Regulation of Complexed and Free Catenin Pools by Distinct Mechanisms
DIFFERENTIAL EFFECTS OF Wnt-1 AND v-Src*

(Received for publication, August 2, 1996, and in revised form, November 7, 1996)

Jackie Papkoff Dagger

From SUGEN, Inc., Redwood City, California 94063

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Cadherins are transmembrane receptors with an extracellular domain that participates in homophilic cell to cell adhesion and a cytoplasmic domain that associates with proteins called catenins. Cadherin-mediated adhesion as well as adhesion-independent functions for catenins play important roles in differentiation, development, and malignant transformation. Mechanisms that regulate steady-state catenin levels and cadherin-catenin complex stability are poorly understood, but activities of both the Wnt-1 proto-oncogene and tyrosine kinases are implicated. Here I define, at the biochemical level, distinct mechanisms that modulate steady-state catenin levels. Increased cadherin expression, providing more catenin binding sites, leads to selective stabilization of the cadherin-associated population of alpha - and beta -catenin, but not p120cas. In contrast, expression of Wnt-1 leads primarily to increased stability of the uncomplexed pool of beta -catenin without effect on p120cas. Significantly, the Wnt-1-induced stabilization of uncomplexed beta -catenin is independent of cadherin expression. Transformation by v-Src does not disrupt the catenin-cadherin complex despite the phosphorylation of E-cadherin and beta -catenin on tyrosine. In contrast to the effects of Wnt-1, v-Src does not modulate the uncomplexed population of beta -catenin. p120cas is phosphorylated on tyrosine by v-Src, and this is accompanied by a significant decrease in the level of uncomplexed p120cas as well as a change in behavior of p120cas upon biochemical fractionation. Taken together these data suggest that p120cas and beta -catenin are regulated independently.


INTRODUCTION

The cadherin cell to cell adhesion receptors play an important part in early embryonic development as well as in the morphogenesis of many tissue types and maintenance of the differentiated phenotype (1, 2). The cadherin adhesion system has also been shown to serve as a tumor invasion suppressor and is often defective in many tumor types that exhibit loss of cell to cell contacts and increased invasive and metastatic capability (3-5).

The cadherins are a family of transmembrane proteins that are localized to the adherens type of intercellular junction (6, 7). The extracellular domain of cadherin participates in Ca2+-dependent, homophilic cell to cell adhesion, and the cytoplasmic domain associates with a family of proteins called catenins (6, 7). Three major catenin species have been identified: alpha -catenin, a vinculin family member (8, 9), and two related proteins, beta -catenin and plakoglobin (gamma -catenin) (10). beta -Catenin and plakoglobin are mammalian homologs of a protein originally described in Drosophila called Armadillo (11), whose function is critical for normal segmental pattern formation during development (12). Another mammalian protein, p120cas, that is phosphorylated on tyrosine in response to both v-Src transformation (13) and activation of several receptor tyrosine kinases (14, 15), has recently been included in the armadillo family based on the presence of a repeating 42-amino acid sequence motif that characterizes these proteins (16, 17), as well as the ability of p120cas to associate with cadherin (18-21).

Several lines of evidence have shown that catenin binding to the cytoplasmic domain of cadherin is required for effective cell to cell adhesion (22, 23). beta -Catenin and plakoglobin form a mutually exclusive association with cadherin (24, 25), and alpha -catenin is incorporated into the complex through direct binding to either beta -catenin or plakoglobin but not cadherin (26-28). The ability of alpha -catenin to anchor the cadherin complex to actin filaments of the cell cytoskeleton is thought to be a key event in strengthening the cadherin-mediated adhesive contact (8, 9, 23, 29).

One of the first insights to the regulation of catenin function came from studies in the Drosophila system where it was shown that expression of the segment polarity gene, wingless, leads to the post-transcriptional accumulation of Armadillo protein (30). In mammalian cells, expression of the wingless homolog, the proto-oncogene wnt-1, results in increased steady-state levels of beta -catenin and plakoglobin (24, 31), stabilization of the cadherin-beta -catenin complex, and increased cell to cell adhesion (24). The specific role that cadherin serves in the regulation of steady-state catenin levels by Wnt-1 is not known.

In addition to serving a pivotal role in modulation of cell adhesion, recent evidence suggests that catenins can function in cadherin-independent signaling pathways that regulate differentiation and cell proliferation (32, 33). In Drosophila, it has been proposed that accumulation of cytoplasmic Armadillo protein serves a signaling function resulting in expression of genes required for segmental pattern formation during development (34). In mammalian cells responding to Wnt-1, the increased steady-state levels of catenins is primarily due to a selective increase in the amount of uncomplexed, monomeric beta -catenin and plakoglobin (35). While this free pool can contribute to an increased stability of cadherin-catenin complexes (24), a second adhesion-independent function is likely. The regulation of p120cas by Wnt-1 and the consequences of tyrosine phosphorylation on the regulation of free pools and complexed populations of p120cas have not been examined.

Both beta -catenin and plakoglobin, but not p120cas, associate directly with adenomatous polyposis coli (APC)1 (19, 36, 37), a tumor suppressor protein that is mutated in the majority of colorectal carcinomas (38). Cells homozygous for mutant APC protein exhibit a significant increase in a free, uncomplexed pool of beta -catenin and plakoglobin (39), and accumulating evidence indicates that APC functions in the Wnt-1 signaling pathway to directly regulate the stability of uncomplexed beta -catenin and plakoglobin (35). In another study a mutant form of beta -catenin, lacking the ability to bind alpha -catenin, leads to transformation of NIH3T3 cells upon overexpression (40). Further evidence for an adhesion-independent function for catenins comes from studies in Xenopus. Overexpression of beta -catenin or plakoglobin in fertilized Xenopus oocytes leads to a duplication of the dorsal embryonic axis during development (41, 42). Portions of beta -catenin that lack the alpha -catenin binding site are still active in this assay, whereas overexpression of cadherin has no effect (42, 43).

Several lines of evidence have implicated tyrosine kinases in the modulation of cadherin-catenin complex formation and function. Transformation of cells by the v-Src tyrosine kinase oncogene leads to a disruption of adherens junctions and an increase in invasive properties and metastatic potential (44, 45). These phenotypes correlate with phosphorylation on tyrosine of both cadherin and beta -catenin (46-48). Activation of several receptor tyrosine kinases, including receptors for hepatocyte growth factor (Met) and epidermal growth factor, also leads to the phosphorylation on tyrosine of cadherin, beta -catenin, and plakoglobin (49, 50). Of interest is the fact that the biological activities of these receptors include the dissociation of cell monolayers and induction of cell motility for Met (51) and cell rounding and membrane ruffling for the epidermal growth factor receptor (52, 53). Transformation of human breast epithelial cells by an activated form of Ras indirectly leads to phosphorylation on tyrosine of beta -catenin and p120cas and this correlates with alterations in the adherens junctions and changes in cell morphology (54). These studies implicate phosphorylation on tyrosine as a key regulatory event in cadherin-catenin complex formation and function; however, the biochemical mechanisms by which this is accomplished are unknown, and the role of tyrosine phosphorylation in regulating free catenin pools has not been examined.

In this study I have defined, at the biochemical level, distinct mechanisms that modulate steady-state catenin levels. By increasing the number of cadherin molecules, thus providing more catenin binding sites, the complexed population of alpha - and beta -catenin, but not p120cas, can be selectively increased without affecting the uncomplexed, monomeric catenin pools. In contrast, the proto-oncogene wnt-1 leads to an increased stability of the free pool of beta -catenin but has no effects on alpha -catenin or p120cas. Significantly, the effects of Wnt-1 on beta -catenin are cadherin-independent. In contrast to the effects of Wnt-1, v-Src transformation does not regulate the uncomplexed population of beta -catenin or the cadherin complex with alpha -catenin and beta -catenin, despite the phosphorylation of beta -catenin and cadherin on tyrosine. However, p120cas also becomes heavily phosphorylated on tyrosine by v-Src, and this leads to a decrease in the uncomplexed population as well as a change in distribution of p120cas detected by biochemical fractionation.


EXPERIMENTAL PROCEDURES

Cell Lines, Vectors, and Transfection

L cells were obtained from ATCC. An expression vector pECE/ECad was constructed by subcloning the E-cadherin cDNA (55), generously provided by Rolf Kemler (Max Planck Institute, Freiburg, Germany), into an SV40 expression vector, pECE. A Wnt-1 expression vector, pRSVWnt-1, and pSV2Neo have been described (56, 57). A previously described v-Src expression vector, pMvSrc (58), was kindly provided by David Shalloway (Cornell University, Itnaca, NY). A vector encoding a hygromycin resistance marker, pREP4 was purchased from Invitrogen Corp.

The L cells were transfected with either pSV2Neo alone (control) or were co-transfected with pECE/ECad plus pSV2Neo using LipofectAMINE reagent (Life Technologies, Inc.) as directed by the manufacturer. After 48 h the cells were either harvested for transient expression analysis as indicated or were subjected to drug selection using Geneticin (G418) (Life Technologies, Inc.). Stable clones of transfected cells were selected, grown, and analyzed for E-cadherin protein expression by Western immunoblot. Multiple transfected clones were analyzed with similar results (for example see Fig. 1). Most of he experiments shown in this report were performed with one clonal line, ECad2-2. Two of the E-cadherin-transfected L cell lines, including clone ECad2-2, were co-transfected with pMvSrc plus pREP4, and stable cell lines were selected using hygromycin B (Sigma). v-Src expression was verified by Western immunoblot analysis. Multiple v-Src transfected clones were analyzed with similar results. The experiments shown in this report were performed with either of two cell lines, ECad/Src 6 or ECad/Src 20. For Fig. 4A, cells were observed with an Axiovert 100 microscope using a 10× projective lens, and photographs were taken using Polaroid 57 film.


Fig. 1. Modulation of steady-state catenin levels by E-cadherin expression. L cells (L) were transfected with either a control vector conferring neomycin resistance (N) or with an E-cadherin expression vector (E), and stable cell lines were selected. Protein equivalent aliquots from multiple clones of each transfected cell type were subjected to Western immunoblot analysis with antisera specific for E-cadherin, beta -catenin, alpha -catenin, or p120cas as indicated. An extract from one of the E-cadherin transfected lines was immunoprecipitated with an antibody directed against E-cadherin (IP: ECAD), and the immunoprecipitate was divided into four equal parts and analyzed in parallel with the total protein extracts. The exposure time for the p120cas immunoblot of the E-cadherin immunoprecipitate was 15 times longer than for the other immunoblots.
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Fig. 4. Expression of v-Src in ECad2-2 L cells. ECad2-2 cells were transfected with an expression vector for v-Src and stable cell lines were selected. A, photographs showing morphologies of three v-Src-transformed ECad2-2 cell lines (ECadherin/Src) compared to the control L cells (Neo) or the ECad2-2 cells (ECadherin). B, Western immunoblot analysis, using Src- or phosphotyrosine (PY)-specific antisera, of protein equivalent aliquots from either ECad2-2 cells (-) or ECad/Src cells (+). An immunoprecipitate with a Src-specific antibody from extract of the ECad/Src cells was included on the Src immunoblot.
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Antisera

Polyclonal antisera made in rabbits against beta -catenin- and alpha -catenin-specific peptides were produced and characterized as described (24). The rabbit polyclonal antiserum against cadherin was generated as described (59) and was generously provided by James Nelson (Stanford University, Stanford, CA). A mouse monoclonal antibody against p120cas was purchased from Transduction Laboratories. Monoclonal antibodies 327 and 2-17 against Src protein were kindly provided by Joan Brugge (Ariad Pharmaceuticals). A monoclonal antibody directed against phosphotyrosine, PY-20, was purchased from ICN Biochemicals.

Cell Extraction and Immunoprecipitation

For immunoprecipitations and affinity precipitations, cells were extracted with a buffer consisting of 1% Nonidet P-40, 10 mM sodium phosphate, pH 7.0, 0.15 M sodium chloride, and a mixture of protease and phosphatase inhibitors. Monolayers of tissue culture cells were extracted for 30 min on ice and scraped from the culture dish, and the mixture was centrifuged at 12,000 × g for 15 min at 4 °C. The protein concentration of clarified extracts was determined using a BCA reagent kit (Pierce). Cell extracts used for total protein analysis only were made with immune precipitation buffer or Laemmli sample buffer as needed.

Antibodies for immunoprecipitation were added, as indicated for each figure, to clarified extracts adjusted for protein concentration. Extracts were incubated with antisera at 4 °C for approximately 2 h, and immune complexes were collected by binding to protein A-Sepharose beads (Pharmacia Biotech Inc.), followed by three washes with extraction buffer. The immunoprecipitates were subjected to SDS-PAGE and immunoblot analysis as indicated.

For the subcellular fractionation experiment (Fig. 8), one dish each of ECad2-2 and ECad/Src cells were fractionated into Triton X-100-soluble and -insoluble fractions as described previously (24). Protein equivalent aliquots of each fraction were immunoprecipitated with an antiserum against p120cas, and the immunoprecipitates as well as aliquots of total protein were analyzed by SDS-PAGE and Western immunoblotting as indicated.


Fig. 8. v-Src transformation leads to changes in subcellular distribution of p120cas detected by biochemical fractionation. Dishes of either ECad2-2 cells or ECad/Src cells were fractionated into Triton X-100-soluble (S) and -insoluble (I) fractions. Equivalent aliquots of each fraction were immunoprecipitated with an antibody against p120cas and immunoprecipitates (IP) as well as protein equivalent aliquots of the cell fractions (Total) were subjected to Western immunoblot analysis with antisera specific for either p120cas or phosphotyrosine (PY) as indicated.
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Affinity Precipitation

For affinity precipitation, clarified cell extracts were incubated with Sepharose beads (Pharmacia) to which either control GST protein or a GST-E-cadherin fusion protein was bound. After incubation at 4 °C for 1 h the Sepharose beads were collected by centrifugation in a microcentrifuge, washed three times with immunoprecipitation buffer, and solubilized in Laemmli sample buffer for SDS-PAGE and Western immunoblot analysis. The GST fusion protein for E-cadherin consists of bacterial glutathione S-transferase fused in frame to the entire cytoplasmic domain of E-cadherin (35). The GST-E-cadherin fusion protein and the control GST protein were purified from bacterial lysates.

Pulse-Chase Analysis

For the pulse-chase analysis shown in Fig. 2, replicate cultures of the indicated cell lines were incubated in the absence of methionine for 20 min, pulse-labeled for 30 min with 250 µCi/ml [35S]methionine, washed twice, and incubated in medium containing excess unlabeled methionine for 0, 0.5, 1.0, 2.0, and 4.0 h. At each indicated time point, the cells were extracted with immunoprecipitation buffer and equivalent aliquots of each extract were immunoprecipitated with an antibody against beta -catenin.


Fig. 2. beta -catenin half-life is increased in the presence of E-cadherin. Replicate dishes of either parental L cells (L) or ECad2-2 cells (L/ECadherin) were pulse-labeled for 30 min with [35S]methionine followed by a chase in the absence of label for 0, 0.5, 1, 2, or 4 h. Equivalent aliquots of each cell extract were immunoprecipitated with an antiserum directed against beta -catenin. Immunoprecipitates were analyzed by SDS-PAGE and fluorography.
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Gel Electrophoresis and Western Immunoblot Analysis

For all experiments the washed immune complex pellets were boiled for 5 min in Laemmli sample buffer, divided as appropriate to generate duplicate gels for Western immunoblot analysis, and separated in a 10% SDS-polyacrylamide gel. Gels consisting of 35S-labeled samples were analyzed by fluorography using Amplify as directed by the manufacturer (Amersham Corp.), and gels with non-radioactive samples were electrophoretically transferred to Immobilon-P filter paper (Millipore Corp.) for Western immunoblot analysis.

For Western immunoblots, filters were first incubated in a blocking solution consisting of Tris-buffered saline solution containing 0.1% Tween 20 and either 5% bovine serum albumin fraction V (for phosphotyrosine immunoblots) or 5% nonfat dry milk (for all other immunoblots). Filters were next incubated with antibodies, added in blocking solution at a 1:1000 dilution, for 2 h at room temperature. The filters were washed for 1 h with multiple changes of Tris-buffered saline plus 0.1% Tween-20 and subsequently incubated for 30 min with blocking solution containing either horseradish peroxidase-conjugated protein A (Amersham) for polyclonal antisera or horseradish peroxidase-conjugated anti-mouse imunoglobulin (Amersham) for monoclonal antibodies. The filters were washed as above and developed using an enhanced chemiluminescence (ECL) detection system (Amersham).


RESULTS

Modulation of Steady-state Catenin Levels by E-cadherin Expression

L cells have previously been used for studies of cadherin function since they normally lack detectable expression of classic cadherins and fail to participate in calcium-dependent cell to cell adhesive contacts (60). However, expression of exogenous cadherins in these cells leads to calcium-dependent cell to cell adhesion (60). For this study, L cells were transfected with either an expression vector for E-cadherin or with a control vector conferring only neomycin resistance. Individual transfected cell clones were selected and analyzed for E-cadherin expression by Western immunoblot with a cadherin-specific antibody. As in previous studies, no expression of cadherin was detected in the parental L cell line, whereas the individual E-cadherin transfectants expressed substantial levels of E-cadherin protein (60) (Fig. 1). Steady-state expression levels of catenins in the transfected cells were next examined by Western immunoblot using antibodies specific for each of the catenins. Very low levels of both beta -catenin and alpha -catenin were observed in the parental L cells and the control transfectants. In contrast, all of the cell lines expressing E-cadherin showed markedly increased steady-state levels of both beta -catenin and alpha -catenin (Fig. 1). Both the alpha -catenin and beta -catenin proteins were detected in a complex with E-cadherin (Fig. 1). Unlike alpha - and beta -catenin, little or no plakoglobin (gamma -catenin) protein expression was detected in either the parental L cells or in the E-cadherin-expressing cell lines (data not shown). In addition to the prototype catenins (alpha , beta , and gamma ), the steady-state levels of p120cas, a Src substrate that has recently been included in the armadillo family along with beta - and gamma -catenin (13, 16), were also examined. Fig. 1 shows that p120cas protein is readily detected in the parental L cells and the control transfectants, but in contrast to alpha - and beta -catenin, the steady-state level of p120cas remains essentially unchanged upon introduction of E-cadherin. Recent studies have shown that p120cas can associate with cadherin (18-21). Similarly, p120cas was detected in complex with E-cadherin in L cells (Fig. 1); however, in contrast to results with alpha - and beta -catenin, very little p120cas appears to associate with E-cadherin (see below). These results indicate that the steady-state levels of alpha - and beta -catenin, which are normally very low in L cells, can be significantly increased upon introduction of E-cadherin expression. One clonal line of E-cadherin-transfected L cells, ECad2-2, was used for subsequent analysis.

beta -Catenin Half-life Is Increased in the Presence of E-cadherin

In order to understand the mechanism leading to increased steady-state beta -catenin levels in response to E-cadherin expression, the half-life of beta -catenin was examined in a pulse-chase experiment. Replicate dishes of parental L cells and ECad2-2 cells were briefly pulse-labeled with [35S]methionine followed by a chase in the absence of label. At each indicated time point, cells were harvested and extracts were immunoprecipitated with a beta -catenin-specific antibody. Fig. 2 shows that the half-life of beta -catenin in the parental L cells is very short, with most of the newly synthesized protein gone within 30 min. In contrast, the half-life of beta -catenin is dramatically increased in the presence of E-cadherin, with most of the protein still remaining after 4 h of chase.

Wnt-1, but Not E-cadherin Expression, Increases Uncomplexed Pools of beta -Catenin; Neither Affects p120cas Levels

We previously showed that Wnt-1 expression significantly prolongs the half-life of beta -catenin in AtT20 and C57MG cells (24), primarily by increasing the stability of uncomplexed, monomeric pools of this protein (35). In order to compare the effects of Wnt-1 and E-cadherin on steady-state levels as well as free pools of both beta -catenin and p120cas, parental L cells were transfected either with an expression vector for Wnt-1 or with an expression vector for E-cadherin. Two days following transfection, the steady-state levels of both beta -catenin and p120cas were examined by Western immunoblot analysis of equivalent amounts of cell extract. Fig. 3 shows that both Wnt-1 and E-cadherin are independently capable of inducing an increase in the steady-state level of beta -catenin in L cells, illustrated by Western blot analysis of total cell extracts. The steady-state levels of p120cas were not affected by either Wnt-1 or E-cadherin expression. To examine whether the increased levels of beta -catenin localized to an uncomplexed population, the free pool of beta -catenin was selectively isolated from equivalent amounts of detergent cell extract by affinity precipitation using a purified GST-fusion protein containing the cytoplasmic domain of E-cadherin. We have previously shown that this method allows the identification of free pools of catenins which are available for association with the exogenous binding site, while the endogenous catenin molecules that are already in a complex with APC or cadherin are not selected by this affinity precipitation protocol (35). As shown in Fig. 3, the GST-cadherin fusion protein identifies a free pool of beta -catenin in the Wnt-1-transfected L cells, whereas no free pool is detected in the parental L cells or the E-cadherin transfectants. In contrast, an equivalent amount of uncomplexed p120cas can be isolated with the GST-cadherin fusion protein from extracts of both the Wnt-1 and E-cadherin transfectants as well as the parental L cells.


Fig. 3. Unlike Wnt-1, E-cadherin expression does not increase uncomplexed pools of beta -catenin. Replicate dishes of parental L cells were transfected with either no DNA (0), an expression vector for Wnt-1 (W), or an expression vector for E-cadherin (E). 48 h following transfection the cells were solubilized, and protein equivalent aliquots of cell extract from each were subjected to Western immunoblot analysis with an antibody against beta -catenin or an antibody against p120cas as indicated (panels marked Total). In addition, protein equivalent aliquots of each were subjected to affinity precipitation with a GST-fusion protein consisting of the cytoplasmic domain of E-cadherin, and these affinity precipitates were subjected to Western immunoblot analysis with an antibody against beta -catenin or an antibody against p120cas as indicated (panels marked ECad affinity).
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Expression of v-Src in ECad2-2 Cells

Previous studies have shown that v-Src transformation leads to tyrosine phosphorylation of cadherin and catenins, and it has been suggested that this contributes to the concomitant loss of cell to cell adhesion observed in these v-Src-transformed cells (46-48). In order to examine in biochemical detail the consequences of tyrosine phosphorylation on cadherin-catenin complex formation and free pools of catenins, the ECad2-2 cells were transfected with an expression vector for v-Src and stable cell lines were selected. The v-Src-expressing cells showed morphological alterations characteristic of transformation by this oncogene (Fig. 4A). The biochemical analysis in Fig. 4B shows that v-Src is readily detected in the transfected cells, and these cells have elevated tyrosine phosphorylation of cell proteins as expected. Either of two clones of v-Src-expressing ECad2-2 cells, ECad/Src 6 or ECad/Src 20, were used for further analysis.

Phosphorylation of E-cadherin and beta -Catenin by v-Src: No Effect on Cadherin-Catenin Levels or Complex Formation

In agreement with previous studies (46-48), immunoprecipitation of either beta -catenin or E-cadherin followed by immunoblot analysis with a phosphotyrosine-specific antibody shows that both of these proteins become phosphorylated on tyrosine in the ECad/Src cells but not in the ECad2-2 cells (Fig. 5A). Tyrosine phosphorylation of beta -catenin was observed both directly by immunoprecipitation with a beta -catenin-specific antibody and indirectly by immunoprecipitating beta -catenin in complex with E-cadherin using a cadherin-specific antibody. No phosphorylation of alpha -catenin was observed in either the ECad2-2 or ECad/Src cells (data not shown). Fig. 5B illustrates that tyrosine phosphorylation of E-cadherin and beta -catenin in the ECad/Src cells does not change the level or stoichiometry of the E-cadherin complex with beta -catenin or alpha -catenin. To demonstrate this point, protein equivalent aliquots of both ECad2-2 and ECad/Src cells were compared by immunoprecipitation of E-cadherin, followed by Western immunoblot analysis for E-cadherin, beta -catenin, or alpha -catenin. These data show that the overall level as well as the ratio of E-cadherin to beta -catenin are unchanged in the presence of v-Src (Fig. 5B). Although alpha -catenin did not become phosphorylated on tyrosine in the v-Src-transformed cells, I determined whether tyrosine phosphorylation of beta -catenin or E-cadherin in these cells led indirectly to alterations in the ability of alpha -catenin to associate with beta -catenin and thus integrate into the cadherin-catenin complex. The Western immunoblot shown in Fig. 5B demonstrates that the amount of alpha -catenin in association with E-cadherin, presumably through binding to beta -catenin is unchanged in response to v-Src transformation. Analysis of steady-state levels of E-cadherin, beta -catenin, and alpha -catenin showed no change in the presence of v-Src (data not shown). Pulse-chase analysis further showed that tyrosine phosphorylation does not affect the half-life of beta -catenin (data not shown).


Fig. 5. Phosphorylation of E-cadherin and beta -catenin by v-Src: no effect on cadherin-catenin levels or complex formation. A, Protein equivalent extracts of either ECad2-2 cells (ECadherin) or ECad/Src cells (ECadherin/Src) were immunoprecipitated with either preimmune rabbit serum (Pre), antiserum against beta -catenin (beta Cat), or antiserum against E-cadherin (ECad). Immunoprecipitates were divided in half and subjected to Western immunoblot analysis with either an antiserum against phosphotyrosine (PY) or antisera against E-cadherin plus beta -catenin (ECad & beta Cat). B, protein equivalent aliquots of either ECad2-2 cells (ECad) or ECad/Src cells (ECad/Src) were immunoprecipitated with an antiserum specific for E-cadherin. The immunoprecipitates (IP: ECAD) were subjected to Western immunoblot analysis with antibodies against E-cadherin, beta -catenin, or alpha -catenin as indicated.
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Phosphorylation of p120cas by v-Src: No Effect on Steady-state Levels

p120cas, an armadillo family member, was originally identified as an abundant substrate for tyrosine phosphorylation in v-Src-transformed cells (13). Here I have examined whether this phosphorylation alters the steady-state level of p120cas or its ability to complex with cadherin. Immunoprecipitation of p120cas followed by Western immunoblot with a phosphotyrosine-specific antibody illustrates that p120cas is heavily phosphorylated on tyrosine in the ECad/Src cells but not in the ECad2-2 cells (Fig. 6). Phosphorylation of p120cas on tyrosine has no effect on its steady-state level, illustrated both by examination of p120cas levels in total cell extracts and by immunoprecipitation of p120cas followed by Western immunoblot analysis for p120cas protein (Fig. 6). The amount of p120cas identified in association with E-cadherin is very small by comparison to the overall pool, but there is no apparent change in this population (Fig. 6).


Fig. 6. Phosphorylation of p120cas by v-Src: no effect on steady-state levels or cadherin-p120cas complexes. Protein equivalent aliquots of either ECad2-2 or ECad/Src cells were immunoprecipitated with either an antiserum against p120cas (IP: p120) or an antiserum against E-cadherin (IP: ECad). Immunoprecipitates (IP) as well as protein equivalent aliquots of total cell extract (Total) were subjected to Western immunoblot analysis with either an antiserum against p120cas or against phosphotyrosine (PY) as indicated. Exposure time for the far right blot (IP: ECad, Blot: p120) was 15 times longer than for the other portions of this experiment.
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Tyrosine Phosphorylation Has No Effect on the Uncomplexed Pool of beta -Catenin; the Uncomplexed Pool of p120cas Is Diminished

To examine potential effects of tyrosine phosphorylation on free pools of beta -catenin and p120cas, an affinity precipitation experiment was performed using the GST-cadherin protein followed by Western immunoblotting for either beta -catenin or p120cas. Very little uncomplexed beta -catenin can be detected in ECad2-2 cells, requiring extended exposure times of the Western immunoblot, and this is unchanged by tyrosine phosphorylation upon v-Src transformation (Fig. 7). On the other hand, a substantial free population of p120cas can be identified in the ECad2-2 cells, and this is decreased significantly in the ECad/Src cells (Fig. 7). Also of note is the finding that tyrosine-phosphorylated p120cas can be efficiently isolated from the ECad/Src cell extract by affinity precipitation with the GST-cadherin protein (Fig. 7).


Fig. 7. v-Src transformation does not modulate the free pool of beta -catenin; uncomplexed pools of p120cas are decreased. Protein equivalent aliquots of either ECad2-2 or ECad/Src cells were subjected to affinity precipitation with either a control GST protein (C) or with a GST-cadherin fusion protein (E). These precipitated products were analyzed by Western immunoblot with antisera specific for beta -catenin, p120cas, or phosphotyrosine (PY) as indicated. Detection of uncomplexed beta -catenin in the ECad2-2 and ECad/Src cells required maximal exposure times of the blot.
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Altered Subcellular Fractionation of p120cas upon v-Src Transformation

Since the uncomplexed pool of p120cas is diminished upon v-Src transformation, it was of interest to determine if the subcellular distribution of p120cas was also altered. For this purpose ECad2-2 and ECad/Src cells were fractionated into Triton X-100-soluble and -insoluble fractions, and protein equivalent aliquots of each fraction were immunoprecipitated with an antiserum against p120cas. Western immunoblot analysis of the immunoprecipitates as well as of total protein aliquots from each fraction shows that a small amount of p120cas is detected in a Triton X-100-insoluble fraction from ECad2-2 cells; the majority is in a Triton X-100-soluble fraction (Fig. 8). V-Src transformation leads to a decrease in the Triton X-100-insoluble population of p120cas with a corresponding increase in the Triton X-100-soluble fraction (Fig. 8). Most of the tyrosine-phosphorylated p120cas is localized to a Triton X-100-soluble fraction from ECad/Src cells (Fig. 8).


DISCUSSION

Cadherin-mediated cell to cell adhesion as well as adhesion-independent functions for catenins have been implicated in the modulation of many aspects of multicellular differentiation, cell proliferation, and malignant transformation of epithelial cells (1-5). In this study I have defined, at a biochemical level, distinct mechanisms leading to changes in steady-state catenin levels. Furthermore, I have characterized the impact of tyrosine phosphorylation on regulation of both free pools of catenins as well as cadherin-catenin complex stability.

An increase in availability of beta -catenin binding sites, by increasing the expression level of E-cadherin, leads to an increased stability of alpha - and beta -catenin in complex with cadherin, but notably no increase in the free pool of alpha - or beta -catenin is observed. beta -Catenin associated with cadherin exhibits a greatly extended half-life and appears to be selectively protected from a rapid degradation mechanism that operates in normal cells to restrict the availability of an uncomplexed beta -catenin population (35). Similar results were obtained in another study, where an increase in the steady-state level of plakoglobin was observed upon co-transfection, into L cells, of expression vectors for plakoglobin and the desmosomal cadherins, desmoglein or desmocollin, when compared to expression of the plakoglobin vector alone (61). The increased steady-state level of plakoglobin resulted from a 15-20-fold decrease in plakoglobin protein degradation and correlated with complex formation between plakoglobin and desmoglein or desmocollin. In our studies no increase in endogenous plakoglobin levels was detected in response to the expression of E-cadherin in L cells, despite the fact that plakoglobin can effectively bind to E-cadherin in other cell types (24, 25). This is perhaps due to an inherent inability of our L cell line to express plakoglobin. Taken together these findings suggest that catenin protein stability can be increased upon co-ordinate interaction with other proteins that participate in junctional complex formation.

In contrast to the stabilization of alpha - and beta -catenin by E-cadherin expression, p120cas is readily detectable in the parental L cells lacking cadherin and is not stabilized further upon introduction of E-cadherin. This is consistent with the fact that very little p120cas binds to E-cadherin (20, 21) and thus is not likely to be significantly stabilized by complex formation with an increased availability of cadherin binding sites. Furthermore, in normal cells cadherin may exhibit a greater affinity for beta -catenin than p120cas. Examination of the uncomplexed population of p120cas in L cells revealed that this pool was not affected by E-cadherin expression.

A second distinct mechanism leading to increased steady-state beta -catenin levels is through an increase in uncomplexed, monomeric pools as a consequence of Wnt-1 signal transduction. As a result of increasing the free pools, the steady-state levels of both beta -catenin and plakoglobin are increased by Wnt-1 signaling in several cell types (24, 31, 35), whereas there is no effect of Wnt-1 on alpha -catenin levels (24). I have extended this analysis to show that the regulation of p120cas is not affected by Wnt-1 expression. While p120cas is considered an armadillo family member, based on sequence analysis and cadherin binding, the data presented here indicate that the regulation of p120cas is is independent of the regulation of beta -catenin and plakoglobin since the free pools are not affected by Wnt-1 expression. Here I also show that the mechanism utilized by Wnt-1 to increase the free pool of beta -catenin does not require the presence of E-cadherin since this can occur in parental L cells in the absence of E-cadherin expression. This suggests that the previously observed stabilization of the beta -catenin-cadherin complex by Wnt-1 must be a secondary consequence of the increased free pool of beta -catenin. The mechanism by which Wnt-1 increases free catenin pools is not completely defined, but it appears to modulate a signal transduction pathway that includes APC, in complex with beta -catenin and plakoglobin, and the serine-threonine kinase, GSK3, both of which function in concert to restrict beta -catenin levels in normal cells (32, 35). Since p120cas does not associate with APC (19), this may explain the lack of effect of Wnt-1 on p120cas levels. This would also predict that changes in GSK3 activity would not affect p120cas levels.

Several studies have shown that transformation by the v-src oncogene leads to phosphorylation on tyrosine of E-cadherin and beta -catenin, and it has been proposed that these phosphorylations could lead to a destabilization of the cadherin-catenin complex, resulting in loss of cell to cell adhesion characteristic of the transformed cell (46-48). Here I have examined directly the stability of the cadherin-catenin complex, the half-life of beta -catenin, and the availability of uncomplexed catenin pools in response to v-Src-induced transformation. Transformation of E-cadherin-expressing L cells with v-Src results in tyrosine phosphorylation of E-cadherin, beta -catenin, and p120cas but not alpha -catenin. However, tyrosine phosphorylation appears to have no effect on the steady-state levels of these proteins. Tyrosine phosphorylation also has no effect on the stability or stoichiometry of complexes of E-cadherin with beta -catenin, alpha -catenin, and p120cas. In agreement with our findings, no effect of v-Src was seen on cadherin complexes with either beta -catenin or p120cas in Madin-Darby canine kidney cells (62).

These observations imply that the morphological transformation and apparent loss of cell to cell contact induced by v-Src are not a consequence of disruption of the cadherin-catenin complex itself upon tyrosine phosphorylation. Although alpha -catenin is not phosphorylated on tyrosine nor is it dissociated from the cadherin complex, it is possible that the tyrosine phosphorylation of beta -catenin and cadherin compromises the ability of the associated alpha -catenin to anchor the complex to the cell cytoskeleton and stabilize cell to cell adhesion. Alternatively, since many of the known substrates for v-Src are cytoskeletal-associated proteins (63), tyrosine phosphorylation of one of these other proteins may destabilize the interaction of the cadherin-catenin complex with the cytoskeleton. v-Src transformation also leads to phosphorylation of components of the cell-matrix adhesion system such as integrins and the focal adhesion kinase (63). By phosphorylation of multiple substrates within the cell-cell and cell-matrix adhesion systems, the striking morphological alterations characteristic of v-Src-induced transformation may result from a disruption of the balance between these distinct adhesion systems. Recent evidence in fact shows that changes in cadherin expression are reflected in altered expression of genes encoding fibronectin and integrins and altered cell-matrix adhesion properties (64, 65).

Alternatively, tyrosine phosphorylation of the E-cadherin-beta -catenin complex may be important for other functions of these proteins aside from adhesion. For example, tyrosine phosphorylation of the cadherin-catenin complex may serve to recruit various signal transduction effectors, similar to the consequences of receptor tyrosine kinase phosphorylation upon ligand-induced activation (66). These effectors could include proteins such as receptor tyrosine kinases or the tyrosine phosphatase mu, proteins that have been identified in association with the cadherin-catenin complex (49, 67, 68).

Recent evidence suggests that the uncomplexed populations of catenins can serve a signal transduction function in response to Wnt-1 and Wingless proteins (32-34). In this study I have also examined uncomplexed catenin populations in the v-Src-transformed L cells and found that in contrast to the effects of Wnt-1, there is no change in the free pool of beta -catenin in response to v-Src phosphorylation and transformation. Conversely, Wnt-1 does not appear to regulate free pools of p120cas, whereas the free pool of p120cas is significantly decreased upon v-Src transformation and tyrosine phosphorylation. The tyrosine phosphorylation and corresponding decrease in uncomplexed p120cas is also accompanied by an increase in the Triton X-100-soluble pool of p120cas at the expense of the Triton X-100-insoluble population. This implies that p120cas, upon phosphorylation on tyrosine, may show increased association with other cytoplasmic effector molecules, perhaps including the tyrosine kinase FER (69). Furthermore, the altered detergent solubility of p120cas may suggest an altered subcellular distribution. These findings support the conclusion that, at the biochemical level, p120cas is regulated independently from beta -catenin and plakoglobin. Thus, while both Wnt-1 and v-Src induce transformation-dependent changes in cell morphologies and modulate uncomplexed catenin pools, the mechanisms are different.

Cadherins and catenins participate in the modulation of the epithelial phenotype, changes that have profound consequences for tissue differentiation and malignant transformation. Here I have characterized, at the biochemical level, several mechanisms that regulate the free pools of catenins and the complexes that form with cadherin. Our data support a model whereby uncomplexed catenin populations are tightly regulated and serve a cadherin independent signal transduction function in response to both Wnt-1 and v-Src. These studies contribute to a biochemical understanding of the phenotypes produced by oncogenes, growth factors, and their receptors.


FOOTNOTES

*   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.
Dagger    To whom correspondence should be addressed. Present address: Megabios Corp., 863 A Mitten Rd., Burlingame, CA 94010. Tel.: 415-697-1900 (ext. 260); Fax: 415-652-1999; E-mail: jackie{at}megabios.com.
1    The abbreviations used are: APC, adenomatous polyposis coli; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

Acknowledgments

I thank James Nelson for the antiserum against E-cadherin, Rolf Kemler for the E-cadherin cDNA clone, and David Shalloway for the v-Src expression vector. I thank John Forsman and James Rice for help with the preparation of the GST fusion proteins and Rhea Pugliese for assistance with the v-Src transfections. I am grateful to Beverly Smolich for critical comments on the manuscript.


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