* Cell Biology, Zoological Institute, Technical University, 38092 Braunschweig, Germany; and Division of Cell Biology and
Immunology, National Research Centre for Biotechnology, 38124 Braunschweig, Germany
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Abstract |
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In epithelial cells, -,
-, and
-catenin are
involved in linking the peripheral microfilament belt to
the transmembrane protein E-cadherin.
-Catenin
exhibits sequence homologies over three regions to
vinculin, another adherens junction protein. While
vinculin is found in cell-matrix and cell-cell contacts,
-catenin is restricted to the latter. To elucidate,
whether vinculin is part of the cell-cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and
-catenin. We show that
-catenin colocalizes at cell-cell contacts with endogenous vinculin and also with the transfected vinculin
head domain forming immunoprecipitable complexes.
In vitro, the vinculin NH2-terminal head binds to
-catenin, as seen by immunoprecipitation, dot overlay,
cosedimentation, and surface plasmon resonance measurements. The Kd of the complex was determined to
2-4 × 10
7 M. As seen by overlays and affinity mass
spectrometry, the COOH-terminal region of
-catenin
is involved in this interaction.
Complex formation of vinculin and -catenin was
challenged in transfected cells. In PtK2 cells, intact
-catenin and
-catenin1-670, harboring the
-catenin-
binding site, were directed to cell-cell contacts. In contrast,
-catenin697-906 fragments were recruited to
cell-cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo
-catenin, like vinculin, is
tightly regulated in its ligand binding activity.
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Introduction |
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FORMATION of epithelia depends critically on the
physical interaction between cells. To this end,
both partners develop a series of highly specific,
morphologically well defined structures, i.e., tight junctions, cell-cell adherens junctions, and desmosomes (Koch
and Franke, 1994). Adherens junctions are specified by
transmembrane linker proteins, the cadherins, which mediate the calcium-dependent, homophilic cell-cell adhesion in a wide variety of tissues and species. Truncating the
COOH-terminal cytoplasmic domain of E-cadherin is deleterious for epithelial cell-cell adhesion, which emphasizes the importance of linking these transmembrane proteins
to the peripheral microfilament belt at the cytoplasmic
face of contact sites (Nagafuchi et al., 1994
). This linkage is
mediated by a complex of three cytosolic proteins, named
-,
-, and
-catenin (Ozawa et al., 1989
). The cadherin-
catenin complex, as characterized by immunoprecipitation, contains cadherin/
-catenin and either
- or
-catenin (Hinck et al., 1994
; Näthke et al., 1994
). Recent data
suggest that
-catenin cannot directly bind to cadherin. Instead, this linkage is mediated through either
- or
-catenin (Aberle et al., 1994
, 1996
). More proximally, the link
to membrane-apposed actin filaments probably involves
direct
-catenin-actin interactions (Rimm et al., 1995
),
and experimental evidence indicates that this interaction is
also indispensable for cell-cell adhesion (Hirano et al.,
1992
; Torres et al., 1997
). Alternatively or in addition, an
-catenin/
-actinin/F-actin complex may be formed, as
proposed by Knudsen et al. (1995)
.
Sequence comparison demonstrated that -catenin, a
protein of 906 amino acids, shares homologies over three
extended regions with vinculin (Herrenknecht et al., 1991
;
Nagafuchi et al., 1991
), a structural component of cell-cell
as well as of cell-matrix adherens junctions (for references
see Burridge et al., 1988
; Jockusch et al., 1995
; Jockusch
and Rüdiger, 1996
). This is emphasized in Fig. 1. Vinculin
is a multiligand protein of 1,066 amino acids, known to
bind to actin filaments (Menkel et al., 1994
; Johnson and
Craig, 1995
) and to microfilament-associated proteins like talin (Otto, 1983
; Burridge and Mangeat, 1984
) and
-actinin (Belkin and Koteliansky, 1987
; Wachsstock et al.,
1987
). Like vinculin (Isenberg et al., 1982
; Johnson and
Craig, 1995
),
-catenin binds to and bundles actin filaments, but while in vinculin this activity is confined to the
COOH-terminal domain (Menkel et al., 1994
; Johnson
and Craig, 1995
; Hüttelmaier et al., 1997
),
-catenin contains two actin-binding sites well separated in its sequence (Rimm et al., 1995
). Again like vinculin,
-catenin interacts directly with
-actinin (Knudsen et al., 1995
; Nieset
et al., 1997
). Since the rod-like COOH-terminal domain of
vinculin can also interact with itself, forming homo-oligomeric aggregates (Otto, 1983
; Molony and Burridge,
1985
), and since this region shares a high degree of sequence homology with the corresponding region in
-catenin, it was proposed that
-catenin-vinculin heterologous complexes may be formed, involving the COOH-terminal
region of both molecules (Herrenknecht et al., 1991
; Nagafuchi et al., 1991
; Kemler, 1993
).
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In this study we tested this hypothesis. By a variety of
biochemical methods we demonstrate that vinculin indeed
binds to -catenin, but involves the vinculin head and the
-catenin "tail" domain. Based on colocalization and
coimmunoprecipitation studies, we present data showing
that both are part of the junctional complex in epithelial
cells, thus both contributing to the architecture of cell-cell
contacts. Furthermore, transfection studies with
-catenin
and deletion fragments thereof are in accordance with the
formation of an
-catenin-vinculin complex. Our results
suggest that the ligand-binding activities of both
-catenin
and vinculin are tightly regulated in vivo.
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Materials and Methods |
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Vectors and Plasmids
A full-length mouse -catenin cDNA with a COOH-terminal histidine tag
cloned in pQE60 (Qiagen, Hilden, Germany) was a kind gift of Dr.
Kemler (Max Planck Institue, Freiburg, Germany). The
-catenin
COOH-terminal sequence was amplified from this template by PCR. The
PCR fragment was cloned into pQE60, to obtain pQE-
-cat697-906 with a
COOH-terminal histidine-tag.
For transfection experiments, -catenin,
-cat697-906, and
-cat1-670
coding sequences were amplified from the prokaryotic expression vector
(see above) by PCR. PCR products were ligated into pcDNA-BiP to obtain the eukaryotic expression vectors pc
-catenin-BiP, pc
-cat697-906-BiP, and pc
-cat1-670-BiP. pcDNA-BiP (Rüdiger et al., 1997
) is a modification of the pcDNA3 vector (Invitrogene, Leek, Netherlands) carrying a
cDNA coding for 10 amino acids of the birch profilin sequence to be used
as a COOH-terminal epitope-tag. This tag is specifically recognized by the
monoclonal antibody 4A6 (Rothkegel et al., 1996
; Wiedemann et al., 1996
;
Rüdiger et al., 1997
). All constructs were sequenced by the dideoxy chain termination technique. For the expression of the vinculin head domain
(residues 1-851 of the vinculin sequence; Swissprot P12003) the coding sequence was amplified by PCR from a chicken vinculin cDNA (kind gift of
Dr. B. Geiger, Weizmann Institute of Science, Rehovot, Israel) and cloned
into pcDNA3 with a COOH-terminal FLAG tag to obtain pcDNA-
VH-FLAG.
Expression and Purification of -Catenin and the
Deletion Mutant
-Cat697-906
Escherichia coli M15 (QIAGEN) was transformed with pQE--catenin
and pQE-
-cat697-906. Expression and purification of the proteins was
performed according to Aberle et al. (1994)
with a minor modification.
The bacteria lysis buffer contained 50 mM Na-phosphate, pH 8.0, 500 mM
NaCl, 10 mM 2-mercaptoethanol, 0.5% Triton X-100, 10 µg/ml Pefabloc
(Merck, Darmstadt, Germany), 10 µM pepstatin A, 10 µg/ml leupeptin,
10 µg/ml aprotinin, and 0.5 g/100 ml CheliteP (Serva, Heidelberg, Germany) as a Ca2+-chelator. CheliteP was removed by centrifugation before
applying the sample to the Ni-NTA-Sepharose column (QIAGEN). After
affinity purification on Ni-NTA-Sepharose,
-catenin was further purified by anion exchange chromatography on MonoQ HR 5/5 (Pharmacia Biotech Sverige, Uppsala, Sweden), using a 40-ml linear gradient of 0-0.5 M
NaCl in 0.1 M Tris-HCl, pH 8.0, supplemented with Pefabloc, pepstatin A,
aprotinin, and leupeptin as above. A contaminating, slightly smaller
polypeptide present in some preparations was identified by NH2-terminal
sequence analysis as a proteolytic fragment, comprising residues 57-906.
Purification of Additional Proteins and Proteolytic Cleavage
Purification of chicken gizzard vinculin and -actinin was performed according to Feramisco and Burridge (1980)
. Thermolysin cleavage of
-actinin
and purification of the 27- and 53-kD fragments were carried out according to Pavalko and Burridge (1991)
. Vinculin was digested with V8 protease from Staphylococcus aureus (ICN Biomedicals, Eschwege, Germany),
and the 90- and the 29/27-kD fragments were purified as previously described (Kroemker et al., 1994
).
Purification of chicken gizzard talin was performed according to standard procedures (O'Halloran et al., 1985).
Ligand Interaction Studies
F-actin binding of proteins was assessed by airfuge sedimentation, according to Menkel et al. (1994). Blot overlays were performed as previously
described (Kroemker et al., 1994
). To determine dissociation constants
(Kd) surface plasmon resonance studies were done on a BIACORE 2000 machine (Biacore, Uppsala, Sweden). Roughly 1,000 resonance units
(RU)1 of purified
-catenin were coupled to a CM5 sensor chip (corresponding to 1 ng/mm2) according to the manufacturers' protocol. Vinculin
and the vinculin head domain were then tested for binding and dissociation kinetics were monitored at the concentrations indicated, using flow
rates of 10 µl/min. The kon and koff values were calculated using the BiaEvaluation software. Best fitting of data was obtained by assuming a homogeneous single-site interaction model (A + B = AB).
Affinity Mass Spectrometry and Carboxy-terminal Sequencing
This assay was performed basically as described (Rüdiger et al., 1998). Purified vinculin head fragments (0.7 mg/ml in 10 mM sodium phosphate, pH
7.5) were coupled to MPG Long Chain Alkylamine magnetic beads according to the manufacturer (Controlled Pore Glass, Lincoln Park, NJ).
Purified recombinant
-catenin (0.1 mg/ml in 100 mM NH4HCO3, pH 7.8)
was incubated overnight with endoproteinase Glu-C (V8, 25 µg/ml; Boehringer Mannheim GmbH, Mannheim, Germany). Proteolysis was stopped
by adjusting the pH to 7.0 and addition of diisopropylfluorophosphate
(DFP) to a final concentration of 50 µM. A 10 mg/ml suspension of the
vinculin head-coated magnetic beads was then incubated for 2 h at room temperature with the V8-treated
-catenin in 100 mM NH4HCO3, 150 mM NaCl, 25 mM potassium phosphate, 240 µM DFP, 4% ethanol, pH 7.5, to allow for the binding of peptides. Beads were then washed thoroughly in PBS and finally in water to remove unspecifically bound
-catenin peptides. For COOH-terminal sequence analysis, vinculin head-
coated magnetic beads with bound
-catenin peptides were treated with
carboxypeptidase Y (0.2 mg/ml; Sigma Chemical Co., Deisenhofen, Germany) in 10 mM ammoniumacetate, pH 5.5, for 90 s at room temperature.
The reaction was stopped by preparing the samples for mass spectrometric analysis according to the sandwich technique (Kussmann et al., 1997
).
Mass spectrometric analysis was performed using a Bruker REFLEX
time-of-flight instrument equipped with a Scout-ion source and pulsed ion
extraction. All spectra were recorded at an acceleration voltage of +20 kV
in both the linear and the reflector mode with delayed extraction, to
achieve high mass resolution.
Cell Extraction, In Situ Cross-linking, and Immunoprecipitation
For immunoprecipitation experiments with the vinculin head domain,
transiently transfected PtK2 cells were used. Confluent monolayers of
cells, transfected with pcDNA-VH-FLAG (see below), were washed twice
with PBS and extracted with lysis buffer (1% Triton X-100, 10% glycerol,
50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 10 µg/ml
aprotinin and leupeptin, 5 µg/ml pepstatin, and 1.2 mM Pefabloc) at 4°C
for 10 min. Cells were scraped off the culture dish with a rubber policeman, disrupted by aspiration through a 20-gauge needle, and clarified by
centrifugation. The cell lysate was incubated with 10 µg of antibody to either FLAG-tag, -catenin, or E-cadherin for 2 h at 4°C. Subsequently, 50 µl
of a 50% protein A(G)-Sepharose bead slurry (Sigma Chemical Co.) was
added. After 1 h, the beads were washed four times with lysis buffer and
boiled in SDS-PAGE sample buffer for 10 min to elute proteins bound.
Each sample was split into two aliquots, analyzed by SDS-PAGE under
reducing conditions, and then electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk
powder and incubated for 1 h with primary antibodies (see below). Antibodies were diluted in PBS with 2% BSA. Membranes were sequentially
washed in TBS-T, TBS-T with 500 mM NaCl, or TBS-T with 0.5% Triton,
incubated with HRP- conjugated secondary antibodies for 1 h, and then
extensively washed as above. Chemiluminescence (ECL system; Amersham, Braunschweig, Germany) was used for detection according to the
manufacturer's instructions.
In situ cross-linking of proteins was adopted from Hinck et al. (1994),
as described below. Monolayers of MDBK cells were rinsed twice with
PBS and exposed to 2 ml of PBS containing 200 µg/ml dithiobis(succinimidylproppionate) (DSP; Pierce, Sankt Augustin, Germany) for 20 min at
room temperature on a rocking platform. The reaction was stopped by
washing the cells in PBS and incubating them for 5 min at room temperature in quenching buffer (PBS/50 mM glycine, pH 7.4). Subsequently, the
cells were rinsed twice with PBS and permeabilized for 20 min at 4°C on a
rocking platform in CSK buffer (50 mM NaCl, 10 mM Pipes, pH 6.8, 3 mM
MgCl2, 0.5% Triton X-100, 300 mM sucrose, 20 mM glycine pH 7.4, 1.2 mM Pefabloc, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A). The permeabilized cells were scraped off the culture dish with a
rubber policeman, disrupted by repeated aspiration through a 20-gauge
needle and sedimented in a microfuge for 10 min. The supernatant (soluble fraction) was removed and the pellet (insoluble fraction) was solubilized in 100 µl SDS immunoprecipitation buffer (15 mM Tris, pH 7.5, 5 mM
EDTA, 2.5 mM EGTA, and 1% SDS) at 100°C for 10 min and then diluted to 1 ml with CSK buffer.
For immunoprecipitations, 4-30 µl of the respective antibodies were added to the solubilized insoluble fractions. After 2 h incubation with end-over-end rotation at 4°C, 50 µl of a 50% slurry of protein A(G)-Sepharose (Sigma Chemical Co.) in PBS was added and mixing was continued for 1 h. The samples were washed 4× with TBS-T. To cleave the cross-linker, the immunoprecipitated complexes were finally incubated in sample buffer with 20% mercaptoethanol at 100°C for 10 min. Finally, immunoprecipitates were split into aliquots and analyzed by immunoblotting as above.
Antibodies
A monoclonal antibody directed against the his tag (Dianova, Hamburg,
Germany) was employed to monitor the tagged recombinant -catenin
proteins. Proteins carrying the birch profilin tag were recognized by monoclonal antibody 4A6 (Rothkegel et al., 1996
; Wiedemann et al., 1996
; Rüdiger et al., 1997
). The FLAG tag was detected by monoclonal antibody
M2 purchased from Eastman Kodak (New Haven, CT). Binding of vinculin, its head, and tail fragments was analyzed with the corresponding monoclonal antibodies against epitopes in both domains (As8 [Kroemker et al.,
1994
; Menkel et al., 1994
] and 4E7 [Kroemker et al., 1994
], respectively).
A monoclonal antibody against vinculin (hVIN-1) was purchased from
Sigma Chemical Co., rabbit antibody 14A (vinculin tail specific) and monoclonal antibody 15E7 (vinculin head specific) were raised in our laboratory. Polyclonal rabbit antibody (L1) against recombinant
-catenin was
custom made (Eurogentec, Seraing, Belgium). A polyclonal antibody against
-catenin was purchased from Sigma Chemical Co. A polyclonal rabbit antibody against
-catenin was a kind gift of Dr. R. Kemler (Max
Planck Institute, Freiburg, Germany). Monoclonal antibodies specific for
-catenin and E-cadherin were obtained from Dianova. Polyclonal antibodies against
-actinin were purchased from Sigma Chemical Co. Secondary antibodies included FITC-conjugated AffiniPure Fab Fragment
Goat anti-mouse IgG (H+L) (Dianova), TRITC-conjugated goat anti-
mouse IgG and horseradish peroxidase-coupled rabbit anti-mouse IgG
(Sigma Chemical Co.).
Cell Culture, Transfection, and Fluorescence Analysis
PtK2 (Potorous tridactylis, no. CRL 6494; American Type Culture Collection, Rockville, MD) kidney epithelial cells, grown on glass coverslips for
24 h in DME supplemented with 10% FCS, were transfected with pc-catenin-BiP, pc
-cat1-670, pc
-cat697-906-BiP, or pcDNA-VH-FLAG,
respectively, applied as Ca-phosphate precipitates, according to standard
protocols. 16 h later, the medium was changed and the cells were further
incubated for 24 h before processing them for either immunofluorescence
analysis or immunoprecipitation.
Madin-Darby bovine kidney epithelial cells (MDBK; a kind gift of Dr.
Kartenbeck, Deutsches Krebsforschungszentrum, Heidelberg, Germany)
were grown as described (Kartenbeck et al., 1991; Volberg et al., 1986
).
For fluorescence analysis, cells were fixed in 3.7% paraformaldehyde
for 15 min and extracted with 0.2% Triton X-100 for 30 min. For double
labeling experiments of endogenous -catenin (or transfected
-catenin,
-cat1-670, or
-cat697-906) and either
-catenin or vinculin, the cells
were first incubated with anti-vinculin (anti-
-catenin) followed by an excess of FITC-conjugated goat anti-mouse Fab-fragments (Dianova). After extensive washing (3× 10 min PBS), the cells were incubated with
mAB 4A6 specific for the birch profilin tag sequence, in conjunction with
a TRITC-labeled secondary antibody to detect
-catenin,
-cat697-906, or
-cat1-670, respectively. F-actin was stained with FITC-phalloidin. All
preparations were examined in a Zeiss Axiophot microscope equipped
with epifluorescence. They were photographed on Kodak Tri-X-Pan. Alternatively, images were recorded with a CCD camera (VarioCam; PCO
Computer Optics GmbH, Kehlheim, Germany) and processed using
Adobe Photoshop.
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Results |
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The Transfected Vinculin Head Domain Is Targeted to Cell-Cell Contacts in PtK2 Cells
Vinculin was identified as a major structural component of
cell-matrix and cell-cell adherens junctions (Geiger et al.,
1980). The molecule contains a potent talin-binding site in
its head domain that was suggested to be responsible for
its targeting to cell-matrix contacts (Bendori et al., 1989
;
Jones et al., 1989
). Since talin is not found in cell-cell adherens junctions, we asked for a potential binding partner
of vinculin in these structures. We addressed this question
by transfecting vinculin head and tail domains, respectively, into PtK2 epithelial cells, to identify the domain responsible for cell-cell contact targeting. We used PtK2 epithelial cells, since we obtained high transfection rates and
these cells contain cadherins of the uvomorulin type (Girard and Senecal, 1995
), rendering them suitable for the
study of catenin targeting. In these cells, like in other epithelial cells, endogenous
-catenin colocalizes with
-catenin, E-cadherin, and vinculin at cell-cell contact sites but
is absent from focal contacts, which are rich in vinculin
(not shown). The transfected vinculin tail fragment, detected by an antibody specific for the avian vinculin tail sequence, was seen along stress fibers, at focal contacts and
in association with the peripheral microfilament belt, in
accordance with its actin-binding properties, as described earlier (Hüttelmaier et al., 1997
; Menkel et al., 1994
). The transfected vinculin head fragment was equipped with a
FLAG-tag, to discriminate it from endogenous proteins. It
was also found at focal contacts (Fig. 2 A), possibly due to
its talin-binding site, as discussed above. In addition, the
vinculin head domain was detected at cell-cell contacts
(arrowheads in Fig. 2 A). Since this fragment lacks an actin-binding site and talin is absent from cell-cell contacts,
this observation suggests the existence of a novel binding
partner for the vinculin head present in cell-cell contacts.
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In addition to a detergent-insoluble, cytoskeleton-bound
pool of junctional complexes, epithelial cells contain detergent-soluble structures comprising the same components.
Both types, however, are membrane associated (Hinck et
al., 1994; Näthke et al., 1994
). Based on this assumption,
we tested whether the transfected vinculin head domain
might be present in immunoprecipitates derived from such
complexes in detergent extracts (Fig. 2, B and C). When
the vinculin head domain was precipitated with the anti-FLAG antibody from 1% Triton X-100 lysates of transiently transfected PtK2 cells,
-catenin (Fig. 2 B),
-catenin
(not shown), and also E-cadherin (Fig. 2 C) were coprecipitated. Vice versa, when
-catenin was precipitated with
specific antibodies, the transfected vinculin head domain
was coprecipitated (Fig. 2 B), and when E-cadherin was
precipitated, the transfected vinculin head was found (Fig. 2 C). These data suggest that vinculin is a constituent of
the cadherin-catenin complex in PtK2 epithelial cells.
In Situ Cross-linking Reveals Complexes Containing
Endogenous -Catenin and Vinculin in MDBK Cells
Additional evidence for an in vivo interaction between
vinculin and -catenin was obtained with immunoprecipitation using nontransfected cells. Since adherens junction
proteins engaged in attaching the F-actin to the plasma
membrane are detergent-insoluble as discussed by Hinck
et al. (1994)
and by Itoh et al. (1997)
, intact complexes
cannot be solubilized. Therefore, we used in situ cross-linking to prevent complex dissociation upon cell extraction. Live MDBK cells were incubated with the membrane-permeable cross-linker DSP in vivo and extracted
with 0.5% Triton X-100. The detergent-insoluble cytoskeletal fraction was then solubilized in buffer containing 1%
SDS and these samples were used for immunoprecipitation. Results from these studies are shown in Fig. 3. When
-catenin was precipitated from such samples with a polyclonal antibody, vinculin coprecipitated. Vice versa, when
vinculin was precipitated with a combination of 14A (vinculin tail specific) and 15E7 (vinculin head specific),
-catenin coprecipitated. In addition to vinculin, a smaller protein of ~90 kD was precipitated by the vinculin antibodies.
This protein may correspond to the vinculin head domain,
which is easily generated by proteolysis, as was for example described for aged platelets (Reid et al., 1993
) and is
also seen in extracts of other cells (our own unpublished
observation).
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Thus, we conclude that endogenous vinculin and -catenin are part of the E-cadherin-catenin complex in MDBK
epithelial cells.
-Catenin Interacts with Vinculin
For reasons already discussed in Herrenknecht et al.
(1991) and Nagafuchi et al. (1991)
, a candidate binding
partner for vinculin within the cadherin-catenin complex
might be
-catenin. Based on their assumption, and on our
immunoprecipitation data (see above), we tested this hypothesis in a variety of assays. First, we performed dot
overlays (Fig. 4).
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Recombinant -catenin and its truncated fragment (
-cat697-906) were spotted onto nitrocellulose and incubated with vinculin, its proteolytic 90-kD head or its 27-kD
tail domain, respectively. Binding was monitored with specific monoclonal antibodies. Purified talin,
-actinin, and
its thermolysin-derived fragments served as controls. As
seen in Fig. 4 A, intact vinculin bound strongly to talin, less
well to
-catenin and only faintly to
-cat697-906. The isolated vinculin head strongly decorated intact
-catenin and its COOH-terminal fragment. This reaction was even
stronger than that seen for the
-actinin COOH-terminal
rod domain, a well characterized binding partner of the
vinculin head (Kroemker et al., 1994
; McGregor et al.,
1994
). In contrast, the vinculin tail fragment neither bound
to
-catenin nor to its COOH-terminal part. As shown in
Fig. 4 B, overlays in the reverse order, i.e., with vinculin
and its head and tail fragments dotted onto nitrocellulose, probed with
-catenin, and monitored with the his-tag antibody, corroborated these results:
-catenin binds to vinculin and its NH2-terminal head fragment but not to the
COOH-terminal vinculin tail. Purity controls of the protein preparations employed show that there were no contaminating proteins present that might have mediated this
interaction (Fig. 4 C). Thus, our results imply a direct
binding of the COOH-terminal region of
-catenin to the 90-kD head domain of vinculin. An interaction between
-catenin and
-actinin was not detected (not shown) corroborating other data that demonstrate that this complex
only forms when
-catenin is associated with the catenin-
cadherin complex (Knudsen et al., 1995
; Nieset et al.,
1997
). Talin-
-catenin binding was also not seen, which
may be related to the fact that these two proteins do not
colocalize in cells.
We used cosedimentation to further analyze the interaction of the vinculin head domain with -catenin. As shown
in Fig. 5 A, a mixture of purified
-catenin and the isolated
vinculin head did not sediment in the absence of F-actin,
whereas
-catenin quantitatively sedimented in the presence of F-actin, as reported by Rimm et al. (1995)
. The
vinculin head fragment did not cosediment with F-actin, in
accordance with the localization of the F-actin-binding site within the vinculin tail (Menkel et al., 1994
; Hüttelmaier et al., 1997
). However, in the presence of
-catenin,
the vinculin head cosedimented with F-actin and
-catenin
(Fig. 5 A), indicating that
-catenin mediated the sedimentation of the vinculin head when bound to F-actin. Similar
amounts of vinculin head and
-catenin cosedimented, arguing for the formation of a 1:1 complex. To exclude that
contaminating
-actinin, which contains binding sites for F-actin (Kuhlmann et al., 1992
), the vinculin head (Kroemker et al., 1994
; McGregor et al., 1994
), and
-catenin
(Knudsen et al., 1995
), might mediate this interaction, an
identical mixture of the proteins as used for cosedimentation was analyzed by immunoblotting. Fig. 5 B shows that
the
-catenin, vinculin, and F-actin preparations were free
of
-actinin. Thus, our data indicate the formation of a ternary complex in which the vinculin head is linked to F-actin via
-catenin.
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Finally, we analyzed the kinetic properties of the complex formation between -catenin and the vinculin head
fragment by surface plasmon resonance measurements.
-Catenin was immobilized on the sensor chip surface.
Binding and dissociation of either vinculin or the vinculin
head were monitored in terms of RUs (Fig. 6). When the
isolated vinculin head was used, binding and dissociation was easily monitored in the range of 0.08-1 µM of vinculin
head. The data obtained (Fig. 6 A) were used to calculate
dissociation kinetics, yielding a koff of 8 × 10
4s
1. The kon
value was in the range of 2-4 × 103 M
1s
1, depending on
the fitting method used. Thus, the dissociation constant of
the complex is Kd = 2-4 × 10
7 M. In the case of intact
vinculin, binding was barely detectable (Fig. 6 B). Binding
of intact vinculin accounted for not more than 15 RU,
even at high concentrations (10 µM) and reliable data fitting was impossible.
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Hence, a direct and specific interaction between full-length -catenin and the vinculin head was seen by three
independent biochemical methods.
To characterize the vinculin-binding site in -catenin
more precisely, we performed an affinity mass spectrometric assay. We used magnetic beads coated with the purified
vinculin head domain, as a bait for vinculin-binding peptides derived from
-catenin by cleavage with endoproteinase Glu-C (V8). Mass spectrometric analysis of these
affinity-purified samples detected one peptide of monoisotopic molecular mass 2,490.70 Da. This corresponds within 0.01% to the calculated molecular mass (2,490.40 D) of the
peptide TQTKIKRASQKKHVNPVQALSE comprising
-catenin residues 878-899, which is a predicted V8 cleavage product of
-catenin. To verify the identity of this peptide, we performed COOH-terminal sequence analysis by
carboxypeptidase treatment and mass spectrometric analysis and obtained the sequence QALSE, which confirms
this prediction. Thus, we affinity purified a COOH-terminal
-catenin fragment that strongly and specifically binds
to the vinculin head domain.
Transfected -Cat697-906 Codistributes with Adherens
Junctions and Stress Fibers
To further characterize the putative -catenin-vinculin
complex in epithelial cells, we performed transfection
studies with truncated
-catenin mutants.
The cellular distribution of transfected -catenin in PtK2
cells is seen in Fig. 7. The recombinant
-catenin was
tagged with the BiP tag to discriminate it from the endogenous protein. As expected, the transfected protein targeted to cell-cell contact sites, identified by immunostaining for
-catenin (Fig. 7, a and a') and for vinculin (Fig. 7,
b and b'). Focal contacts, also detected by staining for vinculin (Fig. 7 b'), were not labeled by intact
-catenin (Fig.
7 b). Hence, the recombinant mouse
-catenin recognized
endogenous PtK2 adherens junctions, and the exogenously expressed protein targeted faithfully to its physiological
position, the cell-cell contact site.
|
Similarly, as shown in Fig. 8, transfected -cat1-670,
comprising the
-catenin-binding site (Huber et al., 1997
;
Nieset et al., 1997
; Obama and Ozawa, 1997
; see Fig. 1),
was also predominantly recruited to cell-cell contacts (Fig.
8, a and a', arrowheads) although in this case, an additional nuclear signal was detected (Fig. 8, a and b).
|
In contrast, when the epitope tagged -cat697-906 was
transiently expressed in PtK2 cells, the fragment was not
only found at cell-cell contact sites as identified by colocalization with
-catenin (Fig. 9, a and a'), but also in focal
adhesions. This was deduced from images as seen in Fig. 9
(a-c) and proven by double staining for vinculin (Fig. 9, b
and b'), and for additional focal contact proteins like paxillin, talin, and
-actinin (not shown). Thus, in transfected
cells, the
-catenin COOH-terminal fragment colocalized
with vinculin, in accordance with the assumption that it comprises a binding site for vinculin. Furthermore,
-cat697-906 was also detected along stress fibers, as seen in the double
stain for F-actin (Fig. 9, c and c'). This may be due to its
active F-actin-binding site mapped earlier to the COOH-terminal half of the
-catenin sequence (Rimm et al., 1995
).
The unexpected localization of
-cat697-906 at focal adhesions and along stress fibers was also observed in transfected NIH/3T3 fibroblastic cells and after microinjection of
the purified recombinant fragment into PtK2 and LLC-PK1
cells (not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we addressed the following questions. First,
why are vinculin and -catenin, two structural proteins related in sequence, differentially recruited to cell-matrix
and cell-cell contacts? Second, is vinculin, a recognized
major component of cell-matrix contacts, also part of the
cell-cell junctional complex?
The recruitment of intact vinculin to focal adhesions is
mainly attributed to its rather high affinity for talin, a protein exclusively seen in cell-matrix contacts (Burridge and
Connell, 1983; Drenckhahn et al., 1988
). As had been described earlier (Bendori et al., 1989
; Menkel et al., 1994
)
and was confirmed in this study (not shown), the isolated
vinculin tail domain, when introduced into host cells by either transfection or microinjection, targets primarily to microfilament bundles and to focal adhesions. In contrast,
the transfected head domain displayed no affinity for microfilament bundles but was strongly enriched in focal adhesions, and in addition in cell-cell contacts. Thus, it must
be the head domain that is responsible for vinculin recruitment to cell-cell adherens junctions. To analyze whether
vinculin is part of the cadherin-catenin complex, which
characterizes these junctions, we performed immunoprecipitation studies. Initial attempts to immunoprecipitate
this complex failed, probably because the complex is
linked to the actin cytoskeleton. This suprastructure cannot be solubilized intact by mild detergent treatment, and
higher detergent concentrations destroy the complex. An
analogous conclusion was recently drawn by Itoh et al.
(1997)
for the
-catenin-ZO-1 complex in nonepithelial
L-cells. Here, we circumvented this problem by two alternative approaches. First, we transiently transfected PtK2
epithelial cells with the epitope-tagged vinculin head domain. This domain targets to cell-cell contacts, but it cannot bind to actin, as it lacks an F-actin-binding site (Menkel et al., 1994
; Johnson and Craig, 1995
; Hüttelmaier et
al., 1997
). Consequently, Triton X-100 soluble cadherin-
catenin complexes comprising the vinculin head domain
should be immunoprecipitable (Hinck et al., 1994
; Itoh et
al., 1997
). Indeed we detected this fragment in immunoprecipitates comprising E-cadherin,
-catenin, and
-catenin. Second, we used a membrane-permeable chemical
cross-linker to form detergent-resistant complexes within
MDBK epithelial cells. Under such conditions, we could
immunoprecipitate vinculin-
-catenin complexes from the
Triton-insoluble, cytoskeletal fraction, using either vinculin- or
-catenin-specific antibodies. Thus, both approaches showed that vinculin is part of the cadherin-catenin junctional complex.
Next, we tested for a direct interaction of -catenin and
vinculin. Hetero-oligomer formation between
-catenin
and vinculin had been predicted previously (Herrenknecht
et al., 1991
; Nagafuchi et al., 1991
; Kemler, 1993
) but was
not demonstrated. Since vinculin homo-oligomers are formed
by tail-tail interactions (Molony and Burridge, 1985
; Winkler et al., 1996
), we initially expected a similar topographical arrangement of the molecules in vinculin-
-catenin hetero-oligomers. However, our dot overlays revealed that
the vinculin binding to
-catenin involves the head domain
of the former and the putative tail (
-cat697-906) of the latter. Hence, the binding of the vinculin head to the
-catenin "tail" is a heterologous head-to-tail interaction. Furthermore we found that
-catenin mediated cosedimentation
of the vinculin head with F-actin and we could affinity purify
-cat878-899 from a V8 digest using immobilized vinculin head fragments. Of course, these data do not preclude putative additional binding sites for vinculin in the
NH2-terminal sequence of
-catenin. The kinetic constants for association and dissociation of the
-catenin-vinculin
head complex were determined by surface plasmon resonance studies. The calculated Kd (2-4 × 10
7 M) is in the
range commonly found for the interaction of cytoskeletal components. For example, the affinity of the interaction of
vinculin/talin was reported to Kd = 2-6 × 10
7 M
(Gilmore et al., 1993
). On the other hand, the binding of
vinculin to
-actinin (Otto, 1983
; Wilkins et al., 1983
;
Kroemker et al., 1994
; McGough et al., 1994
) is apparently
of weaker affinity (Kd = 1.3 × 10
5 M; McGregor et al.,
1994
).
In intramolecular interactions of vinculin, the tail region
binds to residues 1-258 of the head (Weekes et al., 1996;
our own unpublished observations), resulting in the closed
conformation of the vinculin molecule (Jockusch and Rüdiger, 1996
). Hence, it seems likely that in the intermolecular interaction described here, the COOH-terminal region of
-catenin that displays the highest degree of
homology between vinculin and
-catenin (see Fig. 1)
binds to the same vinculin domain, yielding heterologous head-to-tail complexes. In addition, the much lower affinity of intact vinculin as compared with its head domain for
-catenin, as seen in our dot overlays and the surface plasmon resonance measurements, suggests that complex formation is controlled by intramolecular interactions in vinculin, as reported for other vinculin ligands like F-actin,
talin,
-actinin, and acidic phospholipids (for references
see Jockusch and Rüdiger, 1996
).
A direct interaction between vinculin and -catenin is
also consistent with the results obtained by transfection
experiments. In transiently transfected PtK2 cells, full-length
-catenin was exclusively found at cell-cell adhesions, whereas endogenous vinculin was seen in both, focal
adhesions and cell-cell contacts. Intact
-catenin might be
recruited to cell-cell contacts due to its high affinity for
-catenin (Kd = 3.8 × 10
8 M), since the
-catenin-binding site had been mapped to residues 48-163 (Obama and
Ozawa, 1997
). In contrast, transfected
-cat697-906, which
lacks the
-catenin binding region, was not only incorporated into cell-cell contacts, but additionally into focal adhesions and along stress fibers. These are the same sites
where the vinculin tail fragment is localized (Menkel et al.,
1994
). Cosedimentation studies of
-cat697-906 with F-actin
demonstrated a direct interaction of both proteins (own
unpublished results). Hence, the localization of
-cat697-906 along stress fibers is explained by its F-actin-binding site, whereas its recruitment to focal adhesions and cell-
cell junctions might be due to an interaction of the transfected protein with endogenous vinculin.
In contrast, both intact vinculin and -catenin did not
bind to stress fibers of cultured cells, even when high levels
of the proteins were introduced by either microinjection
or overexpression (Rodriguez Fernandez et al., 1992 and
this study). This suggests that their actin-binding domains
are silent under these conditions and need to be activated.
Thus, regulation of ligand binding is apparently also common to both proteins. It remains to be seen whether
-catenin, whose overall structural organization is again similar to that of vinculin (Koslov et al., 1997
), is also regulated by a conformational switch and whether PIP2 and
Ser/Thr phosphorylation are involved as had been described
for vinculin (Gilmore and Burridge, 1996
; Schwienbacher
et al., 1996
; Weekes et al., 1996
). Recently, these structural, functional, and regulatory relationships have been
reviewed in detail (Rüdiger, 1998
).
Currently, it remains elusive which of the multiple interactions between junctional proteins are realized in living
cells, whether the cells selectively use a specific type, and
how such specificity might be regulated in epithelial architecture. The findings reported here add new aspects to our
present view on cell-cell adherens junctions as outlined in
Fig. 10. The initial model, deduced from immunoprecipitation studies, postulated binding of the cytoplasmic domain
of E-cadherin to a heterotrimeric complex of -,
-, and
-catenin (Ozawa et al., 1989
; Shore and Nelson, 1991
;
Grunwald, 1993
; Kemler, 1992
). Based on more recent
findings that two distinct complexes, containing E-cadherin and either
- and
-catenin or
- and
-catenin can
be immunoprecipitated, this model was adapted, proposing a coexistence of both complexes in fully polarized epithelial cells (Hinck et al., 1994
; Näthke et al., 1994
). The
most recent observation that
-catenin-cadherin complexes can also directly interact with
-actinin (Knudsen
et al., 1995
; Nieset et al., 1997
), together with the data reported here on complex formation between
-catenin and
vinculin, further expand the model, suggesting a highly
versatile situation at the plasma membrane of cell-cell
contact sites. Actin filaments might associate with
-catenin either directly through its actin-binding site (Rimm
et al., 1995
), or indirectly through
-actinin or vinculin (Knudsen et al., 1995
and this work). Recent reports of
Torres et al. (1997)
and Sehgal et al. (1997)
are consistent
with an essential role for
-catenin in this situation. Torres
et al. (1997)
showed that a gene trap mutation of mouse
-catenin, affecting the COOH-terminal third of the molecule, leads to a loss-of-function phenotype. Homozygous
mice do not develop further than the blastocyst state, due
to disruption of the trophoblast epithelium. Similarly, Sehgal et al. (1997)
demonstrated that an
-catenin mutant
lacking the COOH-terminal 230 amino acids in Xenopus
results in impaired blastomere adhesion and loss of the
blastocoel. These findings emphasize the significance of
our study, since the expression of such mutant
-catenins
lacking both, the COOH-terminal F-actin-binding site and
the vinculin-binding site, must be deleterious for the establishment of adherens junctions in epithelia.
|
![]() |
Footnotes |
---|
Martina Kroemker's present address is Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
Received for publication 25 May 1997 and in revised form 12 March 1998.
Address all correspondence to Dr. Manfred Rüdiger, Cell Biology, Zoological Institute, Technical University Braunschweig, 38092 Braunschweig, Germany. Tel.: 0531-391-3191. Fax: 0531-391-8203. E-mail: m.ruediger{at}tu-bs.deWe thank Drs. R. Kemler and H. Hoschuetzky (Max Planck Institute,
Freiburg, Germany) for providing -catenin cDNA and antibodies, J. Kartenbeck for MDBK cells, C. Schwienbacher for purified talin, K. Schlüter for purified actin, and H. Faulstich (Max Planck Institute, Ladenburg, Germany) for FITC-phalloidin. We are grateful to Dr. B. Haase
(BIACORE) for his support with surface plasmon resonance studies and
to Dr. M. Kiess (Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany) for NH2-terminal sequence analysis. We are indebted
to T. Meßerschmidt for expert technical assistance and photographic artwork.
This study was supported by the Deutsche Forschungsgemeinschaft.
![]() |
Abbreviations used in this paper |
---|
DFP, diisopropylfluorophosphate; DSP, dithiobis(succinimidylproppionate); MDBK, Madin-Darby bovine kidney epithelial cells; PIP2, phosphatidylinositol-4,5-bisphosphate; PtK2, Potourus tridactylus kidney epithelial cells; RU, resonance units.
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