armadillo
Protein interactions of Armadillo homologs: Interaction with cadherins, APC and other junctional components alpha-Catenin is a 102-kDa protein exhibiting homology to vinculin, and it forms complexes with
both cadherins and the tumor-suppressor gene product adenomatous polyposis coli through binding to
beta-catenin or plakoglobin (gamma-catenin). The incorporation of alpha-catenin into the
cadherin-catenin complexes is a prerequisite for expression of the cell-adhesive activity of cadherins (See Drosophila Shotgun).
Amino-terminal residues 48-163 of alpha-Catenin are able to bind to
beta-catenin and plakoglobin. Consistent with the observation that beta-catenin and plakoglobin bind to
the same region of alpha-catenin, beta-catenin competes with the binding of plakoglobin to
alpha-catenin and vice versa. Under the conditions used, beta-catenin binds to alpha-catenin with
higher affinity than does plakoglobin. The affinity of the interaction
between alpha-catenin and beta-catenin or that between alpha-catenin and plakoglobin is moderately
strong. When transfected into L cells expressing
E-cadherin, the amino-terminal region of alpha-catenin (from residue 1 to 226) forms complexes with
beta-catenin supporting the in vitro binding experiment results (Obama, 1997).
In Xenopus laevis development, beta-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis.
Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell-cell adhesion and can modulate beta-catenin signaling. alpha-catenin links beta-catenin to the actin-based
cytoskeleton. To study the role of endogenous alpha-catenin in early development,
deletion mutants of alphaN-catenin have been made. alphaN-catenin has 81.6% identity to the originally described alpha-catenin. alpha-N-catenin has properties similar to alpha-catenin; both bind to the cadherin complex, but alphaN-catenin is more prevalent in the nervous system. The binding domain of beta-catenin has been mapped to the NH2-terminal 210 amino acids of alphaN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the
blastocoel in early embryos and a ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length alphaN-catenin or a mutant
of beta-catenin that lacks the internal armadillo repeats. Coexpression of
alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene, Siamois. Thus, alpha-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of
the intracellular beta-catenin signaling pathway in vivo (Sehgal, 1997).
The cytoplasmic domain of E-cadherin
binds either beta-catenin or plakoglobin: either one can assemble alpha-catenin into
the complex. An alpha-catenin binding site in beta-catenin
and plakoglobin has been identified and it is postulated, based on sequence analysis, that these protein-protein
interactions are mediated by a hydrophobic interaction mechanism. The reciprocal complementary binding site in alpha-catenin has been identified, which
mediates its interaction with beta-catenin and plakoglobin. The N-terminal 146 amino acids are required for this
interaction. A peptide of 27 amino acids within this sequence (amino
acid positions 117-143) is necessary and sufficient to bind either beta-catenin or
plakoglobin. Hydrophobic amino acids within this
binding site are important for the interaction. The results described here give strong support for the idea that these proteins associate by
hydrophobic interactions of two alpha-helices (O. Huber, 1997).
As a component of adherens junctions and the Wnt signaling pathway, ß-catenin binds cadherins, Tcf family transcription factors, and the tumor suppressor APC. The crystal structures of both unphosphorylated and phosphorylated E-cadherin cytoplasmic domain complexed with the arm repeat region of ß-catenin has been determined. The interaction spans all 12 arm repeats, and features quasi-independent binding regions that include helices that interact with both ends of the arm repeat domain and an extended stretch of 14
residues that closely resembles a portion of XTcf-3. Phosphorylation of E-cadherin results in interactions with a hydrophobic patch of ß-catenin that mimics the binding of an amphipathic XTcf-3 helix. APC contains sequences homologous to the phosphorylated region of cadherin, and is likely to bind similarly (Huber, 2001).
The cadherin cytoplasmic domain is not structured in the absence of ß-catenin, and binds in an extended conformation that forms a large interface with ß-catenin. This mode of binding may allow for degrees of regulation that would be impossible for an interaction involving a well-structured ligand. The surface of such a ligand is presented on a relatively rigid scaffold, and local changes can therefore affect the entire interface between the two proteins. In contrast, the interface between a ligand that is unstructured in its unbound state and its partner can be altered locally without affecting the rest of the interface. In this manner, posttranslational modifications like phosphorylation can modulate the interaction in a graded fashion rather than serving as a simple on/off switch (Huber, 2001).
The armadillo repeat domain architecture complements the binding of extended polypeptides by providing a large surface-to-volume ratio and elongated interaction surface. Multiple, quasi-independent interactions provide the possibility of having a minimal 'core' binding region while allowing other interactions to be more dynamic. Separate binding regions can be regulated independently, enabling combinatorial regulation of the interaction and the integration of multiple input signals. In the case of E-cadherin, the extended region III appears to be absolutely required for the interaction with ß-catenin, since destabilizing the interface by mutation of either ß-catenin Lys435 or Lys312 to glutamate destroys binding. In contrast, the enhanced binding of phosphorylated E-cadherin (region IV) or the decreased binding of E-cadherin upon phosphorylation of ß-catenin Tyr654 (region II) demonstrate that the overall affinity can be modulated without completely eliminating the interaction of these two proteins. An extended interface may also make the system somewhat resistant to mutations; for example, none of 18 ß-catenin alanine mutations eliminates binding to E-cadherin (J. von Kries and W. Birchmeier, personal communication to Huber, 2001). Combined, these features create a robust interface subject to regulation; this is likely to be important in determining the mechanical properties and dynamics of subcellular assemblies such as the adherens junction (Huber, 2001).
To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated
with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins have been
identified:
alpha-catenin, ß-catenin and gamma-catenin (also known as plakoglobin). ß-catenin or plakoglobin is associated directly with the cadherin; alpha-catenin
binds to ß-catenin/plakoglobin and serves to link the cadherin/catenin complex to the actin
cytoskeleton. The domains on the cadherin and ß-catenin/plakoglobin that are responsible
for protein-protein interactions have been mapped. However, little is known about the
molecular interactions between alpha-catenin and ß-catenin/plakoglobin or about the
interactions between alpha-catenin and the cytoskeleton. The
yeast two-hybrid system was used to map the domains on alpha-catenin that allow it to associate with
ß-catenin/plakoglobin and with alpha-actinin. A region on alpha-actinin was identified that is
responsible for its interaction with alpha-catenin. Amino acids 6-506 of alpha-catenin interact with amino acids 479-892 of alpha-actinin. Narrowing the alpha-catenin site further, a construct encoding only amino aids 325-394 of alpha-catenin interact with amino acids 470-529 of alpha-actinin. Filamentous actin, rather than monomeric actin is required for alpha-catenin binding. Both amino (aa 1-228) and carboxyl (aa 461-907) terminal fragments of human alpha-catenin bind actin filaments in vitro. alpha-Actinin is known to form anti-parallel dimers. The data showing an interaction betweeen small regions of alpha-catenin and alpha-actinin suggest that one molecule of alpha-catenin may interact with one member of the alpha-actinin dimer. The data indicate that sequences at one spectrin repeat boundary of alpha-actinin are involved in its interaction with alpha-catenin. alpha-Actinin has four such repeats (Nieset, 1997).
The effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, was studied to determine the level
and localization of beta-catenin and to assess its ability to induce lymphocyte enhancer-binding factor 1
(LEF-1)-responsive transactivation. These cadherin derivatives form complexes with beta-catenin and
protect it from degradation. N-cadherin directs beta-catenin into adherens junctions, and the chimeric
protein induces diffuse distribution of beta-catenin along the membrane whereas the cytoplasmic domain of
N-cadherin colocalizes with beta-catenin in the nucleus. Cotransfection of beta-catenin and LEF-1 into
Chinese hamster ovary cells induces transactivation of a LEF-1 reporter, which is blocked by the
N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor/cadherin chimera in
SW480 cells relocates beta-catenin from the nucleus to the plasma membrane and reduces transactivation.
The cytoplasmic tails of N- or E-cadherin colocalize with beta-catenin in the nucleus, and suppress the
constitutive LEF-1-mediated transactivation, by blocking beta-catenin-LEF-1 interaction. Moreover, the 72
C-terminal amino acids of N-cadherin stabilize beta-catenin and reduce its transactivation potential.
These results indicate that beta-catenin binding to the cadherin cytoplasmic tail either in the membrane, or
in the nucleus, can inhibit beta-catenin degradation and efficiently block its transactivation capacity (Sadot, 1998).
beta-catenin is a multifunctional protein found in three cell compartments: the plasma membrane, the cytoplasm and the nucleus. The cell has developed elaborate ways of regulating the level and localization of beta-catenin to assure its specific function in each compartment. One aspect of this regulation is inherent in the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, it was found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with beta-catenin; the association of beta-catenin with LEF-1 is competed out by the E-cadherin cytoplasmic domain. Similarly, LEF-1 and adenomatous polyposis coli (APC) form separate, mutually exclusive complexes with beta-catenin. In Wnt-1-transfected C57MG cells, free beta-catenin accumulates and is able to associate with LEF-1. The absence of E-cadherin in E-cadherin minus embryonic stem (ES) cells also leads to an accumulation of free beta-catenin and its association with LEF-1, thereby mimicking Wnt signaling. beta-catenin/LEF-1-mediated transactivation in these cells is antagonized by transient expression of wild-type E-cadherin, but not of E-cadherin lacking the beta-catenin binding site. The potent ability of E-cadherin to recruit beta-catenin to the cell membrane and prevent its nuclear localization and transactivation has also been demonstrated using SW480 colon carcinoma cells (Orsulic, 1999).
During early chick heart development the expression pattern of N-cadherin, a calcium-dependent cell
adhesion molecule, suggests its involvement in morphoregulation and the stabilization of cardiomyocyte
differentiation. N-cadherin's adhesive activity is dependent upon its interaction with the intracellular
catenins. N-cadherin's association with alpha-catenin and beta-catenin is also believed to be involved in cell
signaling. The restriction of N-cadherin/catenin localization at stage 5+
from a uniform pattern in vivo to specific cell clusters that demarcate areas where mesoderm
separation is initiated, suggests that the N-cadherin/catenin complex is involved in boundary formation
and in the subsequent cell sorting. The latter two processes lead to the specification, separation, and formation of
the somatic and cardiac splanchnic mesoderm. Cells which form patches of N-cadherin/ß-catenin demarcate the dorsal boundary of the cardiac compartment, separating this compartment from the overlying dorsal mesoderm. N-cadherin colocalizes with alpha- and beta-catenin at
the cell membrane before and during the time that its expression becomes restricted to the lateral
mesoderm and continues cephalocaudally into stage 8. These proteins continue to colocalize in the
myocardium of the tubular heart (Linask, 1997).
The observed in vivo expression patterns of
alpha-catenin, beta-catenin, and plakoglobin suggest that this process occurs as these proteins are directly linked with the
developmental regulation of cell junctions; cardiac cells become stably committed and phenotypically
differentiated to eventually form a mature myocardium. Perturbation of N-cadherin using
a function perturbing N-cadherin antibody (NCD-2) inhibits normal early heart development and
myogenesis in a cephalocaudal, stage-dependent manner. A model is proposed whereby myocardial
cell compartmentalization also defines the endocardial population. The presence of beta-catenin
suggests that a similar signaling pathway involving Wnt (wingless)-mediated events may function in
myocardial cell compartmentalization during early vertebrate heart development, as in Drosophila
contractile vessel development. In Drosophila, contractile vessel development requires wingless. Elimination of wingless function for a short time period after gastrulation in Drosophila results in the selective loss of heart precursors. The data presented here are consistent with the likelihood that Wnt signaling may be involved in the regulation of the N-cadherin/ß-catenin-mediated events associated with vertebrate heart development. The Wnt2 gene is expressed in the early mouse heart field of 7.5-8 days of gestation. This coincides with the period of events described here (Linask, 1997).
In epithelial cells, alpha-, beta-, and gamma-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. alpha-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell-matrix and cell-cell contacts, alpha-catenin is restricted to the latter. To determine whether vinculin is part of the cell-cell junctional complex, complex formation and intracellular targeting of vinculin and alpha-catenin were investigated. Alpha-catenin colocalizes at cell-cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to alpha-catenin. The Kd of the complex is 2-4 x 10(-7) M. The COOH-terminal region of alpha-catenin is involved in this interaction. Complex formation of vinculin and alpha-catenin was challenged in transfected cells. In PtK2 cells, intact alpha-catenin and alpha-catenin1-670, harboring the beta-catenin- binding site, are directed to cell-cell contacts. In contrast, alpha-catenin697-906 fragments are recruited to cell-cell contacts, focal adhesions, and stress fibers. These results imply that in vivo alpha-catenin, like vinculin, is tightly regulated in its ligand binding activity (Weiss, 1998).
It is currently unknown which of the multiple interactions between junctional proteins are realized in living cells, whether the cells selectively use a specific type, and how such specificity might be regulated in epithelial architecture. The findings reported here add new aspects to the present view on cell-cell adherens junctions. The initial model, deduced from immunoprecipitation studies, postulated binding of the cytoplasmic domain of E-cadherin to a heterotrimeric complex of alpha-, beta-, and gamma-catenin. Based on more recent findings, that two distinct complexes containing E-cadherin and either alpha- and beta-catenin or alpha- and gamma-catenin can be immunoprecipitated, it is thought that both complexes coexist in fully polarized epithelial cells. The most recent observation that alpha-catenin-cadherin complexes can also directly interact with alpha-actinin, together with the data reported here on complex formation between alpha-catenin and vinculin, further expand the model, suggesting a highly versatile situation at the plasma membrane of cell-cell contact sites. Actin filaments might associate with alpha-catenin either directly through its actin-binding site, or indirectly through actinin or vinculin (Weiss, 1998 and references).
Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal
spreading of colonies followed by disruption of cell-cell junctions and subsequent cell scattering. In Madin-Darby canine
kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream
signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induce the loss
of the adherens junction proteins E-cadherin and beta-catenin from intercellular junctions during cell spreading, and the
HGF/SF response is blocked by dominant-negative N17Ras. Desmosomes and tight junctions are regulated separately
from adherens junctions, because the adherens junctions are not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI
3-kinase), and Rac are required downstream of Ras, because loss of adherens junctions is blocked by the inhibitors
PD098059 and LY294002 or by dominant-inhibitory mutants of either or both MAP kinase kinase 1 or Rac1. All of these inhibitors also
prevent HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110alpha subunit of PI 3-kinase alone
does not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by
Ras are required for adherens junction disassembly and that the disassembly process is essential for the motile response to HGF/SF (Potempa, 1998).
The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a
target of Cdc42 and Rac1, is localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts
expressing E-cadherin (EL cells), and interacts with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induces
the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells,
but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, results in a decrease in E-cadherin-mediated
cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion
through the cadherin-catenin pathway (Kuroda, 1998).
The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the
basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex
assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion
functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two
putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density
lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the
fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells.
Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein,
GP2CAD1, which comprises the extracellular domain of GP2 (an apical membrane protein) and the
transmembrane and cytoplasmic domains of E-cadherin, is efficiently and specifically delivered to
the basal-lateral membrane. Systematic deletion and recombination of specific regions of the
cytoplasmic domain of GP2CAD1 result in delivery of less than 10% of these newly synthesized proteins to
both apical and basal-lateral membrane domains. Significantly, greater than 90% of each mutant protein is
retained in the ER. None of these mutants form a strong interaction with beta-catenin, which
normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin
that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole
cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the
lateral plasma membrane. In this capacity, it is suggested that beta-catenin acts as a chauffeur, to
facilitate transport of E-cadherin out of the ER and the plasma membrane (Chen, 1999).
Protein phosphatase 2A (PP2A) plays central roles in development, cell growth and transformation. Inactivation of the murine gene encoding the
PP2A catalytic subunit Calpha by gene targeting generates a lethal embryonic phenotype. No mesoderm is formed in Calpha minus embryos. During normal early embryonic development Calpha is predominantly present at the plasma membrane whereas the highly
homologous isoform Cbeta is localized to the cytoplasm and nuclei, suggesting the inability of Cbeta to compensate for vital functions of
Calpha in Calpha minusembryos. In addition, PP2A is found in a complex containing the PP2A substrates E-cadherin and beta-catenin. In Calpha minus embryos, E-cadherin and beta-catenin are redistributed from the plasma membrane to the cytosol. Cytosolic concentrations of beta-caenin are
low. These results suggest that Calpha is required for stabilization of E-cadherin/ beta-catenin complexes at the plasma membrane (Gotz, 2000).
These data suggest that PP2A is involved in wnt signaling.
One likely explanation is that PP2A containing the Calpha
subunit binds to and stabilizes the E-cadherin/beta-catenin
complex within the plasma membrane.
PP2A forms a complex with beta-catenin and E-cadherin. It
is likely that PP2A regulates the phosphorylation status of
E-cadherin and beta-catenin at the plasma membrane. In the
cytosol, the activity of PP2A may not be high enough to
offset GSK3 beta-mediated phosphorylation of beta-catenin. Wnt signaling increases cytoplasmic concentrations of
PP2A by releasing it from the plasma membrane into the
cytosol. Increased concentrations of PP2A effectively offset
the GSK3 beta-mediated phosphorylation of beta-catenin thus
preventing its degradation, and promoting its translocation
to the nucleus with subsequent activation of wnt target genes. In
the Calpha knockout situation
both E-cadherin and beta-catenin are no longer stabilized in
the plasma membrane. It is likely that both are phosphorylated and translocated to the cytoplasm. These data show that
cytoplasmic E-cadherin is stable, and suggest that the associated beta-catenin is targeted for degradation resulting in
reduced beta-catenin levels. It is proposed that in the
absence of PP2A Calpha, wnt signaling releases beta-catenin from
the axin/APC/GSK3 beta complex; beta-catenin associates with
the highly abundant cytoplasmic E-cadherin before it
reaches the nucleus, where it is phosphorylated and becomes
degraded. Consequently, no target genes of wnt signaling,
including Brachyury or goosecoid, are transcribed and embryonic development halts (Gotz, 2000).
This model is consistent with the finding that Calpha is mainly
plasma membrane-associated in the inner cell mass, but
this association becomes lost during differentiation.
This model would also explain why the wnt target gene T-brachyury is negatively regulated by E-cadherin: T-brachyury mRNA levels are high in E-cad minus embryonic stem cells (ES) cells, but absent in wild-type ES cells. Basal cytoplasmic levels of non-phosphorylated beta-catenin may be quenched by cytoplasmic or membrane-bound E-cadherin, preventing transcription of T-brachyury in the absence of a wnt signal. In E-cad minus cells, the quencher is missing, allowing low but significant amounts of non-phosphorylated beta-catenin to be translocated into the
nucleus, and to activate transcription of T-brachyury. This is
consistent with the structural organization of beta-catenin
itself; most of its protein-interacting motifs overlap so that
interaction with one partner can block binding of another at
the same time. Using recombinant proteins, it has been found that
E-cadherin and lymphocyte-enhancer factor-1 (LEF-1),
which targets beta-catenin to the nucleus, form mutually exclusive complexes with beta-catenin. This model is also consistent with the known association
of PP2A with axin. Axin is a negative
regulator of embryonic axis formation in vertebrates (Gotz, 2000).
The heterotrimeric PDZ complex containing LIN-2, LIN-7 (Drosophila homolog CG7662) and LIN-10 (Drosophila homolog: CG5678) is known to be involved in the organization of epithelial and
neuronal junctions in Caenorhabditis elegans and mammals. Both C. elegans and mammalian LIN-2 can bind LIN-10 and
LIN-7 independently of the PDZ domains, which are free to recruit cell adhesion molecules, receptors and signaling proteins. Important advances have been made in characterizing the molecular components of the complex, and it has been shown that
adhesion molecules, such as neurexin and syndecan, as well as transporters, receptors and ion
channels, such as the epithelial gamma-aminobutyric acid (GABA) transporter (BGT-1), the N-methyl-D-aspartate (NMDA) receptor and the N-type
Ca2+ channel, interact with the PDZ domains of mammalian LIN-2, LIN-7 and LIN-10. Mammalian LIN-7 PDZ proteins form a complex
with cadherin and ß-catenin in epithelia and neurons. The association of LIN-7 with cadherin and ß-catenin is Ca2+ dependent and is
mediated by the direct binding of LIN-7 to the C-terminal PDZ target sequence of ß-catenin, as demonstrated by means of
co-immunoprecipitation experiments and in vitro binding assays with the recombinant glutathione S-transferase:LIN-7A. The
presence of ß-catenin at the junction is required in order to relocate LIN-7 from the cytosol to cadherin-mediated adhesions, thus
indicating that LIN-7 junctional recruitment is ß-catenin dependent and that one functional role of the binding is to localize LIN-7. Moreover, when LIN-7 is
present at the ß-catenin-containing junctions, it determines the accumulation of binding partners, thus suggesting the mechanism by which ß-catenin mediates the
organization of the junctional domain (Perego, 2000).
Thus LIN-7 is a component of the cadherin-mediated adhesion that is common to the pre- and post-synaptic compartments, as well as to epithelial junctions; but what are the functional consequences of LIN-7-ß-catenin binding to the junctions? LIN-7 causes the selective accumulation of the interacting BGT-1 transporter in the basolateral junctional domain of MDCK cells by means of retentional mechanisms, and the disruption of LIN-7-BGT-1 binding causes intracellular BGT-1 accumulation. By analogy, LIN-7 binding to ß-catenin might control the amount of cytosolic ß-catenin that is free to function as a coactivator of gene expression. Once recruited to the junctions, LIN-7 participates in the organization of this domain by localizing and accumulating binding partners. Only the LIN-7 recruited in the ß-catenin-containing junctions is capable of retaining the epithelial GABA transporter at the surface domain, whereas the transporter accumulates in an intracellular compartment in the cells not containing LIN-7 at the junctions. This result implies that the single PDZ-containing protein LIN-7 may interact with different partners (ß-catenin and BGT-1) in the same cell type. It is not yet clear whether the same LIN-7 isoform forms a single complex containing both ß-catenin and BGT-1, or multiple complexes containing either one or the other, nor whether the composition of these complexes is regulated during the development of cell polarity. Since several LIN-7 isoforms are expressed in epithelia and neurons, and LIN-7 can associate with CASK or other members of the membrane-associated guanylate kinase subfamily (PALs: proteins associated with LIN-7) via the non-PDZ domain, it is possible that LIN-7 forms multiple complexes characterized by a specific molecular composition. However, it is interesting to note that ß-catenin also appears to be a binding partner of the PDZ protein MAGI 1, but whereas the interaction with MAGI 1 occurs mainly under conditions of Ca2+ depletion, the interaction with LIN-7 is disrupted by Ca2+ depletion, thus suggesting that the composition of the complexes may be regulated during the acquisition of cell polarity (Perego, 2000 and references therein).
LIN-7 as a component of the heterotrimeric complex may play a further role in coupling the cadherin-catenin system (via LIN-7) to the cell adhesion molecule neurexin or the matrix receptor syndecan (via CASK). Given that two different components of these adhesion systems (ß-catenin and CASK) may both function as co-activators of gene expression, the link between the various adhesion systems may be important for the integration and processing of information from the junction in order to generate intracellular signaling (Perego, 2000 and references therein).
The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3)
delays the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin are mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin are also delayed in their recruitment to the cell membrane, and NZO-3 expression has striking effects on actin cytoskeleton dynamics. NZO-3
expression does not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin is increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments show that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. It is hypothesized that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin (Wittchen, 2000).
The involvement of the actin cytoskeleton in maintaining TJ integrity and regulating permeability has been well documented; this involvement is underscored by the fact that actin has multiple protein binding partners at the TJ, which themselves interact in various ways. It can be envisioned that this molecular architecture provides the means by which an actin filament network can be recruited to and organized in a functionally relevant manner at the TJ. The actin cytoskeleton is also a major structural and functional element of the AJ, and is present in a bundled actin belt around the apical periphery cell at the level of the AJ. Interestingly, in MDCK/NZO-3 cells, there is a delay in actin recruitment and formation of this perijunctional apical actin ring. Because the amino-terminal half of ZO-3 is responsible for binding F-actin, this may represent one mechanism whereby expression of this construct affects TJ and AJ assembly (Wittchen, 2000).
Not only does expression of the amino terminus of ZO-3 alter the distribution of ß-catenin, but there is also an increase in the TX-100-soluble pool of signaling-active ß-catenin. Presumably the presence of an increased level of the NZO-3 construct at early time points after calcium switch results in a downstream alteration of the E-cadherin/catenins complex at the adherens junction, releasing ß-catenin from a cytoskeletal linkage into the TX-100-soluble pool. A corresponding change in the levels of ß-catenin in the insoluble pool is not observed, although any possible change may be masked by the overall high levels of ß-catenin present in these samples. Normally cytoplasmic ß-catenin levels are strictly regulated via a ubiquitin-mediated proteolysis pathway requiring ß-catenin interaction with the cytoplasmic tumor suppressor APC. Soluble ß-catenin that escapes this targeted proteolysis is capable of translocating to the nucleus where it acts as a transcriptional transactivator in a complex with TCF/LEF family of transcription factors to direct transcription of a variety of genes that promote a proliferative phenotype. Expression of a mutant signaling-active (soluble) form of ß-catenin in MDCK cells has been shown to cause a delay in the establishment of tight confluent cell monolayers compared with control cells, and the cells appear more motile and form less compact colonies when plated at a low density. These results, taken together with data showing that NZO-3 expression delays transepithelial resistance formation after a calcium switch and results in an increased level of signaling-active, soluble ß-catenin in these cells, suggests that NZO-3 might act through ß-catenin to exert its effects on epithelial junctional complex formation. At present it is not known if this action is direct or indirect (Wittchen, 2000).
The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular
differentiation have also been indicated. A new role for the IGF pathway in regulating head formation has been demonstrated
in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and
the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of
head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense
morpholino oligonucleotides drastically reduces head structures. Furthermore, IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of ß-catenin. Thus, the IGF pathway is required for head formation during embryogenesis (Richard-Parpaillon, 2002).
Wnt signaling is involved in numerous developmental
processes, such as dorsal axis formation, patterning of the
central nervous system, and establishment of cell polarity. The pathway is tightly regulated during embryogenesis and it is becoming increasingly clear that crossregulation between Wnt and other signaling pathways contributes to the complexity and specificity of Wnt activity. For
example, retinoid signaling and a specific MAP kinase
pathway (TAK/NLK) can both inhibit Wnt activity. However, previous
evidence for an interaction between the IGF and the Wnt
pathways is limited. IGF-1 stimulation induces a rapid tyrosine-phosphorylation of ß-catenin in a cell line derived from a
human colonic adenocarcinoma. It has also been shown
that the phosphorylation of ß-catenin induced by IGF-1
leads to a dissociation of the pool of ß-catenin, which is
bound to E-cadherin at the plasma membrane, resulting in its relocation to the cytoplasm (Richard-Parpaillon, 2002 and references therein).
However, despite this accumulation of cytoplasmic
ß-catenin, no enhancement of Wnt activity is observed
after stimulation by IGF-1 alone, as determined by using
the Wnt-responsive luciferase reporter construct TOP-FLASH.
Recent structural studies might provide an explanation
for this paradox. It has been shown that the charged
residues involved in this interaction between ß-catenin/E-cadherin
are the same as those required for the ß-catenin/
TCF interaction. Thus, this raises the interesting possibility that
tyrosine-phosphorylation of ß-catenin, which blocks its
association with E-cadherin may also prevent interaction
between this molecule and its downstream effector Tcf.
This is a potential point at which IGF signaling may inhibit
the Wnt pathway. In the future, it will be interesting to
investigate this hypothesis further, and also to determine
whether the PI3K or the MAPK activated by IGF-1R may be
involved in this process (Richard-Parpaillon, 2002).
Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through β-catenin and α-catenin, although the quaternary complex has never been demonstrated. This hypothesis has been tested and it was found that alpha-catenin does not interact with actin filaments and the E-cadherin-β-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and α-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, β-catenin, and α-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated (Yamada, 2005).
Enduring forms of synaptic plasticity are thought to require ongoing regulation of adhesion molecules, such as N-cadherin, at synaptic junctions. Little is known about the activity-regulated trafficking of adhesion molecules. This study demonstrates that surface N-cadherin undergoes a surprisingly high basal rate of internalization. Upon activation of NMDA receptors (NMDAR), the rate of N-cadherin endocytosis is significantly reduced, resulting in an accumulation of N-cadherin in the plasma membrane. β-catenin, an N-cadherin binding partner, is a primary regulator of N-cadherin endocytosis. Following NMDAR stimulation, β-catenin accumulates in spines and exhibits increased binding to N-cadherin. Overexpression of a mutant form of ß-catenin, Y654F, prevents the NMDAR-dependent regulation of N-cadherin internalization, resulting in stabilization of surface N-cadherin molecules. Furthermore, the stabilization of surface N-cadherin blocks NMDAR-dependent synaptic plasticity. These results indicate that NMDAR activity regulates N-cadherin endocytosis, providing a mechanistic link between structural plasticity and persistent changes in synaptic efficacy (Tai, 2007).
Epithelial tubes represent fundamental building blocks of metazoan organisms; however, the mechanisms responsible for their formation and maintenance are not well understood. This study shows that the evolutionarily conserved coiled-coil MAGUK protein Dlg5 is required for epithelial tube maintenance in mammalian brain and kidneys. Dlg5-/- mice develop fully penetrant hydrocephalus and kidney cysts caused by a deficiency in membrane delivery of cadherin-catenin adhesion complexes and loss of cell polarity. Dlg5 travels with cadherin-containing vesicles and binds to syntaxin 4, a t-SNARE protein that regulates fusion of transport vesicles with the lateral membrane domain. It is proposed that Dlg5 functions in plasma membrane delivery of cadherins by linking cadherin-containing transport vesicles with the t-SNARE targeting complex. These findings show that Dlg5 is causally involved in hydrocephalus and renal cysts and reveal that targeted membrane delivery of cadherin-catenin adhesion complexes is critical for cell polarity and epithelial tube maintenance (Nechiporuk, 2007).
Little is known about the architecture of cellular microenvironments that support stem and precursor cells during tissue development. Although adult stem cell niches are organized by specialized supporting cells, in the developing cerebral cortex, neural stem/precursor cells reside in a neurogenic niche lacking distinct supporting cells. This study finds that neural precursors themselves comprise the niche and regulate their own development. Precursor-precursor contact regulates beta-catenin signaling and cell fate. In vivo knockdown of N-cadherin reduces beta-catenin signaling, migration from the niche, and neuronal differentiation in vivo. N-cadherin engagement activates beta-catenin signaling via Akt, suggesting a mechanism through which cells in tissues can regulate their development. These results suggest that neural precursor cell interactions can generate a self-supportive niche to regulate their own number (Zhang, 2010).
Whether Akt might link N-cadherin to β-catenin activation in cortical precursors was investigated . It was found that function-blocking antibodies to N-cadherin or shRNA to N-cadherin led to a significant reduction in phosphorylated (active) Akt in primary cortical precursors. To test the link between Akt activation and phosphorylation of β-catenin at Ser552, Akt was inhibited in neural precursors using triciribine (API-2), a small molecule Akt pathway inhibitor. Triciribine treatment of primary cortical precursors reduced the fraction of cells expressing β-catenin Ser552 in a dose-dependent fashion. Finally, expression of a dominant-negative (kinase-dead) Akt also reduced both baseline β-catenin signaling in high-density primary cortical precursor cultures as well as Wnt-stimulated β-catenin signaling. To confirm whether Akt functions downstream of N-cadherin to mediate β-catenin signaling, myristoylated (active) Akt was coexpressed along with shRNA to N-cadherin and β-catenin signaling was measured by TOP-flash reporters. It was found that that myrAkt rescued β-catenin signaling following N-cadherin knockdown. It was also found that myrAkt alone could increase β-catenin signaling, a finding consistent with the idea that this pathway may exist in parallel with the canonical Wnt signaling pathway. Together, these observations suggest that N-cadherin engagement leads to phosphorylation of Akt and subsequent Akt-mediated phosphoryation and activation of β-catenin (Zhang, 2010).
Mutation of the adenomatous polyposis coli (APC) tumor suppressor stabilizes beta-catenin and aberrantly reactivates Wnt/beta-catenin target genes in colon cancer. APC mutants in cancer frequently lack the conserved catenin inhibitory domain (CID), which is essential for beta-catenin proteolysis. This study shows that the APC CID interacts with alpha-catenin, a Hippo signaling regulator and heterodimeric partner of beta-catenin at cell:cell adherens junctions. Importantly, alpha-catenin promotes beta-catenin ubiquitylation and proteolysis by stabilizing its association with APC and protecting the phosphodegron. Moreover, beta-catenin ubiquitylation requires binding to alpha-catenin. Multidimensional protein identification technology (MudPIT) proteomics of multiple Wnt regulatory complexes reveals that alpha-catenin binds with beta-catenin to LEF-1/TCF DNA-binding proteins in Wnt3a signaling cells and recruits APC in a complex with the CtBP:CoREST:LSD1 histone H3K4 demethylase to regulate transcription and beta-catenin occupancy at Wnt target genes. Interestingly, tyrosine phosphorylation of alpha-catenin at Y177 disrupts binding to APC but not beta-catenin and prevents repression of Wnt target genes in transformed cells. Chromatin immunoprecipitation studies further show that alpha-catenin and APC are recruited with beta-catenin to Wnt response elements in human embryonic stem cells (hESCs). Knockdown of alpha-catenin in hESCs prevents the switch-off of Wnt/beta-catenin transcription and promotes endodermal differentiation. These findings indicate a role for alpha-catenin in the APC destruction complex and at Wnt target genes (Choi, 2013).
Protein interactions of Armadillo homologs: Interaction with desmosomal components Plakoglobin is a major component of both desmosomes and adherens junctions. At these sites it binds to
the cytoplasmic domains of cadherin cell-cell adhesion proteins and regulates their adhesive and
cytoskeletal binding functions. Plakoglobin also forms distinct cytosolic protein complexes that function
in pathways of tumor suppression and cell fate determination. Recent studies in Xenopus suggest that
cadherins inhibit the signaling functions of plakoglobin, presumably by sequestering this protein at the
membrane and depleting its cytosolic pool. To understand the reciprocal regulation between desmosomal
cadherins (desmoglein and desmocollin) and plakoglobin, an attempt was made to identify the binding domains
involved in the formation of these protein complexes. Plakoglobin comprises 13 central repeats flanked by
amino-terminal and carboxyl-terminal domains. Repeats 1-4 are involved in binding
desmoglein-1. In contrast, the interaction of plakoglobin with desmocollin-1a is sensitive to deletion of
either end of the central repeat domain. These plakoglobin repeat sites are overlapped by the binding sites for two adherens junction components,
alpha-catenin and classical cadherins. Competition among these proteins for binding
sites on plakoglobin may therefore account for the distinct composition of adherens junctions and
desmosomes (Witcher, 1996).
Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions. It has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring
intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg),
and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as
well as in binding assays in vitro. In transfected carcinoma cells, a chimeric
protein combining the vesicle-forming transmembrane glycoprotein (synaptophysin) with the complete
human plakoglobin sequence, is sorted to small vesicles, many of which associate with desmosomal
plaques and their attached IFs. The chimeric
plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but
not to endogenous plakoglobin, demonstrating that the binding of plakoglobin to desmosomal
cadherins does not require its soluble state and is strong enough to attach large structures to desmosomes, such as
vesicles. Plakoglobin
associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly
with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of
the so-called armadillo repeats exhibit distinct binding to the desmosomal cadherins, comparable in strength to
that of the entire molecule. In vitro some internal plakoglobin fragments bind even better to the C-domain of
E-cadherin than to the entire molecule, indicating that elements exist in native plakoglobin that interfere with
the interaction of this protein with its various cadherin partners (Chitaev, 1996).
Plakoglobin, a member of the armadillo family of proteins, is a component of intercellular adhesive
junctions. The central domain of plakoglobin comprises a highly conserved series of armadillo repeats
that facilitate its association with either desmosomal or classic cadherins, or with cytosolic proteins
such as the tumor suppressor gene product adenomatous polyposis coli. Sequences in the N- and
C-terminal domains of plakoglobin are less highly conserved, and their possible roles in regulating
plakoglobin's subcellular distribution and junction assembly are still unclear. The role of plakoglobin end domains have been examined by stably expressing constructs lacking the N and/or C terminus of
plakoglobin in A-431 cells. Myc-tagged plakoglobin lacking either end
domain is still able to associate with the desmosomal cadherin desmoglein and incorporate into
desmosomes. In cell lines that express an N-terminal truncation of plakoglobin, an increase in the
cytosolic pool of endogenous and ectopic plakoglobin is observed that may reflect an increase in the
stability of the protein. Deletion of the N terminus does not have a dramatic effect on the structure of
desmosomes in these cells. In contrast, striking alterations in desmosome morphology are
observed in cells expressing C-terminal truncations of plakoglobin. In these cell lines, ectopic
plakoglobin incorporates into desmosomes, and extremely long junctions or groups of tandemly linked
desmosomes which remain well attached to keratin intermediate filaments, are observed. Together,
these results suggest that the plakoglobin end domains play a role in regulating its subcellular distribution,
and that the presence of the C terminus limits the size of desmosomes, perhaps through regulating
protein-protein interactions required for assembly of the desmosomal plaque (Palka, 1997).
Plakoglobin, a protein belonging to the Armadillo-repeat gene family, is the only component that adherens
junctions and desmosomes have in common. Plakoglobin null-mutant mouse embryos die because of severe heart
defects and may exhibit an additional skin phenotype, depending on the genetic background. No differences between wild-type and Plakoglobin embryos are detected at E12.5, when the epidermis consists of a single-layered periderm or, at E15.5 when the first signs of stratification are observed. Later, however, lack of plakoglobin
affects the number and structure of desmosomes, resulting in visible defects when cells are subjected to increasing
mechanical stress, e.g. when embryonic blood starts circulating or during skin differentiation. By analyzing
plakoglobin-negative embryonic skin differentiation in more detail, it has been shown that, in the absence of
plakoglobin, its closest homolog, beta-catenin, becomes localized to desmosomes and associated with
desmoglein. This substitution may account for the relatively late appearance of the developmental defects seen in
plakoglobin null-mutant embryos. Beta-catenin cannot, however, fully compensate for a lack of plakoglobin. In the
absence of plakoglobin, there is reduced cell-cell adhesion, resulting in large intercellular spaces between
keratinocytes, subcorneal acantholysis and necrosis in the granular layer of the skin. Electron microscopic analysis
documents a reduced number of desmosomes, and those present lack the inner dense plaque and have fewer
keratin filaments anchored. This analysis underlines the central role of plakoglobin for desmosomal assembly and
function during embryogenesis (Bierkamp, 1999).
Protein interactions of Armadillo homologs: Interaction with alpha-Catenin and delta-Catenin The Drosophila protein alpha-Catenin (D alpha-Catenin) is part of the adherens junction. It is a 110-kD protein with 60% identity to mouse
alpha E-catenin. D alpha-Catenin is localized to cell-cell contact sites, expressed throughout development, and present in a
wide variety of tissues. When this protein is immunoprecipitated from detergent extracts of
Drosophila embryos or cell lines, several proteins co-precipitate. These include Armadillo which is known to be a Drosophila homolog of beta-Catenin, another
cadherin-associated protein in vertebrates. These results strongly
suggest that Drosophila has a cell adhesion machinery homologous to the vertebrate
cadherin-catenin system (Oda, 1993).
The cadherin-based transmembrane cell-cell adhesive complex is thought to be composed of a
cadherin molecule, a beta-catenin, and an alpha-catenin (which connects the complex to the
cytoskeleton). The precise stoichiometry of this complex remains uncertain. A series of
recombinant molecules and biophysical techniques has been used to assess the multimeric state of human alpha- and beta-catenin in vitro; they have been visualized by electron microscopy after rotary shadowing. Calculated solution molecular masses are 213 kDa for alpha-catenin, 73 kDa for beta-catenin, and 186 kDa for both. This suggests that alpha-catenin exists as a homodimer in solution, beta-catenin is a
monomer, and when both are present, they form alpha/beta-catenin heterodimers. Co-precipitation and
surface plasmon resonance assays localize the site of alpha-catenin dimerization to the NH2-terminal
228 amino acids. This region encompasses a high-affinity (Kd = 100 nM) binding site for beta-catenin
that lies between residues 54 and 157. It is anticipated that the oligomeric state of alpha-catenin and the
relative stoichiometry of the components in the membrane adhesion complex will be dynamic and
regulated by beta-catenin, cell adhesion, and probably other factors as well (Koslov, 1997).
The interaction of cadherin-catenin complex with the actin-based cytoskeleton through alpha-catenin is indispensable for cadherin-based cell
adhesion activity. E-cadherin-alpha-catenin fusion molecules show cell adhesion and cytoskeleton binding
activities when expressed in nonepithelial L cells. Deletion mutants of E-cadherin-alpha-catenin fusion molecules lacking
various domains of alpha-catenin were constructed and introduced
into L cells. Detailed analysis has identified three distinct functional domains of alpha-catenin:
a vinculin/alpha-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assays
reveal that the fusion molecules containing the ZO-1-binding domain, in addition to the adhesion-modulation domain, confer the strong state
of cell adhesion activity on transfectants, although those lacking the ZO-1-binding domain confer only the weak state. The disorganization of
actin-based cytoskeleton by cytochalasin D treatment shifts the cadherin-based cell adhesion from the strong to the weak state. In the epithelial
cells, where alpha-catenin is not precisely colocalized with ZO-1, the ZO-1-binding domain does not completely support the strong state of cell
adhesion activity. These studies show that the interaction of alpha-catenin with the actin-based cytoskeleton through the ZO-1-binding domain is
required for the strong state of E-cadherin-based cell adhesion activity (Imamura, 1999).
The alpha-catenin binding site in plakoglobin (gamma-catenin) consists of a domain of 29 amino acids necessary and
sufficient for binding alpha-catenin. The alpha-catenin binding site is fully encoded within exon 3 of
plakoglobin but only partially represented in Armadillo repeat 1. This suggests that exons rather
than individual Arm repeats encode functional domains of plakoglobin. Site-directed mutagenesis
identified residues in the alpha-catenin binding site indispensable for binding in vitro. Analogous
mutations in beta-catenin and Armadillo have identical effects. These results indicate that single amino
acid mutations in the alpha-catenin binding site of homologs of Armadillo could prevent a stable
association with alpha-catenin, thus affecting cadherin-mediated adhesion (Aberle, 1996).
The classical adherens junction that holds epithelial cells together consists of a protein complex in
which members of the cadherin family linked to various catenins are the principal components.
delta-catenin is a mammalian brain protein in the Armadillo repeat superfamily with sequence similarity
to the adherens junction protein p120(ctn). Delta-catenin can be immunoprecipitated as a
complex with other components of the adherens junction, including cadherin and beta-catenin, from
transfected cells and brain. The interaction with cadherin involves direct contact within the highly
conserved juxtamembrane region of the COOH terminus, where p120(ctn) also binds. In developing
mouse brain, staining with delta-catenin antibodies is prominent toward the apical boundary of the
neuroepithelial cells, in the ventricular zone. When transfected into Madin-Darby canine kidney
(MDCK) epithelial cells delta-catenin colocalizes with cadherin, p120(ctn), and beta-catenin. The Arm
domain alone is sufficient for achieving localization and coimmunoprecipitation with cadherin. The
ectopic expression of delta-catenin in MDCK cells alters their morphology, induces the elaboration of
lamellipodia, interfers with monolayer formation, and increases scattering in response to hepatocyte
growth factor treatment. It is proposed that delta-catenin can regulate adhesion molecules to implement
the organization of large cellular arrays necessary for tissue morphogenesis (Lu, 1999).
In adherens junctions, alpha-catenin links the cadherin-beta-catenin complex to the actin-based cytoskeleton. alpha-catenin is a homodimer in solution, but forms a 1:1 heterodimer with beta-catenin. The crystal structure of the alpha-catenin dimerization domain, residues 82-279, shows that alpha-catenin dimerizes through formation of a four-helix bundle in which two antiparallel helices are contributed by each protomer. A slightly larger fragment, comprising residues 57-264, binds to beta-catenin. A chimera consisting of the alpha-catenin-binding region of beta-catenin linked to the amino terminus of alpha-catenin (57-264) behaves as a monomer in solution, as expected, since beta-catenin binding disrupts the alpha-catenin dimer. The crystal structure of this chimera reveals the interaction between alpha- and beta-catenin, and provides a basis for understanding adherens junction assembly (Pokuttal, 2000).
ZO-1 is an actin filament (F-actin)-binding protein that localizes
to tight junctions and connects claudin to the actin cytoskeleton in
epithelial cells. Claudins are four-transmembrane domain proteins that constitute the backbone of tight junction strands. Claudins constitute a new gene family, the 'claudin' family, which includes at least 16 members. Interestingly, most of the claudin family members end in YV at their COOH termini and thus are good candidates
for the binding partners for PDZ domains. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through ß- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like
adhesion molecule that localizes to AJs and is associated with the
actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin
is an afadin- and vinculin-binding protein that also localizes to AJs.
The nectin-afadin complex has a potency to recruit the
E-cadherin-ß-catenin complex through alpha-catenin in a manner
independent of ponsin. Whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites has been examined by the use of cadherin-deficient L cell lines
stably expressing various components of the cadherin-catenin and
nectin-afadin systems, and alpha-catenin-deficient F9 cell lines. Nectin shows a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 is dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system (Yokoyama, 2001).
Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that α-catenin links the adhesion protein E-cadherin through ß-catenin to the actin cytoskeleton, it has been shown that α-catenin does not bind simultaneously to both E-cadherin-ß-catenin and actin filaments (Yamada, 2005). This study demonstrates that α-catenin exists as a monomer or a homodimer with different binding properties. Monomeric α-catenin binds more strongly to E-cadherin-ß-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that α-catenin is an allosteric protein. Significantly, α-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for α-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion (Drees, 2005).
Epithelial cell-cell junctions are organized by adhesion proteins and the underlying actin cytoskeleton. They provide adaptable interfaces that can respond to signals for cell movement during convergent extension in gastrulation or changes in cell shape during tube formation but also provide a constant permeability barrier between different biological compartments in the body. Analysis of how migrating cells initiate cell-cell adhesion has revealed dramatic changes in membrane dynamics and organization of the actin cytoskeleton. Migrating cells have characteristic forward-moving lamellipodia produced by rapid Arp2/3-nucleated assembly of a branched actin network perpendicular to the leading edge. Upon cell-cell adhesion, lamellipodial activity is reduced over the contacting area, and there is a concomitant reorganization of actin filaments; electron microscopy of simple epithelial cells indicates the formation of bundles of actin filaments parallel to the contacting membranes, an organization very different from that of branched actin in lamellipodia. It is unknown how engagement of cadherins between migrating cells causes these dramatic changes in actin-filament assembly and organization. The intracellular domain of cadherins binds cytoplasmic proteins that are thought to recruit and organize actin filaments. These molecular linkages include high-affinity binding of ß-catenin to the cadherin cytoplasmic domain and a lower-affinity interaction between ß-catenin and α-catenin (Pokutta, 2000). Since α-catenin can also bind actin filaments in vitro (Pokutta, 2002; Rimm, 1995), it is widely accepted that α-catenin bound to the cadherin-ß-catenin complex bridges these components to actin. In addition, many actin binding proteins have been reported to bind α-catenin, including vinculin and α-actinin, suggesting that they could also link the cadherin-catenin complex to the actin cytoskeleton. Furthermore, the Rho family of small GTPases and the actin nucleators formin and the Arp2/3 complex are involved in cell-cell adhesion and may regulate actin dynamics around the cadherin-catenin complex (Drees, 2005 and references therein).
In an accompanying paper (Yamada, 2005), experiments with purified proteins demonstrated that the ternary complex of cadherin-ß-catenin-αcatenin does not bind directly to actin filaments or indirectly through vinculin or α-actinin. Moreover, live-cell imaging showed that the cadherin-catenin complex has dynamics that are very different from those of actin, consistent with the lack of a stable linkage between the cadherin-catenin complex and actin in cells (Drees, 2005).
The inability of α-catenin to bind simultaneously to the cadherin-ß-catenin complex and actin indicates that it may function as a molecular switch, whereby binding to one partner changes the ability to interact with the other. Evidence is provided in this study that these changes are associated with distinct conformational states of α-catenin and that dimerization of α-catenin influences its ability to selectively bind to ß-catenin or actin. Given the highly dynamic properties of the actin network at cell-cell contacts, the role of α-catenin in regulating actin assembly was further examined. α-catenin is shown to suppress actin polymerization by the Arp2/3 complex, suggesting that assembly and clustering of the cadherin-catenin complex at cell-cell contacts may provide a pool of α-catenin that can locally regulate actin dynamics and organization (Drees, 2005).
Cell-cell contacts are considered to be stable structures that maintain the structural integrity of tissues and are thought to be formed by clustering of cell-adhesion proteins through binding between opposed extracellular domains and linkage through the cytoplasmic domain to the underlying actin cytoskeleton. The cadherin cytoplasmic domain binds with high affinity to ß-catenin, which in turn binds with weaker affinity to α-catenin (Pokutta, 2000). Given that α-catenin binds to actin filaments (Pokutta, 2002; Rimm, 1995) and to other actin binding proteins such as vinculin and α-actinin (Hazan, 1997; Knudsen, 1995; Watabe-Uchida, 1998; Weiss, 1998), it was reasonable to assume that α-catenin bound to the cadherin-ß-catenin complex also binds directly or indirectly to actin filaments. However, direct tests of this model failed. In the accompanying paper, it is shown that α-catenin does not bind simultaneously to the cadherin-catenin complex and actin filaments (Yamada, 2005). These results predict that interactions between the cadherin-catenin complex and underlying actin cytoskeleton in cells might be very dynamic rather than being static as has been assumed. Although direct interactions between the cadherin complex and actin filaments were not verified experimentally, there is a considerable body of work concluding that some sort of interaction of actin filaments and the cadherin-catenin complex is important in cell-cell adhesion. Cytochalasin D and latrunculin A, which change the dynamic organization of the actin cytoskeleton, reduce adhesion and weaken cell-cell contacts. However, these drugs have global effects on actin organization that are not restricted to effects on only cell-cell contacts. Genetic deletion of α-catenin potentially provides a more direct approach to disrupt the putative cadherin-actin linkage. Cell-cell adhesion in α-catenin null cells is reduced and can be rescued by re-expression of α-catenin. However, it is noted that cell-cell adhesion occurs in some α-catenin null cells, presumably because there is sufficient cadherin on the cell surface to initiate cell-cell adhesion. αcatenin null cells have also been used to express chimeras between α-catenin-E-cadherin, α-catenin-vinculin and α-catenin-formin-1, all of which partially rescue cell-cell adhesion. However, no direct evidence is presented in those studies that these chimeric proteins bind actin filaments. Moreover, the current findings that the molecular and functional properties of α-catenin are altered upon binding to ß-catenin - in particular, that the αcatenin/ ß-catenin complex cannot bind actin - demonstrate that the use of E-cadherin-α-catenin chimeras cannot recapitulate the behavior of the cadherin-catenin complex at the membrane. It is possible that expression of some of these chimeric proteins could locally change actin dynamics or simply increase the amount of cadherin at the cell surface to a level that can partially rescue cell-cell adhesion (Drees, 2005).
Although it is surprising that a stable linkage does not exist between cadherins and the underlying actin cytoskeleton, cell-cell adhesion is a dynamic process during embryonic development, wound healing and cancer cell metastasis. This may require a more dynamic interaction between cadherin and the actin cytoskeleton rather than the static, stable linkage proposed in previous models. In addition, it is noteworthy that, in most cell types, cadherins are not the only means of cell-cell adhesion. Many other adhesion proteins are expressed, including members of the nectin occludin/claudin, JAM, and desmosomal cadherin families, all of which are thought to interact directly or indirectly with the actin or intermediate filament cytoskeletons and thereby contribute to cell-cell adhesion (Drees, 2005).
There are dramatic changes in membrane and actin dynamics associated with the formation of cell-cell adhesions. Initial cell-cell contact formation is driven by overlapping membrane lamellipodia from contacting cells. These lamellipodia are regulated by actin polymerization and branching induced by the Arp2/3 complex and local activation of the Rho family of small GTPases. However, lamellipodial activity decays as the cadherin-catenin complex accumulates and the contacts mature into stable cell-cell junctions. It is not known what regulates this contact-dependent decrease of membrane activity, which presumably depends upon a decrease in Arp2/3-mediated actin polymerization. It is interesting to note, in this context, that decreased levels of α-catenin result in increased membrane activity in hippocampal neurons, while overexpression of α-catenin suppress membrane activity, indicating that α-catenin directly regulates membrane protrusive activity. Furthermore, keratinocytes from α-catenin knockout mice are characterized by loss of contact inhibition and increased migratory activity (Drees, 2005).
How might changes in both actin assembly and organization (from networks to bundles) that drive the transition from active lamellipodia to quiescent contacts be coordinated during the formation of cell-cell adhesions? The finding that α-catenin suppresses Arp2/3-mediates actin polymerization in a concentration-dependent manner, combined with the actin bundling activity of α-catenin (Rimm, 1995), may provide an explanation. The cytoplasmic α-catenin monomer concentration (0.6 mM) is too low to bind actin significantly and would need to be concentrated to bind actin and to dimerize. A significant increase in the local concentration of α-catenin at the membrane occurs upon accumulation of the cadherin-catenin complex at nascent contacts. This cadherin bound pool of α-catenin can exchange with the cytoplasmic pool; note that the amount of exchange was probably underestimated because it cannot be directly measure d locally at cell-cell contacts. Although the local concentrations of α-catenin and Arp2/3 immediately adjacent to contacting membranes are unknown, a 10-fold increase in local concentration of α-catenin would be sufficient for α-catenin to compete with the Arp2/3 complex for actin filaments. This would suppress formation of branched actin networks and inhibit lamellipodial activity and would also favor formation of α-catenin homodimers that bundle actin filaments (Rimm, 1995), resulting in a reorganization of actin filaments and a change in membrane dynamics underneath the junction. It has also been proposed that formins, which promote formation of linear actin cables, are recruited to the adherens junction by α-catenin. If so, α-catenin would serve as a switch that turns off Arp2/3-mediated branched-actin-network formation required for lamellipodial activity during the initiation of adhesion and turn on αcatenin- mediated bundling of actin filaments and formation of linear cables by formins during maturation of the adherens junction. While further work is needed to test specific tenets of this hypothesis, the results provide new mechanistic insights into many aspects of the local dynamics of actin and membranes associated with cell-cell contacts not accounted for in previous models (Drees, 2005).
β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. Interaction of β-catenin with alpha-catenin is regulated by the phosphorylation of β-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases (see Drosophila Fps oncogene analog). Transfection of these kinases to epithelial cells disrupts the association between both catenins. Whether these kinases are involved in the regulation of this interaction by K-ras was examined. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little alpha-catenin-β-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of β-catenin-alpha-catenin interaction (Piedra, 2003).
The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by alpha- and β-catenin. The phosphorylation state of β-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. The phosphorylation of β-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of β-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. The interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated β-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a β-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains β-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and β-catenin, as well as localization of cadherin and β-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts (Xu, 2004).
β-Catenin plays important roles in cell adhesion and gene transcription, and has been shown recently to be essential for the establishment of a bipolar mitotic spindle. This study shows that β-catenin is a component of interphase centrosomes and that stabilization of β-catenin, mimicking mutations found in cancers, induces centrosome splitting. Centrosomes are held together by a dynamic linker regulated by Nek2 kinase and its substrates C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin. β-catenin binds to and is phosphorylated by Nek2, and is in a complex with Rootletin. In interphase, β-catenin colocalizes with Rootletin between C-Nap1 puncta at the proximal end of centrioles, and this localization is dependent on C-Nap1 and Rootletin. In mitosis, when Nek2 activity increases, β-catenin localizes to centrosomes at spindle poles independent of Rootletin. Increased Nek2 activity disrupts the interaction of Rootletin with centrosomes and results in binding of β-catenin to Rootletin-independent sites on centrosomes, an event that is required for centrosome separation. These results identify β-catenin as a component of the intercentrosomal linker and define a new function for β-catenin as a key regulator of mitotic centrosome separation (Bahmanyar, 2008).
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