy the structural integrity of the SH2 domain as previously reported (36). When expressed in COS-7 cells, CskS109C was incapable of
binding wild-type CagA (Fig. 2).
Accordingly, we concluded that CagA·Csk complex formation is mediated
by the interaction between tyrosine-phosphorylated EPIYA motifs of CagA
and the SH2 domain of Csk.
Activation of Csk by Complex Formation with CagA--
To elucidate
the biological consequences of the CagA-Csk interaction, we decided to
examine the effect of CagA binding on Csk kinase activity. To do so,
Myc-tagged Csk was ectopically expressed in COS-7 cells in the absence
or presence of CagA. Cell lysates were prepared and Csk was
immunoprecipitated from the lysates with anti-Myc (Fig.
3A). The immunopurified Csk
was then subjected to an in vitro kinase assay using
poly(Glu,Tyr) 4:1 as a substrate (34). As shown in Fig. 3
(B and C), Csk kinase activity was strongly
potentiated when Csk formed a complex with CagA. Based on the
observations, we concluded that CagA stimulates Csk kinase activity
through the physical complex formation.
Next, to determine whether CagA indeed activates Csk in cells, we
generated a human c-Src derivative, Src Inactivation of c-Src by CagA--
Once Tyr-530 is
tyrosine-phosphorylated by Csk, c-Src forms a "closed" inactive
structure. In contrast, c-Src changes to an "open" active state by
dephosphorylation of Tyr-530 and autophosphorylation at Tyr-419
(29-31, 39, 40). The above-described observations indicate that
CagA-Csk interaction activates Csk and thereby inhibits c-Src kinase
activity through inhibitory phosphorylation at Tyr-530. To investigate
this, we examined c-Src kinase activity using anti-phospho-Src and
anti-nonphospho-Src antibodies, which detect active c-Src that is
phosphorylated at Tyr-419, and inactive c-Src, which is non-phosphorylated at Tyr-419, respectively. CagA was co-expressed in
AGS cells with the kinase-dead c-Src, Src Inhibition of the Hummingbird Phenotype Induction by Ectopic
Expression of Csk--
Upon expression in AGS cells, CagA induces a
morphological change termed the hummingbird phenotype (14-16).
Induction of the hummingbird phenotype requires activation of SHP-2,
which is induced by CagA-SHP-2 interaction (16). Because tyrosine
phosphorylation of CagA by Src family kinases is an essential
prerequisite for CagA·SHP-2 complex formation, inhibition of Src
family kinases by Csk was suspected to reduce phosphorylation levels of
CagA and thus to inhibit the hummingbird phenotype induction by CagA.
To determine whether Csk acts as a negative regulator of CagA·SHP-2
signaling, we examined the effect of ectopic Csk expression on the
hummingbird phenotype induction by CagA. As shown in Fig. 5, Co-expression of Csk strongly
inhibited the CagA-dependent morphological transformation.
In contrast, the CskS109C mutant, which cannot bind CagA, exhibited
much less activity to inhibit the hummingbird phenotype than the
wild-type Csk did (Fig. 5, A and B). The
observations indicate that, upon complex formation with CagA, Csk
counteracts CagA·SHP-2 signaling by inactivating Src kinases and
competitively inhibiting CagA·SHP-2 interaction. A less but
significant inhibition of the CagA activity by CskS109C, which cannot
bind CagA, may be due to basal kinase activity of the Csk109C mutant,
which dose not require CagA binding.
Effects of CagA and SHP-2 on the Growth of AGS Cells--
The
above-described observations indicate that Csk, once activated by CagA,
inhibits the complex formation between CagA and SHP-2, thereby
down-regulating CagA-dependent activation of SHP-2. The
fact that there is a negative-feedback regulation of CagA activity
raises the possibility that sustained activation of SHP-2 by CagA
causes adverse effects on the colonization of cagA-positive H. pylori in the stomach. To address this possibility, we
examined the long term effect of CagA or SHP-2 on the growth of AGS
cells. To do so, we transfected a cDNA expression vector for CagA,
wild-type SHP-2, or membrane-targeted, constitutively active SHP-2
(Myr-
The growth-inhibitory activity of CagA or SHP-2 may be due to cell
cycle arrest or programmed cell death. To discriminate these two
possibilities, we performed flow cytometry analysis using Annexin V, a
sensitive method to detect apoptosis. At 36 h after transfection
of CagA, wild-type SHP-2, or Myr- CagA is translocated from H. pylori into the attached
gastric epithelial cells via the type IV injection system (14, 17-21). In this work, we found a novel interaction between the translocated CagA and Csk in gastric epithelial cells. We previously demonstrated that CagA undergoes tyrosine phosphorylation and binds an SH2 domain-containing protein-tyrosine phosphatase, SHP-2, in a tyrosine phosphorylation-dependent manner. Upon complex formation,
CagA activates SHP-2 phosphatase activity and thereby initiates a
cellular morphological change termed the hummingbird phenotype
(14-16).
As is the case with SHP-2, the CagA-Csk interaction is strictly
dependent on CagA tyrosine phosphorylation and involves the SH2 domain
of Csk. This indicates that Csk and SHP-2 compete with each other to
bind CagA. In this regard, a predominant fraction of the CagA proteins
expressed in gastric epithelial cells is present in complexes with
SHP-2 (16), suggesting that SHP-2 has higher affinity to bind
phosphorylated CagA than does Csk and/or that SHP-2 is more abundantly
expressed than is Csk in cells. Our present work thus indicates that a
small but significant fraction of tyrosine-phosphorylated CagA proteins
forms complexes with Csk in host cells.
The CagA-Csk interaction described in this work is thought to play an
important role in the regulation of CagA·SHP-2 signaling. Csk has
been well characterized as a negative regulator of Src family kinases;
it phosphorylates the C-terminal tail of c-Src and probably those of
other Src family members as well, resulting in an intramolecular
interaction between this phosphorylated tyrosine residue and the c-Src
SH2 domain that renders c-Src inactive (29-31, 39, 40). Src family
kinases expressed in gastric epithelial cells (such as c-Src, Fyn, Lyn,
and Yes) are responsible for CagA phosphorylation, and this CagA
phosphorylation is an essential prerequisite for CagA·SHP-2 complex
formation as well as subsequent induction of the hummingbird phenotype
(16, 22, 23). Our findings therefore indicate the presence of a
molecular circuitry that regulates the biological activity of CagA
within certain ranges of intensity by controlling
tyrosine-phosphorylation levels of CagA. In this regulation (Fig.
7), the translocated CagA protein first
undergoes tyrosine phosphorylation by Src family kinases, which are
somehow activated in gastric epithelial cells. A predominant fraction
of tyrosine-phosphorylated CagA proteins binds and deregulates SHP-2,
generating signals that give rise to the hummingbird phenotype. Simultaneously, a small but significant fraction of
tyrosine-phosphorylated CagA proteins binds and stimulates Csk, which
in turn phosphorylates and inactivates Src family kinases. Reduced Src
family kinase activity then down-regulates levels of CagA
phosphorylation, followed by diminished CagA·SHP-2 complex formation.
Accordingly, Csk functions as a negative regulator of CagA·SHP-2
signaling. Activated Csk also down-regulates CagA·Csk complex
formation, eventually diminishing Csk kinase activity as well. Such a
transient nature of Csk activation may provoke oscillation in the
intensity of CagA·SHP-2 signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mammalian expression vector. Rat Csk cDNA was C-terminal
Myc-epitope-tagged and cloned into pSP65SR
(Csk-Myc). To disable the
SH2 domain of Csk, serine 109 of Csk was substituted with the cysteine
residue (CskS109C-Myc) by site-directed mutagenesis. A kinase-dead
mutant of human c-Src was made by replacing ATP-binding lysine
298 to alanine (Src
K). A kinase-dead and
autophosphorylation-defective double mutant of human c-Src was made by
replacing lysine 298 and tyrosine 419 with alanine and phenylalanine,
respectively (Src
K
Y). These cDNAs were cloned into
pSP65SR
. Myc-tagged SHP-2 and its constitutively active mutant,
myr-SHP-2
SH2, have been described previously (16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
as
previously described (16). The cagA gene product, CagA,
undergoes tyrosine phosphorylation when expressed in mammalian cells.
Notably, NCTC11637-derived CagA protein possesses five glutamic
acid-proline-isoleucine-tyrosine-alanine (EPIYA) motifs that are potential tyrosine phosphorylation
sites in gastric epithelial cells (Fig.
1A). Complex formation between CagA and SHP-2 is reconstituted in COS-7 cells by co-transfection of
CagA and SHP-2 expression vectors (16). Using this reconstitution system, we screened additional SH2-containing cellular proteins that
are capable of physically interacting with CagA and found that
C-terminal Src kinase (Csk) bound wild-type CagA when co-expressed in
COS-7 cells (Fig. 1B, lanes 1-4). In contrast,
phosphorylation-resistant CagA, in which all of the five
tyrosine-phosphorylatable EPIYA motifs were replaced
with non-phosphorylatable EPIAA, was incapable of binding
Csk. This indicates that the complex formation is entirely dependent on
tyrosine phosphorylation of CagA. Csk is known to inactivate c-Src
protein-tyrosine kinase by phosphorylation tyrosine 530 (Tyr-530) in the C-terminal tail of c-Src (Tyr-527 in avian c-Src). Csk is considered to phosphorylate and inactivate other Src
family kinases as well. Using the co-expression system in COS-7, we
also overexpressed the p85 subunit of phosphatidylinositol 3-kinase,
c-Src, Gab1, Gab2, Shc, Grb2, Crk-II, or Sos, together with CagA.
However, we were not able to detect any specific interaction between
CagA and these proteins in COS-7 cells. Thus, CagA specifically binds
SHP-2 and Csk.2
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Fig. 1.
Physical complex formation between CagA and
Csk in COS-7 and AGS cells. A, schematic view of EPIYA
mutants of CagA used in B. B, expression vector
for HA-tagged wild-type CagA (WT CagA-HA) or HA-tagged EPIYA
mutant of CagA (phosphorylation-resistant (PR), abCCC, or
ABccc) was co-transfected together with expression vector for
Myc-tagged wild-type Csk (Csk-Myc) or control empty vector
into COS-7 cells. Cell lysates were prepared and immunoprecipitated
with a monoclonal antibody to Myc-epitope. Immunoprecipitates
(IP) and total cell lysates (TCL) were subjected
to immunoblotting (IB) with anti-Myc antibody or anti-HA
polyclonal antibody. Positions of CagA and Csk are indicated by
arrows. C, AGS gastric epithelial cells were
transfected with WT CagA-HA or PR CagA-HA. Lysates were
immunoprecipitated with an anti-Csk polyclonal antibody or rabbit
normal IgG. The immunoprecipitates (IP) and the total cell
lysates (TLC) were subjected to immunoblotting with anti-HA
polyclonal antibody that recognizes HA-tagged CagA. Position of CagA is
indicated by arrow.
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Fig. 2.
Requirement of the Csk SH2 domain in
CagA·Csk complex formation. COS-7 cells were co-transfected with
WT CagA-HA and Csk-Myc, CskS109C-Myc, or control empty vector. Lysates
prepared from the transfected COS-7 cells were immunoprecipitated with
a monoclonal antibody to Myc-epitope. Immunoprecipitates
(IP) and total cell lysates (TCL) were subjected
to immunoblotting (IB) with anti-Myc antibody or anti-HA
polyclonal antibody. Positions of CagA and Csk are indicated by
arrows.
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Fig. 3.
Effect of CagA complex formation on Csk
kinase activity. In vitro Csk kinase assay was
performed in presence or absence of CagA. A, COS-7 cells
were transfected with indicated expression vector or control empty
vector. Csk-Myc was immunoprecipitated from the cell lysates with
anti-Myc monoclonal antibody. Immunoprecipitates (IP) and
total cell lysates (TCL) were subjected to immunoblotting
(IB) with anti-Myc antibody or anti-HA polyclonal antibody.
Positions of CagA and Csk are indicated by arrows.
B, immunoprecipitates containing Csk-Myc was incubated with
poly(Glu,Tyr) 4:1 and 20 µM ATP for the indicated times.
Reaction mixtures were subjected to immunoblotting with
anti-phosphotyrosine to detect phosphorylated poly(Glu,Tyr) 4:1.
C, quantitation of the data obtained in B. Data
are plotted as relative Csk kinase activity in each time point in the
presence ( ) or absence (
) of CagA. Controls were obtained from
anti-Myc immunoprecipitates of COS-7 cell lysates prepared by
transfection with empty vector (
). Each value was calculated with
the amounts of Csk-Myc and phosphorylated poly(Glu,Tyr) 4:1, which were
quantitated by using the luminescence image analyzer LAS1000
(FUJIFILM). Relative Csk kinase activities were calculated by defining
Csk kinase activity at 30-min incubation with the substrate in the
absence of CagA as 1. The experiments were performed in triplicates,
and the error bars indicate 2× S.D.
K
Y, in which lysine 298, an ATP-binding site, and tyrosine 419, an autophosphorylation site (39,
40), were respectively replaced by arginine and phenylalanine residues.
The resulting Src
K
Y is enzymatically inactive and is not
autophosphorylated at Tyr-419 because of the mutation, yet it undergoes
tyrosine phosphorylation at Tyr-530 by Csk. The c-Src mutant was
co-expressed together with CagA in AGS cells, and its phosphorylation
levels were examined by anti-phosphotyrosine immunoblotting. As shown
in Fig. 4A, Src
K
Y became
more tyrosine-phosphorylated when CagA was co-expressed in cells.
Because Src
K
Y possesses only one tyrosine phosphorylation site,
Tyr-530, the result indicates CagA stimulated Csk and the activated Csk
phosphorylated Tyr-530 of c-Src.
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Fig. 4.
Elevated inhibitory phosphorylation of c-Src
by CagA expression in AGS cells. A: Upper,
AGS cells were transfected with indicated expression vector or control
empty vector. Lysates were subjected to immunoblotting with anti-c-Src,
anti-phosphotyrosine, or anti-HA. Src K
Y is a kinase-dead mutant
of c-Src that possesses the inhibitory phosphorylation site by Csk
(tyrosine 530) but does not have the autophosphorylation site (tyrosine
419). Positions of Src
K
Y and CagA are indicated by
arrows. Lower, relative amounts of phosphorylated
Src
K
Y at tyrosine 530 are indicated. Each value was calculated
from the immunoblotting data by using a luminescence image analizer
(LAS1000) and defining the value in the absence of CagA as 1. B: Upper, AGS cells were transfected with
indicated expression vector or control empty vector. Lysates were
subjected to immunoblotting with anti-c-Src, anti-phospho-Src,
anti-nonphospho-Src, anti-phosphotyrosine, or anti-HA antibody. Src
K
is a kinase-dead mutant that lacks the ATP-binding site. Positions of
Src
K and CagA-HA are indicated by arrows.
Lower, relative amounts of Src
K, phosphorylated and
non-phosphorylated at tyrosine 419, were calculated from the
immunoblotting data with the use of LAS1000 and defining the values in
the absence of CagA as 1, respectively.
K, in which Lys-298 was
substituted with arginine, and levels of phosphorylation on Tyr-419
were examined using these antibodies. Under conditions in which
comparable amounts of Src
K were expressed, CagA significantly increased the levels of inactive c-Src that was specifically detected by anti-nonphospho-Src, whereas the levels of active c-Src, which were
detected by anti-Phospho-Src, were reciprocally reduced (Fig. 4B). The observation indicated that CagA inactivated c-Src
by activating Csk upon physical complex formation. As the result, the
levels of autophosphorylation at Tyr-419 in Src
K were reduced. Notably, overall tyrosine phosphorylation levels of Src
K, which possesses both Tyr-419 and Tyr-530, slightly increased in the presence
of CagA as determined by anti-phosphotyrosine immunoblotting. This was
most probably due to elevated levels of Src
K phosphorylated at
Tyr-530 by Csk. Because Csk is considered to be the universal negative
regulator for the Src family kinases (29-31, 39, 40), it should
collectively phosphorylate and inactivate the Src family members
expressed in gastric epithelial cells upon complex formation with CagA.
Notably, Src
K underwent autophosphorylation at Tyr-419, yet it is
catalytically inactive. Hence, autophosphorylation of c-Src is an
intermolecular, rather than intramolecular, process caused by c-Src
dimerization as described previously (41).
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Fig. 5.
Effect of Csk on the hummingbird phenotype
induction by CagA. AGS cells were transfected with the indicated
expression vector or control empty vector (n = 3).
A, at 17 h after transfection, cell morphology was
examined by microscope. B, cells showing hummingbird
phenotype were counted in 10 different 0.25-mm2 fields in
each of three dishes. The experiments were performed in triplicates,
and the error bar indicates 2× S.D.
N-SHP-2) (16), together with the puromycin-resistance gene.
After selection of the transfected cells with puromycin, the number of
drug-resistant colonies was counted. As shown in Fig.
6A, expression of CagA induced
strong inhibition of colony formation. Also, expression of wild-type
SHP-2 resulted in a significant reduction of puromycin-resistant colonies, indicating that these proteins are growth-inhibitory. Furthermore, the constitutively active Myr-
N-SHP-2 exhibited stronger activity to reduce the colony number than wild-type SHP-2 did.
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Fig. 6.
Induction of apoptosis by CagA or SHP-2 in
AGS cells. A, AGS cells were co-transfected with
pBabe-puro and indicated expression vector or control empty vector.
Following transfection, cells were selected with puromycin and, on day
10, were fixed and stained. Densities of colonies in each dishes were
quantitated by using a fluorescence image analyzer LAS1000 (FUJIFILM),
and relative values are indicated. Bars represent 2× S.D.
of the three independent experiments. B, at 36 h after
transfection, cells were harvested, incubated with Cy-3-labeled Annexin
V, and subjected to flow cytometric analysis.
N-SHP-2 expression vector, a
significant fraction of the transfected AGS cells was positive for
Annexin V. From these observations, we concluded that ectopic
overexpression of CagA induces apoptosis and that this
CagA-dependent apoptosis is due to deregulation of SHP-2
caused by CagA·SHP-2 complex formation (Fig. 6B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
Fig. 7.
A model for the regulation of CagA·SHP-2
signaling by Csk. CagA·Csk complex attenuates the CagA·SHP-2
signaling by down-regulating complex formation between CagA and SHP-2.
This process involves 1) competitive inhibition of SHP-2 binding to
CagA by Csk, and 2) reduced CagA phosphorylation by inactivation of Src
family kinases, which is caused by the activation of Csk upon CagA
binding. Y, tyrosine; P, phosphorylation,
SH2; SH2 domain.
The biological relevance of down-regulation of CagA·SHP-2 signaling by CagA·Csk has been shown by the observation in the study that sustained activation of SHP-2 by CagA triggers apoptosis in gastric epithelial cells. Given the physiological role of SHP-2 in transmission of mitogenic signals between receptor tyrosine kinase and Ras, the CagA·SHP-2 complex is most likely to stimulate Ras as well as other signaling molecules. Notably, forced expression of activated Ras induces apoptosis due to signaling imbalance provoked by the deregulated Ras activity (42, 43). Deregulated stimulation of SHP-2 by CagA may also cause an inappropriate Ras activation, which eventually triggers programmed cell death. Massive loss of gastric epithelial cells by CagA-dependent apoptosis, which might provoke severe gastro-duodenal ulcers, is obviously disadvantageous for H. pylori in maintaining long term colonization in the stomach.
Attenuation of CagA·SHP-2 signaling by Csk may prevent excess CagA toxicity and enable cagA-positive H. pylori to persistently infect the human stomach for decades yet, at the same time, provoke chronic gastric damage by occasional but transient deregulation of SHP-2 upon CagA injection. Our findings with AGS cells are supported by the observation that cagA-positive H. pylori infection is associated with profound changes in the pattern of epithelial cell turnover in gastric glands. Results of both in vitro and in vivo studies on H. pylori infection indicate that the cagA-positive strain is associated with an increase in apoptosis (44-47). Furthermore, apoptosis was found to be increased in the stomach in patients with cagA-positive, but not cagA-negative, H. pylori strains (48), although the results of some studies have not supported the notion of a pro-apoptotic role of cagA-positive H. pylori (49, 50). Again, the conflicting results may be due to methods used to determine cagA status. Host genetic differences may also play a role in determining cellular response to H. pylori.
A moderate but continuous induction of apoptosis by CagA may underlie
the elevated epithelial cell turnover associated with cagA-positive H. pylori infection (44-50).
Importantly, extra rounds of DNA replication in gastric cells would
increase the chance of genetic mutations. In particular, mutations in
genes, such as p53, that evade apoptosis may change the host
cell response to CagA·SHP-2 signaling from apoptosis to deregulated
proliferation, a cellular situation in which further mutations in
oncogenes and tumor suppressor genes may progressively accumulate.
Indeed, in vivo studies have indicated that H. pylori infection is associated with acquired genetic instability
and induction of p53 point mutations in patients with
chronic gastritis (51, 52). Accordingly, cellular response to CagA,
either apoptosis or proliferation, may depend on the p53 status of host cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Arai for supplying human c-Src cDNA. We also thank Drs. M. Asaka and T. Azuma for help with the work.
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FOOTNOTES |
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* This work was supported by grants-in-aid for science research from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant from the Human Frontier Science Program Organization, and by a grant from the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim.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 should be addressed: Div. of Molecular Oncology, Inst. for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. Tel./Fax: 81-11-706-7544; E-mail: mhata@imm.hokudai.ac.jp.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M208155200
2 H. Higashi, R. Tsutsumi, and M. Hatakeyama, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: SH2, Src homology 2; SHP-2, SH2 domain-containing protein-tyrosine phosphatase-2; Csk, C-terminal Src kinase; HA, hemagglutinin; WT, wild-type.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Marshall, B. J., and Warren, J. R. (1984) Lancet 1, 1311-1315[Medline] [Order article via Infotrieve] |
2. | Dooley, C. P., Cohen, H., Fitzgibbons, P. L., Bauer, M., Appleman, M. D., Perez-Perez, G. I., and Blaser, M. J. (1989) N. Eng. J. Med. 321, 1562-1566[Abstract] |
3. | Parsonnet, J., Friedman, G. D., Vandersteen, D. P., Chang, Y., Vogelman, J. H., Orentreich, N., and Sibley, R. K. (1991) N. Eng. J. Med. 325, 1127-1131[Abstract] |
4. | Cover, T. L., and Blaser, M. J. (1999) Gastroenterology 117, 257-261[Medline] [Order article via Infotrieve] |
5. |
Covacci, A.,
Telford, J. L.,
Del Giudice, G.,
Parsonnet, J.,
and Rappuoli, R.
(1999)
Science
284,
1328-1333 |
6. | Eck, M., Schmausser, B., Haas, R., Greiner, A., Czub, S., and Muller- Hermelink, H. K. (1997) Gastroenterology 112, 1482-1486[Medline] [Order article via Infotrieve] |
7. | Covacci, A., Censini, S., Bugnoli, M., Petracca, R., Burroni, D., Macchia, G., Massone, A., Papini, E., Xiang, Z., Figura, N., and Rappuoli, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5791-5795[Abstract] |
8. | Tummuru, M. K., Cover, T. L., and Blaser, M. J. (1993) Infect. Immun. 61, 1799-1809[Abstract] |
9. |
Censini, S.,
Lange, C.,
Xiang, Z.,
Crabtree, J. E.,
Ghiara, P.,
Borodovsky, M.,
Rappuoli, R.,
and Covacci, A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14648-14653 |
10. | Crabtree, J. E., Taylor, J. D., Wyatt, J. I., Heatley, R. V., Shallcross, T. M., Tompkins, D. S., and Rathbone, B. J. (1991) Lancet 338, 332-335[Medline] [Order article via Infotrieve] |
11. | Kuipers, E. J., Perez-Perez, G. I., Meuwissen, S. G., and Blaser, M. J. (1995) J. Natl. Cancer Inst. 87, 1777-1780[Abstract] |
12. | Blaser, M. J., Perez-Perez, G. I., Kleanthous, H., Cover, T. L., Peek, R. M., Chyou, P. H., Stemmermann, G. N., and Nomura, A. (1995) Cancer Res. 55, 2111-2115[Abstract] |
13. | Parsonnet, J., Friedman, G. D., Orentreich, N., and Vogelman, H. (1997) Gut 40, 297-301[Abstract] |
14. |
Segal, E. D.,
Cha, J., Lo, J.,
Falkow, S.,
and Tompkins, L. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14559-14564 |
15. | Backert, S., Moese, S., Selbach, M., Brinkmann, V., and Meyer, T. F. (2001) Mol. Microbiol. 42, 631-644[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Higashi, H.,
Tsutsumi, R.,
Muto, S.,
Sugiyama, T.,
Azuma, T.,
Asaka, M.,
and Hatakeyama, M.
(2002)
Science
295,
683-686 |
17. |
Asahi, M.,
Azuma, T.,
Ito, S.,
Ito, Y.,
Suto, H.,
Nagai, Y.,
Tsubokawa, M.,
Tohyama, Y.,
Maeda, S.,
Omata, M.,
Suzuki, T.,
and Sasakawa, C.
(2000)
J. Exp. Med.
191,
593-602 |
18. |
Stein, M.,
Rappuoli, R.,
and Covacci, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1263-1268 |
19. |
Odenbreit, S.,
Püls, J.,
Sedlmaier, B.,
Gerland, E.,
Fischer, W.,
and Haas, R.
(2000)
Science
287,
1497-1500 |
20. | Backert, S., Ziska, E., Brinkmann, V., Zimny-Arndt, U., Fauconnier, A., Jungblut, P. R., Naumann, M., and Meyer, T. F. (2000) Cell Microbiol. 2, 155-164[CrossRef][Medline] [Order article via Infotrieve] |
21. | Christie, P. J., and Vogel, J. P. (2000) Trends Microbiol. 8, 354-360[CrossRef][Medline] [Order article via Infotrieve] |
22. | Stein, M., Bagnoli, F., Halenbeck, R., Rappuoli, R., Fantl, W. J., and Covacci, A. (2002) Mol. Microbiol. 43, 971-980[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Selbach, M.,
Moese, S.,
Hauck, C. R.,
Meyer, T. F.,
and Backert, S.
(2002)
J. Biol. Chem.
277,
6775-6778 |
24. |
Yamaoka, Y.,
Kodama, T.,
Kashima, K.,
Graham, D. Y.,
and Sepulveda, A. R.
(1998)
J. Clin. Microbiol.
36,
2258-2263 |
25. | Yamaoka, Y., El-, Zimaity, H. M., Gutierrez, O., Figura, N., Kim, J. G., Kodama, T., Kashima, K., Graham, D. Y., and Kim, J. K. (1999) Gastroenterology 117, 342-349[Medline] [Order article via Infotrieve] |
26. | Freeman, R. M., Jr., Plutzky, J., and Neel, B. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11239-11243[Abstract] |
27. | Feng, G. S., Hui, C. C., and Pawson, T. (1993) Science 259, 1607-1611[Medline] [Order article via Infotrieve] |
28. | Feng, G. S. (1999) Exp. Cell Res. 253, 47-54[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Okada, M.,
and Nakagawa, H.
(1989)
J. Biol. Chem.
264,
20886-20893 |
30. |
Okada, M.,
Nada, S.,
Yamanashi, Y.,
Yamamoto, T.,
and Nakagawa, H.
(1991)
J. Biol. Chem.
266,
24249-24252 |
31. | Nada, S., Okada, M., MacAuley, A., Cooper, J. A., and Nakagawa, H. (1991) Nature 351, 69-72[CrossRef][Medline] [Order article via Infotrieve] |
32. | Imamoto, A., and Soriano, P. (1993) Cell 73, 1117-1124[Medline] [Order article via Infotrieve] |
33. | Nada, S., Yagi, T., Takeda, H., Tokunaga, T., Nakagawa, H., Ikawa, Y., Okada, M., and Aizawa, S. (1993) Cell 73, 1125-1135[Medline] [Order article via Infotrieve] |
34. | Okada, M., and Nakagawa, H. (1988) J. Biochem. (Tokyo) 104, 297-305[Abstract] |
35. | Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanahusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778[Medline] [Order article via Infotrieve] |
36. | Sabe, H., Hata, A., Okada, M., Nakagawa, H., and Hanafusa, H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3984-3988[Abstract] |
37. |
Gregorieff, A.,
Cloutier, J. F.,
and Veillette, A.
(1998)
J. Biol. Chem.
273,
13217-13222 |
38. | Kawabuchi, M., Satomi, Y., Takao, T., Shimonishi, Y., Nada, S., Nagai, K., Tarakhovsky, A., and Okada, M. (2000) Nature 404, 999-1003[CrossRef][Medline] [Order article via Infotrieve] |
39. | Cooper, J. A., and Howell, B. (1993) Cell 73, 1051-1054[Medline] [Order article via Infotrieve] |
40. | Brown, M. T., and Cooper, J. A. (1996) Biochim. Biophys. Acta 1287, 121-149[CrossRef][Medline] [Order article via Infotrieve] |
41. | Barker, S. C., Kassel, D. B., Weigl, D., Huang, X., Luther, M. A., and Knight, W. B. (1995) Biochemistry 34, 14843-14851[Medline] [Order article via Infotrieve] |
42. | Liu, H. S., Chen, C. Y., Lee, C. H., and Chou, Y. I. (1998) Br. J. Cancer 77, 1777-1786[Medline] [Order article via Infotrieve] |
43. |
Navarro, P.,
Valverde, A. M.,
Benito, M.,
and Lorenzo, M.
(1999)
J. Biol. Chem.
274,
18857-18863 |
44. |
Rudi, J.,
Kuck, D.,
Strand, S.,
von Herbay, A.,
Mariani, S. M.,
Krammer, P. H.,
Galle, P. R.,
and Stremmel, W.
(1998)
J. Clin. Invest.
102,
1506-1514 |
45. |
Peek, R. M., Jr.,
Blaser, M. J.,
Mays, D. J.,
Forsyth, M. H.,
Cover, T. L.,
Song, S. Y.,
Krishnan, U.,
and Pietenpol, J. A.
(1999)
Cancer Res.
59,
6124-6131 |
46. |
Jones, N. L.,
Day, A. S.,
Jennings, H. A.,
and Sherman, P. M.
(1999)
Infect. Immun.
67,
4237-4242 |
47. |
Neu, B.,
Randlkofer, P.,
Neuhofer, M.,
Voland, P.,
Mayerhofer, A.,
Gerhard, M.,
Schepp, W.,
and Prinz, C.
(2002)
Am. J. Physiol. Gastrointest. Liver Physiol.
283,
G309-G318 |
48. |
Moss, S. F.,
Sordilo, E. M.,
Abdalla, A. M.,
Makarov, V.,
Hanzely, Z.,
Perez-Perez, G. I.,
Blaser, M. J.,
and Holt, P. R.
(2001)
Cancer Res.
61,
1406-1411 |
49. |
Peek, R. M., Jr.,
Moss, S. F.,
Tham, K. T.,
Perez-Perez, G. I.,
Weng, S.,
Miller, G. G.,
Atherton, J. C.,
Holt, P. R.,
and Blaser, M. J.
(1997)
J. Natl. Cancer Inst.
89,
863-868 |
50. | Rokkas, T., Ladas, S., Liatsos, C., Petridou, E., Papathodorou, G., Theocharis, S., Karameris, A., and Raptis, S. (1999) Dig. Dis. Sci. 44, 487-493[Medline] [Order article via Infotrieve] |
51. |
Nardone, G.,
Staibano, S.,
Rocco, A.,
Mezza, E.,
D'Armiento, F. P.,
Insabato, L.,
Coppola, A.,
Salvatore, G.,
Lucariello, A.,
Figura, N., De,
Rosa, G.,
and Budillon, G.
(1999)
Gut
44,
789-799 |
52. | Murakami, K., Fujioka, T., Okimoto, T., Mitsuishi, Y., Oda, T., Nishizono, A., and Nasu, M. (1999) Scand. J. Gastroenterol. 34, 474-477[CrossRef][Medline] [Order article via Infotrieve] |