(Received for publication, July 27, 1995; and in revised form, November 10, 1995)
From the
Analysis of the calcium-dependent cell adhesion molecule
E-cadherin has led to the identification of catenins, which are
necessary for cadherin function. Growing evidence that cadherins and
catenins are subjected to genetic alterations in carcinogenesis makes
it especially important to understand protein-protein interactions
within the cadherin-catenin complex. Here we report the identification
and analysis of the -catenin binding site in plakoglobin
(
-catenin). Using N- and C-terminal truncations of plakoglobin, we
identified a domain of 29 amino acids necessary and sufficient for
binding
-catenin. The
-catenin binding site is fully encoded
within exon 3 of plakoglobin but only partially represented in
Armadillo repeat 1. This suggests that exons rather than individual Arm
repeats encode functional domains of plakoglobin. Site-directed
mutagenesis identified residues in the
-catenin binding site
indispensable for binding in vitro. Analogous mutations in
-catenin and Armadillo had identical effects. Our results indicate
that single amino acid mutations in the
-catenin binding site of
homologs of Armadillo could prevent a stable association with
-catenin, thus affecting cadherin-mediated adhesion.
Cadherins comprise a family of calcium-dependent, homophilic
cell adhesion molecules that function in establishing tissue integrity
and cell polarity(1) . E-cadherin is expressed on the
basolateral surfaces of epithelial cells and is concentrated at
adherens junctions. Three proteins termed -,
-, and
-catenin are complexed with the cytoplasmic domain of
E-cadherin(2) . Biochemical evidence suggests that
-catenin is identical to the desmosomal component
plakoglobin(3, 4, 5) .
-Catenin and
plakoglobin seem to play a central role in the architecture of two
independent cadherin-catenin complexes (CCC) (
)by linking
E-cadherin to
-catenin(4, 5, 6) . The
catenins are thought to be involved in connecting E-cadherin to the
cortical actin cytoskeleton (7) . For proper adhesive function
of the CCC, both the homophilic interaction of the extracellular domain
and the binding of the catenins to the cytoplasmic domain are
essential. If one component is missing or nonfunctional,
E-cadherin-dependent adhesion is abolished (7, 8, 9) .
The primary sequences of
-catenin and plakoglobin contain a 42-amino acid motif repeated 12
or 13 times, originally identified in the Drosophila segment
polarity gene product Armadillo(10) . Proteins with these
repeats have been grouped together as the Armadillo (Arm) repeat
family. Arm repeats are present in a variety of proteins with diverse
cellular functions(11) . This gene family can be further
divided into two subclasses, true Armadillo homologs (Armadillo,
-catenin, plakoglobin) (12) and more distantly related
proteins (p120
, band 6 protein, APC, smgGDS,
SRP1)(11, 13) .
Disruption of the CCC can
destabilize intercellular junctions, leading to altered cell morphology
and increased invasiveness(14, 15) . For instance,
nonadherent PC9 lung carcinoma cells and invasive colon carcinoma cells
which do not express -catenin recovered adhesiveness after
transfection with
-catenin cDNA(16, 17) . Cells
of the human gastritic cancer cell line HSC-39 express a truncated
-catenin that does not associate with
-catenin and barely
form cell aggregates, but made epithelial-like structures after
transfection with full-length
-catenin(18, 19) .
Genetic studies support a possible role of the CCC in invasion
suppression. For instance, E-cadherin mutations are found in
gynecologic cancers and in 50% of diffuse-type gastritic carcinomas (20, 21) . The gene encoding human plakoglobin has
been mapped to chromosome 17q21, a complex genomic region which is
subjected to genetic alterations in both sporadic and familial breast
carcinomas (22) . Plakoglobin has been shown to be subjected to
loss of heterozygosity in breast and ovarian tumors(23) .
Because plakoglobin links -catenin to E-cadherin, mutations in
important domains of plakoglobin mediating protein-protein interactions
could inhibit cadherin function.
Therefore, we analyzed the
interaction between plakoglobin and -catenin. We demonstrate here
that a 29-amino acid domain (Q29A) located at the N terminus of
plakoglobin (aa 109-137) is necessary and sufficient for high
affinity binding to
-catenin in vitro. Single amino acid
substitutions in the Q29A sequence abolished the binding of
plakoglobin,
-catenin, and Armadillo to
-catenin in
vitro. These amino acids could represent mutational hot spots
during tumor progression, as disruption of the cadherin system leads to
decreased epithelial adhesion.
To insert it into a prokaryotic expression vector, the armadillo cDNA (10) was amplified with forward primer Arm1801 (ACAGGATCCATGAGTTACATGCCAG), which introduces a BamHI site before the start codon, and reverse primer Arm2471R (CCTTGGTGCTCTCCAGATCGTTG). The purified PCR product was digested with BamHI and EcoRI and simultaneously ligated with a 3`-terminal EcoRI-fragment (2481 bp) from clone E16 into a pGEX4T1 digested with BamHI and EcoRI to create pGEX.Arm. Point mutants were created as above, using the primer Arm2610.Tag1 (GGTGGTGGGACTCGGACTGCTTCACAGAGTGGTAATTGCATAGAAC) containing 22 nucleotides of Drosophila armadillo on its 3` end and the Tag1 sequence on its 5` end, and the following mutagenic primers: T128A (AGACGTTGCACTGCCGCGGGCTGTTGGGGAT, SacII); L140A (CACCGCGTGCTTGGCCATTTGTGAC, MscI); Y150A (AGCGTCGTCCTGGGCATTAATCAGATTGACCAC, AsnI); Y150F (AGCGTCGTCCTGGAAATTAATCAGATTGACCAC, AsnI). The mutagenized strand was selectively amplified by PCR using the primers pGEX903 and Tag1. Purified PCR product was subcloned into pGEX.Arm using BamHI and CelII.
SW480 cells (1 10
) were metabolically
labeled with 50 µCi/ml [
S]methionine (1000
Ci/mmol) (Amersham) for 4-12 h, washed twice with
phosphate-buffered saline, and extracted with 1 ml of lysis buffer (10
mM HEPES-NaOH, pH 7.4, 100 mM KCl, 1 mM MgCl
, 2 mM EGTA, 0.2% Triton X-100)
phosphatase inhibitors (1 mM each of NaVO
and NaF)
and protease inhibitors (10 µg/ml of leupeptin and
phenylmethylsulfonyl fluoride, 0.1 unit/ml of
-macroglobulin). Crude extracts were clarified by
centrifugation (4 °C, 16, 000
g, 10 min). The
soluble fraction was precleared with 10% (v/v) glutathione-agarose
beads; 200 µl of precleared supernatant (2
10
cells) was mixed with 800 µl of association buffer and
incubated with 2 µg of recombinant protein for 1 h at room
temperature. Protein complexes were purified with 60 µl of
glutathione-agarose beads for 20 min at 4 °C. Beads were washed
five times with association buffer at room temperature. Proteins were
eluted with Laemmli sample buffer, separated by SDS-PAGE, and analyzed
by fluorography, autoradiography, or immunoblotting. Precipitated
-catenin was quantified with the aid of a Fujix BAS 1000
PhosphorImager.
The primary sequence of plakoglobin can be subdivided into
three domains according to sequence homology among homologs of
Armadillo. Depending on the alignment, the central region consists of a
modular structure of 12 or 13 hydrophobic Armadillo repeats (Fig. 1)(11, 12) . In order to study the
interaction between plakoglobin and -catenin, various domains of
plakoglobin were expressed as bacterial fusion proteins carrying at the
N terminus either GST or MBP. Recombinant
-catenin carries six
histidine residues (6
His) as a Tag sequence at the C terminus.
The fusion partner was used as a Tag for both purification from crude
lysates and affinity isolation of reconstituted protein complexes. The
presence of a GST- or MBP-Tag alters the electrophoretic mobility of
the recombinant proteins in comparison with their native counterparts
(by 26 or 42 kDa, respectively). The identity of the fusion partner in
a given deletion mutant had no effect on the interaction with
-catenin. Fusion proteins seemed to be folded correctly, because
they were recognized by specific antibodies in immunoprecipitation
experiments (not shown) and they interacted with their endogenous
binding partners in lysates of SW480 human colon carcinoma cells (Fig. 2).
Figure 1:
Schematic
representation of GST- and MBP-fusion proteins of human plakoglobin
used. Their ability to interact with -catenin is indicated at the right. The numbers refer to the amino acids of
plakoglobin according to the published sequence (GenBank M23410). GST, glutathione S-transferase; MBP,
maltose-binding protein; ECT, cytoplasmic domain of mouse
E-cadherin; R1, plakoglobin Arm repeat 1; E3,
plakoglobin sequence encoded by exon 3.
Figure 2:
Mapping of the -catenin binding site
in plakoglobin with C-terminal truncations. A, interaction of
recombinant plakoglobin with endogenous
-catenin. The indicated
recombinant proteins were incubated with
S-labeled lysates
of SW480 cells as described under ``Materials and Methods.''
Proteins bound to the GST-tagged plakoglobin proteins were separated by
SDS-PAGE and processed for fluorography. The prominent protein at 46
kDa binds to the glutathione-agarose beads. B, an analogous
gel was blotted onto nitrocellulose and developed with
anti-
-catenin antibody. C, fine mapping of the
-catenin binding site. The indicated GST-fusion proteins were
incubated with 6
His-tagged
-catenin in reconstitution
assays. Protein complexes were affinity-purified with
glutathione-agarose and separated by SDS-PAGE. Molecular weight markers
are indicated on the left. CBB, Coomassie Blue
staining.
In order to identify an N-terminal
boundary of the -catenin binding site, deletion mutants were
constructed lacking parts of the N terminus but extending to the C
terminus of plakoglobin. The pMAL-C2 vector, encoding the MBP, was
chosen for these constructs, because a flexible spacer of 20 amino
acids renders the MBP unlikely to overlap the putative binding site at
the N terminus of the truncated plakoglobin proteins. Purified
MBP-plakoglobin fusion proteins were incubated with 6
His-tagged
-catenin and affinity-isolated with amylose-agarose
beads. The precipitates were immunoblotted with plakoglobin-specific or
-catenin-specific antibodies (Fig. 3, A and B). Plako109C bound strongly to
-catenin. In contrast,
Plako120C, Plako139C, and Plako
128/139 (a construct carrying an
in-frame deletion of the amino acid cluster mapped with C-terminal
truncated proteins) were unable to interact with
-catenin. The
results with N-terminal deletion mutants of plakoglobin suggest that
amino acids between 109 and 120 are necessary for interaction with
-catenin. In summary, N- and C-terminal mapping narrowed down the
N- and C-terminal borders of the
-catenin binding region to amino
acids 109-137 of plakoglobin (Q29A).
Figure 3:
Mapping of the -catenin binding site
with N-terminal deletions of plakoglobin. MBP-fusion proteins of
plakoglobin were incubated with
-catenin carrying a 6
His-Tag in reconstitution assays as indicated. Protein complexes were
affinity purified with amylose-agarose beads and separated by SDS-PAGE.
Subsequent immunoblots were developed with anti-plakoglobin antibody (A) or anti-
-catenin antibody (B). The double
bands in A are due to proteolytic degradation of the
MBP-plakoglobin fusion proteins. Molecular weight markers are indicated
on the left.
Figure 4:
A sequence encoded by exon 3 of
plakoglobin but not Arm repeat 1 is sufficient to mediate the
interaction with -catenin. A, GST-tagged chimeric
proteins were incubated with recombinant
-catenin as indicated.
Protein complexes were purified with glutathione-agarose beads,
separated by SDS-PAGE, and stained with Coomassie Blue. B,
duplicate gels were blotted and developed with antibodies specific for
-catenin. C, schematic localization with respect to
full-length plakoglobin of Arm repeat 1, the
-catenin binding site
Q29A, and amino acids encoded by exon 3.
Figure 5:
A peptide corresponding to amino acids
109-137 of plakoglobin is sufficient to bind to -catenin. A, GST-tagged peptides of plakoglobin were incubated with
recombinant
-catenin in association buffer as indicated. Protein
complexes were purified with glutathione-agarose beads, separated by
SDS-PAGE, and stained with Coomassie Blue. GST migrates at the same
molecular weight as GST-Plako109-129, because the polylinker of
pGEX4T1 adds additional amino acids. B and C, the
synthetic peptide PlakoQ29A competes with full-length plakoglobin to
bind
-catenin. GST-tagged plakoglobin was incubated with
recombinant
-catenin and the indicated amounts of the peptide Q29A
(numbers indicate the protein concentration in µg/ml). Protein
complexes of plakoglobin were purified with glutathione-agarose and
immunoblotted with antibodies specific for plakoglobin (B) and
-catenin (C). D, sequence comparison of the
-catenin binding site in homologs of Armadillo. The position of
the sequence within the full-length protein is given at the left. Residues identical to those of human plakoglobin are
shaded in black. Alignment was performed by the Clustal
alignment algorithm.
The interaction between the synthetic
peptide Q29A and -catenin was also studied using surface plasmon
resonance detection(31) . This technique measures real-time
association and dissociation of molecules on a sensor surface and
allows estimates of kinetic binding constants. The biotinylated peptide
Q29A was immobilized on a streptavidin-coated sensor surface.
Concentration-dependent binding of
-catenin was observed and used
to calculate kinetic rate constants (not shown). The association rate
constant k
was 2.0
10
±
2.9
10
M
s
(n = 5). The dissociation phase was biphasic,
probably due either to heterogeneity in the biological material or to
rebinding of
-catenin during the dissociation phase. Therefore,
two dissociation rate constants were obtained: k
1
1.4
10
± 2.3
10
s
and k
2 2.1
10
± 4.6
10
s
(n = 5). The apparent
dissociation equilibrium constant (K
= k
/k
) was 7.0
10
M for k
1 and 1.1
10
M for k
2.
The values for the K
obtained from the biosensor
agree well with the peptide concentration needed to inhibit binding of
-catenin to plakoglobin (3
10
M
10
K
).
Figure 6:
Single amino acid substitutions in
proteins of the armadillo gene family abolish their
interaction with -catenin in vitro.A,
full-length plakoglobins carrying single point mutations in the
-catenin binding site were assayed with recombinant
-catenin
as indicated. Reconstituted protein complexes were separated by
SDS-PAGE and stained with Coomassie Blue. GST-tagged plakoglobin and 6
His-tagged
-catenin have approximately the same molecular
weight. B, extracts of metabolically labeled SW480 cells were
incubated with GST-fusion proteins of plakoglobin,
-catenin, and
Armadillo, either wild type or carrying identical mutations at
corresponding amino acids. Protein complexes were affinity-purified
with glutathione-agarose beads. Bound proteins were separated by
SDS-PAGE and processed for fluorography.
To demonstrate that
the -catenin binding site is conserved in members of the armadillo gene family, point mutations were introduced in the
-catenin binding site of mouse
-catenin and Drosophila Armadillo (Fig. 6B). Wild type
-catenin and
Armadillo were able to interact with
-catenin from lysates of
metabolically labeled SW480 cells. As with plakoglobin, the mutants
T120A,
Y142A, armT128A, and armY150A showed strongly reduced
binding. Again, replacement of the tyrosine with phenylalanine restored
the binding. The amount of metabolically labeled
-catenin in the
precipitates was used to quantify the ability of a given mutant protein
to interact with
-catenin. The results of these measurements are
summarized in Fig. 7A. The data suggest that identical
amino acids in all homologs of Armadillo are required for the stable
interaction with
-catenin via a not yet known hydrophobic
interaction mechanism.
Figure 7:
A, quantitative analysis of the alanine
mapping of the -catenin binding site in plakoglobin,
-catenin, and Armadillo. In the chart above, only the amino acids
differing from the wild type sequence are shown. The relative
percentage of a given mutant protein associating with metabolically
labeled
-catenin in comparison with wild type plakoglobin is shown
on the right. The values represent the mean of four
independent measurements using a PhosphorImager. Individual values
obtained from a given mutant varied less than 20%. Numbers higher than
100 indicate an increased stability of the heterodimeric complex during
the washing procedure. B, schematic representation of a
partial sequence (aa 121-133) of the
-catenin binding site
as a helical wheel. All hydrophobic amino acids which affect the
binding (black circles) are located on one side of the
putative helix.
Biochemical analysis of eukaryotic cells has already provided
important information on the molecular organization of the
cadherin-catenin complex (CCC). To investigate the interactions of
catenins, which are necessary for the adhesive function of E-cadherin,
we expressed components of the CCC as fusion proteins. We have
previously assembled the CCC in vitro with recombinant
proteins(4) . Here we have extended this study and add new
information about the interaction between plakoglobin and
-catenin. N- and C-terminal truncated proteins defined a 29-amino
acid sequence in plakoglobin (Q29A, aa 109-137) necessary and
sufficient for binding to
-catenin.
The corresponding motif in
other homologs of Armadillo also serves as a binding domain for
-catenin. The homologous sequence in
-catenin is located
between amino acids 118 and 146, which fits well with our previous
study identifying amino acids between 120 and 151 of
-catenin as
necessary for interaction with
-catenin(4) . The human
gastritic cancer cell line HSC-39 has a homozygous in-frame deletion in
-catenin that removes amino acids 28-134, the N-terminal
part of the
-catenin binding site. This cell line shows extremely
weak cadherin-mediated adhesion, because the truncated
-catenin
cannot interact with
-catenin(18, 19) . Using
amino acid exchanges in mouse
-catenin and Drosophila Armadillo, we confirm that this motif mediates the interaction
with
-catenin in homologs of plakoglobin.
Although highly
conserved in homologs of Armadillo, the Q29A motif is absent in more
distantly related proteins containing Armadillo repeats, indicating
that such proteins (e.g. p120, APC, band 6
protein) (11, 13) cannot be associated with
-catenin by the same mechanism as are homologs of Armadillo.
Indeed, previous studies have shown that APC and p120
do
not directly interact with
-catenin(32, 33) .
Based on the alanine mapping, it is also worth noting that the part of
the Q29A sequence derived from Arm repeat 1 does not match the
consensus sequence for Armadillo repeats (11) and cannot be
substituted by any other published Armadillo repeat to reconstitute a
functionally
-catenin binding site.
We have established the
partial genomic structure of the human plakoglobin (23) and
mouse -catenin genes(34) . The region of plakoglobin
encoded by exon 3 (aa 72-156) constitutes a defined protein
domain sufficient for interaction with
-catenin. In contrast, Arm
repeat 1 (aa 123-165) contains only the C-terminal portion of the
binding region and is not by itself sufficient to bind to
-catenin. This could also be observed in vivo with
adhesion-deficient HSC-39 cells, in which the truncated
-catenin
has an almost complete copy of Arm repeat 1(19) . This finding
argues that exon-encoded protein domains rather than individual repeats
could be carriers of function in plakoglobin. It favors the idea that
individual Arm repeats could assemble together into a scaffold-like
structure, which might serve to place functional amino acids at the
right place.
Alanine mapping of the -catenin binding site
identified mostly hydrophobic amino acids as indispensable for
interaction with
-catenin in vitro. The implicated
residues are spaced appropriately for an
-helical interaction
mechanism (Fig. 7B). Further analysis of the potential
secondary structure using three different software packages (DNAstar,
MacVector, and GCG) resulted in different predictions, probably due to
the conserved proline residue in the center of the
-catenin
binding site. Assuming a stabilization of
-catenin-plakoglobin
dimers through hydrophobic forces, one should expect a similar motif in
-catenin.
Exchanges of critical hydrophobic amino acids into
hydrophilic residues could affect the interaction between Armadillo
homologs and -catenin in vivo. This, in turn, would
probably disturb cadherin-mediated adhesiveness, leading to a
destabilization of intercellular junctions which might play an
important role in the progression of cancer from an adhesive to an
invasive state.