N-cadherin-Catenin Complexes Form Prior to Cleavage of the Proregion and Transport to the Plasma Membrane*

James K. Wahl IIIDagger, Young J. Kim, Janet M. Cullen, Keith R. Johnson, and Margaret J. Wheelock

From the University of Nebraska Medical Center, College of Dentistry and Eppley Cancer Center, Omaha, Nebraska 68198

Received for publication, November 10, 2002, and in revised form, February 24, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that beta -catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120ctn can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cadherins comprise a family of calcium-dependent cell-cell adhesion proteins that play important roles in the embryonic development and maintenance of normal tissue architecture. As the transmembrane component of cellular junctions, the cadherins are composed of three segments, i.e. an extracellular domain comprised of five homologous repeats that mediates adhesion, a single pass transmembrane domain, and a conserved cytoplasmic domain that interacts with catenins to link cadherins to the actin cytoskeleton (1-5). The catenins were first identified as proteins that co-immunoprecipitated with cadherins and were termed alpha -catenin, beta -catenin, and gamma -catenin (plakoglobin) according to their mobility on SDS-PAGE. Either beta -catenin or plakoglobin binds directly to the cadherin and to alpha -catenin, whereas alpha -catenin associates directly and indirectly with actin filaments (6-9). The ability of cadherins to simultaneously self-associate and link to the actin cytoskeleton allows strong cell-cell adhesion.

In addition to catenins, p120ctn, which was originally identified as a Src substrate, binds to the cytoplasmic domain of cadherins and has been suggested to play a role in regulating the adhesive activity of cadherins (10-12). p120ctn binds to the juxtamembrane domain of cadherins, a domain that has been implicated in cadherin clustering and cell motility (13-16). It is thought that p120ctn influences the strength of cadherin-mediated adhesion, perhaps by influencing the organization of the actin cytoskeleton (17-19). The goal of the present study was to further understand the sequence of events that leads to the formation of a functional cadherin-catenin-p120ctn complex.

Cadherins are synthesized as precursor proteins that must be proteolytically cleaved to generate functional, mature proteins (20, 21). All of the classical cadherins have similar proteolytic cleavage sites within the proregion, suggesting that each is processed by proteases with similar specificities. Ozawa and Kemler (21) showed that mutant forms of E-cadherin missing the proteolytic cleavage sites were transported to the cell surface when transfected into cadherin-negative cells but were not active in cell-cell adhesion. These precursor forms could be converted to active molecules by exogenous cleavage of the proregion at the cell surface. An emerging idea in the cadherin field is that cadherin family members promote cell type-specific phenotypes. For example, we have presented evidence suggesting that N-cadherin expression can promote motility in epithelial cells, whereas E-cadherin suppresses motility in the same cells (22-24). Thus, it is important to examine the activity and processing of cadherins in cells that endogenously express these proteins. To facilitate these studies, we developed monoclonal antibodies against the proregion of N-cadherin that would allow us to define the sequence of events that occur during the synthesis, processing, and transport to the cell surface of an endogenous N-cadherin-catenin-p120ctn complex.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- HeLa, VA13, and HT1080 cells were obtained from ATCC (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT). A431D cells have been described (25).

Detergent Extraction of Cells-- Confluent monolayers were rinsed three times with phosphate-buffered saline and extracted in TNE extraction buffer (10 mM Tris acetate, pH 8.0, 0.5% Nonidet P-40, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate). The cells were placed on ice, scraped, and triturated vigorously for 10 min. Insoluble material was pelleted by centrifugation at 14,000 × g for 15 min at 4 °C, and the supernatant was used immediately for immunoprecipitations or stored at -70 °C.

Detergent-free Extraction of Cells-- Confluent monolayers of cells were rinsed three times with phosphate-buffered saline and scraped from the flask in TE buffer (10 mM Tris acetate, pH 8.0, 1 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride). The cells were Dounce homogenized on ice by using five strokes of the tight pestle. The membrane fraction was removed by centrifugation at 14,000 × g for 15 min at 4 °C, and the resulting supernatant was used immediately for immunoprecipitations or stored at -70 °C.

Antibodies-- We produced antibodies specific for human proN-cadherin by immunizing mice with a maltose-binding protein (MBP)1 fusion. The upstream primer 5'-GCG AAT TCG CCA TGG TCT CTG TAG AGG CTT CTG G-3' and the downstream primer 5'-GCC TCG AGT TAA CCT CTC TTC TGC CTT TGT AG-3' were used to PCR amplify nucleotides 269-682 of human N-cadherin (GenBankTM accession NM_001792) encoding amino acids 22-159. The PCR product was digested using EcoRI and XhoI and ligated to pMAL-C2 (New England Biolabs, Beverly, MA) prepared by digestion with EcoRI and SalI. The PCR product was sequenced and found to encode the correct amino acid sequence. Hybridomas secreting antibodies specific for the proregion of N-cadherin were generated as described previously (26, 27). Epitope mapping of the anti-proN-cadherin antibodies was performed by testing their ability to bind to deleted fusion proteins. The PCR product was truncated at either the endogenous HindIII site or the endogenous PstI site to create two MPB fusion proteins. The former included amino acids 22-92 and the latter amino acids 22-77 of N-cadherin. Recombinant fusion proteins were resolved by SDS-PAGE, and anti-proN-cadherin antibodies were tested by immunoblot analysis.

Anti-beta -catenin (5H10), anti alpha -catenin (1G5), anti-plakoglobin (11E4), anti-N-cadherin cytoplasmic domain (13A9), anti-c-Myc tag (9E10), and anti-N-cadherin extracellular domain (8C11) have been described (23, 26-28). Rabbit anti-beta -catenin was purchased from Sigma. Anti-p120ctn was purchased from BD Transduction Laboratories (Lexington, KY). Rabbit anti calnexin was purchased from Stressgen (Victoria, British Columbia, Canada) and rabbit anti mannosidase II was purchased from Chemicon (Temecula, CA).

Immunoprecipitation and Immunoblot-- All polypropylene tubes were rinsed with 0.1% Nonidet P-40 and dried prior to use in immunoprecipitations. The indicated volume of cell extract was added to 300 µl of hybridoma-conditioned medium and gently mixed at 4 °C for 30 min. 50 µl of packed anti-mouse IgG affinity gel (ICN Pharmaceuticals, Costa Mesa, CA) was added, and mixing continued for 30 min. Immune complexes were washed five times with TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, and 2 mM sodium orthovanadate). After the final wash, the packed beads were resuspended in 50 µl of 2× Laemmli sample buffer (29), boiled for 5 min, and the proteins were resolved by SDS-PAGE. Proteins were electrophoretically transferred overnight to nitrocellulose membranes and blocked in 5% nonfat dry milk in TBST for 45 min. Blocking solution was removed by washing for 15 min followed by 2× 5 min in TBST. Hybridoma conditioned media was used at 1:100 dilution in TBST for 1 h. Membranes were washed 15 min followed by 2× 5 min in TBST. Membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories) at 1:10,000 for 1 h. Secondary antibody was removed by washing 15 min followed by 4× 5 min in TBST. Immunoreactive bands were detected using Super Signal Pico substrate (Pierce).

Immunofluorescence-- Cells were grown on glass coverslips to 80% confluence and fixed in 1% paraformaldehyde for 30 min and permeabilized in methanol at -20 °C for 5 min. For experiments comparing permeabilized cells to non-permeabilized cells, the coverslips were fixed in 1% paraformaldehyde for 10 min with or without methanol treatment. After three 5 min washes in serum-free culture medium, the coverslips were blocked in 10% goat serum in culture medium for 30 min. Coverslips were incubated with primary antibodies for 20 min, washed 3× 5 min with culture medium, and incubated in fluorescein isothiocyanate or rhodamine-conjugated secondary antibody for 20 min. Coverslips were washed 3× 5 min in culture medium and briefly rinsed in distilled water prior to mounting in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Images were collected on a Zeiss Axiovert 200 M equipped with an ORCA-ER (Hamamatsu) digital camera. Images were collected and processed using OpenLab software from Improvision Inc. (Boston, MA)

Metabolic Labeling-- For pulse-chase experiments, HeLa cells grown in a 25-cm2 flask were cultured in DMEM lacking methionine and cysteine and supplemented with 1% dialyzed fetal bovine serum (Hyclone) for 4 h. Cells were pulsed with media containing 250 µCi of Tran35S-label (ICN Pharmaceuticals) for 15 min and chased for the indicated times with DMEM supplemented with 10% fetal bovine serum.

For 33P labeling, HeLa cells were cultured overnight in DMEM lacking phosphate and supplemented with 10% dialyzed fetal bovine serum. Fresh media containing 1 mCi of H333PO4 was then added for 2 h. Following labeling, Nonidet P-40 cell extracts were prepared and immunoprecipitated with the appropriate antibody. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. Nitrocellulose sheets were exposed to Biomax MR1 film (Kodak) at -70 °C using an intensifying screen (Transcreen LE, Kodak)

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Monoclonal Antibodies Specific for the Proregion of Human N-cadherin-- To facilitate the examination of the processing that is required to generate mature N-cadherin from proN-cadherin, we developed monoclonal antibodies specific for the proregion of human N-cadherin. The proregion (Fig. 1A) of human N-cadherin was fused to MBP, expressed in bacteria, affinity purified, and hybridoma cell-lines secreting antibodies specific for the proregion of N-cadherin were produced (Fig. 1B). Epitope mapping using truncated fusion protein showed that the epitope recognized by monoclonal antibodies 10A10 and 19D8 resides in the carboxyl-terminal portion of the proregion. The 10A10 and 19D8 antibodies each recognized a fusion protein containing amino acids 22-159 but did not recognize a fusion protein containing amino acids 22-77 or amino acids 22-92. Immunoblot analysis of HeLa cell lysates using anti N-cadherin proregion antibodies revealed two distinct bands that migrated slower than mature N-cadherin on SDS-PAGE (compare lanes 1 and 2 in Fig. 1C with lane 3). Lanes 1 and 2 contain ~3 times as much protein as lane 3. The 13A9 antibody recognized both the processed and unprocessed forms of N-cadherin, and a faint band corresponding to the unprocessed form can be seen in lane 3. When we loaded sufficient protein to clearly identify proN-cadherin using the 13A9 antibody, the lane was overloaded and the bands smeared together, making it difficult to separate them from one another. The results with 13A9 show that the vast majority of the N-cadherin in HeLa cells at steady state lacks the proregion. Immunoblot analysis of HT1080 and VA13, two other cell-lines expressing endogenous N-cadherin, produced similar results (data not shown).


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Fig. 1.   Production of antibodies against the proregion of human N-cadherin. A, schematic diagram of human N-cadherin, including the proregion (pro), the 5 extracellular repeats (EC1-5), the transmembrane domain (TM), and the cytoplasmic domain. Two previously characterized antibodies were used in this study, namely 13A9, which binds to the cytoplasmic domain of N-cadherin, and 8C11, which binds to the extracellular domain between EC3 and EC4. B, MBP fused to the proregion of human N-cadherin was used as an antigen to make monoclonal antibodies. MBP-fusion proteins containing amino acids 22-159 of N-cadherin (lane 1), amino acids 22-77 of N-cadherin (lane 2), and amino acids 22-92 of N-cadherin (lane 3) were resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-maltose-binding protein, 10A10, or 19D8. Monoclonal antibodies 10A10 and 19D8 each bound to a region included in amino acids 92-159. 19D8 also cross-reacts with a bacterial protein that co-migrates with the fusion protein corresponding to amino acids 22-77 of N-cadherin. C, HeLa cell extract was separated by SDS-PAGE and transferred to nitrocellulose, and the membrane was blotted with antibodies specific for proN-cadherin, 10A10 (lane 1), and 19D8 (lane 2) or the cytoplasmic domain of N-cadherin, 13A9 (lane 3).

Immunolocalization of proN-cadherin in HeLa Cells-- Immunofluorescence analysis of HeLa cells using the anti-proN-cadherin antibody produced a staining pattern that closely resembled that seen for the endoplasmic reticulum protein calnexin (Fig. 2, D-F) and the Golgi marker, mannosidase II (Fig. 2, G-I). No proN-cadherin staining was observed at cell borders (Fig. 2, A, D, and G). This suggests that the majority of proN-cadherin is localized to the endoplasmic reticulum and Golgi complex and that proN-cadherin is not transported to the plasma membrane. In contrast, HeLa cells stained with an antibody directed against the cytoplasmic domain of N-cadherin (13A9) that recognizes both mature N-cadherin and proN-cadherin stained mainly cell-cell borders with a small amount of staining in the endoplasmic reticulum/Golgi area (Fig. 2B).


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Fig. 2.   ProN-cadherin co-localizes with endoplasmic reticulum and Golgi apparatus proteins. HeLa cells were fixed in 1% paraformaldehyde, permeabilized in methanol at -20 °C, and stained with antibodies specific for proN-cadherin, 10A10 (panel A), and 19D8 (panel D and G), mature N-cadherin, 13A9 (panel B), the endoplasmic reticulum marker calnexin (panel E), or the Golgi marker mannosidase II (panel H). Panel C is a phase contrast image of the cells in panel B. Panel F is a merged image of panels D and E. Panel I is a merged image of panels G and H. The scale bar in panel A is 1 µm.

To further demonstrate that proN-cadherin is restricted to the cytosol, we compared the staining pattern of permeabilized and non-permeabilized HeLa cells using an anti-proN-cadherin antibody or an antibody that recognizes all forms of N-cadherin. Non-permeabilized HeLa cells showed no immunostaining using the anti-proN-cadherin antibody 10A10 (Fig. 3A). An antibody specific for the cytoplasmic domain of N-cadherin (13A9) was also negative on non-permeabilized cells (Fig. 3D). The fact that cells were present in panels A and D is shown by the corresponding DAPI staining in panels B and E, respectively. Staining with an anti N-cadherin antibody directed against the extracellular domain (8C11) showed that mature N-cadherin was present on the cell surface (Fig. 3G). HeLa cells permeabilized by methanol treatment and immunostained using 10A10 revealed cytoplasmic staining localized to ER and Golgi compartments (Fig. 3C), whereas 13A9 showed typical cell border staining (Fig. 3F).


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Fig. 3.   ProN-cadherin is not present on the surface of HeLa cells. HeLa cells grown on glass coverslips were fixed in 1% paraformaldehyde and stained with anti-proN-cadherin (panel A), anti N-cadherin cytoplasmic domain (panel D), or anti N-cadherin extracellular domain (panel G). Alternatively, cells were permeabilized after fixation and stained with anti-proN-cadherin (panel C) or anti N-cadherin cytoplasmic domain (panel F). Panels B and E are DAPI staining of panels A and D respectively to show the location of the cells. The scale bar in panel G is 1 µm.

ProN-cadherin Is Removed by a Single Proteolytic Event-- Because proN-cadherin must be cleaved to generate a functional cadherin, we sought to detect the cleaved proregion of N-cadherin using immunoblots of HeLa cell extracts. The cleaved proregion of N-cadherin was undetectable on straight immunoblots, perhaps because it is not very abundant (data not shown). However, when anti-N-cadherin proregion antibodies were used to immunoprecipitate HeLa cell extracts, and the immunoprecipitated proteins were subsequently immunoblotted using the same antibody, the proregion was detected as a single band at ~15kDa (Fig. 4). Anti-proN-cadherin antibodies (10A10 and 19D8) each immunoprecipitated a peptide of ~15kDa, which is consistent with the proregion of N-cadherin being ~135-140 amino acids. The 10A10 and 19D8 antibodies themselves contain multiple immunoglobulin chains that migrate between 25 and 68 kDa, which were identified by resolving the antibody in the absence of HeLa cell extract (Fig. 4, lanes 1 and 2).


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Fig. 4.   Immunoprecipitation of the proregion of N-cadherin. Anti-proN-cadherin antibodies were used to immunoprecipitate buffer alone (lanes 1 and 2) or HeLa cell extract (lanes 3 and 4). The immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-proN-cadherin (10A10). Note that 10A10 and 19D8 each immunoprecipitated a protein that migrated at 15 kDa, consistent with the entire proregion of human N-cadherin.

Catenins Co-immunoprecipitate with proN-cadherin-- beta -catenin, plakoglobin, and p120ctn each directly bind to the cytoplasmic domain of N-cadherin (11, 28). alpha -catenin is localized to the cadherin-catenin complex via binding beta -catenin or plakoglobin. We sought to determine at which point the catenins assemble on N-cadherin by examining the pool of proN-cadherin in HeLa cells. ProN-cadherin was immunoprecipitated from HeLa cell extract, and the immunoprecipitate was resolved by SDS-PAGE. Immunoblot analysis using antibodies specific for alpha -catenin, beta -catenin, p120ctn (Fig. 5A), or plakoglobin (data not shown) demonstrated that each catenin co-immunoprecipitated with proregion-containing N-cadherin, suggesting that all four catenins can assemble on N-cadherin before the proregion is cleaved. HeLa cells express two isoforms of p120ctn, and both isoforms were associated with proN-cadherin (Fig. 5A).


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Fig. 5.   Catenins associate with proN-cadherin. A, HeLa cell extract was immunoprecipitated (IP) with anti-proN-cadherin monoclonal antibody (Pro-region aby) 10A10. The immunoprecipitate (left lane) and HeLa cell extract (right lane) were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies specific for alpha -catenin, beta -catenin, or p120ctn. B, 900 µl of HeLa cell extract was immunoprecipitated with anti-proN-cadherin (19D8; left lane), and 150 µl of HeLa cell extract was immunoprecipitated with anti-mature N-cadherin (13A9; right lane). Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti alpha -catenin (1G5) and anti N-cadherin (13A9). C, cytosolic HeLa extract prepared in the absence of Nonidet P-40 was immunoprecipitated with anti alpha -catenin (alpha -catenin aby; 1G5) or no antibody (no aby). The immunoprecipitation was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies specific for beta -catenin or N-cadherin. Cytosolic and membrane extracts were also immunoblotted to show the absence of N-cadherin in the cytosolic extract.

To estimate the stoichiometric ratio of cadherin to catenin, we immunoprecipitated HeLa cell extracts with either anti-proN-cadherin or anti-N-cadherin antibodies such that the resulting immunoblot signals for alpha -catenin were roughly equal. To accomplish this, different amounts of cell extract were used for each immunoprecipitation. The nitrocellulose membrane was immunoblotted simultaneously for alpha -catenin and N-cadherin using the 13A9 antibody that recognizes all forms of N-cadherin (Fig. 5B). When roughly equal amounts of alpha -catenin were present in each immunoprecipitation, the N-cadherin signals were approximately equal. This result indicates that proN-cadherin-catenin complexes are stoichiometrically similar to mature cadherin-catenin complexes, suggesting that the majority of the proregion containing cadherin in HeLa cells is complexed with alpha -catenin. This result clearly demonstrates that assembly of the complex does not require removal of the proregion.

It has been suggested that beta -catenin binds to E-cadherin co-translationally, but alpha -catenin binds at the time of proteolytic cleavage or after the complex has been transported to the plasma membrane (30, 31). The data presented in Fig. 5B suggest that both beta -catenin and alpha -catenin can associate with unprocessed N-cadherin. One possibility is that beta -catenin and alpha -catenin form a complex in the cytosol before either one associates with N-cadherin. To determine whether beta -catenin and alpha -catenin could associate with one another in the absence of cadherin, we Dounce homogenized HeLa cells in the absence of detergent to generate cadherin-free cytosolic extract. We then analyzed the cytosolic extract for complexes of beta -catenin and alpha -catenin by co-immunoprecipitation experiments. Fig. 5C shows that beta -catenin co-immunoprecipitated with alpha -catenin in these extracts. Reciprocal experiments showed that alpha -catenin co-immunoprecipitated with beta -catenin (not shown), and immunoblots confirmed that there was no cadherin in the cytosolic extract (Fig. 5C). Although these data do not prove that beta -catenin and alpha -catenin form dimers before they associate with N-cadherin, when considered in conjunction with the knowledge that alpha -catenin co-immunoprecipitates with proN-cadherin, they are consistent with the hypothesis that beta -catenin and alpha -catenin could load onto the cadherin as a dimer.

Catenins Co-localize with Unprocessed Cadherin-- The quantity of unprocessed N-cadherin in HeLa cells is very small. We can detect it in immunofluorescence using anti-proN-cadherin, because proN-cadherin is discretely localized within the endoplasmic reticulum and the Golgi complex and because the antibody detects only the unprocessed N-cadherin. It was not possible to use HeLa cells to examine the co-localization of unprocessed N-cadherin and catenins, because the catenin antibodies recognize all the catenins, including those complexed with mature N-cadherin. Because most of the N-cadherin in these cells is mature (Fig. 1C), the majority of the immunofluorescence signal corresponding to catenins is at the cell surface, which tends to obscure any signal that may be in the cytosol. During the course of a separate project, we prepared a series of N-cadherin-E-cadherin chimeras (23) and introduced these constructs into the cadherin-negative cell line A431D. The protein encoded by one construct called N/E5a-myc (23) was highly expressed by A431D cells but was not processed and not transported to the plasma membrane. Presumably, the failure of N/E5a-myc to leave the endoplasmic reticulum was due to inappropriate protein folding. A431D cells do not stably express catenins until they are transfected with a cadherin, and the majority of the stable catenin they express after transfection is associated with the transfected cadherin (25). Thus, in A431D cells expressing the N/E5a-myc chimera, the technical complication of cell surface catenin is not a problem for immunofluorescence experiments.

A431D cells expressing the N/E5a-myc chimera were lysed, and the extracts were used for immunoblot analysis. Antibodies against the N-cadherin proregion (10A10 and 19D8), the N-cadherin extracellular domain (8C11), or the C-terminal Myc tag (9E10) were used to identify the chimera. In contrast to the results with HeLa cells (Fig. 1C), each antibody identified one band with the same mobility (Fig. 6A). Thus, the majority of the N/E5a-myc cadherin contained the proregion and was unprocessed. Immunofluorescence staining of the transfected A431D cells revealed that the N/E-chimeric cadherin was present in a reticular staining pattern consistent with a protein that is trapped in the endoplasmic reticulum. Cells expressing the N/E5a-myc chimera also stably expressed beta -catenin, which co-localized with the cadherin (Fig. 6, E-G) and with the endoplasmic reticulum marker, calnexin (Fig. 6, B-D). In addition, alpha -catenin (Fig. 6, H-J) and p120ctn (Fig. 6, K-M) were stably expressed in the transfected cells, and these cytoplasmic proteins were also found in a reticular staining pattern that co-localized with beta -catenin and, thus, with the N/E5a-myc chimeric cadherin. These data confirm that the catenins and other cytoplasmic proteins of the adherens junction can assemble with cadherins prior to cadherin localization at the plasma membrane.


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Fig. 6.   Catenins co-localize with proN-cadherin. A, cell extracts prepared from A431D cells transfected with the N/E5a-myc chimeric cadherin were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against the N-cadherin proregion (10A10 and 19D8), the N-cadherin extracellular domain (8C11), and the C-terminal Myc tag (9E10). B-L, transfected cells were fixed in 1% paraformaldehyde, permeabilized in methanol at -20 °C, and co-stained with mouse anti beta -catenin (panel B) and rabbit anti calnexin (panel C); mouse anti N-cadherin (panel E) and rabbit anti beta -catenin (panel F); mouse anti alpha -catenin (panel H) and rabbit anti beta -catenin (panel I); and mouse anti p120ctn (panel K) and rabbit anti beta -catenin (panel L). Panels D, G, J, and M are corresponding merged images. The scale bar in panel B is 1 µm. Note that not all the cells in this experiment were transfected with the chimeric cadherin. Thus, panel C includes cadherin-negative and cadherin-positive cells, which are all positive with the calnexin antibody. Likewise, panel L includes cadherin-negative and cadherin-positive cells, which are all positive for p120ctn.

The Cytoplasmic Domain of N-cadherin Is Phosphorylated Prior to the Removal of the Proregion-- Anti-proN-cadherin antibodies recognized two major bands in immunoblots of HeLa cell extract (Fig. 1). Thus, we performed pulse-chase experiments to examine the synthesis of proN-cadherin and determine which band was the earlier precursor form (Fig. 7A). The faster migrating form of proN-cadherin was labeled during a 15-min pulse with radioactive amino acids (lane 1). After a 10-min chase, the slower migrating form began to appear (lane 2), and by 30 min the majority of the protein was in this form (lane 4). By 60 min nearly all the signal had been chased to the slower migrating form of proN-cadherin, and the overall signal had begun to diminish due to removal of the proregion by protease cleavage (lane 5).


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Fig. 7.   Characterization of two proN-cadherin species. A, HeLa cells were starved for 4 h in methionine- and cysteine-deficient media and then pulsed for 15 min in deficient media supplemented with 35S-labeled methionine and cysteine. Cultures were chased with complete media for the indicated times, and cells were extracted in TNE buffer. Anti-proN-cadherin (10A10) was used to immunoprecipitate proN-cadherin from labeled extracts. The immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was exposed to autoradiographic film at -70 °C. B, HeLa cells were cultured in phosphate-deficient media overnight and then labeled with 33PO4-containing media for 2 h. Cells were extracted in TNE buffer and immunoprecipitated with 10A10. The immunoprecipitate was resolved by SDS-PAGE, transferred to nitrocellulose, and exposed to film (autorad.; lane 2) prior to immunoblotting with anti N-cadherin (anti-N-cad blot; 13A9; lane 1). C, HeLa cell extract was immunoprecipitated with anti-beta -catenin, anti-plakoglobin, and anti-p120ctn. Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-proN-cadherin (10A10).

To characterize the difference between the two proN-cadherin bands, we metabolically labeled HeLa cell cultures using 33P and immunoprecipitated proN-cadherin. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose; the 33P labeled bands were identified by autoradiography of the nitrocellulose membrane prior to immunoblotting with anti-proN-cadherin antibody (Fig. 7B, lane 2). From this experiment we determined that the slower migrating band was phosphorylated, whereas the faster migrating band was not phosphorylated. These data indicate that N-cadherin is phosphorylated while in the endoplasmic reticulum or Golgi complex prior to removal of the proregion.

It has been suggested that phosphorylation of the cytoplasmic domain of cadherin regulates catenin binding. Thus, we used beta -catenin, plakoglobin, and p120ctn antibodies to co-immunoprecipitate proN-cadherin and anti-proN-cadherin antibodies to detect which proN-cadherin species are co-immunoprecipitated with each catenin (Fig. 7C). All three catenins were associated with the slower migrating phosphorylated form of proN-cadherin, whereas only p120ctn was associated with the faster non-phosphorylated form. Thus, p120ctn appears to bind N-cadherin in its most immature state, although beta -catenin and plakoglobin bind after the cadherin has been phosphorylated.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The goal of the present study was to investigate endogenous N-cadherin processing and cadherin-catenin complex formation. We chose to use HeLa cells that endogenously express N-cadherin and catenins and form N-cadherin-containing cell-cell junctions. To monitor the processing of N-cadherin, we generated monoclonal antibodies specific for the proregion of N-cadherin. Immunoblot analysis of HeLa cell extracts with the proN-cadherin antibodies revealed two major bands. Immunoblots of extracts prepared from other cells expressing endogenous N-cadherin (HT1080 and VA13) gave similar results (data not shown). Metabolic labeling of HeLa cell cultures with 33P identified the faster migrating form of proN-cadherin as a non-phosphorylated form and the slower migrating form as a phosphorylated form. In pulse-chase experiments, the earliest (non-phosphorylated) form of proN-cadherin that we could detect rapidly chased into the slower migrating (phosphorylated) form. It was this slower migrating form that co-immunoprecipitated with beta -catenin and plakoglobin. In addition to the experiments with HeLa cells, the experiments with the N/E5a-myc chimeric cadherin showed that catenins could associate with cadherins that are in the endoplasmic reticulum. Taken together, these data suggest that the phosphorylation of proN-cadherin and the subsequent assembly of the cadherin-catenin complex occurs while the cadherin is in the endoplasmic reticulum and Golgi complex. Fig. 8 presents a model depicting the sequence of events leading to N-cadherin-catenin complex localization at the plasma membrane.


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Fig. 8.   Model of N-cadherin processing and complex formation. N-cadherin is synthesized in the endoplasmic reticulum where p120ctn binds to the cytoplasmic juxtamembrane domain. The cytoplasmic domain is then phosphorylated by casein kinase II (CKII), which leads to the binding of beta - and alpha -catenin. The proregion is removed by furin protease, and the complex is transported to the plasma membrane.

The proregion of cadherins must be removed in order to generate functional adhesion molecules (21). Recently, Ozawa (32) used L cells to show that the proregion of E-cadherin not only prevented cell aggregation (which requires the formation of adhesion dimers) but also prevented the formation of lateral dimers. The fact that we found catenins complexed with proN-cadherin suggests that the catenins can load onto the monomeric form of this cadherin. Because our pulse-chase experiments showed that the earliest detectible form of proN-cadherin chases into a slower migrating form that co-immunoprecipitates all the catenins, it is likely that the proN-cadherin-catenin complex forms in the endoplasmic reticulum.

The furin subgroup of subtilisin-like proprotein convertases is thought to be responsible for cadherin processing. E-cadherin has been shown to be a furin protease substrate in a baculovirus system, and the consensus sequence for furin protease in E-cadherin is identical to that of N-cadherin (20). The furin convertases have been shown to be proteolytically active in the trans-Golgi network (33). Interestingly, our immunofluorescence experiments using anti-proN-cadherin antibodies revealed a staining pattern consistent with proN-cadherin localization to the ER and Golgi network. In addition, immunoprecipitation experiments showed that a 15-kDa fragment, corresponding to the cleaved proregion, was present in HeLa cell extracts, suggesting that the proregion of N-cadherin can be removed in one step, most likely by the furin convertases. The detection of the 15-kDa pro-peptide raises the possibility that it could be secreted and may have some yet to be identified function outside the cell. In permeabilization experiments, proN-cadherin was not present at the plasma membrane of HeLa cells, consistent with the proregion of N-cadherin being removed prior to transport to the plasma membrane.

Studies focusing on E-cadherin-catenin complex formation in L cells suggested that alpha -catenin was added to the cadherinbeta -catenin complex after the cadherin proregion was removed (30). In addition, studies on the assembly of E-cadherin complexes in Madin-Darby canine kidney (MDCK) cells suggested that alpha -catenin was added to the adherens junction complex after localization at the plasma membrane (31). Our results demonstrate that this is not the case for N-cadherin in HeLa cells. Immunoprecipitations showed that beta -catenin, plakoglobin, p120ctn, and alpha -catenin were all found in a complex with proN-cadherin. The studies with E-cadherin raise the possibility that, although we could find the catenins associated with proN-cadherin, only a small fraction of the total proN-cadherin is actually complexed with the catenins. However, the experiment shown in Fig. 5 demonstrates that the association of alpha -catenin with proN-cadherin is not a rare event; the alpha -catenin-proN-cadherin complex has the same stoichiometry as the alpha -catenin-mature N-cadherin complex. The cadherin-beta -catenin-alpha -catenin complex has been estimated to have molar ratios of 1:1:1 (30), a conclusion that is supported by recent structural studies (34, 35). It is likely that proN-cadherin binds the catenins in the same ratios as does mature N-cadherin.

To date, p120ctn association with unprocessed cadherin has not been investigated. Our data show that p120ctn, but not beta -catenin or plakoglobin, can bind to non-phosphorylated proN-cadherin, which is the earliest form of N-cadherin we can detect. Taken together, our results suggest a model where, following synthesis, proN-cadherin associates immediately with p120ctn. Following phosphorylation of the cadherin, possibly by casein kinase II (36), beta -catenin or plakoglobin then associates with the cadherin (Fig. 8). Our data also show that beta -catenin-alpha -catenin complexes can form in the cytosol, raising the possibility that beta -catenin and alpha -catenin may simultaneously load onto proN-cadherin. The complex is then transported to the plasma membrane where linkage to the actin cytoskeleton occurs. The mechanism of cadherin-catenin complex transport to the plasma membrane is unknown, although recent work suggests a microtubule-dependent mechanism for the formation of N-cadherin cell-cell contacts (37). Understanding the sequence of events leading to the formation of the N-cadherin-catenin complex and its localization at the plasma membrane will help clarify the regulation of N-cadherin mediated cell-cell adhesion and identify new mechanisms for controlling N-cadherin-mediated cell-cell adhesion.

    ACKNOWLEDGEMENTS

We thank Jill Nieset and Jennifer Oiler for expert technical assistance in preparing proN-cadherin fusion proteins and generating the constructs used in this study.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM51188 (to M. J. W.) and DE12308 (K. R. J.).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: University of Nebraska Medical Center, College of Dentistry and Eppley Cancer Center, 987696 Nebraska Medical Center, Omaha, NE 68198-7696. Tel.: 402-559-3893; Fax: 402-559-3739; E-mail: Jwahl@unmc.edu.

Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M211452200

    ABBREVIATIONS

The abbreviations used are: MBP, maltose-binding protein; DMEM, Dulbecco's modified Eagle's medium; DAPI, 4,6-diamidino-2-phenylindole.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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