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Identification of a Novel β-Catenin-Interacting Protein
Biochemical and Biophysical Research Communications 273, 712–717 (2000) doi:10.1006/bbrc.2000.3002, available online at http://www.idealibrary.com on
Identification of a Novel -Catenin-Interacting Protein 1 Aie Kawajiri,* ,2 Naohiro Itoh,* ,2 Masaki Fukata,* ,† Masato Nakagawa,* Masaki Yamaga,* Akihiro Iwamatsu,‡ and Kozo Kaibuchi* ,† ,3 *Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101, Japan; †Department of Cell Pharmacology, Nagoya University School of Medicine, 65 Tsurumai, Showa, Nagoya, 466-8550, Japan; and ‡Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5 Fukuura, Kanazawa, Yokohama, 236-0004, Japan
Received May 13, 2000
Cadherin is a well-known cell– cell adhesion molecule, and it binds to -catenin, which in turn binds to ␣-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell– cell adhesion. Here we purified two novel -catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)–catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313 which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain and p150 to be the same as KIAA0705 which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM), which binds -catenin and KIAA0313 in the yeast twohybrid system and overlay assay, respectively (Ide et al., Biochem. Biophys. Res. Commun. 256, 456 – 461, 1999; Ohtsuka et al., Biochem. Biophys. Res. Commun. 265, 38 – 44, 1999). -Catenin was coimmunoprecipitated with KIAA0313 in Madin–Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and -catenin were partly colocalized at sites of cell– cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with -catenin through KIAA0705 in vivo at sites of cell– cell contact. © 2000 Academic Press
Abbreviations used: GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; aa, amino acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PBS, phosphatebuffered saline; MDCKII, Madin–Darby canine kidney II. 1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (1999) and by grants from the program Research for the Future of the Japan Society for the Promotion of Science, the Human Frontier Science Program, and Kirin Brewery Company Limited. 2 These authors contributed equally to this article. 3 To whom correspondence should be addressed at Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101, Japan. Fax: 0743-72-5449. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Key Words: -catenin; KIAA0313; KIAA0705; Rap1 GEF.
Cell– cell adhesion is dynamically rearranged in various situations including the establishment of epithelial polarity and developmental morphogenesis (1–3). Cadherin is a calcium-dependent cell– cell adhesion molecule that binds to -catenin, which in turn binds to ␣-catenin (cadherin/catenins complex) (4, 5). This cadherin/catenins complex is linked to the actin cytoskeleton, and the linkage is essential for proper cadherin function (5). However, little is known about the regulatory mechanism underlying cadherin-mediated cell– cell adhesion. Recent studies showed that the Rho family of small GTPases, Cdc42, Rac1, and Rho, participate in the regulation of cadherin-mediated cell– cell adhesion (6 –9). We recently found that IQGAP1, an effector of Cdc42 and Rac1, negatively regulates cadherinmediated cell– cell adhesion and that activated Cdc42 and Rac1 positively regulate cadherin-mediated cell– cell adhesion by inhibiting IQGAP1 function (10, 11). Dimerization of cadherin is another crucial contributor to the cadherin-mediated cell– cell adhesion. The mechanism, however, underlying dimer formation remains to be elucidated. Recent studies suggest that the membrane-proximal region of the cadherin cytoplasmic domain, which is separate from the distal -catenin-binding domain, regulates cadherin dimerization and activity. The membrane-proximal region inhibits dimerization and adhesive activity (12). Given that p120 ctn, a member of the Armadillo/-catenin family, binds to the membrane-proximal region of the cadherin cytoplasmic domain (12, 13) and inhibits cadherin activity in some carcinoma cell lines (14, 15), p120 ctn might modulate the cadherin activity. However, it remains to be clarified whether cadherin-mediated
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cell– cell adhesion is regulated only by Cdc42/Rac1/ IQGAP1 system and p120 ctn. Here, to further understand the mechanism underlying the cadherin-mediated cell– cell adhesion, we purified two novel -catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using GST–catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313, which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain, and p150 to be KIAA0705, which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM) (16). In this study, we focused on the association of KIAA0313 with -catenin in vivo, and found that -catenin could be coimmunoprecipitated with KIAA0313 and colocalized with KIAA0313 at sites of cell– cell contact. MATERIALS AND METHODS Materials and chemicals. EL cells and MDCKII cells were kindly provided by Drs. A. Nagafuchi and Sh. Tsukita (Kyoto University, Kyoto, Japan). The cDNAs encoding mouse ␣-catenin, mouse -catenin, and mouse E-cadherin were kindly provided by Drs. A. Nagafuchi and Sh. Tsukita (Kyoto University, Kyoto, Japan). The cDNA encoding KIAA0313 was kindly provided by Drs. T. Nagase and N. Nomura (Kazusa DNA Research Institute, Kisarazu, Japan). Polyclonal antiKIAA0313-N and anti-KIAA0313-C antibodies were generated against recombinant GST–KIAA0313-N (aa 1–250) and GST–KIAA0313-C (aa 935–1499), respectively. Anti--catenin antibody was purchased from Transduction Laboratories (Lexington, KY). Anti-ZO-1 antibody was purchased from CHEMICON International Inc. (Temecula, CA). All materials used in the nucleic acid study were purchased from Takara Shuzo Co. Ltd. (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources. Plasmid constructions. pGEX4T-1-cytoplasmic domain of mouse E-cadherin (aa 734 – 884), -mouse ␣-catenin (aa 1–906), and -mouse -catenin (aa 1–781) were produced as described previously (10). To obtain the GST–KIAA0313-N (aa 1–250) and GST–KIAA0313-C (aa 935–1499) as antigens, we subcloned their cDNA fragments into pGEX4T-1. Preparation of recombinant proteins. The expression and purification of various GST-fusion proteins were done as previously described (17). GST– cytoplasmic domain of E-cadherin, GST–␣-catenin, and GST–-catenin affinity column chromatography. The affinitypurification was performed essentially as described (17). Briefly, bovine brain cytosol was passed through a column of glutathione beads to remove endogenous GST. Then, the passed fraction was loaded onto a column of glutathione beads (200 l) coated with GST– cytoplasmic domain of E-cadherin, GST–␣-catenin, or GST–-catenin (each 6.5 nmol). The columns were washed with 2 ml (10 volumes) of buffer A (20 mM Tris/HCl at pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, and 10 M [p-amidinophenyl]-methanesulfonyl fluoride). The proteins bound to the affinity columns were eluted three times by the addition of 660 l (3.3 vol) of buffer A containing 500 mM NaCl. Mass spectral analysis. The 500 mM NaCl eluate from the GST– -catenin affinity column was dialyzed against distilled water and concentrated by freeze-drying. The concentrated samples were electrophoresed on a 6% polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Problot, Applied Biosystems). The immobilized proteins were reduced, S-carboxy-
FIG. 1. Purification of -catenin-interacting proteins. (A) Bovine brain cytosol was loaded onto glutathione-Sepharose columns coated with the indicated GST-fusion proteins. The bound proteins were eluted by the addition of buffer A containing 500 mM NaCl. Aliquots of the eluates were resolved by SDS–PAGE followed by silver staining. Arrowheads from the top denote the positions of p250, p180, p150, p120, and p110, respectively. (B) Aliquots of the eluates were resolved by SDS–PAGE followed by immunoblotting with antiKIAA0313-N antibody. The arrow denotes the position of KIAA0313. The results are representative of three independent experiments. methylated, and digested in situ with Achromobacter protease I (a Lys-C) (18). Molecular mass analyses of Lys-C fragments were performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using a PerSeptive Biosystem Voyager-DE/RP (19). Identification of proteins was carried out by comparison between the molecular weights determined by MALDITOF/MS and theoretical peptide masses of the proteins registered in the NCBInr. Cell culture. EL cells were grown in Dulbecco’s modified Eagles medium (DMEM) containing 10% fetal bovine serum and 100 g/ml of G418 at 37°C in an air–5% CO 2 atmosphere at constant humidity (20). MDCKII cells were grown in DMEM containing 10% calf serum at 37°C in an air–5% CO 2 atmosphere at constant humidity. Coimmunoprecipitation of -catenin with KIAA0313. Immunoprecipitation was performed as described previously (10). Briefly, subconfluent MDCKII or EL cells were harvested and lysed with lysis buffer (20 mM Tris/HCl at pH 7.4, 50 mM NaCl, 10 M [p-amidinophenyl]-methanesulfonyl fluoride, 10 g/ml leupeptin, 0.5% [w/v] Triton X-100, 1 mM CaCl2). The lysates and bovine brain cytosol were clarified by centrifugation at 100,000g for 30 min at 4°C. The soluble supernatants were incubated with 20 g of anti-KIAA0313-N antibody and incubated for 1 h at 4°C. The immunocomplexes were then precipitated with 40 l of protein A Sepharose 4B. The immunocom-
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FIG. 2. Structure of KIAA0313 and KIAA0705. (A) Schematic drawing showing the localization of domains and the percent amino acid sequence identity between KIAA0313 and Epac. The GenBank accession no. of KIAA0313 is AB002311. REM: Ras exchange motif, PDZ: named after three proteins, PSD-95/SAP90, Dlg-A, and ZO-1, RA: Ras association domain, GEF: guanine nucleotide exchange factor domain, DEP: named after three proteins, Disheveled, Egl-10, and Pleckstrin. (B) Schematic drawing showing the localization of domains and the percent amino acid sequence identity between KIAA0705 and S-SCAM. The GenBank accession no. of KIAA0705 is AB014605. WW, domain consisting of two separate tryptophans and invariant proline; GK, guanylate kinase-like domain. plexes were washed five times with lysis buffer, eluted by boiling in sample buffer for SDS–PAGE, and subjected to immunoblot analysis with anti-KIAA0313-N and anti--catenin antibodies. Immunofluorescence analysis. Subconfluent MDCKII cells were fixed with ice-cold 100% methanol for 15 min on ice. After having been washed with ice-cold PBS, the cells were then incubated with anti-KIAA0313-N polyclonal antibody for 1 h at room temperature. The cells were washed with PBS, and subsequently incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit Ig Ab for 1 h at room temperature. Next, the cells were incubated with anti-catenin monoclonal or anti-ZO-1 monoclonal antibody for 1 h at room temperature. After washes with PBS, the cells were incubated with Texas Red-conjugated anti-mouse Ig Ab for -catenin or antirat Ig Ab for ZO-1 for 1 h at room temperature. Fluorescent images were taken with a multidimensional microscopy system (DeltaVision SA3.1; Applied Precision, Inc., Issaquah, WA) built around a Zeiss Axiovert S100-2TV (Carl Zeiss, Oberkochen, Germany) and equipped with a Photometrics PXL-2 cooled charge-coupled device (CCD) camera containing a Kodak KAF1400 chip (Photometrics, Tucson, AZ). A Zeiss 63 X plan-Apochromat oil immersion objective was used. The out-of-focus information in the raw data was removed by threedimensional constrained iterative deconvolution using software supplied with the DeltaVision.
RESULTS Cadherin-mediated cell– cell adhesion is dynamically regulated during various cellular processes, in-
cluding those involved in development. However, how cadherin-mediated cell– cell adhesion is regulated remains to be clarified. To clarify the mechanism underlying the cadherin-mediated cell– cell adhesion, we sought to identify molecules interacting with E-cadherin, ␣-catenin, and -catenin by using affinity columns containing immobilized GST– cytoplasmic domain of E-cadherin, GST–␣-catenin, and GST–-catenin. Bovine brain cytosol was applied onto affinity beads coated with GST– cytoplasmic domain of E-cadherin (aa 734 – 884), GST–␣-catenin (aa 1–906) or GST–catenin (aa 1–781). After the beads had been washed with buffer A, the bound proteins were eluted by the addition of 500 mM NaCl in buffer A. Proteins with molecular masses of about 180 kDa (p180) and 150 kDa (p150) were specifically detected in the eluate from the GST–-catenin affinity column, but not in that from the GST, GST– cytoplasmic domain of E-cadherin, or GST–␣-catenin affinity column (Fig. 1A). Proteins with molecular masses of about 120 kDa (p120) and 110 kDa (p110) were specifically detected in the eluate from the affinity column containing the GST– cytoplasmic domain of E-cadherin. A protein with a molecular mass of about 250 kDa (p250) was specifically detected in the
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FIG. 3. In vivo interaction of KIAA0313 with -catenin. MDCKII or EL cells were lysed with lysis buffer. These lysates or bovine brain cytosol were incubated in the absence or presence of control rabbit IgG or anti-KIAA0313-N antibody. Then, the immunocomplexes were precipitated with protein A–Sepharose 4B. The immunocomplexes were subjected to immunoblot analysis using antiKIAA0313-N and anti--catenin antibodies. The arrow denotes the position of KIAA0313; and the arrowhead that of -catenin. The results are representative of three independent experiments.
eluate from the GST–␣-catenin affinity column. The identities of p110, p120 and p250 are currently under investigation. To determine the molecular identity of p180 and p150, we subjected the proteins to mass spectral analysis as described (18, 19). Such analysis revealed p180 to be identical to KIAA0313. We also confirmed that p180 was specifically recognized by anti-KIAA0313-N antibody only in the eluate from the GST–-catenin affinity column (Fig. 1B). KIAA0313 has a cyclic AMP (cAMP) binding motif, a PDZ domain, a Ras association (RA) domain, a putative Rap1 GEF domain, a prolinerich region that possibly interacts with WW domain or SH3 domain, and a C-terminal consensus motif for binding to PDZ domains (Fig. 2A). The domain organization of KIAA0313 is similar to that of Epac/cAMPGEF, which is a GEF for Rap1 (21, 22), except for its PDZ domain and C-terminal region. Very recently, it was reported that KIAA0313 is a GEF for Rap1A, Rap1B, and Rap2 (23–25). p150 was identified as a product of KIAA0705 which has a high degree of sequence similarity to S-SCAM (Fig. 2B), which was identified as a SAPAP (SAP90/PSD-95-associated protein)-interacting protein (16) and is a scaffolding protein that interacts with many molecules such as NMDA receptor 2C, ␦-catenin, -catenin, ZO-1, PKU-␣, rat Sec8, synaptic vesicle protein SV2, NET1 (Rho GEF), rat elongation factor 1␣ (EF-1␣) (26), and KIAA0313 (23). KIAA0705 as well as S-SCAM has two WW domains and five PDZ domains but lacks the N-terminus PDZ domain and guanylate kinase (GK)like domain of S-SCAM. Next we examined whether KIAA0313 interacts with -catenin in vivo. When KIAA0313 was immunoprecipitated from MDCKII cells, -catenin was coimmunoprecipitated with it (Fig. 3). Similar results were
obtained when bovine brain cytosol and EL cells, which are L cells stably expressing E-cadherin, were used instead of MDCKII cells. Therefore, it is most likely that KIAA0313 interacts with -catenin in vivo, although it is not known whether this interaction is direct or not. In MDCKII cells, E-cadherin and ZO-1, the latter of which is one of the peripheral components of the cell– cell adhesion molecule, were also coimmunoprecipitated with KIAA0313 (data not shown). These results suggest that KIAA0313 functions at sites of cell– cell contact. When we next examined the localization of KIAA0313 in MDCKII cells, we found the protein to be localized at sites of cell– cell contact and partly colocalized with -catenin and ZO-1 (Fig. 4). These results are consistent with the results shown in Fig. 3. Finally we examined the expression of KIAA0313 in several rat tissues by immunoblot analysis. Various rat tissue extracts (150 g of protein for each) were subjected to SDS–PAGE followed by immunoblotting with anti-KIAA0313-N antibody. KIAA0313 was especially abundant in brain, and weakly expressed in heart, lung, liver, and stomach (Fig. 5). Essentially identical results were obtained when the other antibody, which had been generated against the C-terminus of KIAA0313 (aa 935–1499), was employed. Thus, it is likely that KIAA0313 is mainly expressed in brain but also is expressed in various other tissues.
FIG. 4. Colocalization of KIAA0313 with -catenin and ZO-1 at sites of cell– cell contact in MDCKII cells. Subconfluent MDCKII cells were doubly stained with anti-KIAA0313-N antibody and either anti--catenin or ZO-1 antibody, followed by FITC-conjugated antirabbit polyclonal antibody for KIAA0313 and Texas red-conjugated anti-mouse monoclonal antibody for -catenin or Texas redconjugated anti-rat monoclonal antibody for ZO-1. The results are representative of three independent experiments. Bar, 10 m.
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DISCUSSION The dynamic rearrangement that occurs in cadherinmediated cell– cell adhesion plays essential roles in various processes including morphogenesis and tumorigenesis. Despite numerous attempts to clarify the regulation of cadherin-mediated cell– cell adhesion, its mechanism is not fully understood. In this study, we purified two novel -catenin-interacting proteins that we showed to be identical to KIAA0313 and KIAA0705. KIAA0313 and KIAA0705 have a high degree of sequence similarity to Epac and S-SCAM, respectively (Fig. 2). Here, we focused on the association of KIAA0313 with -catenin in vivo, and found that -catenin was coimmunoprecipitated with KIAA0313 and colocalized with KIAA0313 at sites of cell– cell contact. Very recently, it was reported that KIAA0313 binds to S-SCAM (23), and has a GEF activity for Rap1A, Rap1B, and Rap2 (23–25). Since KIAA0705 has a high degree of sequence similarity to S-SCAM (Fig. 2), which binds to -catenin in the yeast two-hybrid system (26), there is a great possibility that KIAA0313 associates with -catenin through KIAA0705. KIAA0705 might recruit KIAA0313 at sites of cell– cell contact, thereby increasing the amount of the GTP-bound form of Rap1 (active form of Rap1) at sites of cell– cell contact (Fig. 6). Further studies are required to elucidate the physiological role of the interaction of -catenin with KIAA0705 and KIAA0313. Rap1 GTPase, belonging to the Ras superfamily, is involved in growth control (27). Posern et al. showed that Rap1 is activated during cell adhesion to coated and uncoated tissue culture plates (28). Tsukamoto et al. showed that the GTP-bound form of Rap1 is required for cell-substratum adhesion and that SPA-1 (Rap1 GTPase-activating protein) negatively regulates the cell-substratum adhesion (29). Taken together, the data suggest that E-cadherin-mediated cell– cell adhesion as well as cell-substratum adhesion might induce activation of Rap1, and, if so, KIAA0313 might be involved in this activation process.
FIG. 6. Model for the relationship among the cadherin/catenins complex, KIAA0705, and KIAA0313. Very recently, it was reported that KIAA0313 binds to S-SCAM (23) and has a GEF activity for Rap1A, Rap1B, and Rap2 (23–25). Since KIAA0705 has a high degree of sequence similarity to S-SCAM which binds to -catenin in the yeast two-hybrid system (26), there is a great possibility that KIAA0313 associates with -catenin through KIAA0705. KIAA0705 might recruit KIAA0313 at sites of cell– cell contact, thereby increasing the amount of the GTP-bound form of Rap1 at sites of cell– cell contact.
In conclusion, we showed that KIAA0313 interacts with -catenin in vivo, possibly through KIAA0705. The dynamic regulation of cadherin-mediated cell– cell adhesion plays important roles in various cellular processes such as compaction of early embryogenesis, gastrulation, cell scattering, and synaptic formation between neurons. It is plausible that the interaction of KIAA0313 with -catenin is involved in regulation of the above phenomena. The next challenge we are facing is to clarify the physiological role of the interaction of -catenin with KIAA0705 and KIAA0313. ACKNOWLEDGMENTS We thank Drs. A. Nagafuchi and Sh. Tsukita (Kyoto University, Kyoto, Japan) for providing MDCKII cells, EL cells, and cDNAs of E-cadherin, -catenin, and ␣-catenin and Drs. T. Nagase, N. Nomura, and the Kazusa DNA Research Institute for providing the cDNA of KIAA0313 and support from a cDNA Research Program.
REFERENCES
FIG. 5. Tissue distribution of KIAA0313. The indicated rat tissue extracts (150 g of protein for each) were subjected to SDS–PAGE followed by immunoblotting with anti-KIAA0313-N antibody. The arrow denotes the position of KIAA0313. KIAA0313 was especially abundant in brain, and weakly expressed in heart, lung, liver, and stomach. Essentially identical results were obtained when antibody against the C-terminus of KIAA0313 (aa 935–1499) was employed. 716
1. Takeichi, M. (1995) Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7, 619 – 627. 2. Gumbiner, B. M. (1996) Regulation of cadherin adhesive activity. Cell 84, 345–357. 3. Adams, C. L., and Nelson, W. J. (1998) Cytomechanics of cadherin-mediated cell– cell adhesion. Curr. Opin. Cell Biol. 10, 572–577. 4. Ozawa, M., Baribault, H., and Kemler, R. (1989) The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 8, 1711–1717. 5. Tsukita, Sh., Tsukita, Sa., Nagafuchi, A., and Yonemura, S. (1992) Molecular linkage between cadherins and actin filaments in cell– cell adherens junctions. Curr. Opin. Cell Biol. 4, 834 – 839.
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6. Braga, V. M., Machesky, L. M., Hall, A., and Hotchin, N. A. (1997) The small GTPases rho and rac are required for the establishment of cadherin-dependent cell– cell contacts. J. Cell Biol. 137, 1421–1431. 7. Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H., and Takai, Y. (1997). Regulation of cell– cell adhesion by rac and rho small G proteins in MDCK cells. J. Cell Biol. 139, 1047–1059. 8. Kuroda, S., Fukata, M., Fujii, K., Nakamura, T., Izawa, I., and Kaibuchi, K. (1997) Regulation of cell– cell adhesion of MDCK cells by Cdc42 and Rac1 small GTPases. Biochem. Biophys. Res. Commun. 240, 430 – 435, doi:10.1006/bbrc.1997.7675. 9. Kaibuchi, K., Kuroda, S., Fukata, M., and Nakagawa, M. (1999) Regulation of cadherin-mediated cell– cell adhesion by the rho family GTPases. Curr. Opin. Cell Biol. 11, 591–596. 10. Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin-mediated cell– cell adhesion. Science 281, 832– 835. 11. Fukata, M., Kuroda, S., Nakagawa, M., Kawajiri, A., Itoh, N., Shoji, I., Matsuura, Y., Yonehara, S., Kikuchi, A., and Kaibuchi, K. (1999) Cdc42 and Rac1 regulate the interaction of IQGAP1 with -catenin. J. Biol. Chem. 274, 26044 –26050. 12. Ozawa, M., and Kemler, R. (1998) The membrane-proximal region of the E-cadherin cytoplasmic domain prevents dimerization and negatively regulates adhesion activity. J. Cell Biol. 142, 1605–1613. 13. Yap, A. S., Niessen, C. M., and Gumbiner, B. M. (1998) The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn. J. Cell Biol. 141, 779 –789. 14. Aono, S., Nakagawa, S., Reynolds, A. B., and Takeichi, M. (1999) p120(ctn) Acts as an inhibitory regulator of cadherin function in colon carcinoma cells. J. Cell Biol. 145, 551–562. 15. Ohkubo, T., and Ozawa, M. (1999) p120(ctn) binds to the membrane-proximal region of the E-cadherin cytoplasmic domain and is involved in modulation of adhesion activity. J. Biol. Chem. 274, 21409 –21415. 16. Hirao, K., Hata, Y., Ide, N., Takeuchi, M., Irie, M., Yao, I., Deguchi, M., Toyoda, A., Sudhof, T. C., and Takai, Y. (1998) A novel multiple PDZ domain-containing molecule interacting with N-methyl-D-aspartate receptors and neuronal cell adhesion proteins. J. Biol. Chem. 273, 21105–21110. 17. Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1. J. Biol. Chem. 271, 23363–23367.
18. Iwamatsu, A. (1992) S-carboxymethylation of proteins transferred onto polyvinylidene difluoride membranes followed by in situ protease digestion and amino acid microsequencing. Electrophoresis 13, 142–147. 19. Jensen, O. N., Podtelejnikov, A., and Mann, M. (1996) Delayed extraction improves specificity in database searches by matrixassisted laser desorption/ionization peptide maps. Rapid Commun. Mass Spectrom. 10, 1371–1378. 20. Nagafuchi, A., Ishihara, S., and Tsukita, S. (1994) The roles of catenins in the cadherin-mediated cell adhesion: Functional analysis of E-cadherin–␣-catenin fusion molecules. J. Cell Biol. 127, 235–245. 21. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J. L. (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474 – 477. 22. Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) A family of cAMP-binding proteins that directly activate Rap1. Science 282, 2275–2279. 23. Ohtsuka, T., Hata, Y., Ide, N., Yasuda, T., Inoue, E., Inoue, T., Mizoguchi, A., and Takai, Y. (1999) nRap GEP: A novel neural GDP/GTP exchange protein for rap1 small G protein that interacts with synaptic scaffolding molecule (S-SCAM). Biochem. Biophys. Res. Commun. 265, 38 – 44, doi:10.1006/bbrc.1999.1619. 24. Liao, Y., Kariya, K., Hu, C. D., Shibatohge, M., Goshima, M., Okada, T., Watari, Y., Gao, X., Jin, T. G., Yamawaki-Kataoka, Y., and Kataoka, T. (1999) RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans. J. Biol. Chem. 274, 37815–37820. 25. de Rooij, J., Boenink, N. M., van Triest, M., Cool, R. H., Wittinghofer, A., and Bos, J. L. (1999) PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2. J. Biol. Chem. 274, 38125–38130. 26. Ide, N., Hata, Y., Deguchi, M., Hirao, K., Yao, I., and Takai, Y. (1999) Interaction of S-SCAM with neural plakophilin-related Armadillo-repeat protein/delta-catenin. Biochem. Biophys. Res. Commun. 256, 456 – 461, doi:10.1006/bbrc.1999.0364. 27. Bos, J. L. (1998) All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral. EMBO J. 17, 6776 – 6782. 28. Posern, G., Weber, C. K., Rapp, U. R., and Feller, S. M. (1998) Activity of Rap1 is regulated by bombesin, cell adhesion, and cell density in NIH3T3 fibroblasts. J. Biol. Chem. 273, 24297–24300. 29. Tsukamoto, N., Hattori, M., Yang, H., Bos, J. L., and Minato, N. (1999) Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion. J. Biol. Chem. 274, 18463–18469.
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Identification of a Novel -Catenin-Interacting Protein 1 Aie Kawajiri,* ,2 Naohiro Itoh,* ,2 Masaki Fukata,* ,† Masato Nakagawa,* Masaki Yamaga,* Akihiro Iwamatsu,‡ and Kozo Kaibuchi* ,† ,3 *Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101, Japan; †Department of Cell Pharmacology, Nagoya University School of Medicine, 65 Tsurumai, Showa, Nagoya, 466-8550, Japan; and ‡Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5 Fukuura, Kanazawa, Yokohama, 236-0004, Japan
Received May 13, 2000
Cadherin is a well-known cell– cell adhesion molecule, and it binds to -catenin, which in turn binds to ␣-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell– cell adhesion. Here we purified two novel -catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)–catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313 which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain and p150 to be the same as KIAA0705 which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM), which binds -catenin and KIAA0313 in the yeast twohybrid system and overlay assay, respectively (Ide et al., Biochem. Biophys. Res. Commun. 256, 456 – 461, 1999; Ohtsuka et al., Biochem. Biophys. Res. Commun. 265, 38 – 44, 1999). -Catenin was coimmunoprecipitated with KIAA0313 in Madin–Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and -catenin were partly colocalized at sites of cell– cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with -catenin through KIAA0705 in vivo at sites of cell– cell contact. © 2000 Academic Press
Abbreviations used: GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; aa, amino acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PBS, phosphatebuffered saline; MDCKII, Madin–Darby canine kidney II. 1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (1999) and by grants from the program Research for the Future of the Japan Society for the Promotion of Science, the Human Frontier Science Program, and Kirin Brewery Company Limited. 2 These authors contributed equally to this article. 3 To whom correspondence should be addressed at Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101, Japan. Fax: 0743-72-5449. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Key Words: -catenin; KIAA0313; KIAA0705; Rap1 GEF.
Cell– cell adhesion is dynamically rearranged in various situations including the establishment of epithelial polarity and developmental morphogenesis (1–3). Cadherin is a calcium-dependent cell– cell adhesion molecule that binds to -catenin, which in turn binds to ␣-catenin (cadherin/catenins complex) (4, 5). This cadherin/catenins complex is linked to the actin cytoskeleton, and the linkage is essential for proper cadherin function (5). However, little is known about the regulatory mechanism underlying cadherin-mediated cell– cell adhesion. Recent studies showed that the Rho family of small GTPases, Cdc42, Rac1, and Rho, participate in the regulation of cadherin-mediated cell– cell adhesion (6 –9). We recently found that IQGAP1, an effector of Cdc42 and Rac1, negatively regulates cadherinmediated cell– cell adhesion and that activated Cdc42 and Rac1 positively regulate cadherin-mediated cell– cell adhesion by inhibiting IQGAP1 function (10, 11). Dimerization of cadherin is another crucial contributor to the cadherin-mediated cell– cell adhesion. The mechanism, however, underlying dimer formation remains to be elucidated. Recent studies suggest that the membrane-proximal region of the cadherin cytoplasmic domain, which is separate from the distal -catenin-binding domain, regulates cadherin dimerization and activity. The membrane-proximal region inhibits dimerization and adhesive activity (12). Given that p120 ctn, a member of the Armadillo/-catenin family, binds to the membrane-proximal region of the cadherin cytoplasmic domain (12, 13) and inhibits cadherin activity in some carcinoma cell lines (14, 15), p120 ctn might modulate the cadherin activity. However, it remains to be clarified whether cadherin-mediated
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cell– cell adhesion is regulated only by Cdc42/Rac1/ IQGAP1 system and p120 ctn. Here, to further understand the mechanism underlying the cadherin-mediated cell– cell adhesion, we purified two novel -catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using GST–catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313, which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain, and p150 to be KIAA0705, which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM) (16). In this study, we focused on the association of KIAA0313 with -catenin in vivo, and found that -catenin could be coimmunoprecipitated with KIAA0313 and colocalized with KIAA0313 at sites of cell– cell contact. MATERIALS AND METHODS Materials and chemicals. EL cells and MDCKII cells were kindly provided by Drs. A. Nagafuchi and Sh. Tsukita (Kyoto University, Kyoto, Japan). The cDNAs encoding mouse ␣-catenin, mouse -catenin, and mouse E-cadherin were kindly provided by Drs. A. Nagafuchi and Sh. Tsukita (Kyoto University, Kyoto, Japan). The cDNA encoding KIAA0313 was kindly provided by Drs. T. Nagase and N. Nomura (Kazusa DNA Research Institute, Kisarazu, Japan). Polyclonal antiKIAA0313-N and anti-KIAA0313-C antibodies were generated against recombinant GST–KIAA0313-N (aa 1–250) and GST–KIAA0313-C (aa 935–1499), respectively. Anti--catenin antibody was purchased from Transduction Laboratories (Lexington, KY). Anti-ZO-1 antibody was purchased from CHEMICON International Inc. (Temecula, CA). All materials used in the nucleic acid study were purchased from Takara Shuzo Co. Ltd. (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources. Plasmid constructions. pGEX4T-1-cytoplasmic domain of mouse E-cadherin (aa 734 – 884), -mouse ␣-catenin (aa 1–906), and -mouse -catenin (aa 1–781) were produced as described previously (10). To obtain the GST–KIAA0313-N (aa 1–250) and GST–KIAA0313-C (aa 935–1499) as antigens, we subcloned their cDNA fragments into pGEX4T-1. Preparation of recombinant proteins. The expression and purification of various GST-fusion proteins were done as previously described (17). GST– cytoplasmic domain of E-cadherin, GST–␣-catenin, and GST–-catenin affinity column chromatography. The affinitypurification was performed essentially as described (17). Briefly, bovine brain cytosol was passed through a column of glutathione beads to remove endogenous GST. Then, the passed fraction was loaded onto a column of glutathione beads (200 l) coated with GST– cytoplasmic domain of E-cadherin, GST–␣-catenin, or GST–-catenin (each 6.5 nmol). The columns were washed with 2 ml (10 volumes) of buffer A (20 mM Tris/HCl at pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, and 10 M [p-amidinophenyl]-methanesulfonyl fluoride). The proteins bound to the affinity columns were eluted three times by the addition of 660 l (3.3 vol) of buffer A containing 500 mM NaCl. Mass spectral analysis. The 500 mM NaCl eluate from the GST– -catenin affinity column was dialyzed against distilled water and concentrated by freeze-drying. The concentrated samples were electrophoresed on a 6% polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Problot, Applied Biosystems). The immobilized proteins were reduced, S-carboxy-
FIG. 1. Purification of -catenin-interacting proteins. (A) Bovine brain cytosol was loaded onto glutathione-Sepharose columns coated with the indicated GST-fusion proteins. The bound proteins were eluted by the addition of buffer A containing 500 mM NaCl. Aliquots of the eluates were resolved by SDS–PAGE followed by silver staining. Arrowheads from the top denote the positions of p250, p180, p150, p120, and p110, respectively. (B) Aliquots of the eluates were resolved by SDS–PAGE followed by immunoblotting with antiKIAA0313-N antibody. The arrow denotes the position of KIAA0313. The results are representative of three independent experiments. methylated, and digested in situ with Achromobacter protease I (a Lys-C) (18). Molecular mass analyses of Lys-C fragments were performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using a PerSeptive Biosystem Voyager-DE/RP (19). Identification of proteins was carried out by comparison between the molecular weights determined by MALDITOF/MS and theoretical peptide masses of the proteins registered in the NCBInr. Cell culture. EL cells were grown in Dulbecco’s modified Eagles medium (DMEM) containing 10% fetal bovine serum and 100 g/ml of G418 at 37°C in an air–5% CO 2 atmosphere at constant humidity (20). MDCKII cells were grown in DMEM containing 10% calf serum at 37°C in an air–5% CO 2 atmosphere at constant humidity. Coimmunoprecipitation of -catenin with KIAA0313. Immunoprecipitation was performed as described previously (10). Briefly, subconfluent MDCKII or EL cells were harvested and lysed with lysis buffer (20 mM Tris/HCl at pH 7.4, 50 mM NaCl, 10 M [p-amidinophenyl]-methanesulfonyl fluoride, 10 g/ml leupeptin, 0.5% [w/v] Triton X-100, 1 mM CaCl2). The lysates and bovine brain cytosol were clarified by centrifugation at 100,000g for 30 min at 4°C. The soluble supernatants were incubated with 20 g of anti-KIAA0313-N antibody and incubated for 1 h at 4°C. The immunocomplexes were then precipitated with 40 l of protein A Sepharose 4B. The immunocom-
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FIG. 2. Structure of KIAA0313 and KIAA0705. (A) Schematic drawing showing the localization of domains and the percent amino acid sequence identity between KIAA0313 and Epac. The GenBank accession no. of KIAA0313 is AB002311. REM: Ras exchange motif, PDZ: named after three proteins, PSD-95/SAP90, Dlg-A, and ZO-1, RA: Ras association domain, GEF: guanine nucleotide exchange factor domain, DEP: named after three proteins, Disheveled, Egl-10, and Pleckstrin. (B) Schematic drawing showing the localization of domains and the percent amino acid sequence identity between KIAA0705 and S-SCAM. The GenBank accession no. of KIAA0705 is AB014605. WW, domain consisting of two separate tryptophans and invariant proline; GK, guanylate kinase-like domain. plexes were washed five times with lysis buffer, eluted by boiling in sample buffer for SDS–PAGE, and subjected to immunoblot analysis with anti-KIAA0313-N and anti--catenin antibodies. Immunofluorescence analysis. Subconfluent MDCKII cells were fixed with ice-cold 100% methanol for 15 min on ice. After having been washed with ice-cold PBS, the cells were then incubated with anti-KIAA0313-N polyclonal antibody for 1 h at room temperature. The cells were washed with PBS, and subsequently incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit Ig Ab for 1 h at room temperature. Next, the cells were incubated with anti-catenin monoclonal or anti-ZO-1 monoclonal antibody for 1 h at room temperature. After washes with PBS, the cells were incubated with Texas Red-conjugated anti-mouse Ig Ab for -catenin or antirat Ig Ab for ZO-1 for 1 h at room temperature. Fluorescent images were taken with a multidimensional microscopy system (DeltaVision SA3.1; Applied Precision, Inc., Issaquah, WA) built around a Zeiss Axiovert S100-2TV (Carl Zeiss, Oberkochen, Germany) and equipped with a Photometrics PXL-2 cooled charge-coupled device (CCD) camera containing a Kodak KAF1400 chip (Photometrics, Tucson, AZ). A Zeiss 63 X plan-Apochromat oil immersion objective was used. The out-of-focus information in the raw data was removed by threedimensional constrained iterative deconvolution using software supplied with the DeltaVision.
RESULTS Cadherin-mediated cell– cell adhesion is dynamically regulated during various cellular processes, in-
cluding those involved in development. However, how cadherin-mediated cell– cell adhesion is regulated remains to be clarified. To clarify the mechanism underlying the cadherin-mediated cell– cell adhesion, we sought to identify molecules interacting with E-cadherin, ␣-catenin, and -catenin by using affinity columns containing immobilized GST– cytoplasmic domain of E-cadherin, GST–␣-catenin, and GST–-catenin. Bovine brain cytosol was applied onto affinity beads coated with GST– cytoplasmic domain of E-cadherin (aa 734 – 884), GST–␣-catenin (aa 1–906) or GST–catenin (aa 1–781). After the beads had been washed with buffer A, the bound proteins were eluted by the addition of 500 mM NaCl in buffer A. Proteins with molecular masses of about 180 kDa (p180) and 150 kDa (p150) were specifically detected in the eluate from the GST–-catenin affinity column, but not in that from the GST, GST– cytoplasmic domain of E-cadherin, or GST–␣-catenin affinity column (Fig. 1A). Proteins with molecular masses of about 120 kDa (p120) and 110 kDa (p110) were specifically detected in the eluate from the affinity column containing the GST– cytoplasmic domain of E-cadherin. A protein with a molecular mass of about 250 kDa (p250) was specifically detected in the
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FIG. 3. In vivo interaction of KIAA0313 with -catenin. MDCKII or EL cells were lysed with lysis buffer. These lysates or bovine brain cytosol were incubated in the absence or presence of control rabbit IgG or anti-KIAA0313-N antibody. Then, the immunocomplexes were precipitated with protein A–Sepharose 4B. The immunocomplexes were subjected to immunoblot analysis using antiKIAA0313-N and anti--catenin antibodies. The arrow denotes the position of KIAA0313; and the arrowhead that of -catenin. The results are representative of three independent experiments.
eluate from the GST–␣-catenin affinity column. The identities of p110, p120 and p250 are currently under investigation. To determine the molecular identity of p180 and p150, we subjected the proteins to mass spectral analysis as described (18, 19). Such analysis revealed p180 to be identical to KIAA0313. We also confirmed that p180 was specifically recognized by anti-KIAA0313-N antibody only in the eluate from the GST–-catenin affinity column (Fig. 1B). KIAA0313 has a cyclic AMP (cAMP) binding motif, a PDZ domain, a Ras association (RA) domain, a putative Rap1 GEF domain, a prolinerich region that possibly interacts with WW domain or SH3 domain, and a C-terminal consensus motif for binding to PDZ domains (Fig. 2A). The domain organization of KIAA0313 is similar to that of Epac/cAMPGEF, which is a GEF for Rap1 (21, 22), except for its PDZ domain and C-terminal region. Very recently, it was reported that KIAA0313 is a GEF for Rap1A, Rap1B, and Rap2 (23–25). p150 was identified as a product of KIAA0705 which has a high degree of sequence similarity to S-SCAM (Fig. 2B), which was identified as a SAPAP (SAP90/PSD-95-associated protein)-interacting protein (16) and is a scaffolding protein that interacts with many molecules such as NMDA receptor 2C, ␦-catenin, -catenin, ZO-1, PKU-␣, rat Sec8, synaptic vesicle protein SV2, NET1 (Rho GEF), rat elongation factor 1␣ (EF-1␣) (26), and KIAA0313 (23). KIAA0705 as well as S-SCAM has two WW domains and five PDZ domains but lacks the N-terminus PDZ domain and guanylate kinase (GK)like domain of S-SCAM. Next we examined whether KIAA0313 interacts with -catenin in vivo. When KIAA0313 was immunoprecipitated from MDCKII cells, -catenin was coimmunoprecipitated with it (Fig. 3). Similar results were
obtained when bovine brain cytosol and EL cells, which are L cells stably expressing E-cadherin, were used instead of MDCKII cells. Therefore, it is most likely that KIAA0313 interacts with -catenin in vivo, although it is not known whether this interaction is direct or not. In MDCKII cells, E-cadherin and ZO-1, the latter of which is one of the peripheral components of the cell– cell adhesion molecule, were also coimmunoprecipitated with KIAA0313 (data not shown). These results suggest that KIAA0313 functions at sites of cell– cell contact. When we next examined the localization of KIAA0313 in MDCKII cells, we found the protein to be localized at sites of cell– cell contact and partly colocalized with -catenin and ZO-1 (Fig. 4). These results are consistent with the results shown in Fig. 3. Finally we examined the expression of KIAA0313 in several rat tissues by immunoblot analysis. Various rat tissue extracts (150 g of protein for each) were subjected to SDS–PAGE followed by immunoblotting with anti-KIAA0313-N antibody. KIAA0313 was especially abundant in brain, and weakly expressed in heart, lung, liver, and stomach (Fig. 5). Essentially identical results were obtained when the other antibody, which had been generated against the C-terminus of KIAA0313 (aa 935–1499), was employed. Thus, it is likely that KIAA0313 is mainly expressed in brain but also is expressed in various other tissues.
FIG. 4. Colocalization of KIAA0313 with -catenin and ZO-1 at sites of cell– cell contact in MDCKII cells. Subconfluent MDCKII cells were doubly stained with anti-KIAA0313-N antibody and either anti--catenin or ZO-1 antibody, followed by FITC-conjugated antirabbit polyclonal antibody for KIAA0313 and Texas red-conjugated anti-mouse monoclonal antibody for -catenin or Texas redconjugated anti-rat monoclonal antibody for ZO-1. The results are representative of three independent experiments. Bar, 10 m.
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DISCUSSION The dynamic rearrangement that occurs in cadherinmediated cell– cell adhesion plays essential roles in various processes including morphogenesis and tumorigenesis. Despite numerous attempts to clarify the regulation of cadherin-mediated cell– cell adhesion, its mechanism is not fully understood. In this study, we purified two novel -catenin-interacting proteins that we showed to be identical to KIAA0313 and KIAA0705. KIAA0313 and KIAA0705 have a high degree of sequence similarity to Epac and S-SCAM, respectively (Fig. 2). Here, we focused on the association of KIAA0313 with -catenin in vivo, and found that -catenin was coimmunoprecipitated with KIAA0313 and colocalized with KIAA0313 at sites of cell– cell contact. Very recently, it was reported that KIAA0313 binds to S-SCAM (23), and has a GEF activity for Rap1A, Rap1B, and Rap2 (23–25). Since KIAA0705 has a high degree of sequence similarity to S-SCAM (Fig. 2), which binds to -catenin in the yeast two-hybrid system (26), there is a great possibility that KIAA0313 associates with -catenin through KIAA0705. KIAA0705 might recruit KIAA0313 at sites of cell– cell contact, thereby increasing the amount of the GTP-bound form of Rap1 (active form of Rap1) at sites of cell– cell contact (Fig. 6). Further studies are required to elucidate the physiological role of the interaction of -catenin with KIAA0705 and KIAA0313. Rap1 GTPase, belonging to the Ras superfamily, is involved in growth control (27). Posern et al. showed that Rap1 is activated during cell adhesion to coated and uncoated tissue culture plates (28). Tsukamoto et al. showed that the GTP-bound form of Rap1 is required for cell-substratum adhesion and that SPA-1 (Rap1 GTPase-activating protein) negatively regulates the cell-substratum adhesion (29). Taken together, the data suggest that E-cadherin-mediated cell– cell adhesion as well as cell-substratum adhesion might induce activation of Rap1, and, if so, KIAA0313 might be involved in this activation process.
FIG. 6. Model for the relationship among the cadherin/catenins complex, KIAA0705, and KIAA0313. Very recently, it was reported that KIAA0313 binds to S-SCAM (23) and has a GEF activity for Rap1A, Rap1B, and Rap2 (23–25). Since KIAA0705 has a high degree of sequence similarity to S-SCAM which binds to -catenin in the yeast two-hybrid system (26), there is a great possibility that KIAA0313 associates with -catenin through KIAA0705. KIAA0705 might recruit KIAA0313 at sites of cell– cell contact, thereby increasing the amount of the GTP-bound form of Rap1 at sites of cell– cell contact.
In conclusion, we showed that KIAA0313 interacts with -catenin in vivo, possibly through KIAA0705. The dynamic regulation of cadherin-mediated cell– cell adhesion plays important roles in various cellular processes such as compaction of early embryogenesis, gastrulation, cell scattering, and synaptic formation between neurons. It is plausible that the interaction of KIAA0313 with -catenin is involved in regulation of the above phenomena. The next challenge we are facing is to clarify the physiological role of the interaction of -catenin with KIAA0705 and KIAA0313. ACKNOWLEDGMENTS We thank Drs. A. Nagafuchi and Sh. Tsukita (Kyoto University, Kyoto, Japan) for providing MDCKII cells, EL cells, and cDNAs of E-cadherin, -catenin, and ␣-catenin and Drs. T. Nagase, N. Nomura, and the Kazusa DNA Research Institute for providing the cDNA of KIAA0313 and support from a cDNA Research Program.
REFERENCES
FIG. 5. Tissue distribution of KIAA0313. The indicated rat tissue extracts (150 g of protein for each) were subjected to SDS–PAGE followed by immunoblotting with anti-KIAA0313-N antibody. The arrow denotes the position of KIAA0313. KIAA0313 was especially abundant in brain, and weakly expressed in heart, lung, liver, and stomach. Essentially identical results were obtained when antibody against the C-terminus of KIAA0313 (aa 935–1499) was employed. 716
1. Takeichi, M. (1995) Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7, 619 – 627. 2. Gumbiner, B. M. (1996) Regulation of cadherin adhesive activity. Cell 84, 345–357. 3. Adams, C. L., and Nelson, W. J. (1998) Cytomechanics of cadherin-mediated cell– cell adhesion. Curr. Opin. Cell Biol. 10, 572–577. 4. Ozawa, M., Baribault, H., and Kemler, R. (1989) The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 8, 1711–1717. 5. Tsukita, Sh., Tsukita, Sa., Nagafuchi, A., and Yonemura, S. (1992) Molecular linkage between cadherins and actin filaments in cell– cell adherens junctions. Curr. Opin. Cell Biol. 4, 834 – 839.
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6. Braga, V. M., Machesky, L. M., Hall, A., and Hotchin, N. A. (1997) The small GTPases rho and rac are required for the establishment of cadherin-dependent cell– cell contacts. J. Cell Biol. 137, 1421–1431. 7. Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H., and Takai, Y. (1997). Regulation of cell– cell adhesion by rac and rho small G proteins in MDCK cells. J. Cell Biol. 139, 1047–1059. 8. Kuroda, S., Fukata, M., Fujii, K., Nakamura, T., Izawa, I., and Kaibuchi, K. (1997) Regulation of cell– cell adhesion of MDCK cells by Cdc42 and Rac1 small GTPases. Biochem. Biophys. Res. Commun. 240, 430 – 435, doi:10.1006/bbrc.1997.7675. 9. Kaibuchi, K., Kuroda, S., Fukata, M., and Nakagawa, M. (1999) Regulation of cadherin-mediated cell– cell adhesion by the rho family GTPases. Curr. Opin. Cell Biol. 11, 591–596. 10. Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin-mediated cell– cell adhesion. Science 281, 832– 835. 11. Fukata, M., Kuroda, S., Nakagawa, M., Kawajiri, A., Itoh, N., Shoji, I., Matsuura, Y., Yonehara, S., Kikuchi, A., and Kaibuchi, K. (1999) Cdc42 and Rac1 regulate the interaction of IQGAP1 with -catenin. J. Biol. Chem. 274, 26044 –26050. 12. Ozawa, M., and Kemler, R. (1998) The membrane-proximal region of the E-cadherin cytoplasmic domain prevents dimerization and negatively regulates adhesion activity. J. Cell Biol. 142, 1605–1613. 13. Yap, A. S., Niessen, C. M., and Gumbiner, B. M. (1998) The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn. J. Cell Biol. 141, 779 –789. 14. Aono, S., Nakagawa, S., Reynolds, A. B., and Takeichi, M. (1999) p120(ctn) Acts as an inhibitory regulator of cadherin function in colon carcinoma cells. J. Cell Biol. 145, 551–562. 15. Ohkubo, T., and Ozawa, M. (1999) p120(ctn) binds to the membrane-proximal region of the E-cadherin cytoplasmic domain and is involved in modulation of adhesion activity. J. Biol. Chem. 274, 21409 –21415. 16. Hirao, K., Hata, Y., Ide, N., Takeuchi, M., Irie, M., Yao, I., Deguchi, M., Toyoda, A., Sudhof, T. C., and Takai, Y. (1998) A novel multiple PDZ domain-containing molecule interacting with N-methyl-D-aspartate receptors and neuronal cell adhesion proteins. J. Biol. Chem. 273, 21105–21110. 17. Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1. J. Biol. Chem. 271, 23363–23367.
18. Iwamatsu, A. (1992) S-carboxymethylation of proteins transferred onto polyvinylidene difluoride membranes followed by in situ protease digestion and amino acid microsequencing. Electrophoresis 13, 142–147. 19. Jensen, O. N., Podtelejnikov, A., and Mann, M. (1996) Delayed extraction improves specificity in database searches by matrixassisted laser desorption/ionization peptide maps. Rapid Commun. Mass Spectrom. 10, 1371–1378. 20. Nagafuchi, A., Ishihara, S., and Tsukita, S. (1994) The roles of catenins in the cadherin-mediated cell adhesion: Functional analysis of E-cadherin–␣-catenin fusion molecules. J. Cell Biol. 127, 235–245. 21. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J. L. (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474 – 477. 22. Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) A family of cAMP-binding proteins that directly activate Rap1. Science 282, 2275–2279. 23. Ohtsuka, T., Hata, Y., Ide, N., Yasuda, T., Inoue, E., Inoue, T., Mizoguchi, A., and Takai, Y. (1999) nRap GEP: A novel neural GDP/GTP exchange protein for rap1 small G protein that interacts with synaptic scaffolding molecule (S-SCAM). Biochem. Biophys. Res. Commun. 265, 38 – 44, doi:10.1006/bbrc.1999.1619. 24. Liao, Y., Kariya, K., Hu, C. D., Shibatohge, M., Goshima, M., Okada, T., Watari, Y., Gao, X., Jin, T. G., Yamawaki-Kataoka, Y., and Kataoka, T. (1999) RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans. J. Biol. Chem. 274, 37815–37820. 25. de Rooij, J., Boenink, N. M., van Triest, M., Cool, R. H., Wittinghofer, A., and Bos, J. L. (1999) PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2. J. Biol. Chem. 274, 38125–38130. 26. Ide, N., Hata, Y., Deguchi, M., Hirao, K., Yao, I., and Takai, Y. (1999) Interaction of S-SCAM with neural plakophilin-related Armadillo-repeat protein/delta-catenin. Biochem. Biophys. Res. Commun. 256, 456 – 461, doi:10.1006/bbrc.1999.0364. 27. Bos, J. L. (1998) All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral. EMBO J. 17, 6776 – 6782. 28. Posern, G., Weber, C. K., Rapp, U. R., and Feller, S. M. (1998) Activity of Rap1 is regulated by bombesin, cell adhesion, and cell density in NIH3T3 fibroblasts. J. Biol. Chem. 273, 24297–24300. 29. Tsukamoto, N., Hattori, M., Yang, H., Bos, J. L., and Minato, N. (1999) Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion. J. Biol. Chem. 274, 18463–18469.
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