FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2110052730> ?p ?o ?g. }

• W2110052730 endingPage "1228" @default.
• W2110052730 startingPage "1220" @default.
• W2110052730 abstract "Cell-cell adhesive events affect cell growth and fate decisions and provide spatial clues for cell polarity within tissues. The complete molecular determinants required for adhesive junction formation and their function are not completely understood. LIM domain-containing proteins have been shown to be present at cell-cell contact sites and are known to shuttle into the nucleus where they can affect cell fate and growth; however, their precise localization at cell-cell contacts, how they localize to these sites, and what their functions are at these sites is unknown. Here we show that, in primary keratinocytes, the LIM domain protein Ajuba is recruited to cadherin-dependent cell-cell adhesive complexes in a regulated manner. At cadherin adhesive complexes Ajuba interacts with α-catenin, and α-catenin is required for efficient recruitment of Ajuba to cell junctions. Ajuba also interacts directly with F-actin. Keratinocytes from Ajuba null mice exhibit abnormal cell-cell junction formation and/or stability and function. These data reveal Ajuba as a new component at cadherin-mediated cell-cell junctions and suggest that Ajuba may contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton and as such contribute to the formation or strengthening of cadherin-mediated cell-cell adhesion. Cell-cell adhesive events affect cell growth and fate decisions and provide spatial clues for cell polarity within tissues. The complete molecular determinants required for adhesive junction formation and their function are not completely understood. LIM domain-containing proteins have been shown to be present at cell-cell contact sites and are known to shuttle into the nucleus where they can affect cell fate and growth; however, their precise localization at cell-cell contacts, how they localize to these sites, and what their functions are at these sites is unknown. Here we show that, in primary keratinocytes, the LIM domain protein Ajuba is recruited to cadherin-dependent cell-cell adhesive complexes in a regulated manner. At cadherin adhesive complexes Ajuba interacts with α-catenin, and α-catenin is required for efficient recruitment of Ajuba to cell junctions. Ajuba also interacts directly with F-actin. Keratinocytes from Ajuba null mice exhibit abnormal cell-cell junction formation and/or stability and function. These data reveal Ajuba as a new component at cadherin-mediated cell-cell junctions and suggest that Ajuba may contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton and as such contribute to the formation or strengthening of cadherin-mediated cell-cell adhesion. phosphate-buffered saline 1,4-piperazinediethanesulfonic acid glutathione S-transferase mouse embryo fibroblast Cell-to-cell adhesion is important for tissue morphogenesis. During development, cell-cell contacts provide spatial clues for cell polarity and sorting, thereby ensuring proper cellular organization within tissues. Cell surface adhesion receptor proteins direct cell-cell adhesion. The cadherins, for example, are a superfamily of receptors that display calcium-dependent adhesion between the same types of proteins (i.e. homophilic interaction). E-cadherin is one of the best studied cell-cell adhesion proteins. In epithelia, E-cadherin has an important role in the generation and maintenance of the cell morphology, polarity, and function (1Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (681) Google Scholar, 2Steinberg M.S. McNutt P.M. Curr. Opin. Cell Biol. 1999; 11: 554-560Crossref PubMed Scopus (247) Google Scholar). At adhesive contacts, E-cadherin receptors also provide cytosolic actin filaments with points of attachment to the membrane, from which tension and reorganization of the cortical cytoskeleton are initiated. E-cadherin-mediated adhesion triggers redistribution of membrane, cytoskeletal, and cytosolic signaling proteins to sites of cell-cell contacts, giving rise to multiprotein signaling complexes (1Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (681) Google Scholar). Much investigation has been directed at understanding how these supramolecular protein complexes are formed, what proteins make up the functional complex, and what their contribution is to the strength of junction formation and remodeling of the cytoskeletal network. Proteins of the catenin family indirectly mediate the binding of actin filaments to cadherin receptors. β-Catenin (or γ-catenin/plakoglobin) associates directly with the cadherin tail, and then α-catenin bridges the β-catenin-cadherin complex to actin filaments (1Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (681) Google Scholar). α-Catenin is an essential component of the cadherin complex (1Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (681) Google Scholar). It not only binds and bundles actin (3Rimm D.L. Koslov E.R. Kebriaei P. Cianci C.D. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8813-8817Crossref PubMed Scopus (627) Google Scholar) but also provides docking sites for other cytoskeletal proteins that may contribute to the cytoskeletal reorganization at junctions, such as vinculin and α-actinin (reviewed in Ref. 4Provost E. Rimm D. Curr. Opin. Cell Biol. 1999; 11: 567-572Crossref PubMed Scopus (145) Google Scholar). Cytosolic proteins containing LIM domains have also been observed to localize at cell-cell contact sites (5Crawford A.W. Beckerle M.C. J. Biol. Chem. 1991; 266: 5847-5853Abstract Full Text PDF PubMed Google Scholar, 6Petit M.M.R. Fradelizi J. Golsteyn R.M. Ayoubi T.A.Y. Menichi B. Louvard D. Van de Ven W.J.M. Friederich E. Mol. Biol. Cell. 2000; 11: 117-1129Crossref PubMed Scopus (119) Google Scholar, 7Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (113) Google Scholar, 8Vasioukhin V. Bauer C. Yin M. Fuchs E. Cell. 2000; 100: 209-219Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar). LIM domains contain two tandemly repeated zinc fingers implicated in protein-protein interactions. They are found in a wide variety of proteins present in the nucleus or cytoplasm or that shuttle between these two cellular compartments (reviewed in Ref. 9Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (473) Google Scholar). The LIM domain-containing protein family can be subdivided into different subfamilies according to sequence homology within the LIM domains, the number of LIM domains, and their organization within the proteins (9Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (473) Google Scholar). The Zyxin subfamily of cytosolic LIM proteins is characterized by the presence of three related LIM domains at the COOH terminus and unique PreLIM regions, which are rich in proline residues (10Schmeichel K.L. Beckerle M.C. Mol. Biol. Cell. 1997; 8: 219-230Crossref PubMed Scopus (89) Google Scholar). Within this family, there have been five mammalian members described: Zyxin (5Crawford A.W. Beckerle M.C. J. Biol. Chem. 1991; 266: 5847-5853Abstract Full Text PDF PubMed Google Scholar), LPP (11Petit M. Mols R. Schoenmakers E. Mandahl N. Van De Ven W. Genomics. 1996; 36: 118-129Crossref PubMed Scopus (181) Google Scholar), Trip6 (12Yi J. Beckerle M.C. Genomics. 1998; 49: 314-316Crossref PubMed Scopus (70) Google Scholar), Ajuba (13Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar), and LIMD1 (14Kiss H. Kedra D. Yang Y. Kost-Alimova M. Kiss C. O'Brien K.P. Fransson I. Klein G. Imreh S. Dumanski J.P. Hum. Genet. 1999; 105: 552-559Crossref PubMed Scopus (54) Google Scholar). The cellular function of these proteins is largely unknown. In fibroblasts, they localize to sites of attachment to the substratum (focal adhesion) and can associate with the actin cytoskeleton (6Petit M.M.R. Fradelizi J. Golsteyn R.M. Ayoubi T.A.Y. Menichi B. Louvard D. Van de Ven W.J.M. Friederich E. Mol. Biol. Cell. 2000; 11: 117-1129Crossref PubMed Scopus (119) Google Scholar, 15Crawford A.W. Michelsen J.W. Beckerle M.C. J. Cell Biol. 1992; 116: 1381-1393Crossref PubMed Scopus (198) Google Scholar,16Yi J. Kloeker S. Jensen C.C. Bockholt S. Honda H. Hirai H. Beckerle M.C. J. Biol. Chem. 2002; 277: 9580-9589Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) but have also been observed at cell-cell contact sites in epithelial cells (5Crawford A.W. Beckerle M.C. J. Biol. Chem. 1991; 266: 5847-5853Abstract Full Text PDF PubMed Google Scholar, 6Petit M.M.R. Fradelizi J. Golsteyn R.M. Ayoubi T.A.Y. Menichi B. Louvard D. Van de Ven W.J.M. Friederich E. Mol. Biol. Cell. 2000; 11: 117-1129Crossref PubMed Scopus (119) Google Scholar, 7Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (113) Google Scholar). In fibroblasts, Zyxin and Trip6 appear to affect cell motility (16Yi J. Kloeker S. Jensen C.C. Bockholt S. Honda H. Hirai H. Beckerle M.C. J. Biol. Chem. 2002; 277: 9580-9589Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 17Drees B.E. Andrews K.M. Beckerle M.C. J. Cell Biol. 1999; 147: 1549-1559Crossref PubMed Scopus (90) Google Scholar). In addition, they contain nuclear export signals and shuttle between the nucleus and cytoplasm (6Petit M.M.R. Fradelizi J. Golsteyn R.M. Ayoubi T.A.Y. Menichi B. Louvard D. Van de Ven W.J.M. Friederich E. Mol. Biol. Cell. 2000; 11: 117-1129Crossref PubMed Scopus (119) Google Scholar, 7Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (113) Google Scholar, 18Nix D.A. Beckerle M.C. J. Cell Biol. 1997; 138: 1139-1147Crossref PubMed Scopus (196) Google Scholar). Whereas the significance of Zyxin and LPP nuclear localization is not clear, accumulation of Ajuba in the nucleus plays a role in growth control and differentiation (7Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (113) Google Scholar). Thus, these proteins could be ideal candidates to convey messages from adhesion sites to the nucleus. How these proteins are recruited to these disparate cellular locations and what their cellular functions are at these sites is not well understood. Ajuba is expressed in organs abundant in epithelia, such as skin, kidney, liver, lung, and the genitourinary system (13Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar). Immunofluorescence analysis of embryonal carcinoma cells revealed that, as sheets of contacted cells formed, Ajuba was localized to cell-cell contacts (7Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (113) Google Scholar). Therefore, to determine how Ajuba is recruited to cell junctions in epithelia, we used primary human keratinocytes as an epithelial model. In these primary cells, we found that Ajuba preferentially co-localizes with cadherin adhesive complexes at sites of cell-cell contacts but not at focal adhesions. Recruitment of Ajuba to cell-cell junctions was regulated and occurs through a direct interaction with α-catenin. Ajuba also interacted directly with filamentous actin, suggesting that Ajuba could contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton. Therefore, in addition to its role in the regulation of cell proliferation and differentiation (7Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (113) Google Scholar, 13Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar), Ajuba may also function in the regulation of cell-cell adhesion. Normal human keratinocytes (strain Kb, passages 3–7) were grown in standard medium (1.8 mm calcium ions) (19Rheinwald J.G. Green H. Cell. 1975; 6: 331-344Abstract Full Text PDF PubMed Scopus (3836) Google Scholar) or in low calcium medium (20Hodivala K.J. Watt F.M. J. Cell Biol. 1994; 124: 589-600Crossref PubMed Scopus (205) Google Scholar) (0.1 mm calcium). For induction of cell-cell contacts, confluent keratinocytes grown in low calcium medium were changed to standard medium for different periods of time. COS-7 cells, human breast carcinoma cell line MDA-MB-468, human keratinocyte cell line HaCAT, and human epidermoid carcinoma cell line A-431 were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. Primary cortical glial cells were isolated from rat pups (2 days postnatal) as described (21Banker G. Goslin K. Nature. 1988; 336: 185-186Crossref PubMed Scopus (156) Google Scholar). Primary mouse keratinocyte growth medium contained calcium-free Eagle's minimal essential medium, 0.05 mmCaCl2, 0.4 μg/ml hydrocortisone, 5 μg/ml insulin, 10 ng/ml epidermal growth factor, 2 × 10−9m 3,3′,5-triiodo-l-thyronine, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, 4% Chelex-treated fetal bovine serum. The following monoclonal antibodies were used to detect E-cadherin: mouse monoclonal HECD-1 (22Shimoyama Y. Hirohashi S. Hirano S. Nogushi M. Shimosato Y. Takeichi M. Abe O. Cancer Res. 1989; 49: 2128-2133PubMed Google Scholar) and rat monoclonal ECCD-2 (23Hirai Y. Nose A. Kobayashi S. Takeichi M. Development. 1989; 105: 271-277PubMed Google Scholar) (Santa Cruz Biotechnology, Inc.). Mouse monoclonal antibodies used included anti-N-cadherin (13A9), anti-β1-integrins P5D2 and VM2 (24Dittel B.N. McCarthy J.B. Wayner E.A. LeBien T.W. Blood. 1993; 81: 2272-2282Crossref PubMed Google Scholar, 27Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (650) Google Scholar), and anti-Myc and anti-FLAG epitopes (Sigma). Rabbit polyclonal antiserum used were anti-α-catenin (VB1) (25Braga V.M.M. Hodivala K.J. Watt F.M. Cell Adhesion Commun. 1995; 3: 201-215Crossref PubMed Scopus (58) Google Scholar) (Santa Cruz Biotechnology), anti-Zyxin (B38) provided by M. Beckerle (University of Utah) (26Macalma T. Otte J. Hensler M.E. Bockholt S.M. Louis H.A. Kalff-Suske M. Grzeschik K.-H. von der Ahe D. Beckerle M.C. J. Biol. Chem. 1996; 271: 31470-31478Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and affinity-purified anti-Ajuba (HA35) (7Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (113) Google Scholar). Antiserum to human LPP was kindly provided by M. Petit (6Petit M.M.R. Fradelizi J. Golsteyn R.M. Ayoubi T.A.Y. Menichi B. Louvard D. Van de Ven W.J.M. Friederich E. Mol. Biol. Cell. 2000; 11: 117-1129Crossref PubMed Scopus (119) Google Scholar), and Trip6 antiserum was kindly provided by M. Beckerle (University of Utah) (16Yi J. Kloeker S. Jensen C.C. Bockholt S. Honda H. Hirai H. Beckerle M.C. J. Biol. Chem. 2002; 277: 9580-9589Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Antiserum directed against murine LIMD1 was generated by immunizing rabbits with a baculovirus produced and purified PreLIM region of LIMD1. Secondary antibodies for immunofluorescence were from Jackson Laboratories (Stratech Scientific). Immunofluorescence was performed essentially as described (27Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (650) Google Scholar). Briefly, cells were fixed in 3% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100 in 10% fetal calf serum/PBS1 for 10 min before sequential incubation with the primary and secondary antibodies. For some experiments, simultaneous fixation and permeabilization was performed using 3% paraformaldehyde and 0.5% Triton X-100 for 10 min at room temperature. In addition, prepermeabilization of the cells in 0.5% Triton X-100, 10 mm PIPES, pH 6.8, 50 mmNaCl, 3 mm MgCl2, 300 mm sucrose, 1 mm phenylmethylsulfonyl fluoride prior to fixation was performed (25Braga V.M.M. Hodivala K.J. Watt F.M. Cell Adhesion Commun. 1995; 3: 201-215Crossref PubMed Scopus (58) Google Scholar). Images were collected using a Bio-Rad confocal microscope. Different Ajuba plasmids were microinjected into the nucleus of normal keratinocytes grown in standard medium as small colonies (28Braga V.M.M. Betson M., Li, X. Lamarche-Vane N. Mol. Biol. Cell. 2000; 11: 3703-3721Crossref PubMed Scopus (130) Google Scholar). After 2-h expression, coverslips were double labeled for E-cadherin and the Myc tag. Latex beads (15 μm; Polysciences) were coated with the mouse monoclonal anti-E-cadherin (HECD-1), anti-integrin (VM2), or bovine serum albumin as previously described (27Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (650) Google Scholar). Keratinocytes cultured in low calcium medium were incubated with beads (105 beads/coverslip) resuspended in low calcium medium for 15 min at 37 °C. Coverslips were washed in PBS, fixed in 3% paraformaldehyde, and co-stained with phalloidin and anti-Ajuba or anti-α-catenin antiserum. Amino-terminal hexa-Myc-tagged mammalian expression vectors used were pCS2-mAjuba, pCS2-PreLIM (NH2-terminal PreLIM domain of Ajuba), pCS2-LIM (COOH-terminal three LIM domains of Ajuba) (13Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar), pCS2-hZyxin, pCS2-hLPP, and pCS2-mLIMD1. Bacterial expression plasmids (pGEX vector; Amersham Biosciences) were GST-β-catenin (full-length), GST-A907 (α-catenin full-length), GST-C447 (α-catenin COOH-terminal amino acids), GST-N228 (α-catenin NH2-terminal amino acids (3Rimm D.L. Koslov E.R. Kebriaei P. Cianci C.D. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8813-8817Crossref PubMed Scopus (627) Google Scholar)), and GST-E-cadherin tail (29Lickert H. Bauer A. Kemler R. Stappert J J. Biol. Chem. 2000; 275: 5090-5095Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). We introduced a His-FLAG epitope tag into the baculovirus vector pBacPAK9 (Clontech). All cDNAs were then subcloned to generate NH2-terminal epitope-tagged proteins and sequenced to confirm the proper reading frame. Recombinant baculoviruses were generated using the Clontech BacPAK baculovirus expression system as described by the manufacturer. SF21 insect cells were infected with viruses, and 48 h postinfection cells were collected and lysed, and proteins were purified by passing extracts over Protein A-Sepharose beads containing bound anti-FLAG monoclonal antibody. Following extensive washing of the columns, bound protein was eluted with FLAG peptide (Sigma), dialyzed, and concentrated. For Western blots, cultured cells were homogenized in SDS loading buffer or radioimmune precipitation buffer, sonicated, and boiled. After separation in a SDS-PAGE gel, samples were transferred to membranes, probed with primary antibodies, and revealed by enhanced chemiluminescence (Amersham Biosciences). For pull-down assays, COS-7 cells were transfected by LipofectAMINE (Clontech) with 10 μg of plasmid and incubated overnight at 37 °C. After a wash in PBS supplemented with 1 mm phenylmethylsulfonyl fluoride, cells were scraped and pooled together in solubilization buffer A (10 mm Tris-Cl, pH 7.6, 1% Nonidet P-40, 150 mmNaCl, 5 mm EDTA, 5 mm EGTA, 50 mmsodium fluoride, 1 mm sodium orthovanadate, and protease inhibitors). After solubilization for 1 h at 4 °C on a rotating wheel, the lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. Precleared supernatants were incubated with the different GST fusion proteins bound to the glutathione-agarose beads for 1 h on a wheel at 4 °C. GST was used as a negative control. The beads were then washed four times with solubilization buffer (same composition as above, but with 0.4% Nonidet P-40). Bound proteins were detected by Western blotting using anti-Myc antibodies. COS-7 cells were transfected using Trans-IT LT1 (Panvera, Inc.) with 10 μg of plasmid and incubated for 24 h. Cells were harvested, washed twice in ice-cold PBS, and then lysed in 500 μl of cold G-Buffer (5 mm Tris, pH 8.0, 0.2 mm ATP, 0.5 mm dithiothreitol, and 0.2 mm CaCl2) containing protease inhibitors. To polymerize actin, G-actin stocks (Cytoskeleton, Inc.) were diluted to 0.4 mg/ml in G-Buffer and incubated on ice for 1 h. Polymerization buffer 50× (2.5 m KCl, 100 mmMgCl2, 50 mm ATP) was diluted to 1× in the actin stock for 1 h at room temperature. F-actin (5.5 μm) was mixed with COS lysate or purified protein for 1 h at room temperature. Samples were then centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was removed, and the pellet was resuspended in polymerization buffer. Equivalent amounts of the supernatant and pellet fractions were analyzed by Western blotting with anti-Myc or anti-FLAG antibodies. Cells were harvested, washed twice in ice-cold PBS, and lysed in isotonic lysis buffer A (150 mm NaCl, 20 mm Tris, pH 7.5, 1 mm EDTA, 1% Nonidet P-40) with protease inhibitors. Precleared lysates were incubated with primary antiserum for 1 h at 4 °C, Protein A/G-Sepharose was added and incubated at 4 °C overnight. Immune complexes were pelleted by centrifugation and washed four times with isotonic lysis buffer. Precipitated proteins were identified by Western blots. For immunodepletion experiments, HaCAT cell extracts were prepared as described above, and a sample of preimmunoprecipitation extract was put aside. Extracts were divided into three portions and immunoprecipitated with antibodies against Ajuba, E-cadherin, or α-catenin. Following immunoprecipitation, a portion of the postimmunoprecipitation extract was put aside. Equal amounts of pre- and postimmunoprecipitation extracts were then immunoblotted with anti-Ajuba or anti-Zyxin antibodies. Immune complexes were also immunoblotted to confirm immunoprecipitation of the appropriate protein. Membranes were prepared from cells lysed in hypotonic buffer on ice. Following a low speed spin, the supernatant underwent a high speed spin (100,000 × g) to pellet membranes. Membranes were resuspended in buffer A. His-FLAG-Ajuba, His-FLAG-PreLIM, and His-FLAG-LIM protein (5 μg each) were precleared by incubation with 5 μg of GST and glutathione-agarose beads in isotonic lysis buffer for 1 h at 4 °C. After centrifugation, each protein was incubated with 5 μg of each purified GST fusion protein with E-cadherin tail, β-catenin, full-length α-catenin, α-catenin N228, and α-catenin C447 for 1 h at 4 °C. Glutathione-agarose beads were added and incubated at 4 °C overnight. After centrifugation, pellets were washed four times with isotonic lysis buffer. Precipitated proteins were detected by Western blotting using an anti-FLAG antibody. Prior analyses had demonstrated that Ajuba was expressed in tissues rich in epithelia (13Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar). Thus, we determined and contrasted protein levels of Ajuba and related family members, in primary epithelial cells (mouse keratinocytes) and primary mesenchymal cells (mouse embryonic fibroblasts (MEFs)) (Fig. 1 a). Surprisingly, the level of Ajuba present in epithelial cells greatly exceeded that in MEFs (by 10–20-fold), whereas for other family members expression in MEFs was greater than (e.g. LPP, Trip6, and Zyxin) or equivalent to (e.g. LIMD1) epithelial cells. Thus, of the Zyxin family of cytosolic LIM proteins, Ajuba was preferentially expressed in epithelial cells. This analysis also demonstrated that the Ajuba antiserum did not cross-react with other family members. The multiple bands detected with the Ajuba antiserum represent differential serine/threonine phosphorylation of Ajuba (data not shown). Next, we determined the subcellular localization of endogenous Ajuba in primary epithelial cells: human keratinocytes and rat glial cells. In keratinocytes, immunostaining for Ajuba co-localized with E-cadherin staining at junctions (Fig. 1, b and c). Whereas E-cadherin staining at the cell surface was uniform, Ajuba staining was more discontinuous. The same junctional localization of Ajuba was observed in primary rat glial cells using anti-N-cadherin and anti-Ajuba antibodies (data not shown). This junctional localization of Ajuba was specific, since preabsorption of the antibody with GST-Ajuba completely abolished staining at cell-cell contacts, while not affecting E-cadherin staining (Fig. 1, d and e). Western blot analysis of subcellular fractions prepared from confluent sheets of the keratinocytes cell line, HaCAT cells, indicated that ∼10% of the cellular Ajuba was present in the membrane fraction (total cell membranes, data not shown). Thus, in primary epithelial cells, Ajuba was found to localize at membranes where neighboring cells contact each other and co-localized with cadherins at these cell-cell junctions. Zyxin, a related family member, localizes at sites of adhesion to substratum (focal contacts) in fibroblasts (5Crawford A.W. Beckerle M.C. J. Biol. Chem. 1991; 266: 5847-5853Abstract Full Text PDF PubMed Google Scholar). To determine whether Ajuba was also recruited to these sites in keratinocytes, cells were fixed and permeabilized simultaneously to facilitate visualization of focal adhesions. Under these conditions, β1-integrin staining was observed at both cell-cell contacts and focal adhesions (Fig. 2, a and e). Whereas Zyxin staining showed strong labeling at focal contacts (Fig. 2 f), only a minor proportion of Ajuba staining co-localized with β1 integrins at these sites (Fig. 2 b). Junctional staining of Ajuba is not well visualized in this focal plane, since it was chosen to optimize β-integrin staining. Ajuba staining was predominantly at the cell junctions and co-localized with E-cadherin (Fig. 2, c and d), whereas only faint labeling of Zyxin was seen at the keratinocyte junctions (Fig. 2,g and h). Thus, in primary keratinocytes, the localization of Ajuba was predominantly at cell-cell junctions and distinct from the localization of Zyxin, which was primarily at focal adhesion sites. Cadherins and cadherin-associated proteins present at cell-cell adhesion sites become detergent-insoluble following recruitment to cell surface adhesive complexes. To determine whether Ajuba also became detergent-insoluble after redistribution to junctions, cell-cell contacts were induced, and the total amount of cadherin and Ajuba staining at junctions was determined (Fig. 3, a and b,fixed). We tested two different extraction conditions of increasing stringency: fixed and permeabilized at the same time (Fig. 3, c and d, fixed/perm) or prepermeabilized before fixation (Fig. 3, e andf, pre-perm; see “Experimental Procedures” for details). Under both extraction conditions, the cytoplasmic staining of E-cadherin and Ajuba was mostly removed, whereas a significant proportion of the two proteins remained insoluble at the keratinocyte junctions (Fig. 3, arrows). This indicated that, under these biochemical conditions, Ajuba and E-cadherin were recruited to the same “compartment” at cell-cell junctions. A time course of induction of cell-cell contacts was studied in keratinocytes. To initiate cell-cell contacts, confluent keratinocytes were switched from low Ca2+ to standard Ca2+-containing medium, and cells were double labeled for E-cadherin and Ajuba (Fig. 4). In control cells (maintained in the absence of cell-cell adhesion), no co-localization of Ajuba and E-cadherin was observed in the cytosol (low calcium medium) (Fig. 4,a–c). However, as early as 5 min after cell-cell adhesion was stimulated by calcium addition, both Ajuba and E-cadherin accumulated at contact sites (Fig. 4, d and e), and this progressively increased over 60 min (Fig. 4, g andh). Merged images indicated that Ajuba and E-cadherin were now co-localized at cell-cell contact sites (Fig. 4, f andi). These results indicated that Ajuba recruitment to cell junctions was regulated by the initiation of E-cadherin-mediated adhesion and temporally followed E-cadherin redistribution to the cell surface. Many other cellular proteins are recruited to E-cadherin-dependent adhesive sites. To determine whether clustering of E-cadherin receptors was sufficient to recruit Ajuba, we used latex beads coated with antibodies against E-cadherin (27Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (650) Google Scholar). This technique has been routinely used to demonstrate the recruitment of specific cytosolic proteins according to the receptor clustered (27Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (650) Google Scholar). We incubated keratinocytes grown in the absence of cell-cell contact with anti-E-cadherin (Fig. 5,a–d), anti-integrin (Fig. 5, e andf), or bovine serum albumin-coated beads (data not shown). Under these conditions, Ajuba was recruited to anti-E-cadherin beads but not substantially to anti-integrin beads (Fig. 5, compared with f) or bovine serum albumin beads (data not shown). Both types of antibody-coated beads were able to recruit actin (Fig. 5, a, c, and e). As a positive control, α-catenin was recruited to anti-E-cadherin-coated beads (Fig. 5 b). Thus, it seems that E-cadherin clustering was sufficient to translocate cytosolic Ajuba to the adhesive complex. To determine what component(s) of the cadherin adhesive complex Ajuba interacted with, GST-fusion proteins comprising components of the cadherin adhesive complex were added to COS cell extracts prepared from cells transiently transfected with Ajuba plasm" @default.
• W2110052730 created "2016-06-24" @default.
• W2110052730 creator A5003470989 @default.
• W2110052730 creator A5005580794 @default.
• W2110052730 creator A5016232722 @default.
• W2110052730 creator A5020112282 @default.
• W2110052730 creator A5020338130 @default.
• W2110052730 creator A5025268046 @default.
• W2110052730 creator A5041062049 @default.
• W2110052730 creator A5054589692 @default.
• W2110052730 creator A5079529198 @default.
• W2110052730 creator A5088714641 @default.
• W2110052730 creator A5090749270 @default.
• W2110052730 date "2003-01-01" @default.
• W2110052730 modified "2023-10-12" @default.
• W2110052730 title "The LIM Protein Ajuba Is Recruited to Cadherin-dependent Cell Junctions through an Association with α-Catenin" @default.
• W2110052730 cites W1509886380 @default.
• W2110052730 cites W1596385135 @default.
• W2110052730 cites W1962281270 @default.
• W2110052730 cites W1980907691 @default.
• W2110052730 cites W1988432188 @default.
• W2110052730 cites W1998776819 @default.
• W2110052730 cites W2003496812 @default.
• W2110052730 cites W2006133276 @default.
• W2110052730 cites W2023080606 @default.
• W2110052730 cites W2026728942 @default.
• W2110052730 cites W2036832250 @default.
• W2110052730 cites W2044850263 @default.
• W2110052730 cites W2049043712 @default.
• W2110052730 cites W2058170261 @default.
• W2110052730 cites W2058416613 @default.
• W2110052730 cites W2061987693 @default.
• W2110052730 cites W2067236137 @default.
• W2110052730 cites W2067671393 @default.
• W2110052730 cites W2074018262 @default.
• W2110052730 cites W2077743844 @default.
• W2110052730 cites W2088574304 @default.
• W2110052730 cites W2093279875 @default.
• W2110052730 cites W2102391005 @default.
• W2110052730 cites W2111683138 @default.
• W2110052730 cites W2118857009 @default.
• W2110052730 cites W2118934341 @default.
• W2110052730 cites W2120492537 @default.
• W2110052730 cites W2133307942 @default.
• W2110052730 cites W2137079116 @default.
• W2110052730 cites W2143646667 @default.
• W2110052730 cites W2146140843 @default.
• W2110052730 cites W2150518441 @default.
• W2110052730 cites W2155515810 @default.
• W2110052730 cites W2169019804 @default.
• W2110052730 cites W2342443123 @default.
• W2110052730 doi "https://doi.org/10.1074/jbc.m205391200" @default.
• W2110052730 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12417594" @default.
• W2110052730 hasPublicationYear "2003" @default.
• W2110052730 type Work @default.
• W2110052730 sameAs 2110052730 @default.
• W2110052730 citedByCount "146" @default.
• W2110052730 countsByYear W21100527302012 @default.
• W2110052730 countsByYear W21100527302013 @default.
• W2110052730 countsByYear W21100527302014 @default.
• W2110052730 countsByYear W21100527302015 @default.
• W2110052730 countsByYear W21100527302016 @default.
• W2110052730 countsByYear W21100527302017 @default.
• W2110052730 countsByYear W21100527302018 @default.
• W2110052730 countsByYear W21100527302019 @default.
• W2110052730 countsByYear W21100527302020 @default.
• W2110052730 countsByYear W21100527302021 @default.
• W2110052730 countsByYear W21100527302022 @default.
• W2110052730 countsByYear W21100527302023 @default.
• W2110052730 crossrefType "journal-article" @default.
• W2110052730 hasAuthorship W2110052730A5003470989 @default.
• W2110052730 hasAuthorship W2110052730A5005580794 @default.
• W2110052730 hasAuthorship W2110052730A5016232722 @default.
• W2110052730 hasAuthorship W2110052730A5020112282 @default.
• W2110052730 hasAuthorship W2110052730A5020338130 @default.
• W2110052730 hasAuthorship W2110052730A5025268046 @default.
• W2110052730 hasAuthorship W2110052730A5041062049 @default.
• W2110052730 hasAuthorship W2110052730A5054589692 @default.
• W2110052730 hasAuthorship W2110052730A5079529198 @default.
• W2110052730 hasAuthorship W2110052730A5088714641 @default.
• W2110052730 hasAuthorship W2110052730A5090749270 @default.
• W2110052730 hasConcept C104317684 @default.
• W2110052730 hasConcept C137620995 @default.
• W2110052730 hasConcept C142853389 @default.
• W2110052730 hasConcept C1491633281 @default.
• W2110052730 hasConcept C149402561 @default.
• W2110052730 hasConcept C15744967 @default.
• W2110052730 hasConcept C185592680 @default.
• W2110052730 hasConcept C201508349 @default.
• W2110052730 hasConcept C24284526 @default.
• W2110052730 hasConcept C30145163 @default.
• W2110052730 hasConcept C542102704 @default.
• W2110052730 hasConcept C5432534 @default.
• W2110052730 hasConcept C54355233 @default.
• W2110052730 hasConcept C62478195 @default.
• W2110052730 hasConcept C86339819 @default.
• W2110052730 hasConcept C86803240 @default.
• W2110052730 hasConcept C95444343 @default.

• next