Leukemia cells (K562) that grow as non-adhesive
single cells and have no endogenous cadherin were transfected with an
E-cadherin expression vector, and cell clones stably expressing
E-cadherin on their surface were established. The expression of
E-cadherin induced the up-regulation of catenins, and E-cadherin became
associated with catenins. The transfected cells grew as floating
aggregates. Cell aggregation was
Ca2+-dependent and was inhibited by
E-cadherin antibodies. The aggregates dissociated into single cells on
the addition of pervanadate. Pervanadate caused a dramatic augmentation
of the phosphorylation of E-cadherin,
-catenin, and
-catenin
(plakoglobin), but
-catenin was not detectably phosphorylated. After
pervanadate treatment,
-catenin and
-catenin migrated more slowly
on gel electrophoresis, suggesting changes in their conformations due
to eventual changes in their phosphorylation levels. In the treated
cells, a significant amount of
-catenin was dissociated from the
E-cadherin·catenin complex. Aggregates of cells expressing an
E-cadherin chimeric molecule covalently linked with
-catenin were
not dissociated on pervanadate treatment, supporting the idea that the
dissociation of
-catenin from the complex underlies the observed
E-cadherin dysfunction.
 |
INTRODUCTION |
Cadherins are a family of structurally and functionally related
molecules that mediate Ca2+-dependent cell-cell
interactions in a homophilic manner (1). They are subdivided according
to their primary structures into several groups, such as the classical
cadherins and the protocadherins (2). The classical cadherins,
including the E-, P-, and N-cadherins, are highly conserved
transmembrane glycoproteins with similar domains for homophilic
binding, Ca2+-binding, and interaction with intracellular
proteins termed catenins (
-,
-, and
-catenins) (1, 2).
The function of cadherins depends on their association with the actin
cytoskeleton. Deletion or truncation of the cytoplasmic domain of
cadherin results in a loss of function despite their continued
expression on the cell surface (3). The interaction of cadherins with
the cytoskeleton is mediated by
-,
-, and
-catenins (4, 5).
Consistent with the role of the catenins in the linking of cadherins to
the cytoskeleton, a loss of the cadherin function has been correlated
with alterations in catenins (6-8). Furthermore, cell-cell adhesion
could be restored by transfection of these cells lacking
- or
-catenin with the respective cDNA (8-10). In vitro
and in vivo experiments have shown that
- and
-catenin
bind directly to the cytoplasmic domain of E-cadherin, whereas
-catenin binds to
- or
-catenin (11-18). The
-catenin-binding site of both
- and
-catenin is the
amino-terminal part (11, 12, 15, 17). The central core region,
consisting of thirteen copies of the so-called Armadillo repeat, is
involved in the association of both
- and
-catenin with cadherin
(11-14, 18, 19). The core of both is also involved in complex
formation with the APC tumor suppressor protein (12, 13, 15) and, in
the case of
-catenin (plakoglobin), with desmogleins and
desmocollins, the desmosomal cadherins (19-21). The region of
-catenin responsible for
- and
-catenin binding was recently
identified as the amino-terminal region of the molecule (22, 23).
-Catenin mediates the interaction between the cadherin·catenin
complex and the actin cytoskeleton through its association with
-actinin (23, 24) and actin filaments (25). The complex formation
is, however, a dynamic process including the exchange of catenins
(26).
p120cas is a protein that is tyrosine phosphorylated in cells
transformed with Src and in response to growth factors such as epidermal growth factor.1
Like
- and
-catenin, p120cas possesses 11 copies of the
Armadillo repeat (27) and also associates with E-cadherin·catenin
complexes (28-30).
Cells have to regulate the strength of their adhesiveness, and
post-translational modifications such as tyrosine phosphorylation may
be one mechanism of regulation. In v-src transfected cells,
-catenin
in the cadherin·catenin complex is preferentially
tyrosine-phosphorylated, and the increased tyrosine phosphorylation of
-catenin is apparently associated with a dysfunction of cadherin
(31-33). Treatment of adenocarcinoma cells with growth factors results
in the accumulation of phosphorylated tyrosine residues on
- and
-catenin, concomitant with a loss of cadherin-mediated adhesion
(34). These data suggest that the degree of tyrosine phosphorylation of
-catenin is a critical parameter controlling cadherin function,
possibly by regulating the association of cadherin with the
cytoskeleton. In the case of Ras-transformed mammary cells,
p120cas seems to displace
-catenin for cadherin binding upon
elevated tyrosine phosphorylation of these proteins (35). The
dissociation of tyrosine-phosphorylated
-catenin from N-cadherin has
also been reported (36). Our knowledge on the dysfunctional state of
cadherin induced by the tyrosine phosphorylation of
-catenin, however, remains fragmentary. In this study, we have examined the
consequence of tyrosine phosphorylation of
- and
-catenin on
cadherin function and the composition of the cadherin·catenin complex. We find that the tyrosine phosphorylation of
- and
-catenin induced by pervanadate treatment of cells does not result
in the dissociation of these proteins, instead causing a significant decrease in the amount of
-catenin in the E-cadherin·catenin complex.
 |
EXPERIMENTAL PROCEDURES |
Chemicals and Antibodies--
Sodium orthovanadate, okadaic
acid, and cytochalasin D were purchased from Sigma. Monoclonal
antibodies against
-,
-, and
-catenins were purchased from
Transduction Laboratories. DECMA-1, a monoclonal antibody to
E-cadherin, and monospecific antibodies to
-catenin were described
previously (37, 38). A monoclonal antibody to phosphotyrosine (4G10)
was obtained from Upstate Biotechnology.
Cells and Transfection--
Human leukemia K562 cells (kindly
provided by Dr. K. Sekiguchi, Research Institute, Osaka Medical Center
for Maternal and Child Health) were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. The E-cadherin
cDNA encoding the wild-type or mutant proteins, E
C37 (5) or
ED134A (39), was cloned into a mammalian expression vector, pCAGGS neo
(40) (a gift from Dr. K. Yamamura, Kumamoto University). For the
expression of an E-cadherin-
-catenin chimeric protein, the
ClaI-EcoRV fragment of E-cadherin cDNA that
encodes the 71 amino acid, including catenin-binding domain of
E-cadherin, was replaced with a 2469-base pair
Eco47III-EcoRV fragment that encodes the
carboxyl-terminal two-thirds of
-catenin (amino acids 301-906).
K562 cells (5 × 106) were transfected with the
expression vectors (10 µg) by electroporation using a Bio-Rad Gene
Pulser set at 280 V and 960 microfarads.
Immunoprecipitation and Immunoblotting--
For immunoblot
analysis, cells (1 × 105) were boiled for 5 min in
Laemmli SDS gel sample buffer, run on 8% polyacrylamide gels, and then
electroblotted onto nitrocellulose membranes. The membranes were
blocked with 5% nonfat milk in phosphate-buffered saline and then
incubated with monoclonal antibodies and finally peroxidase-conjugated antibodies (Jackson ImmunoResearch Laboratories). After washing with
the buffer containing 0.1% Tween 20, the protein bands were visualized
with an ECL detection kit (Amersham Corp.). Immunoprecipitation was
carried out as described previously (4) with the following modifications. Cells were lysed in 10 mM Tris-HCl buffer,
pH 7.6, containing 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM CaCl2, 0.1 mM sodium orthovanadate, 1 mM
phenylmethylsufonyl fluoride, 10 µg/ml leupeptin, and 25 µg/ml
aprotinin. The E-cadherin·catenin complex was collected with either
rabbit anti-E-cadherin or
-catenin antibodies, both of which had
been preabsorbed to protein A-Sepharose CL4B. The immune complex was
washed with the same buffer four times and then boiled in the SDS-PAGE
sample buffer.
Cell Aggregation and Dissociation--
The cell aggregation
assay was performed as described previously (5) except that the cells
were passed through Pasteur pipettes several times to obtain single
cells. For the cell dissociation assay, cells (2 × 106) were cultured overnight in 60-mm plastic dishes and
then incubated for another 1 h after the addition of reagents
dissolved in a minimum volume of an appropriate solvent.
 |
RESULTS |
Expression of E-Cadherin on K562 Leukemia Cells--
A commonly
used method devised by Takeichi and co-workers (41) to examine
potential homophilic adhesive properties of surface molecules includes
the transfection of mouse L fibroblasts with the cDNA of interest
and analysis of the adhesive properties of the transfectants. Although
L cells have no endogenous cadherin activity, they exhibit adhesiveness
and form aggregates after overnight culture in suspension, suggesting
that they have cell-cell adhesion molecules other than cadherin. To
determine the conditions that modulate the cadherin activity of the
preformed cell-cell contacts, L cells were less suitable because of
their cadherin-independent adhesiveness. A human leukemia cell line,
K562, was chosen for analysis because it expresses no endogenous
cadherin and grows in suspension as non-adhesive single cells. K562
cells transfected with an E-cadherin expression vector and selected for
G418 resistance exhibited two different phenotypes. One type grows as
aggregates and the other as single cells. On immunofluorescence
staining with E-cadherin antibodies, the clones growing as aggregates
were found to be positive for E-cadherin expression, whereas those growing as single cells were negative (not shown).
The aggregation of K562 cells expressing E-cadherin (EK cells) was
inhibited in the presence of the E-cadherin antibody, DECMA-1 (Fig.
1, A and E). The
aggregation of EK cells is Ca2+-dependent since
no aggregation was observed in the presence of 5 mM EGTA
(data not shown). Recently, cadherin-mediated adhesion was postulated
to have two states (42). The aggregates in the weak state are easily
dissociated into single cells on passage several times through Pasteur
pipettes, whereas the aggregates in the strong state are hardly
affected by the same treatment. According to this definition, the state
of EK cell aggregates is weak, because they were dissociated into
single cells when passed several times through a Pasteur pipette.
Absent expression in K562 cells of ZO-1, which binds to
-catenin and
actin filaments (43), may explain why EK cells remain in the weak
state. The aggregates treated with cytochalasin D (1 µg/ml) to
disrupt the actin cytoskeleton did not dissociate into single cells,
although the cells became rounded and the aggregates seemed to be more decompacted (data not shown).

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Fig. 1.
Aggregation of K562 cells expressing
wild-type E-cadherin but not mutant E-cadherin polypeptides. K562
cells expressing the wild-type E-cadherin (EK cells,
panels A and E); nonfunctional E-cadherins,
i.e. either ED134A (ED134AK, panels B
and F), with an amino acid substitution in the
Ca2+-binding motif; or E C37 (E C37K, panels C
and G), with a deletion in the cytoplasmic domain, or
K562 cells transfected with the control vector (NK,
D and H) were allowed to aggregate for 1 h in the absence (A, B, C, and
D) or presence (E, F, G,
and H) of an anti-E-cadherin monoclonal antibody.
Bar, 50 µm.
|
|
To confirm that the observed aggregation of K562 cells expressing
E-cadherin was dependent on E-cadherin, two additional cell clones
expressing mutant forms of E-cadherin were established. ED134AK cells
express E-cadherin with an amino acid substitution in the
Ca2+-binding motif (ED134A), i.e. with an
aspartic acid at residue 134 being replaced by alanine, and therefore
exhibit no adhesive activity in L cells (39). E
C37K cells express
another form of mutant E-cadherin with a deletion in the cytoplasmic
domain (E
C37) (5). This mutant form of E-cadherin cannot associate with catenins because of the deletion and is also nonfunctional when
expressed on L cells. As expected, K562 cells expressing either of
these mutant E-cadherin proteins showed no aggregation (Fig. 1).
Because cadherin expression has been shown to induce the up-regulation
of catenins in L fibroblasts (44, 45), we assessed the steady-state
levels of the
-,
-, and
-catenin proteins in these different
cell lines by immunoblot analysis. Very low levels of
-,
-, and
-catenin were observed in the parental K562 cells, the control
transfectants (NK), and E
C37K cells (Fig. 2). In contrast, cells expressing
E-cadherin with the intact catenin-binding site (EK and ED134AK) showed
markedly increased steady-state levels of
-,
-, and
-catenins
(Fig. 2).

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Fig. 2.
Stabilization of catenins by E-cadherin
expression. K562 cells expressing wild-type E-cadherin
(lanes 1), nonfunctional E-cadherins, i.e. either
ED134A (lanes 2) or E C37 (lanes 3), or K562
cells transfected with the control vector (lanes 4), or parental K562 cells (lanes 5) were subjected to immunoblot
analysis.
|
|
Complex Formation of E-Cadherin in K562
Cells--
Cadherin·catenin complex formation was studied by means
of co-immunoprecipitation experiments. Cells were metabolically labeled with [35S]methionine, and E-cadherin was
immunoprecipitated from cell lysates with E-cadherin antibodies. Three
proteins migrating to positions corresponding to 102, 88, and 82 kDa
were coprecipitated with E-cadherin (120 kDa) in the case of EK cells
(Fig. 3). These coprecipitated proteins
were identified as
-,
-, and
-catenin by subjecting the
immunoprecipitates to immunoblot analysis with the respective
antibodies (data not shown, but see below).

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Fig. 3.
Complex formation of E-cadherin with
catenins. Cells labeled overnight with
[35S]methionine were lysed and then subjected to
immunoprecipitation with an E-cadherin antibody. The immunoprecipitates
were analyzed by SDS-PAGE and fluorography. L cells expressing
E-cadherin (EL) were used as a positive control. The
positions of E-cadherin and -, -, and -catenins are indicated
on the right.
|
|
Dissociation of EK Cell Aggregates on Pervanadate
Treatment--
The addition of pervanadate (0.5 mM sodium
orthovanadate + 1.5 mM H2O2), an
inhibitor of phosphotyrosine phosphatases, to EK cell aggregates formed
on overnight culture resulted in almost complete dissociation of the
aggregates into single cells within 1 h (Fig.
4). Okadaic acid (up to 500 nM), an inhibitor of serine-threonine phosphatase, induced
morphological changes, including blebbing of individual cells, in the
aggregates, but it had no effect on the aggregates of EK cells (not
shown).

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Fig. 4.
Dissociation of EK cell aggregates in the
presence of pervanadate. Cells were allowed to aggregate overnight
(A) and then incubated for 1 h in the presence of
pervanadate (B). Bar, 50 µm.
|
|
Pervanadate Treatment Does Not Affect the Amounts of the Components
of the cadherin·catenin Complex--
We examined whether the amounts
of the components of the cadherin·catenin complex were changed after
pervanadate treatment. Immunoblot analysis showed that there was almost
no difference in the amounts of E-cadherin,
-,
-,
-catenins,
or p120cas between the untreated and treated EK cell aggregates
(Fig. 5). A small but reproducible shift
was detected in the electrophoretic mobility of
- catenin,
-catenin, and p120cas (Fig. 5).

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Fig. 5.
Immunoblot detection of E-cadherin and
catenins. EK cell aggregates treated with pervanadate (+), or
untreated ( ) were lysed in SDS sample buffer and then subjected to
analysis.
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|
Elevated Tyrosine Phosphorylation of
-Catenin and
-Catenin,
and Reduced Association of
-Catenin with the E-Cadherin·Catenin
Complex after Pervanadate Treatment--
We next examined whether
pervanadate treatment affected the association of E-cadherin with
catenins by immunoblot analysis of immunoprecipitates collected with
E-cadherin antibodies. In untreated EK cell aggregates, E-cadherin was
associated with
-,
-, and
-catenin, and no detectable tyrosine
phosphorylation of these proteins was observed after blotting with
anti-phosphotyrosine antibodies (Fig. 6).
After pervanadate treatment, similar amounts of
- and
-catenin
(with modified electrophoretic mobilities of 90 and 84 kDa,
respectively) were found associated with E-cadherin. The amount of
-catenin in the complex was, however, significantly decreased to
less than one-half that of untreated aggregates (Fig. 6). The
anti-phosphotyrosine antibody specifically reacted with three bands, at
120, 90, and 84 kDa, for the treated aggregates. Immunoprecipitation
with E-cadherin antibodies under stringent conditions revealed that
E-cadherin was tyrosine phosphorylated upon pervanadate treatment (data
not shown). A similar analysis of
-catenin collected with
-catenin antibodies revealed that
-catenin was not detectably
phosphorylated (data not shown). Taken together, these results suggest
that, upon increased tyrosine phosphorylation of components of the
cadherin·catenin complex,
-catenin is released from the
complex.

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Fig. 6.
Immunoblot analysis of catenins
co-precipitating with E-cadherin. EK cell aggregates were
incubated for 1 h in the absence ( ) or presence (+) of
pervanadate and then lysed. E-cadherin and the associated proteins were
collected with an E-cadherin antibody. After SDS-PAGE and transfer to
nitrocellulose membranes, the proteins were stained with antibodies
either E-cadherin, -, -, or -catenin, p120cas, or
phosphotyrosine. Under the conditions used, p120cas was not
detected in the immunoprecipitates.
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|
Conversely using anti-
-catenin antibodies, we again collected the
E-cadherin·catenin complex from untreated and pervanadate-treated cells. In untreated EK cell aggregates,
-catenin antibodies
co-precipitated E-cadherin and
- and
-catenins. In treated
aggregates, reduced amounts (less than one-half) of
- and
-catenin were co-precipitated with
-catenin, and consequently
there was a reduced amount of E-cadherin (Fig.
7). The
- and
-catenin
co-precipitated with
-catenin from the untreated and treated
aggregates showed small differences in electrophoretic mobility as
compared with those associated with E-cadherin.

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Fig. 7.
Immunoblot analysis of the proteins
co-precipitating with -catenin. EK cell aggregates were
incubated for 1 h in the absence ( ) or presence (+) of
pervanadate and then lysed. The proteins associated with -catenin
were collected by precipitation with an -catenin antibody. After
SDS-PAGE and transfer to nitrocellulose membranes, the proteins were
stained with either E-cadherin or -, -, or -catenin
antibodies.
|
|
Aggregates of K562 Cells Expressing an E-Cadherin-
-Catenin
Chimera Resist Dissociation by Pervanadate Treatment--
To test our
hypothesis that the dissociation of
-catenin from the
E-cadherin·catenin complex underlies the dysfunction of E-cadherin,
we constructed a cDNA for an E-cadherin-
-catenin chimeric
molecule consisting of (a) the entire extracellular and transmembrane domains of E-cadherin as well as the first 80 amino acids
of its cytoplasmic domain, excluding the region shown to associate with
- or
-catenin (5), and (b) amino acids 301-906 of
-catenin, which include the domains necessary for association with
-actinin and actin (23, 25), but not the domain essential for
association with
- and
-catenins (22, 23) (Fig.
8A). This chimeric cDNA
was transfected into K562 cells. Clones expressing the chimeric protein
on their surface (E-
CNK) were confirmed by immunofluorescence
staining (data not shown), and by immunoblotting with anti-E-cadherin
(Fig. 8B) and anti-
-catenin antibodies (not shown), both reacted
with a protein of 170 kDa, the expected molecular mass of the chimera.
The cells form aggregates (Fig. 8C) in an E-cadherin-dependent manner in that the aggregation was
inhibited by the E-cadherin antibody (data not shown). In contrast to
EK cell aggregates, the preformed aggregates were not dissociated upon
treatment with pervanadate (Fig. 8D).

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Fig. 8.
Resistance to pervanadate of aggregates of
K562 cells expressing an E-cadherin- -catenin chimeric protein.
Schematic representation of an E-cadherin- -catenin chimeric protein
(A). The sequence derived from -catenin was
shaded. K562 cells expressing wild-type E-cadherin
(lane 1) or the chimeric molecule (lane 2) were
subjected to immunoblot analysis with E-cadherin antibodies (B). Samples were run on 7% polyacrylamide gels. Cells
expressing the chimeric protein were allowed to aggregate overnight
(C) and then incubated for 1 h in the presence of
pervanadate (D). Bar, 50 µm.
|
|
 |
DISCUSSION |
The functions of cadherins are regulated from the cytoplasmic
side, and these regulations are thought to be of great importance in
the development of organs and in tumor metastasis. To understand the
mechanisms regulating the cadherin function, especially to identify
intrinsic factors involved in the inactivation of cadherin, we
expressed E-cadherin on K562 leukemia cells. Since the formation of
aggregates by the transfected K562 cells was solely mediated by
E-cadherin, the system seems to be suitable for the analysis of
cellular factors or of changes that lead to inactivation of cadherin.
The aggregates dissociate into single cells on the addition of
pervanadate. In pervanadate-treated cells,
- and
-catenins exhibit increased tyrosine phosphorylation and conformational changes,
and these changes are correlated with reduced association of
-catenin to the E-cadherin·catenin complex. Since the increased tyrosine phosphorylation of
- and
-catenins and E-cadherin did not affect their interactions with each other, one of the molecular events resulting in the inactivation of E-cadherin is apparently the
dissociation of
-catenin from the complex. Consistent with this
idea, the E-cadherin chimeric molecule covalently linked with
-catenin was not inactivated by pervanadate treatment. However, in
these cells, numerous proteins exhibit increased tyrosine
phosphorylation, and it will be important in the future to determine if
any other factors contribute to the inactivation of E-cadherin. With
elevated tyrosine phosphorylation, there is a rapid reorganization of
the actin cytoskeleton and a modulation of intercellular adherens-type junctions in epithelial and endothelial cells (46, 47). Disruption of
the actin cytoskeleton by cytochalasin D did not, however, affect
aggregation.
The correlation between the presence of phosphorylated tyrosine
residues on
- catenin and the loss of cadherin-mediated adhesion is
consistent with reports from other laboratories. The dissociation of
-catenin from the cadherin·catenin complex has not, however, been
reported previously. Instead, the displacement of
-catenin in the
complex by p120cas (35), the dissociation of
-catenin from
the complex (36), and no apparent changes in the composition of the
complex upon tyrosine phosphorylation of
-catenin have been reported
(31-34). Differences in the protein kinases involved in the
phosphorylation of these proteins could explain this discrepancy.
BCR-ABL oncogene product exhibiting increased tyrosine kinase activity
in K562 cells (48) is a candidate for the kinases responsible for the tyrosine phosphorylation of
-catenin and
-catenin in the
pervanadate-treated cells. Association with the actin cytoskeleton and
possible involvement in the cell adhesion of BCR-ABL protein has been
reported (49). Further studies are needed to determine the nature of
the protein-tyrosine kinases responsible for the elevated tyrosine
phosphorylation of E-cadherin and
- and
-catenins in
pervanadate-treated EK cell aggregates.
Besides protein-tyrosine phosphatase 1B-like phosphatase, which has
been shown to be involved in regulation of N-cadherin function in
chicken retina cells (36), several other protein tyrosine phosphatases
have been shown to interact with E-cadherin or
-catenin, including
receptor-type protein tyrosine phosphatase µ (50), a member of the
leukocyte antigen-related protein-related transmembrane tyrosine
phosphatase family (51), and protein-tyrosine phosphatase
(52). The
protein-tyrosine phosphatases involved in keeping the tyrosine
phosphorylation of
- and
-catenins at low levels also should be
identified.
The mechanism by which the tyrosine phosphorylation of
- and
-catenins results in the dissociation of
-catenin from these proteins is unknown at present. In the case of tyrosine phosphorylation of
-catenin induced by epidermal growth factor, the phosphorylated tyrosine residues are located in either the amino- or the
carboxyl-terminal domain of the protein (53). As mentioned above, the
-catenin-binding site of both
- and
-catenins has been
localized to the amino-terminal domain of these proteins (11, 12, 14,
15, 17). Analysis by alanine mapping of the
-catenin binding site of
the proteins has revealed that hydrophobic amino acids are
indispensable for the interaction with
-catenin, suggesting that
hydrophobic interactions stabilize the heterodimeric complex (17). The
amino acid substitution of the tyrosine residue in the site of
- and
-catenin with alanine (Y142A and Y133A, respectively) also strongly
reduces the ability of these proteins to bind to
-catenin (17). The
amino acid sequences surrounding this tyrosine residue seem, however,
not to conform to the consensus sequence of previously identified tyrosine kinases.
An alternative possibility to be considered is that phosphorylation of
tyrosine residues not in the
-catenin binding site induces a
conformational change, which in turn weakens the interaction with
-catenin or masks the binding site. Upon pervanadate treatment resulting in tyrosine phosphorylation,
- and
-catenins show a
slower electrophoretic mobility, suggesting a phosphorylation-induced conformational change of these proteins. It has been reported that
after the treatment of cells with serine-threonine phosphatase inhibitors (okadaic acid and calyculin A),
-catenin was
phosphorylated on serine and to a lesser extent on threonine residues,
and showed a slower electrophoretic mobility (54).
We thank Drs. Kiyotoshi Sekiguchi and
Ken-ichi Yamamura for providing the reagents and Kumiko Sato for
secretarial assistance.