The LIM Protein Ajuba Is Recruited to Cadherin-dependent Cell Junctions through an Association with alpha -Catenin*

Helene MarieDagger §, Stephen J. Pratt||, Martha BetsonDagger , Holly Epple||, Josef T. KittlerDagger , Laura Meek||, Stephen J. MossDagger , Sergey Troyanovsky||, David Attwell§, Gregory D. Longmore||**, and Vania M. M. BragaDagger DaggerDagger

From the Dagger  Medical Research Council Laboratory for Molecular Cell Biology and § Department of Physiology, University College London, Gower Street, London WC1E 6BT, United Kingdom and the || Departments of Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, May 31, 2002, and in revised form, October 8, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 alpha -catenin, and alpha -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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1, 2).

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 (1). 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. beta -Catenin (or gamma -catenin/plakoglobin) associates directly with the cadherin tail, and then alpha -catenin bridges the beta -catenin-cadherin complex to actin filaments (1). alpha -Catenin is an essential component of the cadherin complex (1). It not only binds and bundles actin (3) but also provides docking sites for other cytoskeletal proteins that may contribute to the cytoskeletal reorganization at junctions, such as vinculin and alpha -actinin (reviewed in Ref. 4). Cytosolic proteins containing LIM domains have also been observed to localize at cell-cell contact sites (5-8).

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. 9). 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 (9). 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 (10). Within this family, there have been five mammalian members described: Zyxin (5), LPP (11), Trip6 (12), Ajuba (13), and LIMD1 (14).

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 (6, 15, 16) but have also been observed at cell-cell contact sites in epithelial cells (5-7). In fibroblasts, Zyxin and Trip6 appear to affect cell motility (16, 17). In addition, they contain nuclear export signals and shuttle between the nucleus and cytoplasm (6, 7, 18). 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 (7). 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 (13). Immunofluorescence analysis of embryonal carcinoma cells revealed that, as sheets of contacted cells formed, Ajuba was localized to cell-cell contacts (7). 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 alpha -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 (7, 13), Ajuba may also function in the regulation of cell-cell adhesion.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cells-- Normal human keratinocytes (strain Kb, passages 3-7) were grown in standard medium (1.8 mM calcium ions) (19) or in low calcium medium (20) (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 (21). Primary mouse keratinocyte growth medium contained calcium-free Eagle's minimal essential medium, 0.05 mM CaCl2, 0.4 µg/ml hydrocortisone, 5 µg/ml insulin, 10 ng/ml epidermal growth factor, 2 × 10-9 M 3,3',5-triiodo-L-thyronine, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 4% Chelex-treated fetal bovine serum.

Antibodies-- The following monoclonal antibodies were used to detect E-cadherin: mouse monoclonal HECD-1 (22) and rat monoclonal ECCD-2 (23) (Santa Cruz Biotechnology, Inc.). Mouse monoclonal antibodies used included anti-N-cadherin (13A9), anti-beta 1-integrins P5D2 and VM2 (24, 27), and anti-Myc and anti-FLAG epitopes (Sigma). Rabbit polyclonal antiserum used were anti-alpha -catenin (VB1) (25) (Santa Cruz Biotechnology), anti-Zyxin (B38) provided by M. Beckerle (University of Utah) (26), and affinity-purified anti-Ajuba (HA35) (7). Antiserum to human LPP was kindly provided by M. Petit (6), and Trip6 antiserum was kindly provided by M. Beckerle (University of Utah) (16). 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).

Immunostaining and Microinjection-- Immunofluorescence was performed essentially as described (27). 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 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 1 mM phenylmethylsulfonyl fluoride prior to fixation was performed (25). 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 (28). 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 (27). 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-alpha -catenin antiserum.

Expression Plasmids, GST Fusion Proteins, and Protein Purification-- 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) (13), pCS2-hZyxin, pCS2-hLPP, and pCS2-mLIMD1. Bacterial expression plasmids (pGEX vector; Amersham Biosciences) were GST-beta -catenin (full-length), GST-A907 (alpha -catenin full-length), GST-C447 (alpha -catenin COOH-terminal amino acids), GST-N228 (alpha -catenin NH2-terminal amino acids (3)), and GST-E-cadherin tail (29).

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.

Western Blots and Pull-down Assays-- 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 mM NaCl, 5 mM EDTA, 5 mM EGTA, 50 mM sodium 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.

Actin Co-sedimentation-- 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 mM MgCl2, 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.

Immunoprecipitation and Immune Depletion-- 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 alpha -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.

In Vitro Protein-Protein Interaction-- 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, beta -catenin, full-length alpha -catenin, alpha -catenin N228, and alpha -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ajuba Co-localized with the Cadherin Adhesive Complex at Cell-Cell Contact Sites in Primary Keratinocytes-- Prior analyses had demonstrated that Ajuba was expressed in tissues rich in epithelia (13). 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. 1a). 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).


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Fig. 1.   Ajuba co-localizes with cadherins at cell-cell contacts in primary keratinocytes. a, Western blot analyses of Ajuba/Zyxin family members and alpha -catenin in primary mouse embryonic fibroblasts (MEF) (left column) and primary mouse keratinocytes (right column). Equal amounts of protein were loaded in each lane. b-e, primary skin keratinocytes double immunofluorescence for E-cadherin (b and d) and Ajuba (c and e). In d and e, the same double labeling was done, but the Ajuba and E-cadherin antibodies were first preincubated with GST-Ajuba.

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 (5). 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, beta 1-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. 2f), only a minor proportion of Ajuba staining co-localized with beta 1 integrins at these sites (Fig. 2b). Junctional staining of Ajuba is not well visualized in this focal plane, since it was chosen to optimize beta -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.


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Fig. 2.   Ajuba preferentially localizes at cell junctions, not focal adhesion sites, in epithelial cells. Keratinocytes grown in standard medium were stained for beta 1-integrins (a and e) to visualize focal adhesions and for E-cadherin (c and g) to label cell-cell contacts. Cells were co-stained with either Ajuba antibody (b and d) or Zyxin antibody (f and h). The arrows identify focal contact staining; arrowheads show junctional staining. *, a fibroblast cell from the feeder layer. In a, b, e, and f, the focal plane selected gives optimal focal contact staining. In c and d, stratified keratinocytes are present at the base of the colony obscuring junctional staining.

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 and f, 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.


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Fig. 3.   Ajuba becomes detergent-insoluble following recruitment to cell junctions. Cells grown in low calcium medium (absence of cell-cell adhesion) were transferred to standard medium for 10 min to induce cell-cell contacts. After fixation, cells were stained with E-cadherin (a, c, and e) and Ajuba (b, d, and f) antibodies. To test for Ajuba detergent insolubility, cells were fixed and permeabilized at the same time (c and d) or prepermeabilized before fixation (e and f; see "Experimental Procedures" for details). The arrows indicate junctional staining.

Ajuba's Recruitment to Newly Formed Cell-Cell Contacts Was Rapid, Regulated, and Temporally Associated with E-cadherin Recruitment-- 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 and h). Merged images indicated that Ajuba and E-cadherin were now co-localized at cell-cell contact sites (Fig. 4, f and i). 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.


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Fig. 4.   Ajuba is rapidly recruited to newly formed cell-cell contacts in keratinocytes. Cells grown in low calcium medium (absence of cell-cell adhesion) (a-c) were transferred to standard medium for 5 min (d-f) or 60 min (g-i) to induce cell-cell contacts. Cells were stained for E-cadherin (a, d, and g) and Ajuba (b, e, and h). Merged images are shown in c, f, and i. The arrows in c identify the distinct staining pattern of Ajuba (green) and E-cadherin (red) in the absence of junction formation. The arrows in e identify Ajuba recruited to newly formed cell junctions.

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 (27). This technique has been routinely used to demonstrate the recruitment of specific cytosolic proteins according to the receptor clustered (27). We incubated keratinocytes grown in the absence of cell-cell contact with anti-E-cadherin (Fig. 5, a-d), anti-integrin (Fig. 5, e and f), 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, compare d 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, alpha -catenin was recruited to anti-E-cadherin-coated beads (Fig. 5b). Thus, it seems that E-cadherin clustering was sufficient to translocate cytosolic Ajuba to the adhesive complex.


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Fig. 5.   Cytosolic Ajuba translocates to clustered cell surface E-cadherin adhesive receptors. Latex beads coated with anti-E-cadherin (a-d) or anti-integrin (e and f) antibodies were resuspended in low calcium medium and incubated with keratinocytes grown in the absence of cell-cell contacts. Cells were fixed and stained for filamentous actin (a, c, and e), Ajuba (d and f), or alpha -catenin (b). Cellular staining is diffuse, and no staining at junctions is seen, because cells were grown in low calcium medium, and the confocal plane used to visualize the beads is at the cell apical domain. The arrows indicate the presence of staining at beads, and the arrowheads show the absence of staining at beads.

Ajuba Associated with alpha -Catenin at the Cadherin-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 plasmid (Myc-tagged) (Fig. 6a). Ajuba associated with full-length alpha -catenin and an NH2-terminal portion of alpha -catenin (GST-N228). Compared with control GST alone, there was no association between Ajuba and the GST-E-cadherin cytoplasmic tail, GST-C447 (COOH terminus of alpha -catenin), or GST-beta -catenin.


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Fig. 6.   Ajuba associates with alpha -catenin in vivo and in vitro. a, GST fusion proteins containing full-length alpha -catenin (A907), alpha -catenin NH2-terminal (N228), alpha -catenin COOH-terminal region (C447), beta -catenin, and the E-cadherin cytoplasmic tail were used to precipitate Myc-tagged Ajuba from transfected COS-7 cell extracts. GST was used as a negative control. b, alpha -catenin antibodies were used to immunoprecipitate endogenous alpha -catenin from cells transfected with Myc-tagged Ajuba, Zyxin, LPP, or LMD1. Co-associated proteins were detected by Western blotting with Myc antibody (right). Expression levels of the various transfected proteins are shown by probing cell lysates (one-fortieth of total extract) with Myc antibody (left). c, FLAG-tagged purified Ajuba protein was mixed with various purified GST-alpha -catenin peptides. Glutathione-agarose-bound products were immunoblotted with FLAG antibody to detect Ajuba (right strips). The left panel depicts the purified FLAG-Ajuba protein used. d, left panel, extracts from HaCAT cells, which contain endogenous levels of Ajuba and alpha -catenin, were immunoprecipitated with preimmune serum (lane 1), Ajuba (lane 2), or Zyxin (lane 4) antibodies. In lane 3, one-fortieth of the total cell extract was run. Bound products were immunoblotted for alpha -catenin. Right panels, membrane fractions were prepared from A431 cells. In lane 1, one-tenth of the total membrane extract was run; lane 2, no primary antibody was added; lane 3, E-cadherin immunoprecipitation (four-tenths of total membrane preparation); lane 4, Ajuba immunoprecipitation (four-tenths of total membrane preparation). Bound products were immunoblotted with E-cadherin antibody (upper panel) and alpha -catenin antibody (lower panel). e, cell lysates from human breast carcinoma cells with (+) or without (-) alpha -catenin were immunoblotted for E-cadherin (top panel), alpha -catenin (center panel), and Ajuba (lower panel). Human breast carcinoma cells containing alpha -catenin (+) or without alpha -catenin (-) were transfected with Myc-Ajuba and stained for Myc (upper panels). The lower panels are light images of cells. The arrows indicate junctional staining.

Next, endogenous alpha -catenin was immunoprecipitated from extracts from COS cells transfected with plasmids expressing Myc-tagged Ajuba or, as controls for specificity, family members Zyxin, LPP, and LIMD1 (all Myc-tagged) (Fig. 6b). All transfected Zyxin family members were expressed at similar level, as shown by Myc immunoblots of cell extracts (Fig. 6b, extract). Approximately 2-3% of the total cellular Ajuba co-immunoprecipitated with alpha -catenin (Fig. 6b), whereas less than 0.1% of total cellular Zyxin, LPP, and LIMD1 co-immunoprecipitated with alpha -catenin. This represented a 20-30-fold difference in the association of Ajuba with alpha -catenin compared with related family members. The low level of Ajuba associated with alpha -catenin most likely reflects the large cytoplasmic pools of Ajuba and alpha -catenin that presumably do not interact (see below).

We next asked whether the association between Ajuba and alpha -catenin could occur directly. Baculovirus-produced and -purified FLAG-tagged Ajuba was mixed with purified GST-alpha -catenin protein in vitro, and then glutathione-agarose was added. Bound products were separated and immunoblotted with FLAG antibodies (Fig. 6c). Ajuba interacted directly with full-length alpha -catenin (Fig. 6c, right strips, lane 1) and the NH2-terminal region of alpha -catenin (N228, Fig. 6c, right strips, lane 3) but not with the COOH-terminal region of alpha -catenin (C447, Fig. 6c, right strips, lane 2).

To determine whether Ajuba and alpha -catenin associated in cells expressing endogenous levels of each protein, Ajuba was immunoprecipitated from the human keratinocyte cell line, HaCAT, and the bound products were immunoblotted with anti-alpha -catenin antiserum. Antibodies against Ajuba, but not preimmune serum or Zyxin antibodies, co-immunoprecipitated alpha -catenin (Fig. 6d, left). To determine whether Ajuba associated with alpha -catenin bound to membrane E-cadherin, Ajuba was immunoprecipitated from a membrane fraction of A431 cells, and the products were immunoblotted for the presence of alpha -catenin and E-cadherin (Fig. 6d, right). Approximately 10% of the membrane E-cadherin was present in Ajuba immunoprecipitates. In an alternative approach, immunodepletion of E-cadherin or alpha -catenin from HaCAT membrane preparations resulted in a reduction of Ajuba protein levels while not affecting Zyxin levels (data not shown).

Taken together, these data indicated that of proteins known to be present in the cadherin adhesive complex, Ajuba associated with alpha -catenin but not beta -catenin or the cytoplasmic tail of E-cadherin. This association occurred with membrane E-cadherin-bound alpha -catenin. The association between Ajuba and alpha -catenin was preferentially through the NH2-terminal region of alpha -catenin. Finally, purified Ajuba interacted directly with alpha -catenin protein, in vitro.

Recruitment of Ajuba to Cell Junctions Required alpha -Catenin-- To determine whether alpha -catenin was necessary for the recruitment of Ajuba to E-cadherin-mediated cell-cell contacts, we determined the distribution of Myc-tagged Ajuba following transient transfection of a human breast carcinoma cell line that lacked expression of alpha -catenin protein. As a control, the same cell line in which alpha -catenin had been stably transfected was also examined. Western blot analysis of extracts from these cells demonstrated the absence of alpha -catenin in the parental cells and its presence in the alpha -catenin-transfected cells (Fig. 6e, right). The lack of alpha -catenin and its reintroduction did not affect the levels of E-cadherin or endogenous Ajuba (Fig. 6e, right). In cells lacking alpha -catenin, transfected Myc-Ajuba was predominantly cytosolic (Fig. 6e, right), whereas reintroduction of alpha -catenin resulted in the localization of Myc-Ajuba to the cell surfaces between adherent cells (Fig. 6e, left). These data indicated that the efficient recruitment of Ajuba to cell-cell adhesion sites required the presence of alpha -catenin.

Domains of Ajuba Required for Junctional Recruitment and Association with alpha -Catenin-- To determine which domains of Ajuba directed its recruitment to cell junctions, primary keratinocytes (grown in the presence of cell-cell contacts) were microinjected with plasmids expressing Myc-tagged Ajuba (Fig. 7, a and b), the PreLIM region of Ajuba (Fig. 7, c and d), or the LIM domains of Ajuba (Fig. 7, e and f). After 2 h, cells were fixed and stained for the Myc epitope (Fig. 7, a, c, and e) and E-cadherin (Fig. 7, b, d, and f). In the majority of cells injected with full-length Ajuba or the LIM region, co-localization with E-cadherin at junctions was achieved (72 ± 2.7% and 55 ± 1.7% of injected patches, respectively). In contrast, a minority of cells injected with the PreLIM region showed staining at cell-cell adhesion sites (20 ± 5.7% of injected patches). As previously reported, the cellular distribution of the PreLIM region was mostly cytosolic, whereas in cells injected with the LIM regions, the LIM region accumulates in the nucleus (7). In a minority of cells injected with full-length Ajuba, both cytosolic and nuclear staining was apparent (7). These results suggest that the LIM regions of Ajuba "preferentially" direct its recruitment to cell junctions. Since full-length Ajuba localized to junctions more efficiently than the LIM regions alone, we cannot exclude the possibility that the PreLIM region contributes, in some manner (see "Discussion"), to optimal localization of Ajuba at cell-cell junctions.


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Fig. 7.   Mapping of Ajuba domains required for junctional recruitment and association with alpha -catenin. Plasmids expressing Myc-tagged full-length Ajuba (a and b), PreLIM region (c and d), and LIM region (e and f) were microinjected into the nuclei of normal human keratinocytes. After 2 h, cells were fixed and stained for E-cadherin (b, d, and f) and Myc (a, c, and e). The arrows point to Myc-Ajuba and Myc-LIM staining at junctions (a and e, respectively); the arrowhead demonstrates absence of Myc-PreLIM staining at cell-cell adhesion (c). LIM domains localized to both junctions and nuclei. The images shown are a single confocal plane (1 µm thick). Plasmid-injected cells (200 for each plasmid) were identified, and the number of patches with Myc staining at junction were enumerated and presented as the percentage positive for Myc staining. g, anti-alpha -catenin antibodies were used to immunoprecipitate endogenous alpha -catenin from epithelial cells transfected with empty vector, Myc-tagged Ajuba, Myc-PreLIM region, or Myc-LIM region. Co-precipitated proteins were detected by Western blotting with anti-Myc antibody. The left panel represents 5% of cell extract used for immunoprecipitation. The relative mobility of protein standards is shown on the left (in kDa). The percentage of total protein associated with alpha -catenin was determined and presented as percentage of input associated with alpha -catenin.

To determine which domains of Ajuba directed its association with alpha -catenin, we transfected epithelial cells with Myc-tagged full-length Ajuba, the PreLIM region, or the LIM regions. Cells were lysed, and endogenous alpha -catenin was immunoprecipitated. Bounds products were immunoblotted for the presence of each Ajuba isoform (anti-Myc Western blot) (Fig. 7g). All three isoforms of Ajuba associated with alpha -catenin; however, when the percentage of Myc-tagged Ajuba input protein immunoprecipitated by anti-alpha -catenin antibodies was determined, 10-fold less PreLIM region (0.5%) associated with alpha -catenin compared with full-length Ajuba (5%) and LIM region (7%). Therefore, as observed in the junctional localization experiment (Fig. 7, a-f), full-length Ajuba and the LIM region preferentially associated with alpha -catenin.

Ajuba Interacted Directly with Actin Filaments-- Cadherin-mediated cell adhesion results in the reorganization of the cortical cytoskeleton. Since Ajuba was found to be detergent-insoluble at junctions (Fig. 3) and Ajuba was translocated to E-cadherin-adhesive receptors following receptor clustering (Fig. 5) and other cytosolic LIM-containing proteins co-localize with the cytoskeleton (15) (9), we asked whether Ajuba could interact with actin filaments and, if so, how. Extracts from COS cells expressing Ajuba, PreLIM, or LIM domains of Ajuba (all Myc-tagged) were mixed with freshly polymerized actin. Filamentous actin was then pelleted by high speed centrifugation, and the soluble supernatant and F-actin pellet were analyzed by immunoblotting for the presence of Myc-tagged proteins (Fig. 8a). In the absence of added F-actin, Ajuba was present in only the soluble fraction (Fig. 8a, lanes 1 and 2), whereas the addition of F-actin resulted in a significant proportion of Ajuba recovered in the F-actin pellet (Fig. 8a, lanes 3 and 4). This indicated that Ajuba present in cell extracts could associate with actin filaments. Mapping studies showed that the PreLIM region of Ajuba associated with F-actin, whereas the LIM domains alone did not (Fig. 8a, lanes 5-8 and 9-12). Low speed centrifugation of the reaction mixture containing F-actin did not result in pelleted Ajuba, indicating that although Ajuba associated with F-actin, it did not bundle, or cross-link, actin filaments (data not shown).


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Fig. 8.   Ajuba interacts with actin in vivo and in vitro. a, cells expressing Ajuba, PreLIM, or LIM domains (all Myc-tagged) were lysed and incubated with freshly polymerized actin filaments (+). Controls were incubated without actin (-). Samples were then separated into soluble (s; containing G-actin) or pellet (p; containing F-actin), separated by SDS-PAGE, and immunoblotted with Myc antibody. b, baculovirus-produced and purified FLAG-tagged Ajuba isoforms were incubated in the presence (+) or absence (-) of freshly polymerized actin in vitro. Reactions were then pelleted, and the soluble (s) and pellet (p) fractions were immunoblotted with FLAG antibody. The relative mobility of protein standards is shown on the left of each panel (in kDa).

To determine whether the association between Ajuba and F-actin in cell extracts was direct or indirect, FLAG-tagged Ajuba fragments purified from baculovirus-infected insect cells were mixed with freshly polymerized F-actin, in vitro. The reaction was then pelleted by high speed centrifugation and analyzed by immunoblotting for the FLAG epitope (Fig. 8b). Ajuba and the PreLIM region of Ajuba co-sediment with F-actin, whereas the LIM domains alone did not (Fig. 8b and data not shown). The interaction of the PreLIM region of Ajuba with F-actin was saturated in the presence of 18 nM PreLIM Ajuba (data not shown). Further addition of PreLIM region peptide did not increase the amount detected in the F-actin pellets. As a control, there was no significant sedimentation into a pellet fraction when the different Ajuba fragments were centrifuged in the absence of added F-actin (Fig. 8b, lanes 1 and 2 and lanes 5 and 6). These results indicated that Ajuba interacted directly with filamentous actin, in vitro, and that the PreLIM region of Ajuba directed this association.

Cell Adhesion Is Abnormal in Ajuba Null Primary Keratinocytes-- To determine the functional significance of Ajuba's recruitment to cell-cell junctions, we derived mice lacking the Ajuba gene through homologous recombination.2 Primary skin keratinocytes were isolated from litter-matched newborns (wild type, +/+; and Ajuba null, -/-) derived from breeding of Ajuba heterozygous mice. Western blot analysis of primary keratinocyte cell extracts indicated that no Ajuba protein was present in the Ajuba null cells (Fig. 9c, upper panel) and that deletion of the Ajuba gene did not significantly affect the protein levels of Zyxin (Fig. 9c, middle panel), other Ajuba family members (data not shown), or alpha -catenin (Fig. 9c, lower panel).


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Fig. 9.   Ajuba contributes to cell-cell junction formation or stability and function. Primary keratinocytes were isolated from Ajuba null and wild type newborn littermates. a, once confluent, cells were switched to low calcium medium, and then, to initiate cell-cell junction formation, calcium was added. Cells were fixed and stained for E-cadherin. b, cell aggregation assay. Wild type and Ajuba null keratinocytes were grown to confluence in low calcium medium. Cells were trypsinized, and pellets were washed with PBS. 100,000 cells were plated in 2 ml of keratinocyte growth medium (low calcium) or 0.2 mM CaCl2 keratinocyte growth medium (high calcium) and immediately placed on a shaker at 80 rpm at 37 °C and 5% CO2 for 6 h. Cells were counted under low magnification and classified as nonaggregate (<3 cells/aggregate), small aggregate (3-5 cells/aggregate), medium aggregate (5-10 cells/aggregate), or large aggregate (>10 cells/aggregate). Data presented are the average of two different sets of cells from matched littermates. c, Western blot analysis of cell extracts from wild type (+/+) and Ajuba null (-/-) primary keratinocytes for expression of Ajuba (upper panel), Zyxin (middle panel), and alpha -catenin (lower panel). Equal amounts of protein were loaded in each lane.

Once a confluent layer of keratinocytes was achieved, a calcium switch experiment was performed, and cell junction integrity was assessed through staining for E-cadherin (Fig. 9a). In wild type cells at 4 h following calcium addition, E-cadherin was present at cell surfaces between adherent cells, and cell-cell adhesion had formed (Fig. 9a). In Ajuba null keratinocytes, however, at 4 h following calcium addition there remained significant gaps between cells despite the normal recruitment of E-cadherin to cell surfaces (Fig. 9a). These gaps persisted at longer times following calcium addition, until keratinization of the cell layer obscured any further follow-up. This same result was observed with Ajuba null keratinocytes derived from five different litters.

In another approach to assess the cell-cell adhesive function of Ajuba null keratinocytes, wild type or Ajuba null cells were trypsinized from plates and mixed in low or high calcium-containing media, and rotation was applied. Cell aggregate size and numbers were scored and tabulated (Fig. 9b). In low calcium medium (calcium-independent aggregation), there was no difference in the size or number of aggregates formed between the two cell types. However, in high calcium medium, which measures calcium-dependent adhesive events (e.g. E-cadherin mediated), the cell aggregates formed by Ajuba null cells were smaller. With Ajuba null keratinocytes, no aggregates of >10 cells were observed, and the number of aggregates containing 5-10 cells was dramatically reduced compared with wild type keratinocytes.

These results indicated that Ajuba contributes to cell-cell junction formation, stabilization of newly formed cell-cell junctions, or both. In agreement, calcium-dependent cell-cell adhesion of Ajuba null keratinocytes was dramatically decreased compared with wild type cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Of the proteins comprising the Zyxin family of cytosolic LIM proteins, the cellular distribution of Ajuba was distinctive. Its level of expression in epithelial cells greatly exceeded that detected in mesenchymal fibroblast cells, whereas for other family members, although present in epithelial cells, their level of expression in fibroblasts was greater, implying a unique role for Ajuba in epithelial cell biology. In primary epithelial cells we show, biochemically and by immunofluorescence, that Ajuba co-localizes with the cadherin adhesive complex at cell-cell contacts. In contrast to Zyxin, which predominantly localized to focal adhesion sites, Ajuba was predominantly at cell-cell junctions in keratinocytes. The previously reported localization of Zyxin at primary keratinocyte junctions (8) might result from either cross-reaction of the anti-Zyxin antibody used with LPP or other family members3 or represent a minor proportion of cellular Zyxin.

Whereas other family members (e.g. Zyxin and LPP) have been reported to localize to cell-cell contacts in epithelial cells (5, 6, 8), their precise localization and how they localize to these sites is not known. Herein, we show that Ajuba is recruited to membrane cadherin adhesive complexes at adherens junctions through an association with alpha -catenin. The following results support this conclusion. First, in pull-down experiments from cell lysates, Ajuba associated with alpha -catenin but not with beta -catenin or the E-cadherin cytoplasmic tail. Second, Ajuba co-immunoprecipitated with alpha -catenin in total cell extracts from cells expressing endogenous levels of each protein. Third, Ajuba interacted with alpha -catenin present at the cell membrane E-cadherin adhesive complex as Ajuba antibodies co-immunoprecipitated E-cadherin and alpha -catenin from membrane preparations from cells expressing endogenous levels of each protein. Fourth, immunodepletion of E-cadherin and alpha -catenin resulted in a reduction in Ajuba protein levels while not affecting the levels of Zyxin. Finally, Ajuba interacted directly with alpha -catenin, in vitro. Importantly, the recruitment of Ajuba to sites of cell-cell adhesion was severely delayed in epithelial cells not expressing alpha -catenin and could be rescued by reintroduction of alpha -catenin into these cells. Other LIM proteins, such as Paxillin and Zyxin, associate with the alpha -catenin-related proteins alpha -actinin or vinculin, but at focal adhesion sites in fibroblast cells (15, 30). In keratinocyte cell extracts, we did not observe any association between vinculin, which is also present at cell-cell contacts in epithelia, and Ajuba (data not shown). Ajuba does not interact with alpha -actinin, since it lacks the binding site present in Zyxin (31).

The amount of total cell Ajuba that associated with alpha -catenin was small (2-3%). We suggest that this is because the association between Ajuba and alpha -catenin occurs at the cell membrane cadherin adhesive complex and not within the cytosol. In support of this contention, subcellular fractionation of sheets of contacted epithelial cells demonstrates that ~10% of the total cellular Ajuba is membrane-associated (data not shown). The majority of cellular alpha -catenin in keratinocytes, like Ajuba, is cytosolic and not membrane-associated (data not shown) (32). Indeed, under our experimental conditions, E-cadherin antibodies co-immunoprecipitate a small proportion of the total cell alpha -catenin (see Fig. 6d), and we observed that Ajuba antibodies co-immunoprecipitated E-cadherin and alpha -catenin from membrane preparations. However, not all membrane cadherin adhesive complexes contained Ajuba, since antiserum directed against Ajuba immunoprecipitated ~10% of the membrane E-cadherin, and Ajuba staining of keratinocytes junctions was not uniformly continuous with the E-cadherin staining pattern (Fig. 1c). Therefore, it is likely that, in addition to alpha -catenin, Ajuba interacts with other cell membrane-associated proteins.

Mapping studies indicated that full-length Ajuba is most efficiently recruited to junctions. The majority of cells injected with the LIM region localized protein to the junctions (but the number was less than that observed in cells injected with full-length Ajuba), despite the large pool of LIM regions that accumulate and are trapped in the nucleus (7). A minority of PreLIM region-injected cells exhibited junctional staining, suggesting that the PreLIM region may also contribute to junctional localization. Therefore, we suggest that the LIM regions predominantly direct Ajuba to cell junctions and that the PreLIM region, through its association with F-actin, stabilize Ajuba recruited to the cell junctions. In agreement with this hypothesis, full-length Ajuba and the LIM regions better associate with alpha -catenin in co-immunoprecipitation experiments. The PreLIM region does co-immunoprecipitate with alpha -catenin, however, but it is 10-fold less efficient. Similarly, targeting of Zyxin to focal adhesion sites in fibroblasts is mediated primarily by its LIM domains, but PreLIM sequences also contribute (17, 34). Further mapping studies will determine whether a single LIM domain of Ajuba can direct its association with junctions or whether multiple LIM domain are required, as is the case with localization of Zyxin to focal adhesion sites (34).

In pull-down experiments and in vitro, Ajuba associates with full-length alpha -catenin and its NH2-terminal region but not its COOH-terminal region. The NH2-terminal region of alpha -catenin interacts with beta -catenin bound to the cadherin cytoplasmic tail, whereas the COOH-terminal region directs its association with the actin cytoskeleton (3, 4). In this scenario, the COOH-terminal portion of alpha -catenin is left free to associate with actin filaments. In addition to Ajuba, alpha -catenin also interacts with other cytoskeletal proteins such as alpha -actinin, vinculin, and spectrin via different NH2-terminal sequences (4, 35).

In addition to alpha -catenin, we also found that Ajuba interacts with F-actin in cell extracts and directly, in vitro. Mapping studies demonstrate that the PreLIM region of Ajuba interacts with F-actin, whereas the LIM domains alone do not. In contrast, Zyxin also co-localizes with actin filaments in vivo but cannot bind directly to F-actin. Rather, it interacts with F-actin indirectly through an association with alpha -actinin, which directly binds F-actin (15). Therefore, Ajuba, through its association with alpha -catenin at cadherin adhesive complexes and with F-actin, may contribute to the coupling of cadherin adhesive complexes to the actin cytoskeleton. As such, Ajuba may function in the formation of adherens junction or the strengthening of adherens junctions. In support of this hypothesis, primary keratinocytes lacking Ajuba exhibit abnormalities in cell-cell junction formation or stability, and calcium-dependent cell-cell adhesion is reduced in Ajuba null keratinocytes.

Ajuba is present in the cytosol, associates with actin stress fibers in fibroblasts (data not shown) and directly with actin filaments in vitro (reported herein), is recruited to adherens junctions (reported herein), and shuttles into the nucleus (7). Thus, like beta -catenin, there may exist selective cellular pools of Ajuba that are directed to these different sites (33). Alternatively, different signals or post-translational modifications of Ajuba may direct its subcellular localization between these sites. Ajuba was rapidly recruited from a cytoplasmic pool, distinct from E-cadherin, to junctions in response to calcium-initiated adhesion and closely followed the time course observed for cadherin receptors. This implies that recruitment of Ajuba to junctions is regulated. These possibilities require further investigation.

Our data raise other exciting possibilities for future investigations. There is a functional interaction between Rho small GTPases family members and their targets and LIM family members such as LIM-kinase and paxillin (36, 37). Since the stability of cadherin-dependent adhesion requires the activity of small GTPases, it will be of interest to determine whether Ajuba affects their activity. In this respect, it will be important to determine how binding to cadherin complexes modulates Ajuba function and vice versa. We have shown that Ajuba is present at the cadherin adhesive complex at cell-cell junctions, yet Ajuba also shuttles into the nucleus, where it can regulate cell growth and differentiation (7). beta -Catenin, another component of the cadherin adhesive complex also shuttles into the nucleus and modulates cellular growth. Since Wnt signals regulate cellular beta -catenin levels and therefore signaling responses, it will be important to determine whether Wnt signals likewise affect Ajuba function. Since cadherin-mediated adhesion is implicated in contact-dependent inhibition of growth and differentiation (2), future work will determine whether Ajuba, like beta -catenin, participates in these important regulatory pathways in epithelia.

    ACKNOWLEDGEMENTS

We thank D. Rimm, M. Takeichi, J. Sttapert, M. Wheelock, M. Petit, and M. Beckerle for generously providing reagents and Yungfeng Feng for baculovirus-purified Ajuba.

    FOOTNOTES

* This work was funded by The Cancer Research Campaign and the Medical Research Council (to V. B.), National Institutes of Health Grant CA85839 (to G. L.), the Sigrid Juselius Foundation of Finland (to G. L.), and the Wellcome Trust (to D. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The first two authors contributed equally to this work.

** An established investigator of the American Heart Association (Grant 9940116N). To whom correspondence may be addressed: Depts. of Medicine and Cell Biology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, Missouri 63110. E-mail: longmorg@medicine.wustl.edu.

Dagger Dagger A Medical Research Council Senior fellow. To whom correspondence may be addressed: Cell and Molecular Biology Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Bldg., London SW7 2AZ, UK. Tel.: 020-7594-3233; Fax: 020-7594-3015; E-mail: v.braga@ic.ac.uk.

Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M205391200

2 S. Pratt and G. Longmore, manuscript in preparation.

3 M. Beckerle, personal communication.

    ABBREVIATIONS

The abbreviations used are: PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; MEF, mouse embryo fibroblast.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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