(Received for publication, February 5, 1996; and in revised form, February 27, 1996)
From the
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, we have sought 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. Our results show that 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. The binding sites for two adherens junction components,
-catenin and classical cadherins, overlap these sites. Competition
among these proteins for binding sites on plakoglobin may therefore
account for the distinct composition of adherens junctions and
desmosomes.
Adherens junctions and desmosomes connect cells together and
anchor the cytoskeleton to the plasma membrane. Desmogleins (Dsg) ()and desmocollins (Dsc) form the adhesive transmembrane
components of desmosomes, whereas classical cadherins perform this
function in adherens junctions. Their genes display a highly conserved
and unusual genomic structure, suggesting that they arose from a common
ancestor(1, 2, 3, 4) . Moreover,
their protein products share considerable sequence similarity and a
common repetitive organization of their ectodomain (for references see
review in (5) ). Recent crystal structure analyses of the
amino-terminal extracellular repeat of N-cadherin have shown that the
sequences that are highly conserved bind calcium ions and mediate
cadherin dimerization in the plane of the
membrane(6, 7) . Desmosomal and classical cadherins
differ principally in the sequences of their cytoplasmic segment (for
references see reviews in (5) and (8) ). Within this
domain, however, they share a small region of sequence similarity that
interacts with plakoglobin, an 86-kDa cytoplasmic
protein(9, 10, 11, 12, 13) .
Plakoglobin is the only component common to both desmosomes and
adherens junctions; as such it is uniquely positioned to provide a
pivotal point of control in the adhesive
mechanism(8, 14) . At adherens junctions, plakoglobin
forms a link between classical cadherins and -catenin, which in
turn connects the complex to the actin cytoskeleton both directly and
through
-actinin(15, 16, 17, 18) . The
role of plakoglobin at desmosomes is less clear; however, it is
essential for desmosome formation and may promote intermediate filament
association with the membrane(19, 20, 21) .
Recent studies have revealed a wide range of cellular functions for
plakoglobin in addition to its role in adhesive cell-cell junctions.
For example, both plakoglobin and its close relative -catenin are
implicated in pathways controlling cell proliferation, differentiation,
and motility by their association in the cytosol with APC, the product
of a tumor suppressor gene linked to colon
cancer(15, 22, 23, 24, 25) .
Furthermore, recent studies suggest their involvement in developmental
pathways. Armadillo, the Drosophila homologue of plakoglobin
and
-catenin, participates in a signaling pathway that determines
segment polarity via post-transcriptional up-regulation in response to wingless expression(26, 27) . A similar
up-regulation of plakoglobin and
-catenin can be produced in
vertebrate cells by expression of Wnt-1, a homologue of wingless(28, 29) . Ectopic expression of
plakoglobin,
-catenin, or Wnt-1 in Xenopus embryos each produce duplication of the embryonic axis, suggesting
that they operate in a common conserved signaling
pathway(30, 31, 32, 33) .
Intriguingly, in the case of plakoglobin, this phenotype is suppressed
by co-expression of the Dsg1 cytoplasmic domain, indicating that
desmosomal cadherins play a significant role in modulating
plakoglobin's signaling functions(33) . As a step toward
understanding the reciprocal regulation between these proteins, we have
sought to define the regions of plakoglobin involved in their
associations. Our results show that the site of Dsg1 interaction is
relatively discrete, involving the first three to four repeats of the
central domain of plakoglobin. In contrast, the association of Dsc1a
with plakoglobin is sensitive to even small deletions at either end of
the central repeat domain.
Bovine Dsc1a was constructed by replacing the 488-base pair MscI-NheI fragment (bases 2516-2904) of Dsc1b (GB accession number M61750) (35) with an reverse transcription-PCR generated 443-base pair alternatively spliced fragment from the corresponding region of Dsc1a. Sequencing of the PCR-generated segment and flanking sequences confirmed identity to GB accession number X56967. A HindIII-NsiI fragment including the entire Dsc1a coding sequence was subcloned into the HindIII/BstXI site of CDM8.
Human plakoglobin (GB accession number Z68228) (36) was cloned into the EcoR 1 site of the p163/7 mammalian expression vector(37) . The complete coding sequence of human plakoglobin generated by PCR using the oligonucleotides CCGGAATTCATGGAGGTGATGAACCTGATG and CCGGAATTCCTAGGCCAGCATGTGGTC was subcloned into the EcoR1 site of the pGBT9 vector (Clonetech, Palo Alto, CA) in frame with the GAL4 DNA-binding domain. All plakoglobin mutants were constructed by PCR using 5` primers consisting of a three-base spacer followed by an EcoR1 restriction site and six bases conforming to Kozak's rules for the optimal sequence of an initiating methionine (CCGGAATTCGCCGCCATG), followed by 15 nucleotides encoding plakoglobin sequence beginning with the amino acid indicated (Fig. 1). For some mutants, the sequence GAGCAGAAGCTGATCAGCGAGGAGGACCTG, encoding the epitope EQKLISEEDL derived from c-myc was introduced between the initiating ATG codon and the plakoglobin sequence. 3` primers comprised a three-base spacer, an EcoR1 restriction site, a stop codon, and 15 nucleotides encoding the plakoglobin sequence ending with the amino acids indicated (Fig. 1). The deleted forms of human plakoglobin described above (Fig. 1) were subcloned into the EcoR1 site of p163/7 and pGBT9 vectors.
Figure 1: Schematic representation of deletion mutants of human plakoglobin. The diagram at the top represents the amino- and carboxyl-terminal variable domains of plakoglobin flanking the 12 central 42-amino acid arm repeat regions of plakoglobin. The lines below show deletion mutants. The numbers indicate the initial or final amino acids (according to GenBank sequence Z68228) encoded by each construct.
All clones generated by PCR were fully sequenced
by the dideoxy chain termination method. All constructs were found to
have sequences identical to the parent cDNA clones with the exception
of the plakoglobin mutant C, which contained a single mutation
resulting in an alteration in residue 580 from isoleucine to serine.
The product of this construct showed no significant difference from
that of the original full length cDNA in its ability to associate with
Dsg1 or Dsc1a.
Xenopus -catenin (GB accession number
U47624) was cloned by screening a Xenopus stage 17
gt11
library with mouse
-E-catenin cDNA. A full-length clone containing
the complete open reading frame was subcloned into the pCS2 vector
containing a CMV promoter, which was a kind gift of the Hal Weintraub
laboratory. The deduced amino acid sequence shows 93% identity to
murine
-catenin.
The ability of these fusion proteins to interact, thereby
activating transcription of a HIS3 reporter gene under the control of
the GAL1 promoter, was also tested in the HF7c strain by growth on
his plates
(SD-trp
leu
his
).
All cotransformants, including controls, grew on these plates due to
basal levels of HIS3 expression (data not shown). Incorporation of 20
mM 3-aminotriazole, however, reduced this background
significantly in all controls except those expressing full-length
plakoglobin. Growth in the presence of 20 mM 3-aminotriazole
was observed in cells expressing the full-length plakoglobin construct
as well as with mutants (
4-C,
8-C,
11-C,
C,
N/
C,
N,
N-2) but not with (
1-C,
N-6,
N-9,
N-13) (Fig. 2).
Figure 2:
Yeast two-hybrid growth assay of Dsg1 and
plakoglobin interactions. Growth of HF7c colonies co-expressing
plakoglobin mutants (as indicated) fused to the DNA-binding domain and
the desmoglein cytoplasmic domain (DSG) or a truncated form lacking the
plakoglobin binding region (DSGC) fused to the GAL4 activation
domain is shown. Growth on
SD-trp
-leu
-his
plates is shown on the left and replicas on
SD-trp
-leu
-his
+
20 mM 3-aminotriazole on the right.
Figure 3: Specificity of antibodies. Autoradiogram showing TX-100-soluble extracts of metabolically labeled 293T cells transfected with control CDM8 vector lacking an insert (lanes 1, 5, and 9), bovine Dsc1a (lanes 2, 6, and 10), bovine Dsg1 (lanes 3, 7, and 11), and human plakoglobin (Pg, lanes 4, 8, and 12), which were divided equally into three aliquots and precipitated with the following antibodies: anti-plakoglobin (anti-Pg) (lanes 1-4), anti-desmosomal cadherin (anti-Dsc1a/Dsg1) (lanes 5-8), and anti-Dsg1 (lanes 9-12). Note all antibodies recognize their cognate antigens and show no cross-reaction with other overexpressed proteins.
Figure 4:
Immunoprecipitation of PECAM, Dsg1, Dsc1a,
and Plakoglobin Complexes. Autoradiograms of immunoprecipitations of
TX-100 soluble extracts of metabolically labeled 293T cells transfected
with: PECAM (lanes 1 and 2) and cotransfected with
PECAM and plakoglobin (lanes 3 and 4); Dsg1 and
plakoglobin (lanes 5-10); and Dsc1a and plakoglobin (lanes 11-13) precipitated with monoclonal antibody:
HEC7-specific for PECAM (lanes 1 and 3); and
polyclonal antibodies: anti-plakoglobin (Pg, lanes
2, 4, and 6-9); anti-Dsg1 (
Dsg, lane 5) and anti-desmosomal cadherin
antibody (anti-Dsg/Dsc, lanes 10-13). Note the
lack of interaction between PECAM and plakoglobin (lanes 3 and 4) but complex formation between Dsg 1 and plakoglobin (lane 5). Lanes 6-9 show cells metabolically
labeled for 15 min followed by chase periods indicated above the lanes.
Anti-plakoglobin antibodies coimmunoprecipitate plakoglobin complexed
to the desmoglein precursor (lane 6) and mature Dsg1 (lane
9). Lanes 10-13 show immunoprecipitates with a
constant amount of anti-Dsg1/Dsc1a from 293T cells transiently
cotransfected with 5 µg of plakoglobin (lanes 10-13) together
with 5 µg of Dsg1 (lane 10) or 5, 2.5, and 10 µg,
respectively, of Dsc1a indicated above the lanes. Note that equivalent
amounts of desmoglein and desmocollin coprecipitate different amounts
of plakoglobin and that the stoichiometry of the desmocollin complex
remains constant.
Figure 5: Co-immunoprecipitation of Dsg1 with full-length and mutant forms of plakoglobin sequentially deleted from the amino terminus. Autoradiogram of immunoprecipitations with the anti-plakoglobin (Pg) antibody (lanes 1-8) or anti-Dsg1 antibody (lanes 1`-8`) of TX-100-soluble fractions of metabolically labeled 293T cells transiently cotransfected with Dsg1 and the plakoglobin constructs indicated above the lanes.
Figure 6:
Co-immunoprecipitation of Dsg1 with
full-length and mutant forms of plakoglobin sequentially deleted from
the carboxyl terminus. Autoradiogram of immunoprecipitates with
anti-Dsg1 antibody (lanes 1-5 and 6`-9`)
and anti-myc antibody (lanes 6-9 and 12-14) of TX-100 soluble fractions of metabolically
labeled 293T cells transiently cotransfected with the plakoglobin
constructs indicated above the lanes and either Dsg1 (lanes
1-9 and 6`-9`) or -catenin (lanes
10-14).
Figure 7: Co-immunoprecipitation of Dsc1a with full-length and mutant forms of plakoglobin from transiently transfected 293T cells. Autoradiogram of immunoprecipitates with anti-desmosomal cadherin antibody (anti-Dsg/Dsc) of TX-100 soluble fractions of metabolically labeled 293T cells transiently cotransfected with the plakoglobin constructs indicated above the lanes and Dsc1a. Arrowheads indicate where the plakoglobin (Pg) mutants would migrate as judged from sister gels (data not shown).
Plakoglobin belongs to a family of functionally diverse
proteins (41, 42) that contain varying numbers of
42-amino acid sequences termed ``arm'' repeats after
Armadillo, the protein in which they were first
observed(26, 43) . Five of these proteins
(plakoglobin, -catenin, Armadillo, desmosomal band six protein and
p120) associate with cadherins, but plakoglobin is the only one known
to associate with both classical cadherins at adherens junctions and
desmosomal cadherins at
desmosomes(9, 10, 11, 12, 13, 44, 45, 46, 47, 48) .
Definition of its interactions are therefore crucial to our
understanding of the structure and function of the two major vertebrate
cell-cell adhesive junctions.
Plakoglobin is also found in the
cytoplasm, where it forms a homodimer of unknown function (14, 49) (for alternative view, see (24) );
like -catenin, plakoglobin can form cytosolic complexes with the
APC tumor suppressor gene product, implicating it in growth
control(15, 22, 23, 24, 25) .
Studies in Xenopus suggest that cytosolic pools of plakoglobin
participate in the transmission of developmental patterning signals
that, intriguingly, are suppressed by Dsg1 expression(33) .
From this it has been inferred that desmosomal cadherins influence
signaling pathways governing patterning by sequestering plakoglobin at
the membrane, and it is reasonable to surmise that they may also affect
plakoglobin's cytosolic role in growth control. Thus, definition
of cadherin-plakoglobin interactions is important for our understanding
of the connections between these processes and cell adhesion.
Our
results show that plakoglobin binds to the precursor and mature forms
of Dsg1, analogous to the early association reported to occur between
-catenin and E-cadherin(50) . The plakoglobin binding site
for Dsg1 involves a relatively discrete region of amino-terminal
repeats. Specifically, our results indicate that the head and first
three repeats are sufficient for Dsg1 interaction. Amino-terminally
deleted mutants show that although removal of the head domain lessens
binding slightly, progressive removal of the first and second repeats
severely compromises Dsg1 association, and further deletion of the
third and fourth repeats eliminates interaction. Taken together, these
data suggest that the head and repeats 1 and 2 cooperate to enhance
Dsg1 association with the third and possibly fourth repeats. The first
and third repeats of plakoglobin are very similar to those of
-catenin and Armadillo, so it is rather surprising to find them
engaged in an association considered unique to plakoglobin. The second
repeat, however, is less conserved and may therefore impart specificity
to the complex.
In contrast to the localized region of plakoglobin
sufficient to bind Dsg1, Dsc1a association is impaired by deletions of
either the carboxyl-terminal repeats or repeat 2. These results are
compatible with two possibilities. In the first model, Dsc1a
association would require simultaneous binding to both ends of the
repeat domain. Alternatively, the interaction may occur over a region
between repeats 2-13 in a manner that is conformationally
sensitive to deletions at either end. Interestingly, this region
overlaps binding sites for E- and N-cadherin(15, 40) .
Likewise, the Dsg1 binding site lies next to and may involve that for
catenin(15, 40) . It is therefore likely that
binding of desmosomal cadherins will sterically hinder the association
of other partners, accounting for the distinct complexes formed by
plakoglobin with
-catenin and classical cadherins at adherens
junctions and with Dsg1 and Dsc-1a at desmosomes.
Considerably more plakoglobin binds to Dsg1 than to Dsc1a. Furthermore, overexpression of the Dsg1 cytoplasmic domain produces a dominant negative effect on desmosome formation, whereas expression of the analogous region of Dsc1a does not(21) . Given the capacity of Dsg1 for binding plakoglobin and the significance of this association for both the formation of desmosomes and suppression of cytosolic roles of plakoglobin, naturally occurring mutations in the interactive domains of Dsg1 and plakoglobin are likely to have marked consequences not only for cell adhesion but also for proliferation and differentiation.
The interactions of plakoglobin with its partners are similar in
several respects to those described for another arm protein, the
regulatory A subunit of phosphatase 2A (PP2A-A)(51) . For
example, the catalytic C subunit binds to a discrete site at one end of
PP2A-A, as do Dsg1 and -catenin on plakoglobin. There is clear
evidence for competition and cooperation among some of the binding
partners of PP2A, whereas others bind independently(51) . Our
results show that Dsg1 and Dsc1a can interact with plakoglobin
separately. Whether they can bind simultaneously to a single
plakoglobin molecule awaits the development of Dsc-1-specific
antibodies. This question relates to an important potential role for
plakoglobin in organizing cadherins into an adhesive zipper.
A model has been proposed for PP2A-A in which each repeat forms two amphipathic helices that interact along their hydrophobic faces(51) . Consecutive helix pairs stack to form a concertina-like rod with interrepeat and intrarepeat loops projecting from opposite surfaces of the rod. A similar arrangement of secondary structures may exist for plakoglobin, because residues required to form the putative amphipathic helices are conserved in all arm proteins(42) . Despite the potential similarities between PP2A-A and plakoglobin in their secondary structures and topology of their intermolecular associations, biochemical analyses suggest that they differ in their tertiary structure; PP2A-A is a rod-like molecule, whereas plakoglobin behaves as a globular protein(49, 51) . As Dsc1a association requires both ends of the repeat domain of plakoglobin, one can envisage a horseshoe, rather than a rod-like shape for plakoglobin. Dsc1a would bind directly to both ends or to a central conformationally dependent binding pocket created by intramolecular association of the ends. Clearly these models are highly speculative, and crystal structure analyses to resolve the structure of plakoglobin are currently underway.
The emerging model of arm proteins is one in which individual repeats provide the principal binding sites for particular partners and neighboring repeats contribute to the specificity and stability of these associations. Partners recognize partially overlapping regions and may compete or cooperate for binding. Thus distinct tripartite complexes with different functions are formed, integrating cellular processes as diverse as adhesion, proliferation, and differentiation. Definition of the binding sites on plakoglobin now permits the development of minimally altered mutants deficient in cadherin binding functions to test the outcome of uncoupling membrane and cytosolic functions of this protein.