From the Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan
Received for publication, January 31, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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Gab-1 is a multiple docking protein that
is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met,
hepatocyte growth factor/scatter factor receptor, and epidermal growth
factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of
cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine
phosphorylation of Gab-1, whereas the expression of E-cadherin
specifically induced tyrosine phosphorylation of Gab-1. A relatively
selective inhibitor of Src family kinases reduced cell-cell
adhesion-dependent tyrosine phosphorylation of Gab-1,
whereas expression of a dominant-negative mutant of Csk increased it.
Disruption of cell-cell adhesion, which reduced tyrosine
phosphorylation of Gab-1, also reduced the activation of
mitogen-activated protein kinase and Akt in response to cell-cell
adhesion. These results indicate that E-cadherin-mediated cell-cell
adhesion induces tyrosine phosphorylation by a Src family kinase of
Gab-1, thereby regulating the activation of Ras/MAP kinase and
phosphatidylinositol 3-kinase/Akt cascades.
The cell junction plays essential roles in various cell functions,
including cell adhesion and migration (1-5). In polarized epithelial
cells, the cell-cell junction shows a specialized membrane structure
comprising tight junctions,
AJs,1 and desmosomes. AJs
play a particularly important role because the formation of AJs
subsequently leads to the formation of other cell junctions. At AJs,
cadherin, a Ca2+-dependent homophilic cell
adhesion molecule, plays a fundamental role in cell-cell adhesion (2,
5, 6). Cadherin forms Ca2+-dependent homophilic
cis dimerization and trans interaction, which
cause cell-cell adhesion (7-11). The cytoplasmic tail of cadherin
interacts with In addition to its role in cell-cell adhesion, the cadherin-catenin
system may play an important role in the signal transduction system.
Gab-1 is a docking protein that undergoes tyrosine phosphorylation and
subsequently binds Grb2, SHP-2, and the p85 subunit of
PI3-kinase in response to various growth factors such as HGF/SF, epidermal growth factor, insulin, and insulin-like growth factor (31,
32). Gab-1 also contains a pleckstrin homology domain in its
NH2-terminal region; this domain mediates protein
interaction with cellular membranes, possibly by binding to
polyphosphoinositides (33, 34). Gab-1 furthermore contains a
c-Met-binding domain, which may mediate protein-protein interactions by
binding to phosphotyrosine-containing motifs of the cytoplasmic portion
of c-Met (32). Gab-1 has recently been demonstrated to be localized at
cell-cell adhesion sites of MDCK cells (35). The pleckstrin homology
domain of Gab-1 or PI3-kinase activity is required for the proper
membrane localization of Gab-1 (35). We and others have demonstrated
recently that both E-cadherin and c-Met are colocalized at cell-cell
adhesion sites of MDCK cells (36, 37). In addition, HGF/SF or a phorbol ester, TPA, induces disruption of cell-cell adhesion, which is accompanied by endocytosis of both E-cadherin and c-Met. Thus, Gab-1
and the cadherin-catenin system could interact with each other.
In this study, we show that cell-cell adhesion stimulates tyrosine
phosphorylation of Gab-1 and that this effect may be mediated through
E-cadherin and a Src family kinase. Thus, cell-cell adhesion as well as
growth factors regulates the activation of the downstream signaling
pathway of Gab-1.
Antibodies--
The FLAG-tagged human cDNA of Gab-1 was
provided by Dr. T. Hirano (Osaka University, Suita, Japan) (38). MDCK
cells were supplied by Dr. W. Birchmeier (Max-Delbruck Center for
Molecular Medicine, Berlin, Germany). Human recombinant HGF/SF was
provided by Dr. T. Nakamura (Osaka University, Suita, Japan). The
anti-Gab-1 rabbit polyclonal Ab was obtained from Upstate Biotechnology
(Lake Placid, NY) and also provided by Dr. T. Hirano (Osaka
University). An anti-FLAG mouse mAb (M2) was from Eastman Kodak. The
horseradish peroxidase-conjugated anti-phosphotyrosine mAb (PY20) was
obtained from Santa Cruz Biotechnology. (Santa Cruz, CA). The
anti-tyrosine-phosphorylated form of MAP kinase rabbit polyclonal Ab,
the anti-MAP kinase rabbit polyclonal Ab, the
anti-serine-phosphorylated form of Akt rabbit polyclonal Ab, and the
anti-Akt rabbit polyclonal Ab were obtained from New England BioLabs
(Beverly, MA). Other materials and chemicals were obtained from
commercial sources.
Cell Culture--
MDCK cells, mouse mammary tumor MTD-1A cells,
and mouse keratinocyte 308R cells were maintained at 37 °C in a
humidified atmosphere of 10% CO2 and 90% air in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
(Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml
streptomycin. L, CL, and EL cells were kindly supplied by Drs. S. Tsukita and A. Nagafuchi (Kyoto University, Kyoto, Japan). These cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. For transient transfection experiments, L,
CL, and EL cells were seeded at a density of 1 × 105
cells/dish onto 10-cm dishes and transfected with 1 µg of pCMV vector
containing the FLAG-tagged Gab-1 cDNA by the use of LipofectAMINE and PLUS Reagent (Life Technologies, Inc.) at 24 h after seeding. The recombinant adenovirus, Ax1CATcsk- Immunoprecipitation and Immunoblotting--
All cultured cells
(in a 10-cm plate) were frozen in liquid nitrogen and then lysed on ice
in 1 ml of an ice-cold lysis buffer (20 mM Tris-HCl at pH
7.6, 140 mM NaCl, 2.6 mM CaCl2, 1 mM MgCl2, 1% (v/v) Nonidet P-40, and 10%
(v/v) glycerol) containing 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, and 1 mM sodium vanadate.
Whole cell lysates were centrifuged at 10,000 × g at 4 °C for 15 min, and the resulting supernatants were subjected to
immunoprecipitation and immunoblotting. Briefly, the supernatants were
incubated at 4 °C with the anti-Gab-1 poyclonal Ab bound to protein
G-Sepharose beads (2 µg of Ab/20 µl of beads) (Amersham Pharmacia
Biotech) for 4 h, after which the beads were washed twice with 1 ml of WG buffer (50 mM Hepes-NaOH at pH 7.6, 150 mM NaCl, and 0.1% (v/v) Triton X-100) and resuspended in
an SDS sample buffer. SDS-polyacrylamide gel electrophoresis and
immunoblotting with various Abs were performed using an ECL detection
kit (Amersham Pharmacia Biotech).
Phosphorylation of GST-Gab-1 in Vitro--
A GST fusion protein
containing the COOH-terminal half of Gab-1 (amino acids 421-694) was
generated and purified using glutathione-Sepharose beads (Amersham
Pharmacia Biotech). GST fusion proteins (0.1 µg), which were
immobilized on glutathione-Sepharose beads, were washed twice with
kinase assay buffer (50 mM Hepes-NaOH at pH 7.6, 3 mM MnCl2, 10 mM MgCl2,
1 mM dithiothreitol) and incubated with partially purified
Src kinase (Upstate Biotechnology) at 24 °C for 30 min in 50 µl of
a kinase assay buffer in the absence or presence of 10 mM
ATP. The reaction mixtures were then centrifuged at 10,000 × g at 4 °C for 5 min, and the resulting supernatants were
mixed with the SDS sample buffer, boiled, and subjected to SDS-PAGE.
The extent of tyrosine phosphorylation of GST proteins was analyzed by
immunoblotting with the horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20.
Cell-Cell Adhesion-dependent Tyrosine Phosphorylation
of Gab-1--
MDCK cells were serum starved in the culture medium
containing 2 mM Ca2+ for up to 24 h, after
which the whole cell lysates were prepared and subjected to
immunoprecipitation with the anti-Gab-1 polyclonal Ab. Immunoblotting
of the resulting immunoprecipitates with the anti-phosphotyrosine mAb
PY20 revealed that Gab-1 was tyrosine phosphorylated in the
serum-starved MDCK cells (Fig.
1A). When the Ca2+
concentration in the culture medium was switched from 2 mM
to 2 µM, the MDCK cells gradually detached from each
other, and dissociation of cells was complete at 4 h after the
Ca2+ switch as reported previously (36, 37 and data not
shown). The tyrosine phosphorylation of Gab-1 was decreased in a time
of incubation-dependent manner and almost undetectable at
4 h after the Ca2+ switch from 2 mM to 2 µM (Fig. 1A). After MDCK cells were incubated with 2 µM Ca2+ for 4 h, the
concentration of Ca2+ in the culture medium was increased
to 2 mM. The dissociated cells then gradually formed
cell-cell adhesion (data not shown), which was accompanied by tyrosine
phosphorylation of Gab-1 (Fig. 1A). The same filter was
reprobed with the anti-Gab-1 polyclonal Ab to confirm that a similar
amount of Gab-1 was present in each lane (Fig. 1A).
Stimulation of MDCK cells with HGF/SF caused spreading and subsequent
scattering of the cells as described previously (40, 41). The
HGF/SF-induced dissociation of cells also decreased the tyrosine
phosphorylation of Gab-1 in the culture medium containing 2 mM Ca2+ (Fig. 1B). In addition to
MDCK cells, tyrosine phosphorylation of Gab-1 was also observed in
mouse mammary tumor MTD-1A cells and mouse keratinocyte 308R cells,
both of which were serum starved in the culture medium containing 2 mM Ca2+ (Fig. 2).
Furthermore, the marked decrease in the extent of tyrosine phosphorylation of Gab-1 was observed in response to the disruption of
cell-cell adhesion of these two cell lines by the low Ca2+
treatment (Fig. 2). Stimulation of 308R cells with HGF/SF also caused
scattering of the cells and reduced the tyrosine phosphorylation of
Gab-1 (data not shown). These results suggest that cell-cell adhesion
reversibly induces the tyrosine phosphorylation of Gab-1.
Involvement of E-Cadherin in Cell-Cell
Adhesion-dependent Tyrosine Phosphorylation of
Gab-1--
When the concentration of Ca2+ in the culture
medium was increased from 2 µM to 2 mM in the
presence of anti-E-cadherin mAb, which blocks the adhesion activity of
E-cadherin, no recovery of cell-cell adhesion resulted in a loss of the
tyrosine phosphorylation of Gab-1 in MDCK cells (Fig.
3A). In addition, minimal
tyrosine phosphorylation of Gab-1 was observed in both L cells that did not express E-cadherin and CL cells that expressed claudin, an integral
membrane protein responsible for tight junction strand formation (42),
whereas it was evident in EL cells that expressed human E-cadherin (43)
in the culture medium containing 2 mM Ca2+
(Fig. 3B). It has been shown that when MDCK cells
preincubated at 2 µM Ca2+ for 2 h are
incubated further with TPA, a tight junction-like structure is
observed, and ZO-1, but not E-cadherin or Involvement of a Src Family Kinase in the Cell-Cell
Adhesion-dependent Tyrosine Phosphorylation of
Gab-1--
To investigate further the mechanism by which cell-cell
adhesion induces the tyrosine phosphorylation of Gab-1, we next
examined the effects of
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine, a compound reported to act as a relatively selective inhibitor of Src
family kinases (45).
4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine markedly decreased the tyrosine phosphorylation of Gab-1 in the culture
medium containing 2 mM Ca2+ (Fig.
4A). We also examined the
effect of overexpression of a kinase-inactive mutant of Csk (Csk- Effects of Cell-Cell Adhesion on the Downstream Signaling of
Gab-1--
We next examined the effects of cell-cell adhesion on the
downstream signaling of Gab-1. Decreasing the Ca2+
concentration of the medium induced a marked reduction of the tyrosine
phosphorylation of Gab-1 (Fig.
5A). We then determined the
activation of MAP kinase by immunoblotting the whole cell lysates with
the anti-tyrosine-phosphorylated MAP kinase polyclonal Ab. The
significant activation of MAP kinase in MDCK cells without any
stimulation was observed when cells were cultured in the culture medium
containing 2 mM Ca2+ (Fig. 5B). In
contrast, reduction of the Ca2+ concentration in the medium
inhibited MAP kinase activation markedly compared with that observed in
the 2 mM Ca2+ medium (Fig. 5B).
Tyrosine-phosphorylated Gab-1 binds PI3-kinase (48, 49), which
subsequently activates Akt/PKB kinase, a downstream target of
PI3-kinase (50). Thus, we next examined the effect of reduction of
medium Ca2+ on Akt activation, as determined by
immunoblotting the whole cell lysates with the
anti-serine-phosphorylated Akt polyclonal Ab. The significant
activation of Akt was observed in MDCK cells without any stimulation
when cells were cultured in the culture medium containing 2 mM Ca2+, whereas the reduction of the medium
Ca2+ concentration inhibited Akt activation markedly (Fig.
5C). These data suggest that cell-cell adhesion is involved
in the activation of MAP kinase and Akt presumably through the tyrosine
phosphorylation of Gab-1.
In the present study, we have demonstrated that cell-cell adhesion
reversibly induces the tyrosine phosphorylation of Gab-1 in cultured
epithelial cells. The extent of the tyrosine phosphorylation of Gab-1
is increased markedly in EL cells compared with that observed in L
cells, whereas the anti-E-cadherin mAb markedly reduces the tyrosine
phosphorylation of Gab-1 in MDCK cells. Thus, cell-cell adhesion
induces the tyrosine phosphorylation of Gab-1 in an
E-cadherin-dependent manner. Several proteins such as
We have also explored the mechanism by which cell-cell adhesion
stimulates the tyrosine phosphorylation of Gab-1.
4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine, a relatively selective inhibitor of Src family kinases, reduces the
tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increases its response. These data
suggest that a Src family kinase, at least in part, contributes to the
tyrosine phosphorylation of Gab-1 in response to cell-cell adhesion. In
fact, Src family kinases have been found at AJs (51). In addition,
tyrosine phosphorylation of AJ proteins, such as We have also demonstrated that the tyrosine phosphorylation of Gab-1 by
cell-cell adhesion correlates well with the activation of MAP kinase
and Akt activation in response to cell-cell adhesion. Activation of Akt
by the formation of E-cadherin-mediated cell-cell junctions has also
been demonstrated in MDCK cells (52). It has been reported that
cell-cell adhesion, which is mediated by E-cadherin, can promote cell
survival in a variety of cell types (53, 54). Compelling evidence
suggests that both Ras/MAP kinase and PI3-kinase/Akt cascades are
involved in cell survival (55). Thus, our present results provide an
interesting mechanism whereby E-cadherin-mediated cell-cell adhesion
stimulates the tyrosine phosphorylation of Gab-1, which then induces
the activation of both Ras/MAP kinase and PI3-kinase/Akt cascades. This
mechanism could be involved in cell-cell adhesion-dependent
cell survival.
It remains unknown how the E-cadherin-mediated cell-cell adhesion
induces the Src family kinase-mediated tyrosine phosphorylation of
Gab-1. It is possible that the E-cadherin-mediated cell-cell adhesion
induces the activation of a Src family kinase. In contrast, the
E-cadherin-mediated cell-cell adhesion promotes the recruitment of this
kinase to the place where Gab-1 is localized. In fact, it has been
shown that the binding of extracellular matrix to integrin also induces
tyrosine phosphorylation of several proteins by Src, which is recruited
to integrin-based focal adhesions through its binding to FAK, another
focal adhesion-associated tyrosine kinase (56). Further efforts are
clearly necessary to clarify the molecular mechanism by which the
E-cadherin-mediated cell-cell adhesion leads to the tyrosine
phosphorylation of Gab-1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, which in turn interacts with
-catenin.
-Catenin interacts directly with F-actin (12).
-Catenin furthermore interacts with other F-actin-binding proteins,
-actinin and vinculin, through the NH2-terminal half (13-16), and
ZO-1 through the COOH-terminal half (17). The linkage of cadherin to
the actin cytoskeleton strengthens cadherin-based cell-cell adhesion (2, 5, 6, 18).
-Catenin has been shown to play a crucial role in the Wnt-mediated
signaling pathway (19). In addition, the cadherin-catenin system may be
closely linked to tyrosine phosphorylation of proteins, which exist at
cell-cell adhesion sites. In fact,
-,
-, and
-catenins and
p120ctn, which all associate directly or
indirectly with the cytoplasmic region of E-cadherin, are known to be
tyrosine phosphorylated in v-src-transformed cells
(20-22) or in response to epidermal growth factor and HGF/SF (23). The
cadherin-catenin system has been shown to form a complex with the
epidermal growth factor receptor or the c-Erb-B-2 gene product (24,
25). Moreover, protein tyrosine phosphatases, such as LAR-PTP, PTPµ,
PTP1B-like phosphatase, and SHP-2, have been shown to associate
with the cadherin-catenin system (26-28). Increased tyrosine
phosphorylation of catenins and p120ctn is
correlated with the decrease of cell adhesion activity which occurs
upon cell transformation or mitogenic growth factor stimulation (20-23). In contrast, it has been shown recently that cell-cell adhesion induces tyrosine phosphorylation of these junctional proteins
in nontransformed cells (29, 30). However, it remains to be determined
whether tyrosine phosphorylation of cadherin-associated proteins is
involved in the regulation of cell-cell adhesion.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K, was obtained from Dr. M. Okada (Osaka University). MDCK cells were infected with the adenovirus
vector at the indicated multiplicity of infection as described
previously (39).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cell-cell adhesion-dependent
tyrosine phosphorylation of Gab-1 in MDCK cells. A,
MDCK cells were treated as indicated in the figure. The whole cell
lysates were then subjected to immunoprecipitation (IP) with
the anti-Gab-1 polyclonal Ab ( Gab-1), and the
resulting immunoprecipitates were subjected to immunoblotting with the
horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20
(
PY) (upper panel). The same blot
was reprobed with the anti-Gab-1 polyclonal Ab (lower panel)
to ensure that similar amounts of endogenous Gab-1 were present in each
lane. High, MDCK cells were deprived of serum for 12 h
in medium containing 2 mM Ca2+. Low,
MDCK cells were deprived of serum in the medium containing 2 mM Ca2+ for 12 h and subsequently cultured
in medium containing 2 µM Ca2+ for the
indicated time. Low
High, cells pretreated
with 2 µM Ca2+ for 4 h were cultured
further in the medium containing 2 mM Ca2+ for
the indicated time. B, serum-deprived MDCK cells were
incubated in the presence or absence of 10 ng/ml HGF/SF for 0-18 h.
The whole cell lysates were then subjected to immunoprecipitation with
the anti-Gab-1 polyclonal Ab, and the resulting immunoprecipitates were
subjected to immunoblotting with either the horseradish
peroxidase-conjugated anti-phosphotyrosine mAb PY20 (upper
panel) or the anti-Gab-1 polyclonal Ab (lower panel).
The results shown are representative of three independent
experiments.
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Fig. 2.
Cell-cell adhesion-dependent
tyrosine phosphorylation of Gab-1 in MTD-1A cells or 308R cells.
MTD-1A (MT) cells or 308R cells were treated as indicated in
the figure. The whole cell lysates were then subjected to
immunoprecipitation (IP) with the anti-Gab-1 polyclonal Ab
( Gab-1), and the resulting immunoprecipitates
were subjected to immunoblotting with either the horseradish
peroxidase-conjugated anti-phosphotyrosine mAb PY20
(
PY, upper panels) or the
anti-Gab-1 polyclonal Ab (lower panels). High,
cells were deprived of serum for 12 h in the medium containing 2 mM Ca2+. Low, cells were deprived of
serum in the medium containing 2 mM Ca2+ for
12 h and subsequently cultured in the medium containing 2 µM Ca2+ for 4 h. The results shown are
representative of three independent experiments.
-catenin, accumulates
there (44). The addition of TPA after the incubation of MDCK cells with
a low concentration of Ca2+ failed to re-increase the
tyrosine phosphorylation of Gab-1 (Fig. 3C). Thus, these
data suggest that E-cadherin mediates the cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.
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Fig. 3.
E-Cadherin-dependent tyrosine
phosphorylation of Gab-1. A, serum-deprived MDCK cells
were cultured in the medium containing 2 µM
Ca2+ for 4 h and then cultured further in the medium
containing 2 mM Ca2+ for 2 h in the
presence or absence of the anti-E-cadherin mAb Arc-1
( E-Cadherin). The whole cell lysates were then
subjected to immunoprecipitation (IP) with the anti-Gab-1
polyclonal Ab (
Gab-1), and the resulting
immunoprecipitates were subjected to immunoblotting with either the
horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20
(
PY, upper panel) or the anti-Gab-1
polyclonal Ab (lower panel). B, L, CL, and EL
cells were transfected with pCMVFLAG-Gab-1. At 48 h after transfection, these
cells were serum deprived in the medium containing 2 mM
Ca2+, and the whole cell lysates prepared from the cells
were subjected to immunoprecipitation with anti-FLAG mAb
(
FLAG). The resulting immunoprecipitates were
subjected to immunoblotting with either the horseradish
peroxidase-conjugated anti-phosphotyrosine mAb PY20 (upper
panel) or the anti-Gab-1 polyclonal Ab (lower panel).
C, MDCK cells were treated as indicated in the figure. The
whole cell lysates were then subjected to immunoprecipitation with the
anti-Gab-1 polyclonal Ab, and the resulting immunoprecipitates were
subjected to immunoblotting with either the horseradish
peroxidase-conjugated anti-phosphotyrosine mAb PY20 (upper
panel) or the anti-Gab-1 polyclonal Ab (lower panel).
Low
High, MDCK cells were deprived of serum
in the medium containing 2 mM Ca2+ for 12 h and subsequently cultured in the medium containing 2 µM
Ca2+ for 4 h. The cells were cultured further in the
medium containing 2 mM Ca2+ for 2 h.
Low
TPA, cells pretreated with 2 µM Ca2+ for 4 h were cultured further in
the medium containing 2 µM Ca2+ and 100 nM TPA for 2 h. The results shown are representative
of three independent experiments.
K)
on the tyrosine phosphorylation of Gab-1 in MDCK cells. Csk is a
Src-like tyrosine kinase that inhibits the activity of Src family
kinases by catalyzing the phosphorylation of a COOH-terminal tyrosine
residue (46, 47). Recombinant adenovirus vectors were used to express
Csk-
K in MDCK cells as described previously (39). This mutant Csk
has been shown to act in a dominant-negative manner (39). MDCK cells were infected with either Ax1CAT-lacZ or Ax1CAT-csk-
K (Fig.
4B). The extent of the tyrosine phosphorylation of Gab-1 was
increased markedly by the expression of Csk-
K in the culture medium
containing 2 mM Ca2+ compared with that in the
control cells (Fig. 4B). Partially purified Src kinase
tyrosine phosphorylated the GST-COOH-terminal region of Gab-1 (amino
acid 421-694), which contained five tyrosine residues (Tyr-447,
Tyr-472, Tyr-589, Tyr-627, and Tyr-659) (data not shown). Therefore,
these results suggest that a Src family kinase mediates the cell-cell
adhesion-dependent tyrosine phosphorylation of Gab-1.
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Fig. 4.
Effect of an inhibitor for Src family kinases
or expression of a dominant-negative mutant of Csk on the tyrosine
phosphorylation of Gab-1. A, serum-deprived MDCK cells
were cultured at 2 µM Ca2+ for 4 h with
or without 1 µM
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine
(Src Inhibitor), a compound reported to act as a relatively
selective inhibitor of Src family kinases, for 30 min, and then
cultured further at 2 mM Ca2+ for 2 h. The
whole cell lysates were then subjected to immunoprecipitation
(IP) with the anti-Gab-1 polyclonal Ab
( Gab-1), and the resulting immunoprecipitates
were subjected to immunoblotting with either the horseradish
peroxidase-conjugated anti-phosphotyrosine mAb PY20
(
PY, upper panel) or the anti-Gab-1
polyclonal Ab (lower panel). B, MDCK cells were
infected with either Ax1CAT-lacZ (Control) or
Ax1CAT-csk-
K (CSK-
K) at 100 multiplicity of
infection. At 48 h after the transfection, the infected MDCK cells
in the meduim containing 2 mM Ca2+ were
subjected to immunoprecipitation with the anti-Gab-1 polyclonal Ab, and
the resulting immunoprecipitates were subjected to immunoblotting with
either the horseradish peroxidase-conjugated anti-phosphotyrosine mAb
PY20 (upper panel) or the anti-Gab-1 polyclonal Ab
(lower panel). The results shown are representative of three
independent experiments.
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Fig. 5.
Effects of cell-cell adhesion on the
downstream signaling of Gab-1. A, serum-deprived MDCK cells
were treated as indicated in the figure. The whole cell lysates were
then subjected to immunoprecipitation (IP) with the
anti-Gab-1 polyclonal Ab ( Gab-1), and the
resulting immunoprecipitates were subjected to immunoblotting with the
horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20
(
PY, upper panel) or the anti-Gab-1
polyclonal Ab (lower panel). High, MDCK cells
were deprived of serum for 12 h in the medium containing 2 mM Ca2+. Low, MDCK cells were
deprived of serum in the medium containing 2 mM
Ca2+ for 12 h and subsequently cultured in the medium
containing 2 µM Ca2+ for 4 h.
B, the whole cell lysates were subjected to immunoblotting
with either the anti-tyrosine-phosphorylated-MAP kinase polyclonal Ab
(
p-MAPK, upper panel) or the
anti-MAP kinase polyclonal Ab (
MAPK,
lower panel). C, the whole cell lysates were also
subjected to immunoblotting with either the
anti-serine-phosphorylated-Akt polyclonal Ab
(
p-Akt, upper panel) or the
anti-Akt polyclonal Ab (
Akt, lower
panel). The results shown are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin,
-catenin, p120ctn, which are all
located at AJs, have also been shown to be tyrosine phosphorylated
(20-22), although the mechanism underlying their tyrosine phosphorylation is not fully understood. Thus, Gab-1 might be
a new member of the group of proteins that are localized at AJs and
undergo tyrosine phosphorylation in response to cell-cell adhesion.
-catenin,
-catenin, and p120ctn, has been shown to be
increased markedly in response to cell-cell adhesion in mouse
keratinocytes (30). In contrast, in fyn-deficient keratinocytes, the tyrosine phosphorylation of these AJ proteins has
been shown to be decreased markedly, and structural and functional abnormalities at the cell-cell adhesions similar to those caused by
tyrosine kinase inhibitors have been observed (30). Thus, Src family
kinases may generally contribute to regulate the tyrosine phosphorylation of AJ proteins in response to cell-cell adhesion.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. W. Birchmeier (Max-Delbruck Center for Molecular Medicine) for providing MDCK cells and the anti-canine E-cadherin mAb, Drs. S. Tsukita and A. Nagafuchi (Kyoto University) for L and EL cells, Dr. T. Hirano (Osaka University) for the human Gab-1 cDNA and the anti-Gab-1 polyclonal Ab, Dr. T. Nakamura (Osaka University) for HGF/SF, and Dr. M. Ichihashi (Kobe University) for encouragement.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, Sports, and Culture, Japan.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.
The first two authors contributed equally to this work.
§ Present address: Howard Hughes Medical Institute, Dept. of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.
¶ Present address: Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371 8512, Japan.
To whom correspondence should be addressed. Tel.:
81-6-6879-3410; Fax: 81-6-6879-3419; E-mail:
ytakai@molbio.med.osaka-u.ac.jp.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M100909200
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ABBREVIATIONS |
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The abbreviations used are: AJ(s), adherens junction(s); HGF/SF, hepatocyte growth factor/scatter factor; PI3-kinase, phosphatidylinositol 3-kinase; MDCK, Madin-Darby canine kidney; TPA, 12-O-tetradecanoylphorbol 13-acetate; Ab, antibody; mAb, monoclonal Ab; MAP, mitogen-activated protein; GST, glutathione S-transferase.
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REFERENCES |
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