* Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106-4960; Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47097; § Department of Cell Biology,
Neurobiology and Anatomy, University of Cincinnati, Cincinnati, Ohio 45267;
Department of Pathology, Yale University, New
Haven, Connecticut 06510-8023; and ¶ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208
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Abstract |
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There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPµ associates with the cadherin-catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977- 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPµ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPµ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPµ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPµ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPµ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513-1517) have asserted that the association we observed between PTPµ and the cadherin-catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPµ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPµ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPµ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPµ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.
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Introduction |
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THE cadherins are a major family of calcium-dependent, homophilic cell adhesion molecules that are
concentrated at specialized contact points in the cell
termed adherens junctions (for review see Gumbiner,
1996). The cadherins are transmembrane proteins that
possess an extracellular segment, characterized by the
presence of calcium-binding motifs, and an intracellular
segment that is highly conserved between members of the
family (for review see Takeichi, 1995
). The intracellular
segment serves as the site of interaction with proteins
termed catenins (
-,
-, and
-catenin) (for review see
Gumbiner, 1995
). It appears that
-catenin and
-catenin/
plakoglobin, which are related to the product of the segment polarity gene armadillo, bind directly to the cytoplasmic segment of cadherin, whereas
-catenin, which is related to the cytoskeleton-associated protein vinculin, binds
to
/
catenin and functions to link the complex to the actin cytoskeleton (for review see Gumbiner, 1995
). The intracellular, catenin-binding segment of the cadherins is essential for adhesion; mutations in this segment can disrupt
adhesion even in the presence of an intact extracellular
segment (Nagafuchi and Takeichi, 1988
; Ozawa et al.,
1989
). Thus, cadherin-mediated adhesion requires the intact cadherin-catenin complex and association with the actin cytoskeleton.
Mutations have been detected in components of the
cadherin-catenin complex in several tumors, and destabilization of cadherin-mediated adhesion has been linked
with invasion and malignant progression (for reviews see
Birchmeier and Behrens, 1994, Birchmeier, 1995
). In addition, the junctions in normal cells are dynamic and tyrosine phosphorylated rapidly and reversibly (Volberg et
al., 1991
). There is now a growing body of evidence to link
the loss of adhesive function and the destabilization of adherens junctions with changes in the state of phosphorylation of tyrosyl residues in components of the cadherin-
catenin complex (for reviews see Birchmeier and Behrens,
1994
; Brady-Kalnay and Tonks, 1995
). Expression of the protein tyrosine kinase (PTK)1 v-Src causes aberrant tyrosine phosphorylation that results in disruption of adherens junctions, in the absence of an effect on desmosomes
and tight junctions (Warren and Nelson, 1987
). Similarly, treatment of MDCK cells with vanadate, a broad specificity inhibitor of members of the protein tyrosine phosphatase (PTP) family of enzymes, results in tyrosine phosphorylation of proteins at adherens junctions and the
deterioration of junctional structures (Volberg et al.,
1992
). Furthermore,
-catenin was observed to be heavily phosphorylated on tyrosyl residues in rat fibroblasts transformed by v-Src, coincident with changes in cell-cell aggregation (Matsuyoshi et al., 1992
). In addition, these effects were abrogated by the PTK inhibitor herbimycin A
and promoted by the PTP inhibitor vanadate. Interestingly, a temperature-sensitive mutant of v-Src destabilized cadherin-dependent adhesion at the permissive temperature, coincident with tyrosine phosphorylation of E-cadherin,
-catenin, or cytoskeletal components (Behrens et
al., 1993
; Takeda et al., 1995
). The EGF receptor and
Met, the receptor for scatter factor, phosphorylate components of the cadherin-catenin complex, and the EGF receptor has been observed to bind directly to
-catenin and
to associate with the cadherin-catenin complex in epithelial
cells (Hoschuetzky et al., 1994
; Ochiai et al., 1994
; Shibamoto et al., 1994
). These observations suggest that the integrity of adherens junctions is regulated in part at the level
of reversible tyrosine phosphorylation that results from
the coordinated and competing actions of PTKs and PTPs.
Therefore, a prerequisite to understanding fully the significance of tyrosine phosphorylation in the control of cadherin-catenin function will be the identification and characterization of specific PTKs and PTPs that associate with
and modify the phosphorylation status of these proteins.
In examining the physiological significance of the tyrosine phosphorylation of the cadherin-catenin complex,
we have obtained data that implicate the receptor PTP,
PTPµ, as a potential regulator of this complex. PTPµ is
characterized by an extracellular segment that contains
one MAM (Meprin/A5/PTPµ) domain, one immunoglobulin domain, and four fibronectin type III repeats (Gebbink et al., 1991). This combination of motifs suggested
that PTPµ may function in cell-cell adhesion. In fact, we
(Brady-Kalnay et al., 1993
) and others (Gebbink et al.,
1993
; Sap et al., 1994
) demonstrated that PTPµ, and the
structurally related PTP
, participate in homophilic binding interactions. Ectopic expression of recombinant PTPµ
in Sf9 cells induces aggregation of these normally nonadhesive cells (Brady-Kalnay et al., 1993
; Gebbink et al.,
1993
). Subsequently, we determined that the homophilic
binding site within the extracellular segment of PTPµ resides in the immunoglobulin domain (Brady-Kalnay and
Tonks, 1994
). In addition, it has been shown that the
MAM domain plays a role in cell-cell aggregation possibly
by "sorting" of PTPµ from closely related molecules, such
as PTP
, during cell aggregation (Zondag et al., 1995
).
More recent data suggest that one aspect of PTPµ function in vivo may be to affect cell adhesion by regulating
the adhesive properties of the cadherin-catenin complex.
We observed that in the MvLu lung cell line, which expresses PTPµ, catenins, and cadherins endogenously, immunoprecipitates of PTPµ contained cadherins,
-catenin,
and
-catenin (Brady-Kalnay et al., 1995
). In fact, at least
80% of the total cellular cadherins appeared to be associated with PTPµ in MvLu cells. Similarly, complexes between PTPµ and cadherins were detected in rat heart, lung, and brain tissues, where PTPµ is expressed at high
levels (Brady-Kalnay et al., 1995
). The results of binding
studies in vitro suggest that this association results from a
direct interaction between the intracellular segment of
PTPµ and the intracellular domain of E-cadherin (Brady-Kalnay et al., 1995
). Our results raised the possibility that
a component of the cadherin-catenin complex may be an
endogenous substrate for PTPµ. Subsequently, several
other laboratories have reported the observation of interactions between cadherin-catenin complexes and both receptor and nontransmembrane PTPs in a variety of cell
systems (Balsamo et al., 1996
; Fuchs et al., 1996
; Kypta et
al., 1996
; Aicher et al., 1997
; Cheng et al., 1997
).
In this paper, we report the results of a further characterization of the association between PTPµ and cadherin-
catenin complexes. We have identified the cadherins that
associate with PTPµ in vivo from lysates of rat lung as
N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin). We have used a number of systems to characterize
further the association of PTPµ and E-cadherin and have
demonstrated that the COOH-terminal 38 residues of the
cytoplasmic segment of E-cadherin is necessary for binding of PTPµ. Furthermore, we have shown that conditions
that result in tyrosine phosphorylation of E-cadherin also
result in dissociation of PTPµ from the complex. A recent
article from Zondag et al. (1996) argues that PTPµ does not associate with cadherins and suggests that our observations are the result of nonspecific cross-reactivity between BK2, our antipeptide antibody to PTPµ, and cadherins. We present several lines of data to substantiate the
validity of our original observation of the association between PTPµ and the cadherin-catenin complex and to refute the assertion of Zondag et al. that our observation arises from nonspecific antibody cross-reactivity.
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Materials and Methods |
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Expression Vectors and Cell Lines
Expression vectors for wild-type and mutant murine E-cadherins were either generously provided by Masatoshi Takeichi (Kyoto University, Japan) (Nagafuchi and Takeichi, 1988; Nose et al., 1988
) or described in
Chen et al. (1997)
. WC5 cells were derived from neonatal rat cerebellar
cells by transformation with a mutant of Rous sarcoma virus (LA90) that
is temperature-sensitive for transformation (Giotta and Cohn, 1981
). At
the permissive temperature, 33°C, v-Src is active as a PTK, and the cells are
transformed. At 39°C, the v-Src PTK is relatively inactive, and the cells
display an epithelial morphology. WC5 cells were maintained at 33°C in DME (GIBCO BRL, Gaithersburg, MD) containing 10% fetal bovine serum and 10 µg/ml gentamicin. The isolation of stable cell lines expressing
full-length and deletion constructs of E-cadherin has been described
(Chen et al., 1997
). At least two clonal cell lines for each E-cadherin deletion mutant were tested in each of the experiments described. MvLu cells
were cultured as described previously (Brady-Kalnay et al., 1995
). The
cell line MCF10AneoN was generously provided by Bonnie Sloan (Wayne
State University, Detroit, MI) and cultured as described in Kinch et al.
(1997)
. Sf9 cells (CRL 1711; American Type Culture Collection, Rockville, MD) were maintained at 27°C in Grace's Insect Medium Supplemented (GIBCO BRL) containing 10% fetal bovine serum and 10 µg/ml
gentamicin.
Expression in Sf 9 Cells
The following recombinant baculoviruses were used: (a) expressing full-length PTPµ, described previously (Brady-Kalnay et al., 1993); (b) expressing E-cadherin, generated using the BaculoGold Transfection System (Invitrogen Corp., Carlsbad, CA) after ligation of a full-length 2.7-kb
cDNA (recovered by restriction digestion of the pBATEM2 plasmid, generously provided by M. Takeichi) into pVL1392 (Invitrogen Corp.); and
(c) expressing
-catenin, generously provided by R. Kypta (University of
California, San Francisco, CA). Sf9 cells were infected with the recombinant baculoviruses either singly or in pairwise combinations as previously
described (Brady-Kalnay et al., 1993
). Cells were harvested 48 h after infection by centrifugation at 3,000 g for 5 min and processed for immunoprecipitation and immunoblotting as described below.
Antibodies
Hybridoma cells expressing a rat monoclonal antibody against the extracellular domain of E-cadherin, ECCD-2 (Shiroyashi et al., 1986), were
generously provided by Masatoshi Takeichi. Conditioned medium from
these cells was used in our experiments. A mouse monoclonal antibody to
E-cadherin, antibodies to
-catenin, and antiphosphotyrosine antibody
(PY20) were purchased from Transduction Labs (Lexington, KY). In the
course of our experiments involving antibody recognition of E-cadherin
fusion proteins, we determined that the anti-E-cadherin antibody from
Transduction Labs recognized the juxtamembrane half of the intracellular
segment. Pan-cadherin antibodies (monoclonal and polyclonal), which react with the conserved COOH-terminal 24 amino acids of the cadherin cytoplasmic segment, were purchased from Sigma Chemical Co. (St. Louis,
MO). The cadherin-4 antibody (120A) was generously provided by ICOS
Corp. (Seattle, WA). Antibody to N-cadherin (BD7873) was generously
provided by Dr. J. Hemperly at Becton Dickinson Labs (Res. Triangle
Park, NC) and has been described previously (Payne et al., 1996
). Monoclonal antibodies to the intracellular segment of PTPµ (SK series) and
monoclonal antibody BK-2, generated against a peptide derived from the
extracellular segment of PTPµ, have been described previously (Brady-Kalnay et al., 1993
; Brady-Kalnay and Tonks 1994
). Polyclonal antibodies to glutathione S transferase (GST) (Brady-Kalnay et al., 1995
) and the
FG6 monoclonal antibody to PTP1B (Flint et al., 1993
) have been described previously.
Binding Assays In Vitro
The GST fusion protein of the extracellular segment of PTPµ (EXTRA-PTPµ) has been described previously (Brady-Kalnay et al., 1993). Two
GST/E-cadherin fusion proteins were generated that contain either amino
acids 572-631 (the juxtamembrane-half of the cytoplasmic segment, JM
E-cad) or amino acids 648-729 (the COOH-terminal, catenin-binding portion of the intracellular segment, CB E-cad). Proteins were expressed in
Escherichia coli and purified using glutathione Sepharose (Brady-Kalnay
et al., 1995
). In slot blot analyses, purified protein samples were adsorbed
to nitrocellulose mounted in a slot blot apparatus (Bio-Rad Laboratories,
Hercules, CA). The nitrocellulose strip was blocked in 5% nonfat dry milk in TTBS (20 mM Tris-Cl, pH 7.5, 660 mM NaCl, 0.05% Tween-20) and then incubated with primary antibody for 16 h at 4°C. The blot was
washed in TTBS and then developed using horseradish peroxidase-conjugated secondary antibodies and Enhanced Chemiluminescence reagents
(Amersham Corp., Arlington Heights, IL).
Preparation of Samples for Immunoprecipitation
Triton-soluble lysates of rat lung and MvLu cells were prepared as described (Brady-Kalnay et al., 1995). WC5 cells were lysed in 20 mM Tris,
pH 7.5, 2 mM CaCl2, 1% Triton X-100, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM benzamidine, 200 µM phenyl arsine oxide, 1 mM vanadate, and
0.1 mM molybdate). The buffer was supplemented with 150 mM NaCl for
lysis of Sf9 cells. For immunoprecipitation, antibodies were incubated with
protein A or protein G beads (Pharmacia Biotech, Piscataway, NJ) for 2 h
at room temperature and then washed three times with PBS (9.5 mM
phosphate, 137 mM NaCl, pH 7.5) before addition to cell lysates. Purified
monoclonal antibodies were used at 0.6 mg of IgG/ml beads, ascites fluid
was used at 1 mg of IgG/ml beads, and polyclonal serum was used at 3 mg
of IgG/ml beads. Immunoprecipitates were prepared from 200-400 µg of a
Triton-soluble lysate of WC5 cells. The immunoprecipitates were washed
four times in lysis buffer, and the bound material was eluted by addition of
100 µl of 2× sample buffer and heating for 5 min at 95°C. The proteins
were separated by electrophoresis on 6 or 8% SDS polyacrylamide gels and
transferred to nitrocellulose or polyvinyl difluoride for immunoblotting.
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Results |
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PTPµ Interacts with Distinct Members of the Cadherin Superfamily
In our initial paper (Brady-Kalnay et al., 1995), we reported that immunoprecipitates of PTPµ from lysates of
MvLu cells contained components of the cadherin-catenin
complex. Our data indicated that at least 80% of the cadherin in MvLu cell lysates was cleared after immunoprecipitation with antibodies to PTPµ. These results have
been called into question (Zondag et al., 1996
). To address the reproducibility of this observation, we performed a series of immunoprecipitations from lysates of MvLu cells.
We used two different concentrations of anti-PTPµ antibody BK2 or an isotype-matched antibody, FG6, to an unrelated PTP, PTP1B (Flint et al., 1993
) as a negative control. In addition, the pan-cadherin antibody and an antibody
to
-catenin were included as two positive controls. The
relative amounts of antibody heavy chain in each immunoprecipitate are shown in Fig. 1 A. The anti-PTPµ antibody
BK2 coimmunoprecipitated cadherin from MvLu cell lysates, to an extent comparable to that seen with the anticadherin and anti-
-catenin antibodies. In contrast, the
isotype-matched control antibody to PTP1B did not immunoprecipitate cadherin from MvLu cells at either concentration (Fig. 1 B). In addition, after immunoprecipitation with the various antibodies, we immunoblotted the
supernatant that remained to determine the quantity of
cadherin that was not precipitated. The results were in
agreement with the recovery of cadherin in the immunoprecipitates (Fig. 1 B). Cadherin was cleared from the lysates by immunoprecipitation with anti-PTPµ antibody
BK2, to a similar extent as observed in the two positive
control immunoprecipitates with pan-cadherin and anti-
-catenin antibodies (Fig. 1 C). These data confirm that
the majority of cadherin can be recovered in a complex
with PTPµ from MvLu cell lysates. In contrast, control
anti-PTP1B antibodies did not clear cadherin from the supernatant (Fig. 1 C).
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To identify specific cadherins that associate with PTPµ, we performed a series of experiments using extracts of rat lung that express PTPµ endogenously. Immunoprecipitates of PTPµ from rat lung extracts contained three major types of cadherins that were recognized by the pan-cadherin antibody (Fig. 2, arrows). Using antibodies to specific cadherin family members, we determined that PTPµ associated with N-cadherin, E-cadherin, and cadherin-4 (also known as R-cadherin) (Fig. 2).
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Reconstitution of the Interaction between PTPµ and E-cadherin in Sf 9 Cells
A major contention of the paper by Zondag et al. (1996)
was that the association we observed between PTPµ and
cadherin was an artifact arising from nonspecific cross-
reactivity between the anti-PTPµ antibody, BK2, and cadherins. We have now addressed this issue in a variety of
systems.
We have reconstituted the complex in Sf9 cells by expression of the individual components using recombinant
baculoviruses. PTPµ, E-cadherin, and -catenin were expressed singly or in pairwise combinations, and expression
was verified by immunoblotting cell lysates with the appropriate antibodies (Fig. 3, Lysate). E-cadherin was recovered in anti-PTPµ immunoprecipitates, prepared using the BK2 antibody, only from cells in which both proteins
were coexpressed (Fig. 3, BK2 IPs, Ecadherin blots, lane
4). The BK2 antibody did not precipitate E-cadherin from
cell lysates in the absence of PTPµ, thus ruling out the possibility that the antibody recognized E-cadherin nonspecifically (Fig. 3, BK2 IPs, Ecadherin blots, lane 2). Similar
observations were made when we examined immunoprecipitates with the anti-PTPµ antibody BK2 from lysates of
MCF-10A cells, which express large amounts of E-cadherin but do not express detectable levels of PTPµ. Even
in the presence of substantial quantities of E-cadherin and
the inclusion of large quantities of the antibody, BK2 did
not precipitate E-cadherin from lysates of MCF10A cells
(data not shown). When the experiment was repeated using a distinct antibody to PTPµ, SK18, which recognizes an epitope in the intracellular portion of the enzyme, the
same observation was made. E-cadherin was recovered in
anti-PTPµ immunoprecipitates only from lysates of cells
coexpressing the phosphatase together with E-cadherin
and not cells expressing E-cadherin alone (Fig. 3, SK18 IPs).
Furthermore, the formation of a complex was also revealed by the reverse immunoprecipitation/blotting strategy, in that PTPµ was recovered in immunoprecipitates of
E-cadherin, but only from lysates of cells in which both
proteins were expressed (Fig. 3, Ecad IPs, PTPµ blots,
lane 4).
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The data also illustrate that PTPµ did not interact with
-catenin in this system (see Fig. 3, anti-
-catenin blots of
BK2 and SK18 immunoprecipitates and anti-PTPµ blots
of
-catenin immunoprecipitates), whereas
-catenin was
recovered in immunoprecipitates of E-cadherin (Fig. 3,
Ecad IPs and
cat IPs), indicating that the protein was
produced in a conformation appropriate for complex formation. This observation is consistent with our previous
results from blot-overlay assays, which revealed a direct
interaction between PTPµ and E-cadherin in vitro (Brady-Kalnay et al., 1995
) and indicates that the binding of PTPµ
to E-cadherin is not mediated by
-catenin.
These data reveal that PTPµ/E-cadherin complexes are recovered by immunoprecipitation with two distinct antibodies to the phosphatase or with antibody to E-cadherin. In addition, anti-PTPµ antibodies did not precipitate E-cadherin in the absence of the phosphatase. Therefore, it is highly unlikely that this result could be explained by nonspecific antibody cross-reactivity.
The PTPµ/E-cadherin Complex Is Dynamic In Vivo
WC5 is a Rous sarcoma virus-transformed, rat cerebellar
cell line that expresses a temperature-sensitive mutant of
the v-Src PTK. When grown at the nonpermissive temperature (39°C), the Src PTK displays little activity, and the
cells manifest properties of astrocytes (Giotta and Cohn,
1981). In contrast, when grown at the permissive temperature (33°C), Src is active and the cells are transformed.
The expression of endogenous PTPµ, both the unprocessed (200 kD) and proteolytically processed (100 kD)
forms of the enzyme, and ectopically expressed E-cadherin was not affected by switching between permissive
and nonpermissive temperatures (Fig. 4 A).
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We used the WC5 cell line that expresses full-length E-cadherin to assess the effect of tyrosine phosphorylation on the PTPµ/cadherin complex by comparing the extent to which E-cadherin coimmunoprecipitated with PTPµ at the permissive and nonpermissive temperatures. As shown in Fig. 4, PTPµ antibodies coimmunoprecipitated E-cadherin well at the nonpermissive temperature (39°C) but poorly at the permissive temperature (33°C) for the Src PTK (Fig. 4 B), despite the fact that expression of E-cadherin was apparently unaltered at 39°C compared with 33°C (Fig. 4 A). Interestingly, E-cadherin was immunoprecipitated with antiphosphotyrosine antibodies at 33°C but to a lesser extent at 39°C. Under harsh detergent conditions (RIPA buffer), we observed that E-cadherin was tyrosine phosphorylated directly at the permissive temperature (data not shown). Therefore, our data indicate an inverse correlation between the presence of PTPµ in the cadherin-catenin complex and the phosphorylation of tyrosyl residues in E-cadherin. The fact that a complex between PTPµ and E-cadherin was detected at 39°C but not at 33°C, using identical conditions for cell lysis and immunoprecipitation at each temperature, indicates that the complex we observe is dynamic. Furthermore, although the potential existence of a phosphorylation-sensitive, cross-reacting epitope is not formally excluded, these data also indicate that it is unlikely that the complex we detect can be explained by nonspecific antibody cross-reactivity.
Identification of the Catenin-binding Domain within the Intracellular Segment of E-cadherin as the Site of Interaction with PTPµ
In our previous study, we demonstrated that E-cadherin
interacts with PTPµ both in vitro and in vivo. Therefore,
we set out to identify the binding site for PTPµ in E-cadherin. In our initial report, we demonstrated that the intracellular segment of PTPµ interacted directly with the intracellular segment of E-cadherin (Brady-Kalnay et al.,
1995). To determine more precisely the location of the
PTPµ-binding site within the cytoplasmic segment of E-cadherin, we used the WC5 cells, which express PTPµ endogenously but do not express endogenous E-cadherin. We
used a series of WC5 cell lines that express ectopically various forms of E-cadherin containing deletions in the cytoplasmic segment. We tested for the effects of the deletions
in E-cadherin on its ability to associate with PTPµ in a cellular context by immunoprecipitating PTPµ from the various WC5 cell lysates and determining whether E-cadherin
coimmunoprecipitated with the phosphatase. As shown in
Fig. 5 A, WC5 cell lines expressing five deletion mutants of
E-cadherin (Chen et al., 1997
) were used. Specifically,
these included (a) the full-length E-cadherin molecule as a
control (Ecad), (b) a control from which the entire cytoplasmic segment of E-cadherin was deleted (CDD), (c) a
mutant in which the COOH-terminal 38 residues were deleted (CB), (d) a mutant in which the internal domain of
the intracellular segment was deleted (ID), and (e) a mutant in which the juxtamembrane domain of E-cadherin
was deleted (JM).
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The WC5 cell lines expressed each of the E-cadherin deletion mutants to similar levels. Each of the mutants was
precipitated with antibodies to the extracellular segment
of E-cadherin and migrated at the expected molecular
weight (Fig. 5 B). A monoclonal antibody to the cytosolic
PTP, PTP1B (Flint et al., 1993), was used as a negative
control, and although capable of immunoprecipitating PTP1B (data not shown), it did not immunoprecipitate
E-cadherin (Fig. 5 C). Using the BK2 antibody to PTPµ,
which recognizes a peptide sequence in the MAM domain
within the extracellular segment of PTPµ (Brady-Kalnay
and Tonks, 1994
), we observed that E-cadherin mutants
bearing a deletion of the COOH-terminal 38 amino acids did not associate with PTPµ (Fig. 5 D). Thus, PTPµ failed
to coimmunoprecipitate E-cadherin from two distinct
WC5 lines expressing such E-cadherin deletion mutants
(CB1 and CB2) and from a line expressing E-cadherin from which the entire cytoplasmic segment was deleted
(CDD). These results indicate that the COOH-terminal 38 residues of E-cadherin is required for the interaction with
PTPµ.
Originally, our observation of association between
PTPµ and cadherin was founded primarily upon experiments performed with one antibody to the phosphatase,
designated BK2, which recognized an epitope in the extracellular segment of the enzyme. We generated previously
the SK series of monoclonal antibodies to the intracellular domain of PTPµ (Brady-Kalnay et al., 1993). By using histidine-tagged fusion proteins comprising various portions
of the intracellular segment of PTPµ, we identified two
classes of SK antibodies. One class recognized epitopes in
the juxtamembrane segment of the phosphatase whereas
the other recognized epitopes in the first phosphatase domain (data not shown). To extend the scope of our analysis of the association of PTPµ and cadherins, we tested
whether the SK series monoclonal antibodies were able to
immunoprecipitate the PTPµ/E-cadherin complex from
the various WC5 cell lines, concentrating on one with an
epitope in the juxtamembrane segment of PTPµ (SK7) and another that recognized the first PTP domain (SK18).
As shown in Fig. 5, E and F, these antibodies, like BK2,
immunoprecipitated the PTPµ/E-cadherin complex, but
only from cells expressing forms of E-cadherin in which
the COOH-terminal 38 residues were present. Similar
data were obtained with all SK series antibodies tested (data not shown).
The Anti-PTPµ Antibody, BK2, Does Not Cross-react with Cadherins
The BK2 antibody was generated against a peptide derived from the NH2 terminus of human PTPµ (Brady-Kalnay et al., 1994) and did not show cross-reactivity with the
PTPµ-like enzymes PTP and PTP
/PCP-2. The peptide
sequence displayed no obvious similarity to the intracellular segment of cadherin, which contains the site of interaction with PTPµ (Brady-Kalnay et al., 1995
and Fig. 5). Despite the lack of obvious sequence similarity between this
peptide and the cadherins, we addressed the issue of cross-reactivity further in the following experiment.
We examined whether the anti-PTPµ antibody BK2 recognized a GST-E-cadherin fusion protein, purified after
expression in E. coli, in a direct binding assay under nondenaturing conditions in vitro. Our original study demonstrated an interaction between the intracellular segment of
PTPµ and the intracellular segment of E-cadherin (Brady-Kalnay et al., 1995). Furthermore, the data presented here
in Fig. 5 show that the COOH-terminal 38 residues of
E-cadherin were required for association with PTPµ.
Therefore, any potential cross-reacting epitopes would be
in this segment. We tested whether the BK2 antibody
binds to the intracellular segment of E-cadherin under
nondenaturing conditions. We used the following four
GST fusion proteins: (a) GST alone, (b) the juxtamembrane half of the intracellular segment of E-cadherin (JM
E-cad), (c) the COOH-terminal, catenin-binding portion
of the intracellular segment of E-cadherin (CB E-cad),
and (d) the extracellular domain of PTPµ (EXTRA
PTPµ). An antibody to GST recognized all of the fusion
proteins (Fig. 6 A). One of the commercially available antibodies reacted with the juxtamembrane half of E-cadherin (Fig. 6 B), whereas the polyclonal, pan-cadherin antibody
reacted with the catenin-binding half of the E-cadherin intracellular segment (Fig. 6 C). The SK15 antibody, to the
intracellular segment of PTPµ, did not recognize any of
the fusion proteins (Fig. 6 D), whereas antibody BK2 interacted only with the extracellular segment of PTPµ (Fig.
6 E). Thus, under these conditions, the BK2 antibody did
not recognize the intracellular segment of E-cadherin, which contains the binding site for PTPµ (Fig. 5), even
when 10 µg of the purified protein was applied to the nitrocellulose filter to ensure maximal binding of the protein
to the nitrocellulose, and the blot was overexposed. These
data provide further indication that the BK2 antibody
does not recognize E-cadherin nonspecifically.
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Discussion |
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Components of adherens junctions are subjected to rapid,
reversible tyrosine phosphorylation in a cellular context
(Volberg et al., 1991). Tyrosine phosphorylation of the
cadherin-catenin complex has been observed under a variety of conditions, including in response to oncoprotein
PTKs, such as Src (Matsuyoshi et al., 1992
; Behrens et al.,
1993
), or to oncogenic forms of Ras (Kinch et al., 1995
)
and following stimulation of receptor PTKs, such as EGF
receptor and Met (Shibamoto et al., 1994
). In addition, PTKs such as EGF receptor and c-erbB2 have been observed to associate with the cadherin-catenin complex in
vivo (Hoschuetzky et al., 1994
; Ochiai et al., 1994
). The reversibility of tyrosine phosphorylation in vivo depends
upon the coordinated action of both PTKs and PTPs. Therefore, to understand fully the regulation of cadherin
function by reversible tyrosine phosphorylation, it will be
necessary to identify and characterize the phosphatases
that act upon adhesion complexes in vivo. Our observation
of association between a receptor PTP, PTPµ, and cadherins in various tissues and cells is consistent with a role
for this phosphatase in regulating cadherin function and
lends further support to the regulatory importance of tyrosine phosphorylation in cell adhesion.
In this study, we have demonstrated that PTPµ interacted with N-cadherin, E-cadherin, and cadherin-4 (also
called R-cadherin) in extracts of rat lung. Although PTPµ
can interact with several cadherins, it displays a restricted
tissue distribution. Therefore, one would anticipate that, if
regulation of cadherin function by reversible tyrosine
phosphorylation was a general phenomenon, there would
be additional PTPs that function in a manner analogous to
PTPµ in other cell types. Subsequent to our original demonstration of association between PTPµ and the cadherin-
catenin complex (Brady-Kalnay et al., 1995), several reports have appeared that substantiate the general principle
that members of the PTP family may be important regulators of cadherin-mediated adhesion. Association of a number of receptor and nontransmembrane PTPs with different members of the cadherin family in a variety of cell
systems has now been reported. PTP
, a receptor PTP that
is closely related in structure to PTPµ (~75% sequence
identity with the same overall arrangement of structural
motifs), has been shown to associate directly with
-catenin and plakoglobin (Fuchs et al., 1996
). Interestingly,
PTP
displays a much broader expression pattern than
PTPµ (Jiang et al., 1993
) and therefore may interact with cadherin-catenin complexes in many tissues. To date, four
other PTPs have been shown to interact with cadherin-
catenin complexes. Most recently, LAR (Aicher et al.,
1997
) and a novel receptor PTP, termed PTP
(a close relative of PTPs µ and
; Cheng et al., 1997
), were also shown
to associate with
-catenin. These authors observed that
the association with
-catenin, like that involving PTP
(Fuchs et al., 1996
), required the intracellular segment of
the phosphatase (Aicher et al., 1997
; Cheng et al., 1997
). A
LAR-like receptor PTP was found to associate with the
cadherin-catenin complex in PC12 cells, and this association appears to be regulated by nerve growth factor-induced
tyrosine phosphorylation of the PTP itself (Kypta et al.,
1996
). In addition, a PTP1B-like cytoplasmic phosphatase
has been shown to interact with N-cadherin (Balsamo et
al., 1996
). The authors suggest that the association of the PTP with N-cadherin facilitates dephosphorylation of
-catenin, which is required for N-cadherin-mediated adhesion and its association with the actin cytoskeleton.
In contrast to this consensus view of the potential importance of PTPs in regulating the tyrosine phosphorylation of cadherin-catenin complexes in vivo, one report
(Zondag et al., 1996) has questioned the validity of our
original observation. Therefore, we will respond in detail
to the various issues raised in the paper by Zondag et al.,
in an attempt both to resolve this controversy and clarify the various issues. Zondag et al. report the generation of
antibodies to an undefined epitope(s) in the ectodomain
of PTPµ that fail to coimmunoprecipitate cadherin-catenin complexes. After successive rounds of immunoprecipitation with one of these antibodies, 3D7, to "clear" PTPµ
from the cell lysate, the authors subjected the cleared lysate to immunoprecipitation with our antibody BK2. Even though they were unable to detect PTPµ in the cleared lysate by immunoblotting with their antibodies, they still observed cadherin in BK2 immunprecipitates. From this, the
authors concluded that the interaction we observed was
due to nonspecific cross-reactivity between BK2 and cadherin. There are two problems with this experiment and
the conclusion drawn from it. Firstly, the authors did not blot the cleared lysate with BK2 to check whether there
was a pool of PTPµ that was not recognized by their antibodies but was detected by BK2. Secondly, although the
authors made the strong assertion of nonspecific cross-
reactivity between BK2 and cadherin, they failed to demonstrate such cross-reactivity in a direct binding assay.
The following observations refute their argument. First, we used Sf9 cells, which do not contain detectable levels of endogenous PTPµ or E-cadherin, to express these proteins and reconstitute the complex. Through this approach we demonstrated that E-cadherin was only recovered in immunoprecipitates of PTPµ from lysates of cells in which both proteins had been coexpressed. Furthermore, the complex was detected in the reciprocal experiment, in which PTPµ was recovered in immunoprecipitates of E-cadherin, but again only from lysates of cells in which both proteins had been coexpressed. Second, and importantly, BK2 did not recognize E-cadherin in a direct binding assay in vitro, using purified components under nondenaturing conditions. Third, BK2 did not immunoprecipitate E-cadherin from lysates of MCF10A cells, which do not express PTPµ to a level that can be detected by antibody BK2 but express substantial levels of E-cadherin. The authors present data from a similar experiment in COS cells, which they state lacks endogenous PTPµ. They report that cadherin is detected in BK2 immunoprecipitates whether or not PTPµ was expressed ectopically. However, using a number of antibodies to PTPµ, including BK2, we detected expression of this protein in COS cells (data not shown), and thus coprecipitation of cadherin would not be unexpected. The reason for this discrepancy is unclear, although the authors did not test for the presence of PTPµ in COS cell lysates by blotting with BK2. Fourth, in WC5 cells transformed by temperature-sensitive v-Src and expressing E-cadherin ectopically, immunoprecipitates of PTPµ from lysates of cells cultured at the nonpermissive temperature contained coprecipitating cadherin, whereas at the permissive temperature the levels of associated cadherin were reduced substantially (Fig. 4). It is unlikely that BK2 would display cross-reactivity only in lysates from one temperature condition. Finally, we have demonstrated interaction between PTPµ and various members of the cadherin family using different antibodies that recognize at least three distinct epitopes in the phosphatase.
Zondag et al. (1996) presented several additional arguments to question the validity of the association we observed between PTPµ and cadherin. For example, they
cited their failure to detect the phosphatase in anticadherin immunoprecipitates as further evidence that PTPµ
and cadherin do not interact. However, it is important to
note that in these experiments the authors used a pan-cadherin antibody that is directed against the COOH-terminal sequence that contains the segment of E-cadherin that
is required for interaction with PTPµ. Therefore, the absence of PTPµ from these immunoprecipitates could easily be explained by steric hindrance introduced by antibody binding to cadherin. In addition, the authors dismiss
our demonstration of direct interaction in blot-overlay binding studies as the result of production of the cadherin
and PTPµ fusion proteins in bacteria, which, they suggest,
is likely to yield misfolded or denatured protein and result
in a high risk of nonspecific protein-protein interactions.
However, the PTPµ used as probe was produced in insect
Sf9 cells, not bacteria, and was catalytically functional and
therefore was not denatured or misfolded. Furthermore,
we included controls in the experiment to show that, as
would be expected, under identical conditions E-cadherin
bound
-catenin (specifically the NH2-terminal segment and not the COOH-terminal segment of
-catenin) but
not
-catenin. Therefore, it is unlikely that our results can
be explained by nonspecific association or improper protein folding.
The observation that PTPµ could interact with several
cadherins prompted us to investigate the binding site for
PTPµ on the cadherins. For these studies, we used a series
of WC5 rat astrocyte-like cell lines, which express PTPµ
endogenously and express ectopically mutant forms of
E-cadherin that lacked various portions of the cytoplasmic
segment. The results indicated that the COOH-terminal 38 residues, which overlap with the catenin-binding domain, were required for the interaction with PTPµ. A
number of factors suggest that this is likely to be a direct
binding site: (a) We have demonstrated previously that the
intracellular segment of PTPµ interacts directly with the
intracellular segment of E-cadherin in vitro; (b) we have
shown here (Fig. 3) that PTPµ and E-cadherin interact after coexpression in Sf9 cells and, considering the extent of
overexpression achieved in this system, it is unlikely that
the interaction is mediated by an endogenous Sf9 cell protein; and (c) deletions of other portions of the E-cadherin
cytoplasmic segment had little effect on the association
with PTPµ in WC5 cells. Although we did not detect a direct interaction between PTPµ and -catenin in vitro or in
Sf9 cells, we have detected both cadherin and
-catenin in
immunoprecipitates of PTPµ (Brady-Kalnay et al., 1995
).
In light of data suggesting that E-cadherin functions as a
dimer (Brieher et al., 1996
; Nagar et al., 1996
), it is possible that one E-cadherin molecule of the dimer may bind
PTPµ while the other interacts with
-catenin. The observation that the COOH-terminal 38 residues of E-cadherin
are required for interaction with PTPµ raises the possibility that the association may be regulated by
-catenin in
vivo.
In summary, we believe that our data have established convincingly the existence of a complex between PTPµ and various members of the family of cadherins in a number of different cell systems. In addition, we believe that we have presented data to refute convincingly the assertions of Zondag and colleagues (1996) that the association we observe between PTPµ and cadherins is artifactual. Our observations highlight further the potential importance of reversible tyrosine phosphorylation in regulating the adhesive properties of the cadherin family of cell adhesion molecules.
![]() |
Footnotes |
---|
Received for publication 4 June 1997 and in revised form 30 January 1998.
S. Brady-Kalnay is funded by a Junior Faculty Investigator Award from the Case Western Reserve University/University Hospitals Ireland Cancer Center from an ACS Institutional Research Grant (IRG 186), a Case Western Reserve University Research Initiation Grant, and a grant from the American Cancer Society, Ohio Division Inc., Cuyahoga Unit. N.K. Tonks is funded by a grant from the National Institutes of Health (GM55989).We thank Ferdose Sartawi, Jullia Rosdahl, Leif Stordal, and Martha Daddario for technical assistance.
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Abbreviations used in this paper |
---|
GST, glutathione S transferase; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase.
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