From the 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
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ABSTRACT |
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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 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. 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 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 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- 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- 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-
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 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, 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).
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,
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.
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.
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, Ajuba Associated with
Next, endogenous
We next asked whether the association between Ajuba and
To determine whether Ajuba and
Taken together, these data indicated that of proteins known to be
present in the cadherin adhesive complex, Ajuba associated with
Recruitment of Ajuba to Cell Junctions Required
Domains of Ajuba Required for Junctional Recruitment and
Association with
To determine which domains of Ajuba directed its association with
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).
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,
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.
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
The amount of total cell Ajuba that associated with 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 In pull-down experiments and in vitro, Ajuba associates with
full-length In addition to 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
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). -catenin, and
-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
-Catenin (or
-catenin/plakoglobin) associates directly with the cadherin tail,
and then
-catenin bridges the
-catenin-cadherin complex to actin
filaments (1).
-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
-actinin (reviewed in Ref. 4). Cytosolic proteins containing LIM
domains have also been observed to localize at cell-cell contact sites
(5-8).
-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
RESULTS
DISCUSSION
REFERENCES
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.
1-integrins P5D2 and VM2 (24, 27), and
anti-Myc and anti-FLAG epitopes (Sigma). Rabbit polyclonal antiserum
used were anti-
-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).
-catenin antiserum.
-catenin (full-length), GST-A907 (
-catenin full-length),
GST-C447 (
-catenin COOH-terminal amino acids), GST-N228 (
-catenin
NH2-terminal amino acids (3)), and GST-E-cadherin tail
(29).
-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.
-catenin, full-length
-catenin,
-catenin N228, and
-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
View larger version (85K):
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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 -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.
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
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
-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.
View larger version (73K):
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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
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.
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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.
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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.
-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 -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.
-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
-catenin and an NH2-terminal
portion of
-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
-catenin), or
GST-
-catenin.
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Fig. 6.
Ajuba associates with
-catenin in vivo and in
vitro. a, GST fusion proteins containing
full-length
-catenin (A907),
-catenin NH2-terminal
(N228),
-catenin COOH-terminal region (C447),
-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,
-catenin antibodies were used to
immunoprecipitate endogenous
-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-
-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
-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
-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
-catenin antibody
(lower panel). e, cell lysates from
human breast carcinoma cells with (+) or without (
)
-catenin were
immunoblotted for E-cadherin (top panel),
-catenin (center panel), and Ajuba
(lower panel). Human breast carcinoma cells
containing
-catenin (+) or without
-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.
-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
-catenin
(Fig. 6b), whereas less than 0.1% of total cellular Zyxin,
LPP, and LIMD1 co-immunoprecipitated with
-catenin. This represented
a 20-30-fold difference in the association of Ajuba with
-catenin compared with related family members. The low level of Ajuba associated with
-catenin most likely reflects the large cytoplasmic pools of
Ajuba and
-catenin that presumably do not interact (see below).
-catenin
could occur directly. Baculovirus-produced and -purified FLAG-tagged
Ajuba was mixed with purified GST-
-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
-catenin (Fig.
6c, right strips, lane
1) and the NH2-terminal region of
-catenin (N228, Fig. 6c, right strips,
lane 3) but not with the COOH-terminal region of
-catenin (C447, Fig. 6c, right
strips, lane 2).
-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-
-catenin antiserum.
Antibodies against Ajuba, but not preimmune serum or Zyxin antibodies,
co-immunoprecipitated
-catenin (Fig. 6d,
left). To determine whether Ajuba associated with
-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
-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
-catenin from HaCAT
membrane preparations resulted in a reduction of Ajuba protein levels
while not affecting Zyxin levels (data not shown).
-catenin but not
-catenin or the cytoplasmic tail of
E-cadherin. This association occurred with membrane E-cadherin-bound
-catenin. The association between Ajuba and
-catenin was
preferentially through the NH2-terminal region of
-catenin. Finally, purified Ajuba interacted directly with
-catenin protein, in vitro.
-Catenin--
To determine whether
-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
-catenin protein. As a control, the same cell line in
which
-catenin had been stably transfected was also examined.
Western blot analysis of extracts from these cells demonstrated the
absence of
-catenin in the parental cells and its presence in the
-catenin-transfected cells (Fig. 6e, right).
The lack of
-catenin and its reintroduction did not affect the
levels of E-cadherin or endogenous Ajuba (Fig. 6e,
right). In cells lacking
-catenin, transfected Myc-Ajuba was predominantly cytosolic (Fig. 6e, right),
whereas reintroduction of
-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
-catenin.
-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
-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-
-catenin antibodies
were used to immunoprecipitate endogenous
-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
-catenin was determined and presented as percentage
of input associated with
-catenin.
-catenin, we transfected epithelial cells with Myc-tagged full-length Ajuba, the PreLIM region, or the LIM regions. Cells were
lysed, and endogenous
-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
-catenin; however, when the percentage of
Myc-tagged Ajuba input protein immunoprecipitated by anti-
-catenin
antibodies was determined, 10-fold less PreLIM region (0.5%)
associated with
-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
-catenin.
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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).
/
) 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
-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
-catenin (lower
panel). Equal amounts of protein were loaded in each
lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin. The following results support this conclusion. First, in
pull-down experiments from cell lysates, Ajuba associated with
-catenin but not with
-catenin or the E-cadherin cytoplasmic
tail. Second, Ajuba co-immunoprecipitated with
-catenin in total
cell extracts from cells expressing endogenous levels of each protein.
Third, Ajuba interacted with
-catenin present at the cell membrane
E-cadherin adhesive complex as Ajuba antibodies co-immunoprecipitated
E-cadherin and
-catenin from membrane preparations from cells
expressing endogenous levels of each protein. Fourth, immunodepletion
of E-cadherin and
-catenin resulted in a reduction in Ajuba protein
levels while not affecting the levels of Zyxin. Finally, Ajuba
interacted directly with
-catenin, in vitro. Importantly,
the recruitment of Ajuba to sites of cell-cell adhesion was severely
delayed in epithelial cells not expressing
-catenin and could be
rescued by reintroduction of
-catenin into these cells. Other LIM
proteins, such as Paxillin and Zyxin, associate with the
-catenin-related proteins
-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
-actinin, since it lacks the
binding site present in Zyxin (31).
-catenin was
small (2-3%). We suggest that this is because the association between
Ajuba and
-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
-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
-catenin (see Fig.
6d), and we observed that Ajuba antibodies co-immunoprecipitated E-cadherin and
-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
-catenin, Ajuba interacts with other
cell membrane-associated proteins.
-catenin
in co-immunoprecipitation experiments. The PreLIM region does
co-immunoprecipitate with
-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).
-catenin and its NH2-terminal region but not
its COOH-terminal region. The NH2-terminal region of
-catenin interacts with
-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
-catenin is left free to associate with
actin filaments. In addition to Ajuba,
-catenin also interacts with
other cytoskeletal proteins such as
-actinin, vinculin, and spectrin
via different NH2-terminal sequences (4, 35).
-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
-actinin, which directly binds F-actin (15). Therefore, Ajuba, through its association with
-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.
-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.
-Catenin, another component of the cadherin
adhesive complex also shuttles into the nucleus and modulates cellular growth. Since Wnt signals regulate cellular
-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
-catenin,
participates in these important regulatory pathways in epithelia.
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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.
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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.
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.
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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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