Angiomotin is a novel component of cadherin-11/β-catenin/p120 complex and is critical for cadherin-11-mediated cell migration

Abstract

Loss of E-cadherin and up-regulation of mesenchymal cadherins, a hallmark of the epithelial–mesenchymal transition, contributes to migration and dissemination of cancer cells. Expression of human cadherin-11 (Cad11), also known as osteoblast cadherin, in prostate cancer increases the migration of prostate cancer cells. How Cad11 mediates cell migration is unknown. Using the human Cad11 cytoplasmic domain in pulldown assays, we identified human angiomotin (Amot), known to be involved in cell polarity, migration, and Hippo pathway, as a component of the Cad11 protein complex. Deletion analysis showed that the last C-terminal 10 amino acids in Cad11 cytoplasmic domain are required for Amot binding. Further, Cad11 preferentially interacts with Amot-p80 than Amot-p130 isoform and binds directly to the middle domain of Amot-p80. Cad11-Amot interaction affects Cad11-mediated cell migration, but not homophilic adhesion, as deletion of Amot binding motif of Cad11 (Cad11-ΔAmot) did not abolish Cad11-mediated cell–cell adhesion of mouse L cells, but significantly reduced Cad11-mediated cell migration of human C4-2B4 and PC3-mm2 prostate cancer cells and human HEK293T cells. Together, our studies identified Amot-p80 as a novel component of the Cad11 complex and demonstrated that Amot-p80 is critical for Cad11-mediated cell migration.—Ortiz, A., Lee, Y.-C., Yu, G., Liu, H.-C., Lin, S.-C., Bilen, M. A., Cho, H., Yu-Lee, L.-Y., Lin, S.-H. Angiomotin is a novel component of cadherin-11/β-catenin/p120 complex and is critical for cadherin-11-mediated cell migration.

During the epithelial-mesenchymal transition, epithelial cells down-regulate E-cadherin (E-Cad) and express mesenchymal cadherin molecules, a process termed cadherin switch (1). The epithelial–mesenchymal transition confers epithelial cells with a migratory phenotype, which plays a critical role in the metastasis of cancer cells. The mechanism by which aberrant expression of mesenchymal cadherins increases cell migration is not fully understood.

Cadherin-11 (Cad11, also known as osteoblast cadherin, OB-cadherin, CDH11) is a mesenchymal cadherin up-regulated during prostate cancer (PCa) progression (2). Immunohistochemical analyses of clinical PCa specimens showed that Cad11 is not expressed in normal prostate epithelial cells but is up-regulated in castration-resistant PCa samples (3) and in PCa bone metastases (2). Consistently, Cad11 was highly expressed in the bone metastasis–derived PCa cell line, PC3-mm2 (2). We have previously shown that down-regulation of Cad11 in PC3-mm2 decreased metastasis to bone (2). On the cellular level, we found that Cad11 expression increased the migration and invasion of PCa cells and enabled PCa cells to intercalate into an osteoblast monolayer (4). Moreover, deletion of the cytoplasmic (cyto) domain of Cad11 abolished its ability to enhance migration and invasion (4), suggesting that intracellular signaling via the cyto domain mediates Cad11 migration.

The signal transduction pathways of cadherin family proteins are relatively conserved, with β-catenin (β-cat) and p120-catenin (p120) participating in cadherin stability and activities (5, 6). Although both E-Cad and Cad11 bind p120 and β-cat (4–7), these cadherins differ in their functions. Although E-Cad suppresses the migration of tumor cells (7–10), Cad11 expression increases PCa cell migration (4). Therefore, Cad11 probably recruits distinct proteins to the Cad11/β-cat/p120 complex to induce cell migration. The cyto domains of E-Cad and Cad11 share 49% similarity, raising the possibility that cadherin family proteins mediate diverse cellular functions through sequence-specific interaction with unique proteins. In this study, we used a protein subtraction strategy by first removing E-Cad–interacting proteins from cell lysates and then isolating proteins that differentially interact with Cad11. We identified angiomotin (Amot) as a novel Cad11-interacting protein.

Amot is a member of the Motin family, which consists of Amot, Amot-L1, and Amot-L2 (11). They share similar domains, such as a coiled-coil domain (CC domain) in their N-terminal portion and a PDZ-binding motif at their extreme C terminus (11), yet have distinct cellular functions. Amot is known to be involved in multiple cellular functions including cell polarity (12), migration (13), and Hippo pathway (14, 15). Amot is expressed as 2 isoforms through alternative splicing: Amot-p130 (p130) and Amot-p80 (p80). The p130 isoform has a 409 aa extension in its N terminus, which participates in the Hippo pathway to promote cell contact inhibition and attenuate cell proliferation (16). On the other hand, the p80 isoform was shown to regulate apical polarity (12) through interacting with Rich1, a small GTPase-activating protein, and to affect proliferation of Schwann cells in neurofibromatosis through interacting with Merlin (15). Both Rich1 and Merlin bind to the N-terminal CC domains in p80 (12, 15). In addition, the C terminus of p80 contains a PDZ-binding motif that has been shown to promote endothelial cell migration (13) by interacting with PDZ domains of scaffolding proteins, such as MUPP1 and PatJ (17). In this study, we examined the Cad11-Amot interaction and its effect on Cad11 functions.

MATERIALS AND METHODS

Materials

Human kidney 293T (HEK293T) and Phoenix cells were purchased from American Culture Type Collection. Human C4-2B4/Cad11 cell line was generated as described previously (4). Human PC3-mm2, a PC3 subline, was generated by Dr. I. J. Fidler (MD Anderson Cancer Center). The plasmid vectors pAmot-YFP, Amot-p80 (pAmot-3xFlag), Amot mutant 1 (pΔBAR-Flag), Amot mutant 3 (pBAR-Flag), and Amot mutant 5 (pΔEYLI) (12) were kindly provided by Dr. C. Wells (Indiana University). A different set of human Amot deletion mutants, Amot mutant 4 (pCMV-Flag-p80-CC), Amot mutant 2 (pCMV-Flag-p80-ΔCC), and the Amot-p130 (p130-Amot-3xFlag) vectors, as described elsewhere (15), were from Dr. J. L. Kissil (Wistar Institute). The human p80 cDNA in the pCR4-TOPO vector was purchased from Open Biosystems (Pittsburgh, PA). Goat anti-Amot antibody (L-16), anti-Flag antibody, and Cad11 cyto (5B2H5) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma-Aldrich (St. Louis, MO, USA), and Invitrogen (Carlsbad, CA, USA), respectively. Generation of mAb 1A5 and 2C7, against the extracellular domain of human Cad11, were described by Lee et al. (18). The oligonucleotides used were from Sigma-Aldrich; their sequences are listed in Supplemental Table S1.

Construction of Cad11 cyto domain mutants in GST expression vectors

The cyto domain of human Cad11 aa 641–796 was amplified by PCR using full-length human Cad11 as a template. A GST fusion protein expressing 2 copies of Cad11 cyto domain was constructed as follows. Two fragments of cyto domain with different restriction enzyme sites were generated using primers EcoRI-BamHI-cyto-F and EcoRI-cyto-R for fragment 1 and EcoRI-BamHI-cyto-F and NotI-cyto-ter-R for fragment 2 (Supplemental Table S1). The 2 fragments were subcloned into pCR 2.1 TOPO TA (Invitrogen) and confirmed by DNA sequencing. The fragments were digested from the pCR2.1 vectors and ligated in the pGEX-4T-1 plasmid using endonucleases to generate pGEX-Cad11-cyto-2X. The GST fusion proteins containing 2 copies of E-Cad cyto domain was made similarly using the oligos listed in Supplemental Table S1. The mutant Cad11 cyto domain mutants deleting the JMD (GST-Cad11-cyto-ΔJMD-2X) or C-terminal binding sequence (CBS; GST-Cad11-cyto-ΔCBS-2X) domain were constructed using the same 2 fragment method but using the mutant Cad11 cyto domain (4) as template. The CBS deletion mutants were generated using pGEX-Cad11-cyto domain as a template and using the reverse primers listed in Supplemental Table S1 to delete aa from the C terminus. The p3xFlag-Cad11-cyto-2X was generated by restriction digestion of pGEX-Cad11-cyto-2X and subcloned into p3xFLAG-CMV (Sigma-Aldrich).

Isolation of proteins associated with Cad11 cyto domain

GST constructs were expressed in Escherichia coli and purified using glutathione–agarose beads. C4-2B4 cells were collected in cold distilled water with protease inhibitors and homogenized with a Dounce homogenizer. The lysate was mixed with GST-E-Cad-cyto-2X protein immobilized on glutathione–agarose beads and rocked at room temperature for 2 h. The GST-E-Cad-cyto-2X beads were removed, and the supernatant was mixed with GST-Cad11-cyto-2X protein immobilized on glutathione–agarose beads at 4°C overnight. The proteins bound to GST-E-Cad-cyto-2X and GST-Cad11-cyto-2X were resolved on a 4% to 12% gradient NuPage gels (Novex, San Diego, CA). The gel was stained with GelCode (Thermo Fisher Scientific, Waltham, MA, USA), and the proteins associated with Cad11 cyto were identified by mass spectrometry.

Generation of GST-Amot or Amot-His7 proteins

GST-Amot and Amot-His₇ fusion proteins were generated by PCR using pCR4-TOPO-Amot as template and primers Amot-F1 and Amot-R1 (Supplemental Table S1). The PCR product was ligated into the pCR2.1 TOPO TA vector and the sequence confirmed using the Amot oligos Amot F2 to F4 (Supplemental Table S1). The Amot insert was removed from pCR2.1 TOPO TA vector using endonucleases and subcloned into pGEX4T1 or pET28b vectors. GST-Amot and Amot-His₇ proteins were purified using glutathione-agarose or Ni-NTA-agarose, respectively.

Generation of Amot-p80 antibodies

Purified GST-Amot protein was used to immunize rabbits to generate polyclonal anti-human Amot antibody and mice to generate monoclonal antibodies. To affinity purify polyclonal anti-Amot antibody from the rabbit bleeds, freshly purified Amot-His₇ protein was applied on a strip of nitrocellulose membrane and incubated with the rabbit bleed overnight at 4°C. The nitrocellulose strip was washed and the Amot antibodies were eluted using Gentle Elute (Thermo Fisher Scientific).

Direct protein interaction assay

Purified Amot-His₇ protein was incubated with GST-E-Cad cyto-2X or GST-Cad11-cyto-2X. Proteins eluted from the beads were examined by Western blot analysis.

Transfection of mammalian cells

HEK293T were transfected with mammalian expression vectors using polyethylenimine as described previously (19). After 48 h, the transfected HEK293T cell lysates were used for GST pull-down assay.

Immunoprecipitation

Cells were washed twice with ice-cold PBS and lysed in buffer containing 50 mM Tris pH 7.2, 1 mM sodium orthovanadate, 50 mM NaF, 25 mM β-glycerophosphate, protease inhibitors, and 1% Triton X-100, and the cleared lysates were incubated with antibody for 1 h. The protein-antibody complexes were pulled down by protein G-agarose beads and analyzed by SDS-PAGE for Western blot analysis.

Construction of retroviral vectors and cell lines

The Cad11ΔAmot protein was constructed by PCR using pBMN-Cad11 wild-type (WT) as the template and the primers Cad11-EC-forward and CBS-10-reverse (Supplemental Table S1). The Cad11Δβ-cat insert was constructed using the Stratagene site-directed mutagenesis kit, the primers Δβ-cat-Forward and Δβ-cat-Reverse (Supplemental Table S1), and Cad11-WT as the template. After the PCR products were verified by sequencing, the Cad11ΔAmot and Cad11Δβ-cat inserts were digested and ligated into the pBMN-I-GFP and pBMN-I-Neo vectors. These retroviral vectors were transfected into Phoenix cells and the culture media collected for infection of L, C4-2B4, PC3-mm2, and HEK293T cells as described previously (20). GFP-positive cells were selected by fluorescence-activated cell sorting (FACS), and Neo-positive cells were selected by G418.

Generation of PC3-mm2 cells with knockdown of endogenous Cad11 followed with expression of Cad11 mutants

PC3-mm2 cells were transduced with Cad11 shRNA in the lentiviral vector pLKO.1-puro (TRCN0000303363 NM_001797.2-3233s21c1; Sigma-Aldrich). The shRNA sequence is located at the 3′-untranslated region. shRNA transduced PC3-mm2 cells (PC3-shCad11) were selected by puromycin. PC3-mm2 cells transduced with a control pLKO.1-puro vector (PC3-pLKO) were used as a control. PC3-shCad11 cells were then transduced with Cad11 mutants in pBMN-I-GFP vector.

Immunofluorescence staining, FACS, migration, and aggregation assays

Immunofluorescence staining, FACS, migration, and aggregation assays were performed as described in Chu et al. (2), Lira et al. (20), Huang et al. (4), and Lee et al. (18), respectively.

Generation of PC3-mm2 cells overexpressing Amot-p80

To stably overexpress Amot-p80 in PC3-mm2 cells, bicistronic retroviral vector containing cDNA encoding human Amot-p80 with His₇ tag at the C termini was used to infect PC3-mm2 cells. Retroviruses were also generated from pBMN-I-Neo vectors and used as a control. PC3-mm2 cells expressing Amot-p80 were selected by G418.

Generation of C4-2B4 cells with Amot knockdown

To knock down Amot in C4-2B4 cell lines, several shAmot in pGIPZ lentiviral vectors (Addgene, Cambridge, MA) were examined, and shAmot#1 and shAmot#2 were selected for functional studies. C4-2B4 cells infected with pGIPZ lentiviral vector were used as control.

Statistical analyses

Student’s t test (2-tailed, paired) was used for statistical analyses. A P value of less than 0.05 was considered statistically significant. Data are expressed as means ± sd unless otherwise specified.

RESULTS

Amot is associated with Cad11-cyto domain

Because both E-Cad and Cad11 bind p120 and β-cat yet differ in their functions, we used a protein subtraction strategy to remove E-Cad interacting proteins from cell lysates to isolate Cad11-specific interacting proteins. We depleted the C4-2B4 cell cytosolic fraction with GST-E-Cad-cyto-2X then used GST-Cad11-cyto-2X to isolate Cad11-specific interacting proteins (Fig. 1A). Western blot analysis of proteins eluted from the GST-E-Cad-cyto-2X beads showed both β-cat and p120 were pulled down by GST-E-Cad-cyto-2X but not by GST alone (Fig. 1B). The E-Cad-cyto-2X depleted supernatant was then incubated with GST-Cad11-cyto-2X beads. The proteins that were pulled down by Cad11-cyto-2X were resolved on SDS-PAGE, and gel areas marked with a black line were cut out for protein identification by mass spectrometry (Fig. 1C). The prominent protein observed at around 180 kDa was previously identified as clathrin and was therefore excluded from mass spectrometry analysis. Mass spectrometry analysis identified proteins, such as Amot (gi|19111150) and putative NF-κB activating protein (gi|22671723), as candidate Cad11 interacting proteins (Supplemental Table S1).

Figure 1.

Amot associates with Cad11 cyto domain. A) GST-Cad11-cyto-2X, GST-E-Cad-cyto-2X constructs, and procedure for isolation of Cad11 interacting proteins from C4-2B4 cells. B) β-Catenin and p120 were pulled down by GST-E-Cad-cyto-2X. C) Proteins differentially pulled down by GST-Cad11-cyto-2X. Proteins in the region highlighted by the black lines were identified by mass spectrometry. D) Location of 2 peptides (underlined), identified by mass spectrometry, in human Amot-p80 sequence. E) GST-Cad11-cyto-2X, but not GST-E-Cad-cyto-2X, was able to bind Amot-p80. F) Direct interaction between p80-Amot and Cad11 cyto, but not E-Cad cyto, was observed in 3 independent experiments.

Amot was selected for further study. Mass spectrometry identified peptides present in both p80 and p130 Amot isoforms but not Amot-L1 or Amot-L2 (Fig. 1D, underlined). Western blot analysis using anti-Amot antibody showed that GST-Cad11-cyto-2X mainly pulled down p80 and a small amount of p130 in the protein complex (Fig. 1E). GST-E-Cad-cyto-2X protein complex did not contain p80 or p130 (Fig. 1E). We further examined whether Amot binds to Cad11-cyto directly by using purified Amot-His₇ protein from E. coli in pulldown assays. As shown in Fig. 1F, Amot-His₇ protein was pulled down by GST-Cad11-cyto-2X but not by GST or GST-E-Cad-cyto-2X, indicating that Amot binds specifically and directly to Cad11-cyto. Together, these results suggest that Amot is a novel Cad11-interacting protein.

Amot binds to the Cad11 CBS domain

We next examined the sequence in Cad11 cyto domain that interacts with Amot. Previous studies have established that Cad11 has 2 protein binding domains, juxtamembrane domain (JMD) and CBS, which bind p120 and β-cat, respectively (4, 21). To identify the Amot binding domain, we used GST Cad11 WT and mutant cyto constructs (Fig. 2A). GST-Cad11-cyto-ΔJMD-2X bound β-cat but not p120 from HEK293T lysate, whereas GST-Cad11-cyto-ΔCBS-2X bound p120 but not β-cat as expected (Fig. 2B). These cytoplasmic deletion mutants were then used to bind p80-Amot-YFP (Amot-YFP) expressed in HEK293T. Amot-YFP bound to Cad11-cyto-WT-2X and Cad11-cyto-ΔJMD-2X but not Cad11-cyto-ΔCBS-2X, even though a higher concentration of Cad11-cyto-ΔCBS-2X was used (Fig. 2C). Similar results were observed using Flag-tagged p80-Amot (Amot-Flag) expressed in HEK293T cells (Fig. 2D) or Amot-His₇ (Fig. 2E) expressed from E. coli to bind the GST cyto constructs. These observations indicate that the Amot binding site is within the Cad11 CBS domain.

Figure 2.

Amot binding site is within the CBS domain of Cad11. A) GST fusion protein constructs. Two copies of Cad11-cyto domain with deletion in JMD (Cad11-cyto-ΔJMD-2X) or CBS (Cad11-cyto-ΔCBS-2X) were inserted after GST. B) GST-Cad11-cyto-ΔJMD-2X or GST-Cad11-cyto-ΔCBS-2X pulled down β-cat and p120, respectively, from HEK293T cell lysates. Representative of 4 experiments. C) Amot binding to JMD or CBS domain deletion mutants. Cad11-cyto deletion mutants were incubated with HEK293T cells transfected with Amot-YFP fusion protein in GST pulldown assays. Amot was found to bind to Cad11-cyto-WT-2X and Cad11-cyto-ΔJMD-2X, but not Cad11-cyto-ΔCBS-2X. Representative of 4 experiments. D) Flag-tagged Amot-p80 (Amot-Flag) expressed in HEK293T cells was used in GST pulldown assays. Representative of 3 experiments. E) Purified p80-Amot-His was used to show direct interaction with GST-Cad11-cyto-WT-2X but not GST-Cad11-cyto-ΔCBS-2X. Representative of 3 experiments.

Mapping the Amot binding motif within the CBS domain of Cad11

Because both Amot and β-cat bind the Cad11 CBS domain, CBS deletion mutants were used to examine their binding sites (Fig. 3A). The Cad11 CBS mutants were incubated with Amot-YFP expressed in HEK293T cells. As shown in Fig. 3B, deletion of the last 10 aa from Cad11-cyto (cyto 146–156) resulted in loss of Amot binding, suggesting that the Cad11 CBS sequence GSKDTFDDDS (Fig. 3D) is involved in binding Amot. Sequence alignment comparing Cad11, E-Cad, N-cadherin, VE-cadherin, and P-cadherin cyto showed that the GSKDTFDDDS sequence is unique to Cad11 (Fig. 3D). This finding may explain the observation that Amot binds to Cad11-cyto but not E-Cad-cyto.

Figure 3.

Delineating Amot and β-cat binding sequences within the Cad11 CBS domain. A) Diagram of Cad11 deletion mutants within the CBS domain and summary of Amot and β-cat binding to the deletion mutants. B) Deletion of the last 10 amino acids from the C terminus of Cad11-cyto (cyto 1–146) resulted in loss of Amot binding. Representative of 3 experiments. C) β-Cat was found to bind to all cyto mutants except cyto-ΔCBS (cyto 1–99). Representative of 2 experiments. D) Sequence comparison of Cad11, E-Cad, N-cadherin, VE-cadherin, and P-cadherin cyto domains. β-Cat binding sequences are conserved among these cadherins. However, Amot binding sequence is unique in Cad11-cyto domain. An asterisk indicates conservation across cadherins; colon, identical or similar in size or charge; dot, conserved in most cadherins.

When the Cad11 CBS deletion mutants were incubated with HEK293T lysates, β-cat bound to all Cad11-cyto mutants except Cad11-cyto-ΔCBS (1–99) (Fig. 3C), suggesting that the β-cat binding site is near the aa 100–106. The corresponding sequence DSIQIYGYEG (Fig. 3D) coincides with the β-cat binding sequence determined by E-Cad deletion analysis and by crystal structure studies (22, 23). These observations showed that β-cat and Amot bind to adjacent but nonoverlapping sequences in the Cad11 CBS domain.

Middle domain of Amot binds Cad11

Amot contains 2 amphipathic helix–rich CC domains, CC1 and CC2, in the N terminus and a PDZ binding motif, YLI, at the end of the C terminus (24) (Fig. 4A). These domains are present in both p130 and p80. The p130 isoform has a 409 aa extension in its N terminus that contains 2 PPEY and 1 LPTY motifs (14) (Fig. 4A). Previous studies have shown that Amot interacts with various proteins through these domains to modulate different cellular functions. We examined whether Cad11 interacts with Amot through these Amot protein binding domains. GST-Cad11-cyto-2X agarose beads were incubated with a series of 3xFlag tagged Amot mutants expressed in HEK293T cells (Fig. 4A). We found that Cad11-cyto preferentially binds the p80 isoform than p130 (Fig. 4B). As Wells et al. (12) and Ernkvist et al. (25) have previously shown that p80 can form heterodimers with p130, the pulldown of both p80 and p130 by Cad11-cyto-2X from C4-2B4 cells (Fig. 1E) may be due to formation of p80/p130 heterodimers. Truncations of the CC1 domain (mutant 1), deletions of both CC1 and CC2 domains (mutant 2), or deletion of the PDZ motif (mutant 5) did not affect Amot binding to Cad11-cyto-2X (Fig. 4B). These results suggest that Cad11 does not interact with the Amot N terminus CC domains or C terminus PDZ motif. In support of this notion, Amot mutant proteins containing only CC1 (mutant 3) or CC1 + CC2 (mutant 4) domains were not able to interact with Cad11-cyto-2X (Fig. 4B). These results suggest that Cad11 interacts with the middle domain of Amot (Fig. 4C). The p80 middle domain has not been extensively studied. Analysis of the p80 middle domain using the Simple Modular Architecture Research Tool (SMART; http://smart.embl.de/) program did not reveal a recognizable functional motif. Thus, how the middle domain of p80 interacts with Cad11 remains to be determined.

Figure 4.

Cad11 binds to Amot-p80 middle domain. A) Amot mutant constructs and their binding to Cad11-cyto-2X domain. Amot-p130 contains LPTY and 2 PPEY motifs that are absent in Amot-p80. Both p130 and p80 contain 2 CC domains (CC1, CC2) and a C terminus PDZ motif containing amino acid sequence YLI. Five p80 mutants containing various deletions as indicated were used in the study. B) Amot mutant constructs with 3xFlag tags were transfected into HEK293T cells and the cell lysates were used in GST-Cad11-cyto-2X pulldown assays. Representative of 4 experiments. UT, untransfected control. Arrowhead, GST-Cad11-cyto-2X. C) Schematic diagram of Cad11-Amot-p80 interaction. TM, transmembrane domain.

Amot binds to Cad11 in mammalian cells

Next, we examined the interaction of Amot and Cad11 in mammalian cells. We expressed 3xFlag-tagged Cad11-cyto (Cad11-cyto-2X-Flag) and Amot-YFP in HEK293T. Immunoprecipitation of Cad11 with anti-Flag antibody pulled down Amot-YFP while control IgG did not (Fig. 5A), confirming that Amot binds to Cad11 cyto expressed in mammalian cells.

Figure 5.

Interaction of Amot with Cad11 in mammalian cells. A) Plasmids encoding Cad11-cyto-2X-Flag and Amot-YFP were cotransfected into HEK293T cells. Immunoprecipitation of Cad11-cyto-2X-Flag by anti-Flag antibody pulled down Amot-YFP. Representative of 3 experiments. IgG was used as a control. B) Western blot for the expression of Amot using total cell lysates from HEK293, C4-2B4, and PC3-mm2 cells. Representative of 3 experiments. Two exposure times were used to detect Amot signals. A longer 1 h exposure was necessary to detect Amot signals (arrowheads) in PC3-mm2 cells. C) Cad11 is associated with Amot-p80 in mammalian cells. Triton X-100 solubilized C4-2B4/Cad11 or PC3-mm2 cell lysates were immunoprecipitated with anti-Cad11 mAb 1A5. Representative of 5 experiments. Western blot analysis with anti-Amot showed that Amot-p80 is associated with Cad11 in PCa cells. D) Triton X-100 solubilized C4-2B4/Cad11 or PC3-mm2 cell lysates were immunoprecipitated with anti-Amot antibody. Representative of 4 experiments. Western blot analysis with anti-Cad11 showed that Cad11 associates with Amot in PCa cells.

To examine whether Amot interacts with Cad11 in PCa cells, we examined the expression of Amot in C4-2B4 and PC3-mm2 cells using as a control HEK293T cells, which are known to express both p130 and p80. As shown in Fig. 5B, C4-2B4 expressed both p130 and p80 with levels similar to those found in HEK293T. In contrast, PC3-mm2 expressed very low levels of p130 and p80, which were only detectable upon long exposure of the Western blot (Fig. 5B, arrowheads), or using immunoprecipitation (Fig. 5C, D).

Because C4-2B4 does not express Cad11 endogenously, C4-2B4 cells transfected with Cad11 (C4-2B4/Cad11) were used for an immunoprecipitation assay using the anti-Cad11 mAb 1A5 (18). p80, but not p130, was found in the mAb 1A5 immunoprecipitation complex (Fig. 5C, top left), consistent with the observation that Cad11-cyto pulled down p80 (Figs. 1E and 4B). Furthermore, immunoprecipitation of Cad11 from PC3-mm2 cells, which endogenously express Cad11 (2) but very low levels of Amot, also brought down p80 (Fig. 5C, top right).

We also performed reciprocal immunoprecipitation assays. When C4-2B4/Cad11 or PC3-mm2 lysates were immunoprecipitated with anti-Amot antibody, Cad11 was found in the Amot immune complex from C4-2B4/Cad11 cells and PC3-mm2 (Fig 5D). As both p130 and p80 are expressed in C4-2B4/Cad11 cells, anti-Amot antibodies immunoprecipitated both Amot isoforms as expected (Fig 5D, bottom left). In PC3-mm2 cells, which express very low levels of Amot on Western blot analysis (Fig. 5B), anti-Amot antibodies immunoprecipitated proteins with apparent molecular mass of 100 and 80 kDa (Fig. 5D, bottom right). Because it has been reported that the Amot-p130 isoform is susceptible to proteolysis to generate a 100 kDa band (26–28), it is likely that the 100 kDa protein may be a p130 breakdown product generated during the overnight incubation. Together, these observations suggest that Amot associates with Cad11 in PCa cells.

Amot in Cad11-mediated cell adhesion

Next, we examined the role of Amot in Cad11-mediated functions. Because Amot is known to be involved in multiple cellular functions, including cell polarity (12), migration (13), and Hippo pathway (15), we deleted Amot binding sequence from Cad11, rather than knocking down Amot, as one way to investigate the functional outcome of Cad11-Amot interaction. Previous studies have shown that Cad11 expression increases adhesion and migration of PCa cells (4). To examine the role of Amot in Cad11-mediated cell-cell adhesion, Cad11 with deletion of the 10 aa Amot-binding sequence GSKDTFDDDS located at the very C terminus (Cad11-ΔAmot) in the cyto domain was generated (Fig. 6A). Cad11 with deletion of the 10 aa β-cat binding sequence DSIQIYGYEG (Cad11-Δβ-cat) was used as a control. L cells are deficient in adhesion molecules, such as cadherins (29), and therefore are frequently used for aggregation assays. L cells were transduced with bicistronic retroviral vectors containing Cad11-WT or Cad11-ΔAmot and selected by neomycin. L cells transduced with empty vector or Cad11-Δβ-cat were generated in parallel and used as controls. Western blot analysis of L cells with Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat constructs showed the Cad11 proteins were expressed although lower Cad11-Δβ-cat levels were observed (Fig. 6B). FACS analysis on intact cells using monoclonal antibody mAb 2C7 (18) that recognizes the extracellular domain of Cad11 showed cell surface expression of the Cad11 proteins (Fig. 6C). Immunofluorescence analysis further showed that both Cad11-WT and Cad11-ΔAmot were mainly localized at the cell–cell junction in L cells (Fig. 6D), consistent with a role of Cad11 in mediating cell–cell adhesion. Interestingly, an increase in intracellular accumulation of Cad11-Δβ-cat was observed in L cells (Fig. 6D). It has been reported that β-cat plays an important role in membrane targeting of cadherin proteins (30). Deletion of the β-cat binding site in Cad11 may result in the decreased Cad11-Δβ-cat expression observed in Western blot analysis (Fig. 6B), FACS (Fig. 6C), and immunofluorescence imaging (Fig. 6D). Significant changes in cell morphology were also observed in L cells transfected with Cad11-Δβ-cat in that L-Cad11-Δβ-cat cells were more scattered than L-Cad11-WT or L-Cad11-ΔAmot cells (Fig. 6D). This is likely due to a role of β-cat in linking cadherin to cytoskeletal structures (31, 32).

Figure 6.

Cad11 mutants on Cad11-mediated cell adhesion. A) Cad11 WT and mutant constructs. B) Western blot of Cad11 and Amot expression in L cells transfected with Cad11 mutants. Representative of 2 experiments. C) FACS of L cells transfected with Cad11 mutants using anti-Cad11 mAb 2C7, which binds to an epitope on Cad11 extracellular domain. D) Immunofluorescence analysis showing Cad11 and Amot protein expression and cellular localization. Representative of 2 experiments. Green, Cad11; red, Amot; blue, DAPI for DNA. Scale bar, 20 μm. E) Cell aggregation assay showing effects of WT and mutant Cad11 expression on homophilic interaction in L cells. Representative of 3 experiments. *P < 0.05. F) Effects of WT and mutant Cad11 expression on adhesion strength by pipetting (blue arrow) once or twice after 3 h. G) Effects of WT and mutant Cad11 expression on adhesion strength by pipetting (blue arrow) once or twice after 24 h.

A cell–cell aggregation assay showed that both Cad11-ΔAmot and Cad11-Δβ-cat mutants were able to confer cell aggregation activity similar to that of Cad11-WT when the aggregation was measured at 3 h (Fig. 6E). It has been suggested that β-cat interaction with VE-cadherin regulates the steady-state adhesive strength in endothelial cells (33). Thus, we examined the effect of Cad11 deletions on the kinetics of adhesion and stability of adhesion. We found that Cad11 lacking Amot or β-cat binding domain formed aggregates slower than that of Cad11-WT when the time course of Cad11-mediated adhesion was performed, although they reached similar levels of aggregation by 3 h (Fig. 6F). In addition, we found that the aggregates from Cad11 lacking Amot or β-cat binding domain can be more easily dispersed by mechanical disruption, such as pipetting once or twice, compared to that of Cad11-WT (Fig. 6F). When the adhesion assay was performed for 24 h, Cad11-WT showed the highest level of aggregation and remained largely aggregated after pipetting once or twice (Fig. 6G). In contrast, the aggregates from Cad11 lacking Amot or β-cat binding domain were more easily dispersed similar to those observed in the 3 h aggregation assay (Fig. 6G). These observations indicate that Cad11 lacking Amot or β-cat binding domain can form aggregates in suspension. However, these aggregates are poorly adhesive and can be easily dispersed. Thus, Cad11-Amot interaction affects the adhesion strength, but not the adhesion, of Cad11-mediated cell adhesion. Although L-Cad11-Δβ-cat cells showed scattered morphology in 2-D culture (Fig. 6D), Cad11-Δβ-cat was able to mediate cell–cell adhesion in the aggregation assay (Fig. 6E–G), suggesting that cell morphology in 2-dimensional culture and cell aggregation in solution reflect independent cellular events.

Amot in Cad11-mediated cell migration

We then determined the role of Amot on Cad11-mediated migration in PCa cells. C4-2B4 cells were transduced with Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat retroviral vectors. Western blot analysis showed that Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat proteins were expressed (Fig. 7A). Immunoprecipitation of Cad11 proteins in C4-2B4 showed that Cad11-WT and Cad11-Δβ-cat bound p80, while Cad11-ΔAmot did not (Fig. 7B). Cad11-WT and Cad11-ΔAmot bound β-cat while Cad11-Δβ-cat did not, as expected (Fig. 7B). FACS analysis on intact nonpermeabilized cells using mAb 2C7 showed that the Cad11 mutants were localized at the plasma membrane, although the mean fluorescence intensity of Cad11-Δβ-cat was lower than that observed in Cad11-WT and Cad11-ΔAmot (Fig. 7C). Immunostaining further confirmed that all 3 Cad11 proteins were localized on the plasma membrane in C4-2B4 cells (Fig. 7D). We noted high levels of intracellular Cad11-Δβ-cat protein (Fig. 7D), similar to those observed in L cells expressing Cad11-Δβ-cat (Fig. 6D). Interestingly, differences in cell morphology were observed among C4-2B4 cells with Cad11 mutants, although the reason for these morphologic changes is unclear. We then examined the effects of Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat on C4-2B4 cell migration, measured as cells per field in a Boyden chamber migration assay as described previously (4). As shown in Fig. 7E, Cad11-ΔAmot expression significantly reduced Cad11-mediated migration in C4-2B4 cells compared to Cad11-WT cells (127 ± 6 vs. 50 ± 23, P < 0.05).

Figure 7.

Effect of Cad11 mutants on migration of C4-2B4 cells. A) Western blot of Cad11 expression in C4-2B4 cells transfected with Cad11 mutants using anti-Cad11 antibody 5B2H5. B) Immunoprecipitation using anti-Cad11 mAb to show Amot and β-cat binding to Cad11 in Cad11 WT and mutant constructs expressed in C4-2B4 cells. C) FACS analysis of Cad11 expression on cell surface in C4-2B4 cells. D) Immunofluorescence showing Cad11 and Amot protein expression and cellular localization. Scale bar, 20 μm. E) Migratory activity of C4-2B4 cells expressing Cad11 WT or mutants. Representative of 4 experiments. *P < 0.05.

To further examine the effect of Amot in Cad11-mediated migration, we knocked down the endogenous Cad11 in PC3-mm2 cells by an shRNA located at the 3′-untranslated region of Cad11 cDNA in pLKO vector and reexpressed Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat in PC3-shCad11 cells using bicistronic retroviral vector containing GFP. PC3-shCad11 transduced with empty pBMN-I-GFP was used as a control (PC3-shCad11-pBMN). Western blot analysis showed that knockdown of Cad11 in PC3 cells (PC3-shCad11) significantly reduced the endogenous level of Cad11 compared to the control vector transfected PC3-pLKO cells (Fig. 8A). Western blot analysis also confirmed the expression of Cad11 protein in the PC3-shCad11 cells reexpressed with Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat (Fig. 8A). Immunostaining showed that the Cad11 proteins again localized to the plasma membrane (Fig. 8B). The cells were also costained for Amot and β-cat. Expression of Cad11 mutants did not alter the distribution of Amot or β-cat (Fig. 8B). Because both Amot and β-cat have been shown to interact with multiple proteins, expression of Cad11 mutants may not be able to change their distribution within the cells. In addition, we observed differences in cell morphology of Cad11-transduced PC3-shCad11 cells compared to Cad11-transduced L cells (Fig. 6D) and C4-2B4 cells (Fig. 7D), probably attributed to differences in the composition of adhesion molecules present in these cells. These cells were then used to study Cad11 mediated migration. Similar to those observed in C4-2B4 cells, reexpression of Cad11-WT in PC3-shCad11 cells significantly increased the migratory activity compared to PC3-shCad11-pBMN cells (676 ± 98 vs. 160 ± 25, P < 0.05). In contrast, reexpression of Cad11-ΔAmot in PC3-shCad11 cells significantly reduced Cad11-mediated cell migration (676 ± 98 vs. 267 ± 11, P < 0.05) (Fig. 8C). Expression of Cad11-Δβ-cat in PC3-shCad11 cells also increased migration with activity similar to that observed in Cad11-WT (714 ± 21 vs. 676 ± 98) (Fig. 8C).

Figure 8.

Effect of Cad11 mutants on migration of PC3-mm2 and HEK293T cells. A) Western blot of Cad11 WT and mutant proteins in PC3-mm2 cells. PC3-mm2 cells were transduced with Cad11 shRNA in lentiviral vector pLKO.1-puro to knockdown endogenous Cad11. PC3-shCad11 cells were then transduced with Cad11 WT or mutants in bicistronic retroviral vector pBMN to reexpress Cad11 proteins. B) Immunofluorescence analysis of PC3-pLKO, PC3-shCad11, and PC3-shCad11 cells expressing empty vector pBMN, Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat constructs. Cells were stained for Cad11, Amot, and β-cat. Representative of 2 experiments. Scale bar, 20 μm. C) Migratory activity of PC3-mm2 cells reconstituted with Cad11 WT or mutants. Representative of 3 experiments. D) Western blot analysis of HEK293T cells transduced with Cad11 constructs. E) Migratory activity of HEK293T cells expressing Cad11 WT or mutants. Representative of 2 experiments. *P < 0.05.

Finally, we examined whether Amot also plays a role in Cad11-mediated migration in non-PCa cell line. HEK293T cells endogenously express Amot but not Cad11. HEK293T cells were transduced with Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat (Fig. 8D). Deletion of the Amot binding site abolished Cad11-mediated migration in HEK293T-Cad11 cells (50 ± 13 vs. 17 ± 1, P < 0.05) (Fig. 8E), as was also observed in PCa cell lines (Figs. 7E and 8C). Taken together, these observations indicate that Amot plays a role in Cad11-mediated cell migration.

DISCUSSION

We have identified Amot as a novel Cad11 interacting protein and have shown that this interaction plays a role in regulating Cad11-mediated cell migration in PCa cells. In addition, we showed that Amot binding is unique to Cad11, providing a molecular basis for the differences in cell migratory activities observed between Cad11 and E-Cad. A hallmark of the epithelial-mesenchymal transition is a loss of E-Cad expression and an increase in cell migration. During the epithelial–mesenchymal transition, cells switch from expressing E-Cad to mesenchymal cadherins, including Cad11 (1). Thus, our study provides a mechanism by which a cadherin switch promotes cell migration.

How the Cad11-Amot interaction increases cell migration is unknown. Studies on endothelial cell migration by Ernkvist et al. (13) showed that Amot uses its C terminus PDZ motif, specifically the sequence YLI, to bind to the third PDZ domain of the scaffold protein Patj. Patj associates with the guanidine exchange protein Syx through its tenth PDZ domain to form a signaling complex that regulates endothelial cell migration by directing RhoA activity to the leading edge of the cell. Similarly, Wu et al. (34) showed that Amot interacted with Mupp1 to recruit Syx, which activates RhoA to increase endothelial cell migration. As Cad11 binds to Amot through an unknown region of the middle domain in Amot, the PDZ motif in Amot is left available to interact with the PatJ/Syx/RhoA migration complex (13). Thus, it is likely that the Cad11-Amot interaction increases cell migration through the binding of Amot PDZ motif to the PatJ/Syx/RhoA complex. Alternatively, it has been shown that the Amot/Rich1 complex promotes Rac1-mediated activity (12, 15). Amot interacts with Rich1 through N-terminal CC domain. Thus, it is also possible that the Cad11-Amot interaction increases cell migration through the binding of Amot N-terminal CC domain to Rich1/Rac1 complex. Taken together, Amot may serve as an adaptor protein to connect Cad11 to the cytoskeletal migration machinery.

Amot is expressed as 2 different isoforms, p80 and p130, which possess distinct functions. Whereas the shorter p80 isoform enhances cell migration (13, 24), the p130 isoform associates with F-actin and affects cell shape (35). Several lines of evidence indicate that Cad11 preferentially interacts with Amot-p80. First, mass spectrometry analysis of Cad11-cyto interacting proteins identified 2 peptides that belonged to Amot, not Amot-L1 or Amot-L2. Then, GST-pulldown assays using p130 or p80 expressed in HEK293T cells showed that Cad11-cyto interacts mainly with p80. Furthermore, immunoprecipitation analysis found that p80, but not p130, coimmunoprecipitated with Cad11 in both PC3-mm2 and C4-2B4/Cad11 PCa cells. How p80 interacts with Cad11 remains to be determined. The identified Amot binding motif on Cad11 consists of an aspartic acid–rich sequence that may interact with positive charges on Amot. Interestingly, there is a cluster of arginine and histidine residues in the beginning of the middle domain of Amot (i.e., aa 337–342, 358–365, and 404–411), which may contribute to Amot/Cad11 binding. On the other hand, a lysine residue and a phenylalanine residue are uniquely present in the C-terminal Amot binding site of Cad11, but not in other cadherins (Fig. 3D). These residues may play important roles in Cad11-Amot interaction. Further studies will be needed to verify these possibilities.

Because Amot is a novel Cad11 interacting protein, we also considered whether it might have an effect on the recruitment of the other cadherin cytoplasmic partners (i.e., p120 catenin, α-catenin, and β-cat) to Cad11. Using immunofluorescence analysis, we examined the localization of α-catenin, β-cat, and p120 in C4-2B4 cells expressing Cad11-WT, Cad11-ΔAmot, or Cad11-Δβ-cat. We did not detect significant differences in the distribution of p120 catenin, α-catenin, and β-cat in C4-2B4 expressing the various Cad11 mutants (Supplemental Fig. S1). This is likely due to the presence of E-Cad and other cadherins in C4-2B4 cells (18). We performed a similar analysis in PC3-mm2 cells expressing the various Cad11 mutants. We also did not detect significant differences in the distribution of p120 catenin and β-cat in PC3-mm2 expressing the various Cad11 mutants (Supplemental Fig. S2). Furthermore, it has been previously shown that PC3 cells contain a deletion of the gene that encodes α-catenin (36), which may explain the nonspecific diffuse staining pattern for α-catenin in PC3-mm2 cells.

Although most cadherin family proteins interact with β-cat and p120, they are also known to recruit different proteins to the cadherin protein complex to mediate diverse cellular functions. E-Cad has also been shown to interact with presenilin (37) and Hakai (38, 39). Other cadherins also possess their own intracellular partners in addition to β-cat and p120 (40–47). Cadherin family protein interactions with their binding proteins are tightly regulated. For example, Xu et al. (22) show that dephosphorylation of β-cat by PTP1B is required for β-cat binding to N-cadherin. What regulates Cad11/Amot binding is not yet clear. It is possible that posttranslational modifications such as phosphorylation/dephosphorylation of β-cat or Cad11 are involved in regulating the binding of Amot to Cad11. Our studies showed that Amot and β-cat bind to distinct amino acids sequences that are about 40 amino acids apart within the Cad11 CBS domain. Because the molecular weights of both Amot and β-cat are relatively large, we considered that the proximity of their binding sites may cause steric hindrance and that Cad11-Amot interaction may be regulated by Cad11/β-cat status. However, we did not detect an increase in the binding of Amot to Cad11-Δβ-cat or binding of β-cat to Cad11-ΔAmot compared to their binding to Cad11 WT in immunoprecipitation assays (Fig. 7B). How Amot binding to Cad11 is regulated is currently being investigated.

Amots are adaptor proteins that are involved in many cellular functions besides interacting with Cad11. To examine whether Amot has an impact on cell migration independent to Cad11’s function, we altered Amot levels by overexpressing Amot-p80 in PC3-mm2 cells, which express very low levels of Amot, and by Amot knockdown in C4-2B4 cells, which express high levels of Amot. We did not detect differences in cell migration by either increasing Amot-p80 in PC3-mm2 cells (Supplemental Fig. S3A) or knocking down Amot in C4-2B4 cells (Supplemental Fig. S3B). Because C4-2B4 does not express Cad11, these observations suggest that Amot does not have a direct effect on cell migration but is an important adaptor protein for Cad11-mediated cell migration.

Amot-p80 has been shown to play a role as tumor promoter, as its expression enhances endothelial invasion and stabilizes established tubes (48). Knockdown of Amot in NF2 mutant cells inhibited the tumorigenicity of NF2 cells (15). Amot was also shown to be up-regulated in aggressive breast cancer, and high levels of Amot transcript were correlated with shorter overall survival of the patients (49). Consistent with these observations, our studies showed that Amot-p80 increases Cad11-mediated migration of PCa cells. Together, our study identified Amot as a novel Cad11 interacting protein and elucidated the Cad11-Amot interaction as an important component in PCa cell migration.

Glossary

Amot

angiomotin

Cad11

cadherin-11

CBS

C-terminal binding sequence

CC

coiled–coil domain

cyto

cytoplasmic

E-Cad

E-cadherin

FACS

fluorescence-activated cell sorting

JMD

juxtamembrane domain

p120

p120-catenin

PCa

prostate cancer

β-cat

β-catenin

WT

wild type