(Received for publication, August 5, 1994; and in revised form, November 28, 1994)
From the
The tumor suppressor APC protein associates with the
cadherin-binding proteins - and
-catenin. To examine the
relationship between cadherin, catenins, and APC, we have tested
combinatorial protein-protein interactions in vivo, using a
yeast two-hybrid system, and in vitro, using purified
proteins.
-Catenin directly binds to APC at high and low affinity
sites.
-Catenin cannot directly bind APC but associates with it by
binding to
-catenin. Plakoglobin, also known as
-catenin,
directly binds to both APC and
-catenin and also to the
APC-
-catenin complex, but not directly to
-catenin.
-Catenin binds to multiple independent regions of APC, some of
which include a previously identified consensus motif and others which
contain the centrally located 20 amino acid repeat sequences. The APC
binding site on
-catenin may be discontinuous since neither the
carboxyl- nor amino-terminal halves of
-catenin will independently
associate with APC, although the amino-terminal half independently
binds
-catenin. The catenins bind to APC and E-cadherin in a
similar fashion, but APC and E-cadherin do not associate with each
other either in the presence or absence of catenins. Thus, APC forms
distinct heteromeric complexes containing combinations of
-catenin,
-catenin, and plakoglobin which are independent
from the cadherin-catenin complexes.
Mutations in the APC tumor suppressor gene have been linked to inherited and sporadic cancers of the colon(1, 2, 3, 4, 5, 6) . The inherited disorder, known as familial adenomatous polyposis, is characterized by the early onset of numerous polyps in the colon, some of which invariably progress to malignant tumors if not removed. Although the incidence of familial adenomatous polyposis is relatively low, approximately 1 in 10,000 worldwide(7) , the occurrence of APC mutations in colonic tumors, in general, is strikingly high(6, 8, 9) . APC mutations are present in the early stages of colonic tumor progression, preceding the defects identified in the ras, p53, and DCC genes(6) , but the mechanism by which APC gene mutations contribute to cancer progression remains unknown. The recent discovery that the APC gene product associates with catenins (10, 11) suggests it may function in some aspect of cell adhesion.
Catenins associate with
the cytoplasmic domain of the calcium-dependent adhesion molecules N-
and E-cadherin and are essential for cadherin function in cell-cell
adhesion(12, 13, 14) . Recently, - and
-catenins were identified in anti-APC
immunoprecipitates(10, 11) , but cadherins were not,
suggesting that catenins form independent complexes with APC and
cadherins. The binding sites for catenin on these two proteins must
also be different, since there is no significant amino acid sequence
identity between APC and cadherin. The catenin binding site on
E-cadherin has been localized to the carboxyl-terminal 70 amino
acids(15) , whereas a repeated 15-amino acid sequence,
contained between amino acids 1020 and 1169, have been proposed as
-catenin binding sites on APC(11) . A binding site for
-catenin on APC has not been identified. To characterize the
physical complexes of catenins with APC and cadherin, we have utilized
both the yeast two-hybrid system and the association of purified
recombinant proteins in vitro. The data indicate the potential
for multiple distinct heteromeric complexes composed of various
combinations of catenins with either APC or cadherin.
Figure 2:
Constructs and purified proteins. A, schematic representation of the full-length 2843-amino acid
polypeptide chain of wild-type (wt) APC and various engineered
partial constructs. Salient features of the wild-type protein are
indicated at the top. B, Coomassie Blue-stained
4-20% polyacrylamide SDS-gel to which 1 µg of each of the
indicated purified proteins was applied. The molecular weights (
10
) of standard proteins (std) are shown at left.
Baculovirus constructs APC-2, -3, and -4 have been described previously (10) . Baculovirus constructs APC22, APC23, and APC25 were generated by subcloning the AseI-BamHI fragment (APC codons 1121-1337), the NdeI-BamHI fragment (APC codons 1210-1337), or the BamHI fragment (APC codons 1342-2075) from mut 2-3, respectively, into pAcOG, a derivative of pAcC13 (18) in which the polylinker was replaced with a synthetic linker engineered to encode an initiating methionine, the Glu-Glu epitope tag, and a multiple cloning site containing several stop codons.
cDNAs encoding -catenin,
-catenin, and plakoglobin
were cloned from pancreas, fetal brain, and placental cDNA libraries
(Stratagene), respectively, using PCR probes generated against
nucleotides 5-376, 217-762, and 684-1082 of the respective
cDNAs. The
- and
-catenin cDNAs contain the entire open
reading frame(12, 19) , whereas the plakoglobin cDNA
is lacking 121 nucleotides of coding sequence on the 5` end based on
its published sequence(20) . The cDNA encoding the cytoplasmic
151-amino acid domain of E-cadherin was a gift from W. J. Nelson
(Stanford University). For baculovirus expression, the Nhel-KpnI fragment of
-catenin and the AccI-BamHI fragment of
-catenin were subcloned
into pAcOG. For bacterial GST fusion constructs, the EcoRI
fragment of plakoglobin and the BamHI-SalI fragment
of the cytoplasmic domain of E-cadherin were subcloned into pGEX-3X
(Pharmacia Biotech Inc.). For in vitro transcription/translation, the phagemids (pBlueScript SK)
containing the cDNAs for
-catenin,
-catenin, and plakoglobin
were excised from the library, according to the manufacturer's
instructions. The NcoI-SalI fragment of the
cytoplasmic domain of E-cadherin was subcloned pGEM5Z.
cDNAs for
yeast expression were subcloned into either pGBT8, pGADGH, or pGAD424
plasmids containing the trp1 or leu2 gene and a
strong ADH1 promoter encoding either the GAL4 DNA binding domain (amino
acids 1-147) or activation domain (amino acids 768-881)
followed by a multiple cloning site for the in frame generation of
fusion proteins with a nuclear localization signal(21) . The
following APC fragments were subcloned into pGBT8: the APC1 and APC19 EcoRI-BamHI fragments; the APC3 NcoI-EcoRV fragment; the APC4 NcoI-SacI fragment; the APC6 NcoI-XbaI fragment and the APC22, APC23, and APC25 NcoI-NheI fragments. The following catenin or
cadherin fragments were subcloned into pGBT8, pGADGH, or pGAD424: the
-catenin NheI-KpnI fragment; the
-catenin AccI-XbaI fragment; the
-catenin 5` (codons
1-423) and
-catenin 3` (codons 423-781) EcoRI
fragments; the
-catenin
N AflIII-BamHI
fragment (codons 151-781); the plakoglobin EcoRl
fragment; and the E-cadherin-cytoplasmic domain. BamHI-SalI fragment. The control plasmids pGBT8 bcl2
and pGAD bcl2 have been described previously(22) . All
constructs were confirmed by direct DNA sequencing.
In Vitro Binding
Analysis-For estimating binding stoichiometry, 50 ng of
purified APC4 was incubated at 4 °C with 0.1, 0.25, 0.5, 5.0, or 10
µg of purified -catenin in a final volume of 150 µl of
phosphate-buffered saline containing 10 µl of protein A-Sepharose
beads (Sigma). After rocking at 4 °C for 30 min, the beads were
removed by centrifugation and 1 µg of affinity-purified anti-APC3
antibody, and fresh beads were added to the supernatant. After rocking
for an additional 60 min, the beads were recovered by centrifugation
and washed twice with 1 ml each of ice-cold Buffer B (25 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM MgCl
, 0.5% Nonidet P-40.) The beads were eluted in 30
µl each of SDS-PAGE (
)sample buffer and 15 µl of
each sample, along with known quantities of purified
-catenin and
APC, was subjected to SDS-PAGE and electroblotting. The blots were cut
horizontally, and the upper half reacted with affinity-purified
anti-APC3 and the lower half with rabbit polyclonal anti-
-catenin.
The blots were incubated for 1 h in 0.5 µCi/ml
I-protein A (Amersham Corp.) in phosphate-buffered saline
+ 0.05% Tween 20, washed three times and then quantitated using an
Ambis 4000
-scanner. Molar amounts were calculated from protein
mass determined using a standard curve and a molecular mass of 95 kDa
for
-catenin and 220 kDa for APC4. For competition binding, 50 ng
of purified
-catenin was incubated with increasing amounts of
either purified APC2, APC22, or APC23 for 20 min on ice, followed by
the addition of 50 ng of purified APC4, 10 µl of protein
A-Sepharose, and 1 µg of anti-APC3 to a final volume of 150 µl.
After rocking for 60 min at 4 °C, the samples were prepared and
quantitated as described above. For the sake of comparison, molar
ratios in the absence of competitor were normalized to unity.
For
binding of purified proteins to proteins produced by in vitro translation, the indicated cDNAs were first transcribed and
translated in vitro in the presence of
[S]Met using the TNT(TM)-coupled wheat germ
system (Promega). One microgram of purified recombinant protein was
added to 25 µl of precleared lysate along with 10 µl of protein
A-Sepharose and antibody specific to the added protein. Following a 2-h
incubation with rocking at 4 °C, the beads were washed three times
with 1 ml each of buffer B, eluted with 30 µl of SDS-PAGE sample
buffer, and 15 µl was subjected to SDS-PAGE and fluorography.
Binding studies in which only purified proteins were used were
performed in a final volume of 150 µl of phosphate-buffered saline,
0.1% Nonidet P-40 containing 100 ng of each of the indicated proteins.
The reactions were incubated on ice for 30 min followed by the addition
of either 1 µg of anti-APC antibody or 1 µl of
anti-
-catenin antibody and 10 µl of protein A-Sepharose. After
rocking at 4 °C for 1 h the beads were washed three times with
buffer B and then eluted with 30 µl of SDS-PAGE sample buffer.
Figure 1:
Immunoprecipitation of APC-catenin
complexes. APC was immunoprecipitated from detergent lysates of HCT116
and SW480 cells with -APC2 or
-APC3 antibodies and the
precipitates analyzed by SDS-PAGE and immunoblotting. The top panel was probed with
-APC2 and the bottom three panels with antibody specific to the indicated catenin protein. The
positions of the wild-type (wt) and truncated mutant (mut.) APC proteins are indicated at right.
Figure 3:
Titration of APC4 with -catenin. The
titration of APC4 protein with
-catenin is described under
``Materials and Methods.'' The indicated amount of purified
-catenin was incubated with 50 ng of APC4 and the complex isolated
by immunoprecipitation with
-APC3 antibody. Immunoblots of the
precipitates were developed using
I-protein A (inset) and quantitated using a
-scanner.
Figure 4:
Localization of -catenin binding
sites. A, yeast two-hybrid analysis. Yeast were co-transformed
with a plasmid expressing a
-catenin-transactivator fusion protein
and a plasmid expressing the indicated APC fragment or bcl2 as a
negative control, fused to the GAL4 DNA binding domain. A positive
catenin-APC interaction permits survival on media lacking histidine (right panel), whereas all transformants grow on media
supplemented with histidine (left panel). B and D, fluorogram of affinity-precipitated
S-labeled
-catenin. One µg of each of the indicated APC proteins was
added to 25 µl of wheat germ lysate containing radiolabeled
-catenin produced by in vitro translation. APC fragments
were immunoprecipitated and one-half of the sample was analyzed by
SDS-PAGE and fluorography. ``lysate'' indicates 5
µl of input, and the arrow indicates
-catenin. C, estimation of APC-
-catenin binding affinity. A
reaction containing 50 ng of APC4 and 50 ng of
-catenin was
titrated with the indicated concentrations of APC2, APC22, or APC23
protein followed by immunoprecipitation of APC4 with
-APC3
antibody. The ratio of
-catenin to APC4 was estimated by
quantitative immunoblotting. Ratios obtained in the absence of
competitor (ranging from 0.2 to 0.4) were assigned a value of 1.0 and
the remaining ratios normalized, accordingly. The inset shows
the same data plotted to 100 nM competitor.
The weaker interaction of APC23 with -catenin, relative to
APC1, was puzzling, since these two fragments, as well as APC22,
appeared equivalent for
-catenin binding in the yeast two-hybrid
system (Fig. 4A and Table 1). To obtain a better
estimate of the relative affinities of the APC fragments for
-catenin, in vitro competition binding analysis was
performed. Each APC protein fragment was titrated into a binding
reaction containing 50 ng each of
-catenin and APC4, and following
the specific immunoprecipitation of APC4, the amount of associated
-catenin was determined by quantitative immunoblotting (Fig. 4C). APC2 and APC22 competed for
-catenin
binding to APC4 with approximately equal affinities (K
5 nM), whereas APC23 was approximately 20-fold less
effective. Since APC23 contains one of the seven 20-amino acid repeats
located in the central region of APC, we tested an additional fragment,
APC25 (see Fig. 2), that excluded APC23 but still contained six
of these repeats. APC25 formed a stable complex with
-catenin in vitro (Fig. 4D) and in the yeast two-hybrid
system (Table 1). These results indicate that there are multiple
binding sites for
-catenin on APC, three of which were previously
reported to contain a common 15-amino acid consensus sequence (11) and at least two more: one in APC23 and another in APC25.
-Catenin contains 12 copies of an imperfectly repeated amino
acid sequence referred to as the armadillo repeat
sequence(28, 29) . To determine which, if any, of
these repeats mediated
-catenin binding to APC, we independently
expressed the amino- and carboxyl-terminal halves of
-catenin; the
former half containing seven armadillo repeats and the latter half
five. Surprisingly, neither half of the
-catenin molecule
interacted independently with APC (Table 1). The finding that
none of the armadillo repeats independently bind APC suggests that if
the repeats are involved, more than one must be required for high
affinity binding.
Figure 5:
Combinatorial binding of -catenin,
-catenin and APC. The indicated combinations of
-catenin,
-catenin, and APC4 were mixed and then immunoprecipitated (IP) with either anti-APC3 or anti-
-catenin.
Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. The top panels indicate probing with anti-APC and bottom
panels with either anti-
-catenin or anti-
-catenin as
indicated.
It has been suggested that plakoglobin oligomerizes with itself and
possibly with -catenin(30) . Therefore, it is conceivable
that
-catenin might also form homodimers and that this could
account for the superstoichiometric binding to APC. To test for the
oligomerization, we added purified
-catenin containing the Glu-Glu
epitope tag to wheat germ lysates containing radiolabeled, untagged
-catenin or plakoglobin. Immunoprecipitation with anti-Glu-Glu did
not result in the recovery of any radiolabeled
-catenin or
plakoglobin (Fig. 6A). This demonstrates that
-catenin does not directly bind to itself or to plakoglobin.
However, radiolabeled plakoglobin and
-catenin were
affinity-precipitated by Glu-Glu-tagged
-catenin protein, and,
conversely, radiolabeled
-catenin was affinity-precipitated by
purified
-catenin. Plakoglobin was also affinity precipitated by
APC2 protein (Fig. 6B). These results demonstrate that
plakoglobin, like
-catenin, binds directly to both APC and
-catenin. We also found that inclusion of APC2 protein with
plakoglobin and
-catenin permitted the immunoprecipitation of
-catenin by anti-plakoglobin (Fig. 6B). This
suggests that
-catenin can bind to the APC-plakoglobin complex.
The results of additional experiments, however, indicated that
-catenin had considerably higher affinity for unoccupied APC than
for the APC-plakoglobin complex (data not shown).
Figure 6:
Analysis of catenin-catenin interactions. A, direct binding. Purified - or
-catenin was added
to wheat germ lysates containing radiolabeled plakoglobin (PG)
or
- or
-catenin and then recovered by immunoprecipitation
using an antibody directed against an epitope tag present only on the
purified catenins. Precipitates were analyzed by SDS-PAGE and
fluorography. The first three lanes show the crude wheat germ lysates
containing the radiolabeled catenins and the last six lanes show their
affinity precipitation (A.P.) by the purified
- or
-catenin, as designated above the arrow head. B, indirect
binding of plakoglobin and
-catenin. Purified plakoglobin and/or
APC2 protein were incubated with wheat germ lysates containing
radiolabeled
-catenin and the purified proteins recovered with
either anti-APC (
-APC2) or anti-PG (
-PG).
Immunoprecipitates were examined for
-catenin by fluorography (top panel) and for plakoglobin by Western blotting (bottom panel). ``Input'' indicates lysate
for
-catenin or pure protein for
plakoglobin.
Figure 7:
Analysis of cadherin binding interactions. A, direct cadherin binding. The purified proteins, indicated
above the lanes, were added to wheat germ lysates containing
radiolabeled E-cadherin cytoplasmic domain and then recovered by
antibody specific to the protein designated directly above each lane.
Proteins shown in parenthesis were added in addition to the
protein targeted for immunoprecipitation. The first lane is crude
lysate, and control is normal rabbit IgG. B, indirect cadherin
binding. APC4, -catenin, and
-catenin were incubated in the
presence (lanes 2 and 3) or absence (lanes 4 and 5) of purified GST-E-cadherin cytoplasmic domain
followed by immunoprecipitation of APC (lanes 3 and 5) or recovery of GST-E-cadherin with glutathione-agarose (lanes 2 and 4). One-hundred ng of each purified
protein was used in each reaction, and 20 ng of each was applied
directly to lane 1. Immunoblots were developed using
antibodies specific to each of the designated
proteins.
The association of catenins with E-cadherin are essential to
its normal function in the formation of epithelial cell-cell contacts
known as adherens
junctions(15, 31, 32, 33) . The
surprising discovery that the tumor suppressor APC protein also binds
catenins suggests a possible link between APC and E-cadherin. If such a
connection indeed exists, however, it probably does not involve a
physical interaction between these two proteins. The present study
argues strongly against any direct association of E-cadherin with APC
and even argues against their indirect complexation via the catenins.
It is more likely that cadherin and APC serve as separate scaffolds
upon which the catenins assemble in a similar fashion. Both
-catenin and plakoglobin bind directly to these two substrates,
whereas
-catenin binds only through its association with either
-catenin or plakoglobin. Thus, cadherin and APC appear to
represent parallel systems, both utilizing the same set of associated
proteins. A summary of these interactions is presented in Table 2.
Considering that catenins bind both APC and
E-cadherin, it is surprising to find no significant amino acid sequence
identity between APC and E-cadherin. Three copies of a 15-amino acid
consensus binding site for -catenin have been identified between
APC amino acids 1020 and 1169 (11) and, accordingly, we
observed high affinity binding of
-catenin to these sequences.
However, we also observed
-catenin binding to two nonoverlapping
APC fragments (APC23 and APC25) which exclude these sequences but
contain copies of the 20 amino acid repeat. On close comparison of the
15 amino acid sites with the 20 amino acid repeats, we noted the common
invariant feature, (D/E)XXPXX(F/Y)S, shared by these
sequences. Therefore, it is possible that the 20-amino acid repeats
also serve as
-catenin binding sites. Why APC contains multiple
binding sites for
-catenin is not clear. Multiple contacts would
increase binding affinity between the two proteins, but we were unable
to identify reciprocating multiple sites for APC binding on
-catenin. Neither the amino- nor carboxyl-terminal halves of
-catenin would independently bind APC, suggesting that several of
the armadillo repeat sequences may be required to act in unison for
high affinity binding to occur.
The demonstration that plakoglobin
binds to APC, extends further the versatility of this protein.
Plakoglobin, is localized to both desmosomes and adherens junctions (34) through its association with desmogleins (35) and
E-cadherin(12, 36) , respectively. Its association
with APC indicates that plakoglobin may be involved in yet a third
independent system. In addition, plakoglobin, like -catenin,
directly associates with
-catenin. This suggests that plakoglobin
may substitute for
-catenin, at least within the context of
bringing
-catenin into a complex with APC.
The observation that
three proteins known to associate with cadherins also associate with
APC has several implications. The catenins have been proposed to link
cadherins to actin filaments (15) and may therefore confer this
same activity upon APC. Alternatively, APC may function to regulate the
availability of catenin for the cadherins. In response to stimulation
by wnt-1, the intracellular levels of -catenin and plakoglobin
undergo dramatic fluctuations which, in turn, increase both the
stability of the catenin-cadherin complex and cell-cell
adhesion(16, 37) . Alterations in catenin levels occur
post-transcriptionally and could conceivably involve the APC protein.
Finally, armadillo, the Drosophila homolog of
-catenin,
has been proposed to function as a signaling molecule in response to
the wingless ligand(38) . In this regard, it is possible that
APC may serve as an effector target for
-catenin in mammalian
systems. This is particularly interesting when considering the pattern
of APC mutations that are associated with tumor growth. Nearly all of
the somatic mutations result in truncated APC proteins that retain
catenin binding sites (reviewed in (39) and (40) ).
Moreover, germ line mutations that predict truncated APC proteins
containing catenin binding sites are associated with a more aggressive
disease progression than those lacking these
sites(39, 40) . Thus, the mutant APC-catenin complex,
frequently identified in cancer cells, may represent a deregulated or
aberrant signaling complex that contributes directly to tumor growth.