©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The APC Protein and E-cadherin Form Similar but Independent Complexes with -Catenin, -Catenin, and Plakoglobin (*)

(Received for publication, August 5, 1994; and in revised form, November 28, 1994)

Bonnee Rubinfeld Brian Souza Iris Albert Susan Munemitsu Paul Polakis (§)

From the From Onyx Pharmaceuticals, Richmond, California 94806

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The tumor suppressor APC protein associates with the cadherin-binding proteins alpha- and beta-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. beta-Catenin directly binds to APC at high and low affinity sites. alpha-Catenin cannot directly bind APC but associates with it by binding to beta-catenin. Plakoglobin, also known as -catenin, directly binds to both APC and alpha-catenin and also to the APC-beta-catenin complex, but not directly to beta-catenin. beta-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 beta-catenin may be discontinuous since neither the carboxyl- nor amino-terminal halves of beta-catenin will independently associate with APC, although the amino-terminal half independently binds alpha-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 alpha-catenin, beta-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.


INTRODUCTION

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, alpha- and beta-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 beta-catenin binding sites on APC(11) . A binding site for alpha-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.


MATERIALS AND METHODS

Antibodies

Antibodies to APC were raised in rabbits against the purified APC2 and APC3 fragments (see Fig. 2) and affinity purified against these same proteins as described previously(10) . The beta-catenin antibody used for the immunoblot in Fig. 2, and the plakoglobin antibody, were gifts from Drs. W. J. Nelson (Stanford University) and J. Papkoff (Sugen Inc.) and have been described elsewhere(16) . All other experiments involving beta-catenin antibody were performed using a rabbit polyclonal antisera to beta-catenin kindly provided by B. Gumbiner (Sloan-Kettering, New York). The mouse monoclonal antibody recognizing the Glu-Glu epitope tag has been described elsewhere(17) .


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 (times 10) of standard proteins (std) are shown at left.



cDNA Constructs

APC constructs were generated by shuttling restriction fragments into a pGEM vector (Promega) containing a synthetic linker with specific restriction enzyme sites, an initiating methionine, and a stop codon. The restriction fragments were generated using NdeI, BglII, and HindIII-NheI, for APC1 (codons 1-1210), APC19 (codons 957-2075), and APC3 (codons 2130-2843), respectively. Mut 2-3 was generated by in vitro site-directed mutagenesis on APC19 which introduced a stop at APC codon 1137, followed by a BamHI site.

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 alpha-catenin, beta-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 alpha- and beta-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 alpha-catenin and the AccI-BamHI fragment of beta-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 alpha-catenin, beta-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 alpha-catenin NheI-KpnI fragment; the beta-catenin AccI-XbaI fragment; the beta-catenin 5` (codons 1-423) and beta-catenin 3` (codons 423-781) EcoRI fragments; the beta-catenin DeltaN 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.

Protein Purification

Proteins produced in the baculovirus system contain an epitope tag, termed Glu-Glu, and were immunoaffinity-purified as described previously(23) . GST fusion proteins were produced in bacteria grown to mid-log and induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside at 30 °C overnight. Cells were frozen at -70 °C, resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl(2), 10 µg/ml each of leupeptin, pepstatin, and pefabloc, 1 µM aprotinin, and 1 mM dithiothreitol), incubated on ice for 30 min, pulse-sonicated four times in 30-s pulses, and centrifuged. The supernatant was then batch-loaded onto glutathione-agarose beads (Sigma), washed, and eluted with 10 mM glutathione in lysis buffer without dithiothreitol.

Yeast Two-hybrid System

Transformation and growth of yeast cultures were performed essentially as described elsewhere(24) . Briefly, the yeast strain was co-transformed with the indicated plasmids and spread onto both SC-trp,-leu and SC-trp,-leu,-his plates. beta-Galactosidase activity was determined utilizing a filter assay with 5-bromo-4-chloro-3-indoyl beta-D-galactoside as a substrate. Individual plasmid constructions were tested for their ability to transactivate the yeast system. beta-Catenin, plakoglobin, and E-cadherin-cytoplasmic domain, when fused to the GAL4 DNA binding domain (in the pGBT8 plasmid), were found to independently transactivate the yeast system and were therefore not used in further experiments.

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 beta-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(2), 0.5% Nonidet P-40.) The beads were eluted in 30 µl each of SDS-PAGE (^1)sample buffer and 15 µl of each sample, along with known quantities of purified beta-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-beta-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 beta-scanner. Molar amounts were calculated from protein mass determined using a standard curve and a molecular mass of 95 kDa for beta-catenin and 220 kDa for APC4. For competition binding, 50 ng of purified beta-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-beta-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.


RESULTS

Association of Catenins with Mutant and Wild-type APC Protein

It was recently demonstrated that beta-catenin associates with the APC protein(10, 11) , and based on the comparative peptide maps of proteins associated with E-cadherin and APC, it was suggested that alpha-catenin also binds APC(11) . A third protein, termed plakoglobin(20) , which may be identical to -catenin(25) , also associates with cadherins(12) , but its relationship to APC is unknown. To determine whether both catenins and plakoglobin associate with wild-type and mutant APC, we performed immunoblots on APC immunoprecipitates from HCT116 and SW480 cells. Plakoglobin, alpha-catenin, and beta-catenin co-immunoprecipitated with both wild-type and mutant APC (Fig. 1). Since the mutant APC in the SW480 cell is truncated at amino acid 1337(26, 27) , it is not recognized by alpha-APC3, and the catenins were, therefore, not specifically co-immunoprecipitated by this antibody. We next used purified recombinant proteins to examine the direct binding of APC and catenins (Fig. 2B). Obtaining sufficient quantities of purified full-length APC has been technically impractical, so the APC4 fragment was used in these experiments. Stoichiometry of binding was estimated by incubating increasing amounts of beta-catenin with 50 ng of the APC4 fragment. Each reaction was immunoprecipitated with anti-APC3, and the molar amounts of beta-catenin and APC4 were determined by quantitative immunoblotting. Based on the data presented in Fig. 3, the molar ratio of beta-catenin complexed to APC4 was approximated at 1.5:1 at the highest concentration of beta-catenin used. Ratios of 2.1:1 and 1.6:1 were obtained from two additional experiments (data not shown), yielding an average maximum stoichiometry of 1.7:1.


Figure 1: Immunoprecipitation of APC-catenin complexes. APC was immunoprecipitated from detergent lysates of HCT116 and SW480 cells with alpha-APC2 or alpha-APC3 antibodies and the precipitates analyzed by SDS-PAGE and immunoblotting. The top panel was probed with alpha-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 beta-catenin. The titration of APC4 protein with beta-catenin is described under ``Materials and Methods.'' The indicated amount of purified beta-catenin was incubated with 50 ng of APC4 and the complex isolated by immunoprecipitation with alpha-APC3 antibody. Immunoblots of the precipitates were developed using I-protein A (inset) and quantitated using a beta-scanner.



Mutational Analysis of the APC-beta-Catenin Interaction

The binding of multiple beta-catenins to APC is interesting considering the recent identification of a 15-amino acid sequence containing a proposed consensus site for beta-catenin binding(11) . This site is iterated three times between APC amino acids 1020 and 1169 (indicated as beta-catenin binding sites on Fig. 2) and can independently bind beta-catenin(11) . To further define the binding domain, we used a yeast two-hybrid system to test beta-catenin binding of various APC fragments, some containing and some lacking these beta-catenin binding sites. As expected, APC fragments containing amino acids 1-1210 (APC1), 957-2175 (APC19), and 1121-1337 (APC22) all reacted avidly with beta-catenin, whereas fragments containing amino acids 1-1014 (APC6) and 2130-2843 (APC3) did not react at all (Fig. 4A and Table 1). Surprisingly, the APC23 fragment, which lacks the beta-catenin binding sites but contains the first 20-amino acid repeat, gave a strong positive signal with beta-catenin. To verify the binding of APC23 to beta-catenin, we expressed this fragment in sf9 cells and incubated the purified protein with wheat germ lysates containing in vitro translated, radiolabeled untagged beta-catenin and then analyzed anti-Glu-Glu immunoprecipitates for the presence of radiolabeled beta-catenin. Under these conditions, APC23 formed a complex with beta-catenin, but appeared to be less efficient than the APC1 fragment, which contains all three of the consensus binding sites (Fig. 4B).


Figure 4: Localization of beta-catenin binding sites. A, yeast two-hybrid analysis. Yeast were co-transformed with a plasmid expressing a beta-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 beta-catenin. One µg of each of the indicated APC proteins was added to 25 µl of wheat germ lysate containing radiolabeled beta-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 beta-catenin. C, estimation of APC-beta-catenin binding affinity. A reaction containing 50 ng of APC4 and 50 ng of beta-catenin was titrated with the indicated concentrations of APC2, APC22, or APC23 protein followed by immunoprecipitation of APC4 with alpha-APC3 antibody. The ratio of beta-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 beta-catenin, relative to APC1, was puzzling, since these two fragments, as well as APC22, appeared equivalent for beta-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 beta-catenin, in vitro competition binding analysis was performed. Each APC protein fragment was titrated into a binding reaction containing 50 ng each of beta-catenin and APC4, and following the specific immunoprecipitation of APC4, the amount of associated beta-catenin was determined by quantitative immunoblotting (Fig. 4C). APC2 and APC22 competed for beta-catenin binding to APC4 with approximately equal affinities (K(d) 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 beta-catenin in vitro (Fig. 4D) and in the yeast two-hybrid system (Table 1). These results indicate that there are multiple binding sites for beta-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.

beta-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 beta-catenin binding to APC, we independently expressed the amino- and carboxyl-terminal halves of beta-catenin; the former half containing seven armadillo repeats and the latter half five. Surprisingly, neither half of the beta-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.

Association of alpha-Catenin with beta-Catenin and Plakoglobin

Although beta-catenin binds directly to APC with high affinity, it is not clear whether alpha-catenin also binds in this fashion or if it associates indirectly through its binding to another protein. By yeast two-hybrid analysis, alpha-catenin did not react with any APC fragments (Table 1). Also, no specific binding of alpha-catenin to APC could be detected in vitro (Fig. 5). However, when beta-catenin was included in the mixture, alpha-catenin was immunoprecipitated by anti-APC. This was due to the direct association of beta-catenin with alpha-catenin as shown by both the yeast two-hybrid analysis (Table 1) and the association of these two purified proteins in vitro (Fig. 5). Moreover, alpha-catenin binds specifically to the amino-terminal region of beta-catenin. This is demonstrated by its ability to bind to the amino-terminal half, but not the carboxyl-terminal half of beta-catenin, and its inability to bind a deletion mutant lacking 150 amino-terminal residues (Table 1).


Figure 5: Combinatorial binding of alpha-catenin, beta-catenin and APC. The indicated combinations of alpha-catenin, beta-catenin, and APC4 were mixed and then immunoprecipitated (IP) with either anti-APC3 or anti-beta-catenin. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. The top panels indicate probing with anti-APC and bottom panels with either anti-alpha-catenin or anti-beta-catenin as indicated.



It has been suggested that plakoglobin oligomerizes with itself and possibly with beta-catenin(30) . Therefore, it is conceivable that beta-catenin might also form homodimers and that this could account for the superstoichiometric binding to APC. To test for the oligomerization, we added purified beta-catenin containing the Glu-Glu epitope tag to wheat germ lysates containing radiolabeled, untagged beta-catenin or plakoglobin. Immunoprecipitation with anti-Glu-Glu did not result in the recovery of any radiolabeled beta-catenin or plakoglobin (Fig. 6A). This demonstrates that beta-catenin does not directly bind to itself or to plakoglobin. However, radiolabeled plakoglobin and beta-catenin were affinity-precipitated by Glu-Glu-tagged alpha-catenin protein, and, conversely, radiolabeled alpha-catenin was affinity-precipitated by purified beta-catenin. Plakoglobin was also affinity precipitated by APC2 protein (Fig. 6B). These results demonstrate that plakoglobin, like beta-catenin, binds directly to both APC and alpha-catenin. We also found that inclusion of APC2 protein with plakoglobin and beta-catenin permitted the immunoprecipitation of beta-catenin by anti-plakoglobin (Fig. 6B). This suggests that beta-catenin can bind to the APC-plakoglobin complex. The results of additional experiments, however, indicated that beta-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 alpha- or beta-catenin was added to wheat germ lysates containing radiolabeled plakoglobin (PG) or alpha- or beta-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 alpha- or beta-catenin, as designated above the arrow head. B, indirect binding of plakoglobin and beta-catenin. Purified plakoglobin and/or APC2 protein were incubated with wheat germ lysates containing radiolabeled beta-catenin and the purified proteins recovered with either anti-APC (alpha-APC2) or anti-PG (alpha-PG). Immunoprecipitates were examined for beta-catenin by fluorography (top panel) and for plakoglobin by Western blotting (bottom panel). ``Input'' indicates lysate for beta-catenin or pure protein for plakoglobin.



Formation of Independent Complexes of APC and Cadherin with Catenins

All three catenins are immunoprecipitated from cell lysates by antibodies to either E-cadherin (14) or APC (Fig. 1), yet we have been unable to detect cadherins in APC immunoprecipitates or vice versa(10) . To thoroughly examine the possibility of an APC-cadherin interaction, we tested the binding of these proteins in vitro. Purified APC proteins were unable to affinity-precipitate the in vitro translated cytoplasmic domain of E-cadherin (Fig. 7A). The integrity of the E-cadherin was demonstrated by its association with both beta-catenin and plakoglobin. Although these results demonstrate that APC and E-cadherin do not directly associate, it is still conceivable that catenins could bridge an interaction between APC and cadherin. However, the inclusion of beta-catenin with APC still did not permit the affinity precipitation of E-cadherin with APC antibody, even though cadherin bound beta-catenin under these conditions (Fig. 7A). alpha-Catenin does not directly bind E-cadherin (Fig. 7A) or APC (Fig. 5) and therefore could not directly bridge an APC-cadherin interaction either. Moreover, inclusion of alpha- and beta-catenin in a reaction containing cadherin and APC still did not result in complexation of APC and cadherin, even though both these substrates bound catenins under these conditions (Fig. 7B). Finally, APC and E-cadherin did not react when tested in the yeast two-hybrid system (Table 1). These data demonstrate that APC and E-cadherin do not form a stable complex with each other either directly or indirectly in the presence of catenins.


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, beta-catenin, and alpha-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.




DISCUSSION

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 beta-catenin and plakoglobin bind directly to these two substrates, whereas alpha-catenin binds only through its association with either beta-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 beta-catenin have been identified between APC amino acids 1020 and 1169 (11) and, accordingly, we observed high affinity binding of beta-catenin to these sequences. However, we also observed beta-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 beta-catenin binding sites. Why APC contains multiple binding sites for beta-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 beta-catenin. Neither the amino- nor carboxyl-terminal halves of beta-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 beta-catenin, directly associates with alpha-catenin. This suggests that plakoglobin may substitute for beta-catenin, at least within the context of bringing alpha-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 beta-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 beta-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 beta-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 510-222-9700; Fax: 510-222-9758.

(^1)
The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. Jackie Papkoff (Sugen Inc., Redwood City, CA) and W. J. Nelson (Stanford University) for antibodies to alpha-catenin, beta-catenin, and plakoglobin and Dr. Barry Gumbiner (Sloan-Kettering Cancer Center, New York) for antibody to beta-catenin. We are also indebted to David Lowe (Onyx Pharmaceuticals) for the production of recombinant baculovirus and the preparation of infected sf9 cell cultures.


REFERENCES

  1. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., Robertson, M., Sargeant, L., Krapcho, K., Wolff, E., Burt, R., Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., LePaslier, D., Abderrahim, H., Cohen, D., Leppert, M., and White, R. (1991) Cell 66, 589-600 [Medline] [Order article via Infotrieve]
  2. Joslyn, G., Carlson, M., Thliveris, A., Albertsen, H., Gelbert, L., Samowitz, W., Groden, J., Stevens, J., Spiro, L., Robertson, M., Sargeant, L., Krapcho, K., Wolff, E., Burt, R., Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., Le Paslier, D., Abderrahim, H., Cohen, D., Leppert, M., and White, R. (1991) Cell 66, 601-613 [Medline] [Order article via Infotrieve]
  3. Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H., Miyoshi, Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-664 [Medline] [Order article via Infotrieve]
  4. Miyoshi, Y., Ando, H., Nagase, H., Nishisho, I., Horii, A., Miki, Y., Mori, T., Utsunomiya, J., Baba, S., Petersen, G., Hamilton, S. R., Kinzler, K. W., Vogelstein, B., and Nakamura, Y. (1992a) Proc. Natl. Acad. Sci. U. S. A. 89, 4452-4456 [Abstract]
  5. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, I., Koyama, Y., Utsunomiya, J., Baba, S., Hedge, P., Markhan, A., Krush, A. J., Petersen, G., Hamilton, S. R., Nilbert, M. C., Levy, D. B., Bryan, T. M., Preisinger, A. C., Smith, K. J., Su, L., Kinzler, K., and Vogelstein, B. (1991) Science 253, 665-669 [Medline] [Order article via Infotrieve]
  6. Powell, S. M., Zilz, N., Beazer-Barclay, Y., Bryan, T. M., Hamilton, S. R., Thibodeau, S. N., Vogelstein, B., and Kinzler, K. W. (1992) Nature 359, 235-237 [CrossRef][Medline] [Order article via Infotrieve]
  7. Utsunomiya, J. (1990) in Hereditary Colorectal Cancer (Utsunomiya, J., and Lynch, H. T., eds) pp. 3-16, Springer-Verlag, Tokyo
  8. Cottrell, S., Bicknell, D., Kaklamanis, L., and Bodmer, W. F. (1992) Lancet 340, 626-630 [CrossRef][Medline] [Order article via Infotrieve]
  9. Miyoshi, Y., Nagase, H., Ando, H., Ichii, S., Nakatsura, S., Aoki, T., Miki, Y., Mori, T., and Nakamura, Y. (1992b) Hum. Mol. Genet. 1, 229-223 [Abstract]
  10. Rubinfeld, B., Souza, B., Albert, I., Muller, O., Chamberlain, S. C., Masiarz, F., Munemitsu, S., and Polakis, P. (1993) Science 262, 1731-1734 [Medline] [Order article via Infotrieve]
  11. Su, L.-K., Vogelstein, B., and Kinzler, K. W. (1993) Science 262, 1734-1737 [Medline] [Order article via Infotrieve]
  12. McCrea, P. D., Turck, C. W., and Gumbiner, B. (1991) Science 254, 1359-1361 [Medline] [Order article via Infotrieve]
  13. Nagafuchi, A., and Takeichi, M. (1988) EMBO J. 7, 3679-3684 [Abstract]
  14. Ozawa, M., Baribault, H., and Kemler, R. (1989) EMBO J. 8, 1711-1717 [Abstract]
  15. Ozawa, M., Ringwald, M., and Kemler, R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4246-4250 [Abstract]
  16. Hinck, L., Nelson, W. J., and Papkoff, J. (1994) J. Cell Biol. 124, 729-741 [Abstract]
  17. Grussenmyer, T., Scheidtmann, K. H., Hutchinson, M. A., and Walter, G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7952-7954 [Abstract]
  18. Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W. J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042 [Medline] [Order article via Infotrieve]
  19. Nagafuchi, A., Takeichi, M., and Tsukita, S. (1991) Cell 65, 849-857 [Medline] [Order article via Infotrieve]
  20. Franke, W. W., Goldschmidt, M. D., Zimbelmann, R., Mueller, H. M., Schiller, D. L., and Cowin, P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4027-4031 [Abstract]
  21. Hannon, G. H., Demetrick, D., and Beach, D. (1993) Genes & Dev. 7, 2378-2391
  22. Fernandez-Sarabia, M. J., and Bischoff, J. R. (1993) Nature 366, 274-275 [CrossRef][Medline] [Order article via Infotrieve]
  23. Rubinfeld, B., Crosier, W. J., Albert, I., Conroy, L., Clark, R., McCormick, F., and Polakis, P. (1992) Mol. Cell. Biol. 12, 4634-4642 [Abstract]
  24. Spaargaren, M., Martin, G. A., McCormick, F., Fernandez-Sarabia, M. J., and Bischoff, J. (1994) Biochem. J. 300, 303-307 [Medline] [Order article via Infotrieve]
  25. Peifer, M., McCrea, P. D., Green, K. J., Wieschaus, E., and Gumbiner, B. M. (1992) J. Cell Biol. 118, 681-691 [Abstract]
  26. Goyette, M. C., Cho, K., Fasching, C. L., Levy, D. B., Kinzler, K. W., Paraskeva, C., Vogelstein, B., and Stanbridge, E. C. (1992) Mol. Cell. Biol. 12, 1387-1395 [Abstract]
  27. Smith, K. J., Johnson, K. A., Bryan, T. M., Hill, D. E., Markowitz, S., Willson, J. K., Paraskeva, C., Petersen, G. M., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2846-2850 [Abstract]
  28. Peifer, M., Berg, S., and Reynolds, A. B. (1994) Cell 76, 789-791 [Medline] [Order article via Infotrieve]
  29. Riggleman, B., Wieschaus, E., and Schedl, P. (1989) Genes & Dev. 3, 96-113
  30. Kemler, R. (1993) Trends Genet. 9, 317-321 [CrossRef][Medline] [Order article via Infotrieve]
  31. Fujimori, T., and Takeichi, M. (1993) Mol. Biol. Cell 4, 37-47 [Abstract]
  32. Kintner, C. (1992) Cell 69, 225-236 [Medline] [Order article via Infotrieve]
  33. Nagafuchi, A., and Takeichi, M. (1989) Cell Regul. 1, 37-44 [Medline] [Order article via Infotrieve]
  34. Cowin, P., Kapprell, H. P., Franke, W. W., Tamkun, J., and Hynes, R. O. (1986) Cell 46, 1063-1073 [Medline] [Order article via Infotrieve]
  35. Korman, N. J., Eyre, R. W., Kluas-Kovtun, V., and Stanely, J. R. (1989) N. Engl. J. Med. 321, 631-635 [Abstract]
  36. Knudsen, K. A., and Wheelock, M. J. (1992) J. Cell Biol. 118, 671-679 [Abstract]
  37. Bradley, R. S., Cowin, P., and Brown, A. M. C. (1993) J. Cell Biol. 123, 1857-1865 [Abstract]
  38. Peifer, M. (1993) Science 262, 1667-1668 [Medline] [Order article via Infotrieve]
  39. Nakamura, Y. (1993) Adv. Cancer Res. 62, 65-85 [Medline] [Order article via Infotrieve]
  40. Polakis, P. (1995) Curr. Opin. Genet. Dev. 5, in press

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