From the Departments of Structural Biology,
§ Molecular and Cellular Physiology, and
Biochemistry, Stanford University School of Medicine,
Stanford, California 94305
Received for publication, November 15, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Cadherins are single pass transmembrane proteins
that mediate Ca2+-dependent homophilic
cell-cell adhesion by linking the cytoskeletons of adjacent cells. In
adherens junctions, the cytoplasmic domain of cadherins bind to
The formation and maintenance of solid tissues depends upon
specific and regulated intercellular adhesion (1). Cadherins are single
pass transmembrane adhesion proteins that link the cytoskeletons of
adjacent cells in two kinds of intercellular junctions: the adherens
junction and the desmosome. These structures play a critical role in
tissue development, including cell segregation, condensation,
polarization, and differentiation. Cadherin-mediated linkage of
cytoskeletal networks imparts resistance to mechanical stress and
enables concerted motions required by morphogenic processes. Defects in
cadherin-mediated adhesion are associated with several characteristics
of malignant transformation, such as dedifferentiation, high mobility,
and invasive growth (2, 3).
Adherens junctions are sites of cell-cell contact that link the actin
cytoskeletons of adjacent cells (4). Cadherin extracellular domains on
opposing membranes mediate specific
Ca2+- dependent, homotypic interactions. The
cytoplasmic domains bind to The primary structure of Several mechanisms appear to modulate cadherin-based adhesion.
Cadherins associate with As a first step toward understanding the cadherin- Protein Expression and Purification--
Murine
Transformed cells were grown in Super Broth, induced with 0.2 mM isopropyl-1-thio- Cadherin- Endogenous versus Recombinant E-Cadherin-
Cell extracts were prepared by washing 2 × 107 plated
MDCK cells with ice-cold Tris-saline (20 mM Tris-HCl, pH
7.5, 154 mM NaCl) and scraping the cells in 2 ml of DTEB
(0.5% Nonidet P-40, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl) supplemented with protease and phosphatase
inhibitors (0.1 mM Na3VO4, 50 mM NaF, 1 mM Pefabloc (Roche Molecular
Biochemicals), and 10 mg/ml each of leupeptin, aprotinin, pepstatin-A,
chymostatin, and antipain). Insoluble material was removed with a
10-min incubation on ice followed by centrifugation at 20,800 × g for 15 min. The resulting supernatant was "precleared"
by incubating it with immobilized preimmune serum for 45 min at
4 °C, centrifuging it for an additional 5 min at 20,800 × g, and taking the new supernatant.
Precleared extract supernatant was incubated for 2 h at 4 °C
with Sepharose-immobilized polyclonal E-cadherin antibody.
Postincubation Sepharose was washed once with DTEB and twice with
NaCl-supplemented DTEB. All washes were for 10 min at 4 °C and 10 different NaCl concentrations were used. Sepharose beads were
resuspended in DTEB, centrifuged through a 1 M sucrose pad,
washed with additional DTEB, and boiled for 5 min in SDS sample buffer.
After separation on 6.5% SDS-PAGE gels, immunoprecipitated proteins
were transferred to Immobilon-P polyvinylidene fluoride membrane
(Millipore Corp.) and detected with murine monoclonal primary
antibodies specific for E-cadherin and
To compare the behavior of recombinant Tryptophan Fluorescence and Fluorescence
Anisotropy--
Tryptophan fluorescence emission and anisotropy
experiments utilized an SLM 8000 C spectrofluorometer, featuring a
T-format optical pathway and double and single grating excitation and
emission monochromators, respectively. Data were taken at room
temperature from continuously stirred samples. A 290 nm (8 nm bandpass)
excitation wavelength was used for all experiments. Fluorescence
emission spectra were recorded from 300 to 420 nm using a 4-nm emission bandpass and a 2.0-nm wavelength increment with a 1-s integration period. Tryptophan (12 mM), Circular Dichroism--
Circular dichroism data were measured
using an Aviv 60DS spectropolarimeter equipped with a Peltier
temperature control unit (Hewlett-Packard 89100A). The
spectropolarimeter was calibrated with (+)-10-camphorsulfonic acid and
a 1-mm path length was used for all experiments. Spectra of E- and
DE-cadherin cytoplasmic domains were measured in 10 mM
phosphate, pH 7, at 0 °C. These spectra were measured in two parts,
with 25 and 10 µM samples being used for 280-205- and
220-186-nm wavelength ranges, respectively. The two halves of each
spectrum were scaled using the known sample concentrations.
NMR Measurements--
NMR spectra were acquired using a
General Electric GN-Omega instrument operating at 500 MHz and a 680 µM sample of E-cadherin cytoplasmic domain in 5 mM Tris-HCl, pH 8, 10 mM NaCl, 0.35 mM trimethyl silyl propionate, and 50% D2O.
Presaturation was used to suppress the H2O peak, and
trimethyl silyl propionate was employed as the chemical shift standard.
The sample was shimmed briefly before acquisition of one-dimensional
1H NMR spectra of 256 scans each (4096 real points;
spectral width, 7000 Hz) at 50, 25, and 3 °C. NMR data were
processed using Felix, version 2.30, from Biosym Technologies (San
Diego, CA). The free induction decay was processed conservatively by
Fourier transformation without premultiplication. Following phasing,
the base lines were corrected by fitting to a zero order polynomial function.
Poly-L-glutamate versus E-Cadherin Binding
Competition--
GST- Limited Proteolysis of Cadherin- Sequence Analysis--
Cytoplasmic tail sequences from 18 different vertebrate subtypes of classical/type I and atypical/type II
cadherins were analyzed. These sequences are mostly of human origin and
represent 6 type I cadherins (E-cadherin, N-cadherin, P-cadherin,
R-cadherin, M-cadherin, and Xenopus laevis EP-cadherin;
GenBankTM accession numbers Z13009, M34064, X63629, L34059, D83542, and U04707, respectively) and 12 type II cadherins (cadherin-5,
cadherin-6, cadherin-7, cadherin-8, cadherin-9, cadherin-10, cadherin-11, cadherin-12, cadherin-18, cadherin-19, cadherin-20, and
Rattus norvegicus PB-cadherin; GenBankTM
accession numbers X79981, D31784, AJ007611, L34060, AB035302, AF039747,
L34056, L34057, U59325, AJ007607, AF217289, and D83348, respectively)
as described in a recent phylogenetic analysis of the cadherin
superfamily (34). The PEST-FIND program (35) was used with a sequence
window of 10 as implemented at EMBnet Austria. Aliphatic and
instability indices were calculated using the ProtParam tool at the
ExPASy proteomics server. An "aspartic acid index," which compares
the normalized change in total aspartic acid content relative to total
glutamic acid content, is defined as 1.207 (%Asp/%Glu) where 1.207 is
the ratio of the compositional percentage for Glu (numerator) and Asp
as found in SWISS-PROT Release 38.0 and %Asp and %Glu represent the
compositional percentages found in a given cadherin cytoplasmic tail.
Recombinant Cadherin Cytoplasmic Domains Are Not Folded in
Isolation--
Fluorescence, circular dichroism, and proton NMR were
used to characterize the folded state of recombinant E-cadherin and DE-cadherin cytoplasmic domains (rEcyto and
rDEcyto respectively). Ecyto contains a single
tryptophan that is located near the C terminus of the protein.
DEcyto has two tryptophans that are near the N and C
termini, the latter in a position distinct from the tryptophan in
Ecyto. In a folded protein, the tryptophan indole ring is
frequently buried within a hydrophobic core or is otherwise shielded
from solvent, resulting in a blue shift of the fluorescence maximum
relative to free tryptophan (36). The tryptophan emission maximum for
Folded and denatured proteins commonly display significantly different
fluorescence anisotropy values because of the loss of rotational
freedom that occurs when a tryptophan indole ring is buried in a
hydrophobic core.
The fluoresence anisotropy data suggest that the Ecyto and
DEcyto domains are unfolded under native conditions.
However, regions of a folded protein that lack structure, such as large
loops or unstructured N and C termini, might give similar results.
CD spectroscopy was therefore used to probe for the presence
regular protein secondary structure. The spectra measured for
rEcyto and rDEcyto at 0 °C are essentially
identical and feature a single minimum in mean residue ellipticity at
~202 nm (Fig. 1). This spectrum
indicates a lack of secondary structure, which would be expected of an
unstructured polypeptide (37, 38).
To eliminate the possibility that the cytoplasmic tail has a defined
conformation without regular secondary structure, one-dimensional 1H NMR spectra were measured at 3, 25, and 50 °C (Fig.
2). At 50 °C, peaks with the chemical
shift values of random coil peptides (39) are observed at 0.95 ppm ( Recombinant E-Cadherin Cytoplasmic Domain and Endogenous E-Cadherin
Have Similar Electrostatic Complementarity Is Not a Dominant Factor in
E-Cadherin- Instability Is Reflected in the Sequences of the Cadherin
Cytoplasmic Tails--
A protein that is normally unstructured in
solution might be expected to have a higher than average number of
charged/polar residues and a lower than average number of hydrophobic
residues. Analysis of 18 type I and type II cadherins shows that they
contain on average 41% more charged residues (Arg, Asp, Glu, and Lys) and 29% fewer aliphatic residues (Ile, Leu, Met, Phe, and Val) relative to the average amino acid composition of the proteins in the
SWISS-PROT data base (Release 38). Given that the average calculated pI
for the cytoplasmic tails is 4.4, it is not surprising that most of the
increase in charge is from a higher than usual number of aspartic and
glutamic acid residues. In most cases the compositional increase in Asp
far outweighs that of Glu, as reflected in the "Asp index," which
we define as the normalized change in total apartic acid content
relative to total glutamic acid content (Fig.
6). It is interesting to speculate that
this bias is related to the lack of tertiary structure seen in the
domain; the side chain of Asp is less hydrophobic than that of Glu,
making it less likely to form favorable packing interactions with other
residues. The aliphatic index (AI), a measure of hydrophobicity and
thermostability (41), was also calculated from these sequences. This is
a compositional index based upon the sum of the mole percentages of
Ala, Val, Ile, and Leu weighted by the relative side chain volumes.
Proteins from thermophilic bacteria have been found to have a
significantly higher AI value (mean of 92.6, S.D. of 10.6) than
proteins from mesophilic organisms (mean of 78.8, S.D. of 14.5). Thus,
a higher AI value is correlated with higher thermostability. The
sequences from the cadherin cytoplasmic tails have AI values (mean of
64.4, S.D. of 4.2) significantly below that found for mesophilic
proteins.
Cadherins appear to be targeted for degradation when not bound to
We calculated the instability index (II), a measure that is based upon
a correlation between the stability of a protein in vivo and
the frequency of certain dipeptides in its sequence (43), for each of
the 18 sequences analyzed above. Index values greater than 40 are
indicative of metabolic instability, which is defined as an in
vivo half-life of less than 5 h. The average cadherin cytoplasmic tail II value was 56.6 (Fig. 6), and only the cadherin-8 cytoplasmic tail was predicted to be stable.
The cytoplasmic tail is the most highly conserved domain among
type I cadherins (34). With a length of ~150 residues, it is easily
large enough to be an independently folded structural unit. We
expressed the E- and DE-cadherin cytoplasmic domains in E. coli and purified them to homogeneity. Tryptophan fluorescence, circular dichroism, and one-dimensional proton NMR studies all lead to
the same surprising conclusion: rEcyto and
rDEcyto are unfolded in solution. The recombinant domains
appear to have the same biochemical properties as those of endogenous
cadherins. They form 1:1 stoichiometric complexes with The acidic nature of the Electrostatic complementarity between The lack of structure seen in the uncomplexed cadherin tail may be
related to the turnover of cadherins. E-cadherin has a relatively short
half-life (<5 h) in MDCK cells (48). Some of the cadherin tail sequences have low PEST scores (Fig. 6), but
other low scoring PEST sequences are known to be proteolytic degradation signals (35). Moreover, it has been shown that
phosphorylation may activate a latent or low-scoring PEST sequence (35,
49-53). A well studied example is the degradation of I The PEST and Given that the cytoplasmic domains of cadherins are unstructured in the
absence of -catenin, which in turn binds to the actin-associated protein
-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with
-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and 1H NMR measurements indicate that murine E-cadherin
cytoplasmic domain is unstructured. Upon binding to
-catenin,
the domain becomes resistant to proteolysis, suggesting that it
structures upon binding. Cadherin-
-catenin complex stability is
modestly dependent on ionic strength, indicating that, contrary to
previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also
reveals the presence of PEST sequences, motifs associated with
ubiquitin/proteosome degradation, that overlap the previously identified
-catenin-binding site. It is proposed that binding of
cadherins to
-catenin prevents recognition of degradation signals
that are exposed in the unstructured cadherin cytoplasmic domain,
favoring a cell surface population of catenin-bound cadherins capable
of participating in cell adhesion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, which in turn binds to the
actin-associated protein
-catenin (4, 5). An analogous adhesion
system exists in Drosophila, with armadillo and DE-cadherin
the orthologues of
-catenin and E-cadherin, respectively (6).
Deletion mutagenesis studies have mapped the regions of E-cadherin and
-catenin required for association. The C-terminal 72 residues of
E-cadherin are necessary and sufficient for
-catenin binding (7),
and a 30-amino acid stretch within this region has been proposed to be
the "core"
-catenin binding sequence (8).
-catenin consists of an N-terminal region
of 140 amino acids, followed by a 524-residue domain that contains 12 repeats of 42 amino acids known as armadillo (arm) repeats (9) and a
119-residue C-terminal tail. The arm repeat domain is required for
association with cadherins (10, 11). The three-dimensional structure of
the arm repeat domain showed that each arm repeat comprises three
helices, with the repeats packing to form a superhelix of helices (12).
The superhelix features a shallow groove with a positively charged
surface potential. The core
-catenin-binding region of E-cadherin,
which has a calculated pI of 3.3, was proposed to bind within the
positively charged groove presented by the
-catenin armadillo domain
(12).
-catenin shortly after biosynthesis, while
still in the endoplasmic reticulum, and the two proteins move together
to the cell surface, where they associate with
-catenin (13).
Failure of cadherins to associate with
-catenin leads to retention
in the endoplasmic reticulum and degradation of cadherin (14). Adherens
junction formation is also affected by phosphorylation (15, 16). For
example, phosphorylation of serines in the the cadherin cytoplasmic
tail by casein kinase II and glycogen synthase kinase-3
kinases
increases the affinity of cadherin for
-catenin (16). Moreover,
adherens junctions are enriched in protein-tyrosine kinases and
phosphatases, some of which bind the cadherin-catenin complex directly
(17-20). Tyrosine kinases target several adherens junction components
that could modulate junctional stability, including
-catenin (21)
and the arm repeat protein p120ctn (22-24), which binds
the cadherin cytoplasmic domain at a site distinct from
-catenin
(25, 26).
-Catenin also plays a central role in the Wnt/Wg growth factor
signaling pathway that controls cell fate determination during embryogenesis (reviewed in Ref. 27). In this role
-catenin acts as a
transcriptional coactivator when bound to members of the lymphoid
enhancer factor/T-cell factor (Lef/Tcf) transcription factor family
(28, 29). Wnt signaling activates transcription by blocking or slowing
the normally rapid turnover of
-catenin, thereby elevating cytosolic
levels of
-catenin and promoting formation of an active
-catenin-transcription factor complex (27). The cytosolic
concentration of
-catenin is normally maintained below the signaling
threshold by a multi-protein complex that targets
-catenin for
ubiquitination and proteasomal degradation. This protein complex
contains APC, the product of the adenomatous polyposis coli gene, the
serine/threonine kinase GSK3
, and Axin. The
-catenin binding
sequences in LEF-1 and APC are largely electronegative, and recent
mutagenesis studies have indicated that they bind within the positively
charged groove formed by the armadillo repeat region of
-catenin
(30). It is known that cadherin, APC, and LEF-1 compete for binding to
the arm repeat domain of
-catenin (11, 30), and it is likely that
all three ligands bind to overlapping portions of the positively
charged groove. Thus, depending on which ligand is bound,
-catenin
can have one of several distinct fates: adhesion complex component,
transcriptional coactivator, or substrate for proteasomal degradation.
-catenin
interaction, we have used a variety of biophysical and biochemical techniques to characterize the cadherin cytoplasmic tail in the absence
and presence of
-catenin. Our analysis shows that the cadherin
cytoplasmic tail is unstructured in isolation but appears to become
structured upon binding to
-catenin. Comparison of a variety of
cadherin sequences reveals the presence of sequence motifs associated
with proteosomal degradation. It is proposed that these properties are
associated with the regulation of cadherin turnover and cellular adhesiveness.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and
the cytoplasmic domain of murine E-cadherin (Ecyto,
Arg580-Asp728)1
and Drosophila DE-cadherin (DEcyto,
Gln1350-Ile1507) were expressed in
Escherichia coli as C-terminal fusions to glutathione
S-transferase (GST). Expression constructs were designed to
leave a minimal number of additional residues (Gly-Ser-Pro for
-catenin and Gly-Ser for the cadherins) at the N termini after
cleavage of the GST fusion. No additional residues were added to the C
termini. The bacterially expressed, recombinant cadherin cytoplasmic
domains are designated rEcyto or rDEcyto.
-Catenin cDNA in pBluescript SKII+ (31) was digested with
NdeI, and the resulting overhangs were filled with DNA
polymerase I large (Klenow) fragment. Further digestion with
SalI yielded the desired insert, which was ligated into
SmaI-SalI-digested pGEX-KG (32). The sequence
encoding the DE-cadherin cytoplasmic domain was amplified by polymerase
chain reaction from a Drosophila head
ZAP® II cDNA
library (Stratagene, La Jolla, CA) using primers 5'-GCGCCCGGATCCCAGAAGAAGCAGAAGAAT-3' and
5'-GCGCCCGATTTCTTTAGATGCGCCAGCCCTGGTC-3'. The resulting fragment was
digested with EcoRI and BamHI and ligated into
EcoRI-BamHI-digested pGEX-KG. The identity of the
polymerase chain reaction product was verified by DNA sequencing and
its ability to specifically bind armadillo and
-catenin (data not shown). The GST-E-cadherin cytoplasmic domain fusion construct has
already been described (33).
-D-galactopyranoside and
harvested by centrifugation 3 h after induction, and the cell
paste was stored at
70 °C. Thawed cell paste was treated with
protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, and 4 mg/ml pepstatin-A) and deoxyribonuclease I, and
the cells were lysed in a French pressure cell. Fusion proteins were
batch affinity purified from lysates with glutathione-agarose beads
(Sigma). GST-
-catenin fusion protein was obtained by eluting the
protein with a buffer containing 50 mM reduced glutathione. E- and DE-cadherin cytoplasmic domains and
-catenin were obtained by
cleaving the glutathione-agarose-bound fusion proteins with bovine
thrombin (Sigma). Thrombin can cleave
-catenin internally at
Arg90 or Arg95. Thus, digestion of the
GST-
-catenin fusion yields full-length
-catenin and two other
major species. These fragments, which differ only at their N termini,
copurify and are collectively named
76. Anion exchange and size
exclusion chromatography were used to purify all protein products to
near homogeneity.
-Catenin Stoichiometry Experiments--
Mixtures of
rEcyto or rDEcyto and various
-catenin
constructs were incubated for more than 1 h at 4 °C and
injected onto an Amersham Pharmacia Biotech HR 10/30 Superdex 200 size
exclusion column equilibrated with 50 mM Tris-HCl, pH 8, 200 mM NaCl, 20 mM EDTA, and 1 mM
DTT.
-Catenin Binding
Studies--
MDCK cells (type II J) were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum.
Rabbit polyclonal antibody for the E-cadherin cytoplasmic domain was described previously (33). Mouse monoclonal antibodies for E-cadherin and
-catenin were purchased from Transduction Laboratories.
Polyclonal anti-E-cadherin and preimmune sera were covalently coupled
to protein A-Sepharose. In each case, a ratio of 10 ml of serum to 75 ml of a 50% slurry of protein A-Sepharose was incubated for 12 h
at 4 °C. The Sepharose was washed with 0.2 M borate, pH
9.0, and the antibodies were cross-linked to the protein A with
dimethylpimelimidate (Pierce).
-catenin and 0.1 mCi/ml
125I-labeled goat anti-mouse secondary antibody (ICN
Pharmaceuticals, Irvine, CA). Immunoblots were exposed to x-ray film
(X-Omat AR; Eastman Kodak Co.) and quantified using a Molecular
Dynamics Storm 820 PhosphorImager system.
-catenin-E-cadherin complexes
with that of endogenous complex, 1:1 stoichiometric mixtures of the
recombinant proteins were incubated on ice for 16 h and mock
precleared as described above. Immunoprecipitations and washes were
carried out as described above. Immunoprecipitates were separated on
12% SDS-PAGE gels and processed for immunoblotting and quantitation as
described above.
76 (1 mM), and
E- and DE-cadherin cytoplasmic tail (12 mM) samples were
prepared using low ionic strength buffers (10-100 mM
Tris-HCl or HEPES, pH 7.0-8.5, 20 mM NaCl) with 1 mM DTT or 10 mM
-mercaptoethanol present for
76 and DE-cadherin. E-cadherin emission spectra were also measured under denaturing conditions (100 mM Tris-HCl, pH 7.0, 6 M guanidine HCl). Two-channel anisotropy data were
collected using the "single point polarization" option and Schott
WG-335 long pass filters instead of emission monochromators. Data were
collected under native conditions (10 mM Tris-HCl, pH 8.5, 20 mM NaCl, 1 mM DTT) for E-cadherin (12 mM) and DE-cadherin (6, 3, or1.5 mM)
cytoplasmic tails,
76 (2 mM),
-catenin (3.5 mM), bovine serum albumin (BSA, 3 mM), and
tryptophan (12 or 14 mM). Data were also collected under
denaturing conditions (100 mM Tris-HCl, pH 7, 6 M guanidine HCl, and 1 mM DTT) for E- and DE-
(6 mM) cadherin cytoplasmic tails,
76, BSA, and tryptophan.
-catenin fusion protein and
glutathione-agarose were mixed for 45 min at room temperature in a
binding buffer comprising 200 mM Tris-HCl, pH 8.5, 2 mM DTT, and either 100, 200, or 400 mM NaCl.
Poly-L-glutamate (P1818 or P4636, with average molecular masses of roughly 1,000 and 11,000 Da, Sigma) was then added, and the
mixtures were incubated an additional 45 min. E-cadherin was added, and
after another 45-min incubation, the agarose beads were spun down,
washed three times with the appropriate binding buffer, and boiled in
reducing SDS-PAGE sample buffer. The sample supernatants were analyzed
by SDS-PAGE. In each case, after the addition of E-cadherin, the
incubation mixture was 3 µM in GST-
-catenin, 3 µM in E-cadherin cytoplasmic domain, and 0, 3, 9, or 27 mM in poly-L-glutamate. Different E-cadherin
and poly-L-glutamate stocks were made using the appropriate
binding buffers.
-Catenin
Complexes--
Approximately 25 µM recombinant
E-cadherin cytoplasmic domain (rEcyto) alone or mixed in
1:1 stoichiometric amounts with either full-length
-catenin or the
armadillo repeat region (
59) (12) was subjected to limited
proteolysis with subtilisin. A control mixture containing 25 µM rEcyto and 30 µM bovine
serum albumin was also digested. Mixtures were incubated at 4 °C for
1 h and digested for 20 min at room temperature with subtilisin
concentrations ranging from 0.08 to 1.3 µg/ml. Digestions were
carried out in a buffered solution comprising 100 mM CHES,
pH 9.2, 2 mM CaCl2, and 5 mM DTT.
This pH is suboptimal for subtilisin but is required for
59
solubility. In separate experiments, rEcyto-
-catenin complex was digested with endoproteinase Glu-C using concentrations ranging from 0.4 to 27 µg/ml; these reactions were carried out in
50-100 mM Tris-HCl, pH 8.5, 2 mM
CaCl2, and 5 mM DTT.
rDEcyto-
-catenin complex was also digested with
subtilisin as described for rEcyto. Subtilisin digestions
were stopped by adding phenylmethylsulfonyl fluoride to a final
concentration of 8 mM, and endoproteinase Glu-C digestions
were stopped by boiling.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
76, a thrombin-generated fragment of
-catenin (see "Experimental Procedures"), is significantly blue-shifted relative to tryptophan alone, whereas the maxima for Ecyto and
DEcyto are not (Table I).
Thus, the single Ecyto tryptophan and two
DEcyto tryptophans appear to be solvent exposed.
Tryptophan fluorescence data
-Catenin and BSA yield anisotropy values of
~0.085 when folded and ~0.035 when denatured with 6 M
guanidine HCl (Table I). In contrast, recombinant E- and DE-cadherin cytoplasmic domains have anisotropy values comparable with those observed with denatured proteins, and these values do not change significantly when the cadherins are subjected to denaturants (Table
I).
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Fig. 1.
Circular dichroism spectra for recombinant E-
and DE-cadherin cytoplasmic domains. Both samples were prepared
using a 10 mM phosphate pH 7 buffer and measured at
0 °C. Spectra were converted to mean residue ellipticity.
and
protons of Ile, Leu, and Val), 2.10 ppm (
protons of Met),
and 6.81 ppm (Tyr ring protons). Resolved peak doublets at 7.47 and
7.58 ppm are consistent with exposed C7 and C4 Trp protons, as one
would find in an unfolded protein. A broad, very low shoulder between 8 and 9 ppm suggests that amide protons are unprotected and in rapid
exchange with solvent protons and deuterons. These data indicate that
rEcyto is unfolded at 50 °C. Spectra recorded at 25 and
3 °C are similar to that of the 50 °C spectrum (Fig. 2). However,
amide proton peaks appear at 25 °C and become more prominent at
3 °C, with a single small alkyl peak becoming resolved at 0.70 ppm.
The appearance of amide proton peaks may result from secondary
structure formation, or from a lower rate of solvent exchange resulting
from the decreased temperature. Because the amide resonances lack the
chemical shift dispersion that accompanies secondary structure
formation, the latter explanation is more likely. Thus,
rEcyto appears to be largely unstructured at 50, 25, and
3 °C.
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Fig. 2.
Proton NMR spectra for the recombinant
E-cadherin cytoplasmic domain taken at 50, 25, and 3 °C.
Spectra were measured with at 500 MHz using a 680 µM
sample of cadherin cytoplasmic domain in 5 mM Tris-HCl, pH
8, 10 mM NaCl, 0.35 mM trimethyl silyl
propionate, and 50% D2O. The temperature at which each
spectrum was measured is indicated on the left.
-Catenin Binding Properties--
The
-catenin
binding properties of rEcyto were compared with those of
E-cadherin isolated from eukaryotic cells. Full-length
-catenin, as
well as fragments lacking the first ~90 amino acids (
76; see
"Experimental Procedures") or comprising the arm repeats (
59)
(12) were used in these experiments. Size exclusion chromatography (Table II) and native gel electrophoresis
(data not shown) were used to separate mixtures comprising 2:1, 1.5:1,
1:1, 1:1.5, and 1:2 molar ratios of
76 and rEcyto.
Similar experiments were carried out with
rEcyto-
-catenin, rDEcyto-
catenin, and
rEcyto-
59 complexes (data not shown). All experimental
results were consistent with the 1:1 stoichiometry previously derived
using endogenous proteins (13, 40). To compare directly the stability
of recombinant and endogenous E-cadherin-
-catenin complex, we
examined the sensitivity of each complex to salt washes, a common
method of testing protein-protein interactions in cell extracts.
Endogenous E-cadherin-
-catenin complex was immunoprecipitated from
MDCK cell extracts with polyclonal anti-E-cadherin antibodies attached
to Sepharose beads. Recombinant
-catenin-Ecyto complexes
were prepared in parallel and immunoprecipitated using the same buffer
conditions. After washing the immunoprecipitates, the beads were
incubated with buffers containing increasing concentrations of NaCl.
The amount of complex resistant to dissociation was assayed by Western
blots of the post-wash bead-associated proteins with cadherin and
-catenin-specific antibodies. The stability of the recombinant
complex in NaCl was found to be similar to that observed with the
endogenous proteins (Fig. 3). Although
the two curves agree within experimental error, the recombinant complex
was consistently slightly less stable than the endogenous complex. It
is possible that the increased stability of the endogenous complex is
the result of post-translational modifications, such as phosphorylation (16).
-Catenin/E-cadherin complex stoichiometry as determined by size
exclusion chromatography using purified recombinant proteins
76, a fragment of
-catenin extending from residue Arg90
or Arg95 to the C terminus, and recombinant E-cadherin
cytoplasmic domain (rEcyto) were mixed at various
ratios, incubated for more than 1 h at 4 °C, and injected onto
an Amersham Pharmacia Biotech HR 10/30 Superdex 200 column. Peaks were
integrated by photocopying the chart recorder trace, cutting out the
peaks, and weighing them. The peak masses for one molar equivalent of
76 and Ecyto were determined in independent runs (data not
shown).
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Fig. 3.
The stability of recombinant and endogenous
E-cadherin- -catenin complexes as a function of
NaCl concentration. Recombinant (Rec and
RECOMB) Ecyto-
-catenin and endogenous
E-cadherin-
-catenin complexes from MDCK cell extracts were
immunoprecipitated using an immobilized anti-E-cadherin antibody.
Samples were washed with buffers containing different NaCl
concentrations and boiled, and the proteins were separated by SDS-PAGE.
-catenin and E-cadherin were detected by Western blotting with
125I-labeled antibody and quantitated using a
phosphorimaging system. Each plotted data point is the mean
-catenin-E-cadherin ratio from four experiments. The S.D. for each
point is displayed as a bar. Cad, cadherin;
-cat,
-catenin.
-Catenin Complex Stability--
The positively charged
groove presented by
-catenin and the overall negative charge of the
-catenin-binding regions of E-cadherin, LEF-1, and APC suggests that
electrostatic complementarity has a major role in the interaction of
these proteins with
-catenin. As a simple test of this hypothesis,
we examined the salt dependence of the interaction. As described above
(Fig. 3), increasing NaCl concentrations reduce the stability of the
E-cadherin-
-catenin complex. However, roughly 50% of the
cadherin-
-catenin complex remains in NaCl concentrations as high as
2.5 M, suggesting that electrostatics make only a modest
contribution to complex stability. To further gauge the contribution of
electrostatics in complex formation, we tested whether the
poly-L-glutamate polyanion P1818 (~1000 Da; average
length, 10 residues) could act as competitive inhibitor of
rEcyto-
-catenin complex formation. P1818 is a weak competitor of
rEcyto for
-catenin binding when present at a 1000-fold molar excess and an effective competitor when present at a 9000-fold molar excess (Fig. 4). Increasing the
ionic strength of the incubation mixture by adding NaCl significantly
diminishes P1818 competitive inhibition (Fig. 4). The observed
inhibition of cadherin binding by P1818 and its salt dependence are
consistent with a nonspecific and largely electrostatic association of
poly-glutamate with
-catenin. Collectively, these observations
suggest that although electrostatic complementarity plays a role in
cadherin-
-catenin complex formation, it is not a dominant factor in
the stability of the complex.
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Fig. 4.
E-cadherin- -catenin
complex formation in the presence of poly-L-glutamate.
GST-
-catenin fusion protein was bound to glutathione agarose and
incubated with an equimolar amount of Ecyto in the presence
of various amounts of poly-L-glutamate. Postincubation
beads were washed with buffer, and the bound proteins were analyzed by
SDS-PAGE. The experiment was carried out using three different
incubation buffers that differed only in NaCl concentration. The molar
excess of poly-L-glutamate and the concentration of NaCl in
the incubation buffer are shown above the lanes.
The positions of molecular mass standards are marked with
arrows on the left side of the figure and labeled
in kilodaltons. The positions of GST-
-catenin and Ecyto
bands are labeled on the right.
-Catenin Binding Protects Ecyto from
Proteolysis--
The cadherin cytoplasmic domain is unstructured in
isolation, but it could become structured or fold upon binding
-catenin. Compact globular domains typically exhibit some resistance
to proteolytic degradation, so limited proteolysis is one means of ascertaining structural stability. The relatively nonspecific protease
subtilisin and the acid-specific protease endoproteinase Glu-C (V8
protease) both readily digest rEcyto. However, in the presence of
-catenin, what appears to be full-length
rEcyto remains at subtilisin (Fig.
5) and endoproteinase Glu-C (data not
shown) concentrations that completely degrade rEcyto alone
(Fig. 5, lanes 5 and 7 or lanes 12 and
14). Similar protection results were obtained with the
DEcyto-
-catenin complex and subtilisin (data not shown).
59, the armadillo repeat domain of
-catenin (12), is thought to
contain the entire cadherin-binding site. This fragment also protects
rEcyto from degradation but does so less effectively than
full-length
-catenin (Fig. 5, lanes 7 and 9 or
lanes 14 and 16). BSA-rEcyto mixtures
were used to test whether the protection afforded by full-length
-catenin was simply due to the addition of an alternative substrate
for subtilisin. The amount of BSA used in the digests was chosen to
provide the same number of peptide bonds as full-length
-catenin.
Adding BSA protects Ecyto somewhat (Fig. 5, lanes
5, 10, and 11), but the protection is
substantially weaker than that afforded by
-catenin and slightly
weaker than that provided by
59 (Fig. 5, lanes 9 and
11 or lanes 16 and 18).
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Fig. 5.
Limited proteolysis of
Ecyto- -catenin and
Ecyto-
59 complexes. The
E-cadherin cytoplasmic domain alone or in the presence of
-catenin,
59, or BSA was digested with subtilisin for 20 min, the digest was
stopped with phenylmethylsulfonyl fluoride, and the products were
analyzed by SDS-PAGE. The concentration of subtilisin in each digest is
indicated above the lanes, as are the proteins
present in the digest. The positions of molecular mass markers are
indicated with arrows on the left and labeled in
kilodaltons. Every fifth lane is numbered at the bottom for
ease of reference.
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Fig. 6.
Sequence alignment and analysis of cadherin
cytoplasmic domains. The cytoplasmic domains of six type I and
twelve type II cadherins were analyzed with all except those marked as
originating from X. laevis (Xl) or R. norvegicus (Rn) being of human origin. The alignment
includes only the C-terminal PEST region of the cadherins, with the
residue numbers for the mature cadherin-1 protein (CAD 1)
shown above. PEST sequences are shaded, and a box
has been placed around the highly conserved (Leu-Ser-Ser-Leu) motif.
The PEST-FIND score (PEST) for the highlighted sequences, as
well as the instability (II), aliphatic (AI), and
aspartic acid (AAI) indices for the entire cytoplasmic
domain are shown to the right of each sequence. The minimal
-catenin-binding region is indicated at the bottom. A
cluster of phosphorylated serines identified in cadherin-1 is shown
with asterisks.
-catenin (14). Because the PEST sequence motif is correlated with
rapid protein turnover in vivo (35), we used the PEST-FIND program to identify potential PEST sequences within the cadherin cytoplasmic domains. PEST sequences contain Pro, Glu or Asp, and Ser or
Thr and are flanked by but do not contain basic residues (His, Arg, and
Lys). PEST sequences with PEST-FIND scores greater than +5 are
considered the best candidates for being degradation signals, but many
proteins containing sequences with lower PEST scores are known to be
degraded (35). Of the 18 cadherin cytoplasmic domains analyzed, 15 have
positive scoring PEST sequences that contain or overlap the
serine-rich, minimal
-catenin-binding region in the C-terminal half
of the domain; 12 of these sequences score greater than +3, and 4 have
values greater than +5 (Fig. 6). This minimal
-catenin-binding
region of cadherin also has two features frequently found in synthetic
signals that target proteins to the ubiquitin-proteasome system in
Saccharomyces cerevisiae: a high content of serine and
threonine residues and a frequently recurring sequence motif, (bulky
hydrophobic)- (S or T)-(S or T)-(bulky hydrophobic) (42). The
sequence motif Leu-Ser-Ser-Leu is very highly conserved within the
cadherin cytoplasmic tail and is located within the minimal
-catenin-binding region (Fig. 6). It should also be noted that five
of the six type I (E-, N-, R, P-, and EP-cadherins) and two of the
twelve type II (cadherin-8 and cadherin-20) cadherins had positive
scoring PEST sequences in the membrane-proximal, N-terminal half of the
cytoplasmic domain. All of these membrane-proximal type I cadherin PEST
sequences scored greater than +3.5. This region harbors the
p120ctn-binding site (25, 26).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, and
the stability of the rEcyto-
-catenin complex as a
function of salt concentration is comparable with that of endogenous
E-cadherin-
-catenin complex. In addition,
rEcyto and rDEcyto can be
concentrated to 40 and 20 mg/ml,
respectively,2 without the
aggregation that would occur with a "misfolded" protein. Thus, it
is unlikely that the recombinant proteins are simply misfolded in
bacteria, and it is highly probable that endogenous cadherin
cytoplasmic domains are also unfolded.
-Catenin appears to protect full-length rEcyto from
degradation under conditions that completely degrade rEcyto
alone. The
-catenin-binding site of E-cadherin is thought to lie
within the C-terminal 72 residues of the 150-amino acid cytoplasmic
tail, with the minimal binding site encompassing just 30 residues (8). Given the relatively short binding sequence and the unfolded
state of Ecyto, proteolytic protection by sequestration of
the entire domain seems unlikely. The simplest explanation is that
Ecyto adopts a defined conformation upon binding to
-catenin. We interpret the observed difference in protection
provided by full-length
-catenin and
59 as evidence that regions
of
-catenin outside the arm repeat domain interact with E-cadherin.
A structuring of cadherin upon binding to
-catenin may facilitate
interactions between cadherins and other junctional components such as
p120ctn.
-catenin-binding regions of cadherins, APC,
and LEF/Tcf transcription factors led us to propose that these
otherwise unrelated proteins bind as extended polypeptides in the
electrostatically positive groove present in the arm repeat region of
-catenin (12). Indirect support for the groove binding model comes
from the observation that nuclear localization signal peptides bind in
an extended conformation within the groove of the karyopherin
arm
repeat domain (44). Recent site-directed mutagenesis data have shown
that APC and LEF-1 bind within the groove of
-catenin (30). As
cadherins compete with APC and LEF-1 for binding to
-catenin (11,
30), it is likely that cadherin also interacts with this region.
Although the groove appears to be the binding site for
-catenin
ligands, our results indicate that electrostatic complementarity is not
a major contributor to the stability of the cadherin-
-catenin
complex. This is consistent with experimental and theoretical studies
showing that the energetically favorable interactions between
complementary charges do not always compensate for the unfavorable
desolvation of the participating charged groups (45, 46). Although
charged and polar interactions may not have a dominant role in complex
stability, they are generally important for specificity, because the
cost of desolvating polar groups upon burial in an interface must be
offset by electrostatically complementary interactions. In addition,
long range electrostatic interactions may be used to enhance rates of
association, because they can provide attractive forces even during
molecular rotation and realignment (47).
-catenin and Ecyto
may simply be a consequence of maintaining the cadherin cytoplasmic domain in an extended, unstructured state that can readily bind to
-catenin. When unfolded, a typical globular protein has poor solubility properties and is prone to aggregation. Thus, it is not
surprising that the cadherin cytoplasmic domains have compositions skewed toward charged amino acids at the expense of aliphatic residues.
Such a composition is likely to favor an extended conformation, because
charge-charge repulsion will reduce the likelihood of self-association.
-Catenin may present an electrostatically positive surface to
complement the acidic character required to maintain cadherin as an
unstructured protein.
-Catenin associates with
E-cadherin shortly after cadherin synthesis (13), and mutants deficient
for
-catenin binding are retained within the endoplasmic reticulum
and are rapidly degraded (14). We have shown here that the majority of
cadherin cytoplasmic domains contain PEST sequence motifs. The PEST
sequences may function as signals for the degradation of
"catenin-free" or uncomplexed cadherins.
B proteins,
inhibitors of the transcription factor NF-
B (54). The presence and
phosphorylation of PEST sequences in I
Bs is required for a rapid and
constitutive turnover of free I
Bs that may facilitate sustained
NF-
B activity (55-58). Interestingly, a subclass of constitutively
active Ras superfamily members binds to and possibly sequesters the
PEST regions of free I
B proteins, regulating their turnover (59). The E-cadherin PEST sequence is phosphorylated in a serine-rich region
that is highly conserved among type I cadherins and is also present in
type II cadherins (Fig. 6) (8), suggesting that phosphorylation can
activate these degradation signals as well.
-catenin-binding regions of cadherins overlap
extensively, and phosphorylation of the serines in this region has been
shown to increase the affinity of E-cadherin for
-catenin (16). It
is not known when or where in the cell cadherins are phosphorylated.
Binding of cadherins to
-catenin may prevent phosphorylation by
sequestering the PEST serines from kinases. Those cadherin molecules
that have already been phosphorylated will have increased affinity for
-catenin. Upon
-catenin binding, these activated PEST sequences
would again be sequestered, this time from recognition by the
degradation machinery. Either or both of these scenarios would lead to
selective degradation of those cadherin molecules that fail to bind to
-catenin.
-catenin, they are likely to be good substrates for
kinases as well as the cellular protein degradation machinery. Functional adhesion requires cadherins to be linked to the cytoskeleton through
- and
-catenins, so free cadherin molecules at the cell surface might act as competitive inhibitors of adhesion. Cytosolic levels of
-catenin are tightly controlled to prevent inappropriate activation of Wnt-responsive genes. Increasing
-catenin levels by
induction of the Wnt pathway increases the formation of
cadherin-catenin complex and cell-cell adhesion in some cell lines (60,
61). Likewise, the increased level of total
-catenin following
overexpression of cadherin in other cell lines likely reflects
stabilization of
-catenin molecules that would otherwise be turned
over by the Wnt pathway (25, 62-64). These observations indicate that cadherin and
-catenin turnover are coupled. Cadherins that are not
associated with
-catenin may be targeted for degradation by
sequences in their unstructured cytoplasmic domains, reducing the
population of catenin-free cadherins at the cell surface.
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ACKNOWLEDGEMENTS |
---|
We thank Y. Wang and S. Fridman for technical assistance, J. Ames and K. Ng for advice on fluorescence experiments, T. Schwarz for kindly providing the Drosophila head cDNA library, and K. Spink and S. Pokutta for discussions and comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants GM56169 (to W. I. W.) and GM35227 (to W. J. N.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a Jane Coffin Childs Memorial Postdoctoral Fellowship.
** To whom correspondence should be addressed: Dept. of Structural Biology, Stanford University School of Medicine, 299 Campus Dr. West, Stanford, CA 94305. Tel.: 650-725-4623; Fax: 650-723-8464; E-mail: bill.weis@stanford.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M010377200
2 A. H. Huber and W. I. Weis, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
rEcyto, recombinant cytoplasmic domain of E-cadherin;
rDEcyto, recombinant cytoplasmic domain of DE-cadherin;
76, thrombolytic
fragment of
-catenin;
59, armadillo repeat region of
-catenin;
GST, glutathione S-transferase;
DTT, dithiothreitol;
BSA, bovine serum albumin;
CHES, 2-(cyclohexylamino)ethanesulfonic acid;
MDCK, Madin-Darby canine kidney;
AI, aliphatic index.
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