From the Departments of Pathology and Dermatology and
the Robert H. Lurie Cancer Center, Northwestern University Medical
School, Chicago, Illinois 60611 and the
University of New
Mexico Health Sciences Center, College of Pharmacy and Department of
Cell Biology, Albuquerque, New Mexico 87131
Received for publication, March 27, 2001
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ABSTRACT |
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Tyrosine phosphorylation of junctional
components has been proposed as a mechanism for modulating cell-cell
adhesion. Although a correlation exists between the tyrosine
phosphorylation of the adherens junction protein Modulation of adhesive junctions plays a critical role during
development and wound healing (1-3). Moreover, the ability of cells to
down-regulate or alter the adhesive capabilities of their cell-cell
junctions is a contributing factor during tumor metastasis (4).
However, the exact mechanisms that regulate intercellular adhesion
during these processes are not well understood. Tyrosine
phosphorylation of junctional components, and in particular phosphorylation of members of the armadillo family of proteins, has
been proposed as a critical step in modulating cell-cell adhesion. Several lines of evidence suggest that tyrosine phosphorylation of the
cadherin-catenin complex down-regulates cell adhesion and possibly the
association of cadherin-catenin complexes with the cytoskeleton
(5-11). In addition, plakoglobin
(Pg)1 and In certain cell types, tyrosine phosphorylation of Through its ability to bind to either classic or desmosomal cadherin
tails, Pg is a component of both desmosomes and adherens junctions and
serves as a link between members of the cadherin family of proteins and
cytoskeletal associated proteins (3, 17, 18). Therefore, tyrosine
phosphorylation of Pg may affect both classical and desmosomal
cadherin-based adhesion. Although there is evidence suggesting that
tyrosine phosphorylation of Generation of Plakoglobin cDNA Constructs--
Full-length
human plakoglobin cDNA was isolated and subcloned into the
mammalian expression vector LK 444 under the control of the human
The resulting 519-base pair PCR product contains a 5' BglII
and a 3' HindIII restriction site. By using these engineered
sites, the construct was subcloned into the BglII and
HindIII sites of p240 creating a C-terminally Myc-tagged
single point mutant Pg, which was checked by sequence analysis. The
newly engineered Pg construct was subcloned into the
SalI/HindIII sites of the mammalian expression
vector LK 444 generating the vector p730. The triple point mutant Pg
Phe-693, Phe-724, and Phe-729 was generated by PCR amplification
following a similar strategy using the following primers and
p240 as template for the PCR: LNPg143,
5'GCATGATTCCCATCAATGAGCCCTTTGG3' (GATTCCCATC, BsaB1, TTT, Phe-693);
LNPg144,
5'CGCTGAA1GGTGTCGATGGGGAA2GTC3' (GAA1, Tyr-729, GAA2,
Tyr-724); LNPg145, 5'GAGACTTC1CCCATCGACACCTTC2AG3'
(TTC1, Tyr-724, TTC2,
Tyr-729); LN52,
5'GGCCAAGCTTCTACAAGTCCTCTTCAGAAATGAGCTTTTGCTCCACGGCCAGCATGTGGTCTGCAG3' (AAGCTT, HindIII restriction site).
The resulting 219-base pair PCR product contains a 5' BsaB1
and a 3' HindIII restriction site. These engineered sites
were used to subclone the cDNA fragment into the BsaB1
and HindIII sites of p240 creating a C-terminally Myc-tagged
triple point mutant Pg, which was checked by sequence analysis. The
newly engineered Pg construct was subcloned into the
SalI/HindIII sites of the mammalian expression
vector LK 444 generating the vector p731.
Other cDNA Expression Constructs--
The chimeric cadherin
Ecad-Dsg1 comprising the extracellular domain of E-cadherin and the
cytoplasmic domain of Dsg1 was constructed as described previously
(20). A cDNA construct encoding amino acids 1-584 of the
desmoplakin N terminus, C-terminally fused to a FLAG epitope tag, and
termed DP-NTP was previously engineered in a mammalian expression
vector under a cytomegalovirus promoter (19).
Cell Lines and Transfection Methods--
A431 epithelial
cells (a gift from Dr. M. Wheelock, University of Toledo) were cultured
in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin,
and 100 µg/ml streptomycin. A431 cells lines expressing N- and
C-terminal truncations of Pg were described previously (22) and
maintained in media containing 350 µg/ml (active concentration) G418.
A431 clones Pg Antibodies--
The following antibodies used in this study were
previously described: a mouse monoclonal antibody against the c-Myc
epitope tag, 9E10.2 (24); a mouse monoclonal antibody 11E4, directed against the N terminus of plakoglobin (21); a rabbit polyclonal antibody directed against the C terminus of plakoglobin (25); and a
rabbit polyclonal antibody NW161, directed against the N-terminal domain of desmoplakin (19). 11E4 was provided by Dr. M. Wheelock (University of Toledo), and the Pg C-terminal antibody was provided by
Dr. J. Papkoff (Valentis Corporation, Burlingame, CA). The mouse
monoclonal Growth Factor Treatment--
COS-7 cells, 24 h
post-transient transfection, or A431 cell lines grown to 80%
confluence in 60-mm culture dishes or on glass coverslips in 35-mm
culture dishes were rinsed with complete PBS and incubated 12-24 h in
serum-free DMEM containing 0.1% bovine serum albumin (w/v). Cells were
treated with 10 Sequential Detergent Extraction and Immunoprecipitation from A431
Cells--
Cells were grown to 80% confluence on 60-mm culture
dishes, treated with EGF, rinsed in complete PBS, and subjected to
sequential detergent extraction as described previously (22). For
subsequent immunoprecipitation, 1 ml of detergent buffer was used for
extraction (22). Triton X-100-insoluble proteins were solubilized in
100 µl of solubilization buffer (10 mM Tris, pH 7.5, 1%
SDS, 5 mM EDTA, 2 mM EGTA, 0.1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), heated
at 95 °C for 10 min to aid in solubilization, diluted with 900 µl
of dilution buffer (15 mM Tris, pH 7.5, 5 mM
EDTA, 2 mM EGTA, 1% Triton X-100, 1% sodium deoxycholate,
0.1% SDS, 120 mM NaCl, 25 mM KCl, 0.1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), and centrifuged at ~14,000 × g for 30 min
at 4 °C prior to immunoprecipitation. Immunoprecipitations were
conducted using the appropriate antibody as described previously (22).
Samples were subjected to SDS-PAGE on 7.5% gels and subsequent
immunoblot analysis.
Co-immunoprecipitation and Immunoblot Analysis from COS-7
Cells--
Co-immunoprecipitations were carried out as described
previously unless otherwise specified (20, 26). Immune complexes were
released by incubation in reducing Laemmli sample buffer at 95 °C
and were subjected to SDS-PAGE on 7.5% gels and subsequent immunoblot analysis.
Long Term EGF Treatment Results in Shifts in the Subcellular
Distribution of Plakoglobin--
In response to treatment with the
pro-migratory growth factors EGF or hepatocyte growth factor,
epithelial cells such as HT29 or A431 scatter and lose their
intercellular connections (27, 28). Phase contrast microscopy confirmed
the effect of long term EGF treatment on A431 cells (Fig.
1A). In the absence of EGF,
A431 cells remained in compact colonies (Fig. 1A, a). After 24 h of EGF treatment the cells acquired a more motile,
fibroblastic morphology (Fig. 1A, b). In order to determine
the subcellular distribution of the adherens junction and desmosome
component Pg after prolonged EGF treatment, immunofluorescence using a
monoclonal antibody against Pg was performed (Fig. 1A, c and
d). After 24 h of EGF treatment, the cell-cell border
staining typical of Pg (Fig. 1A, c) was shifted to a more
cytoplasmic localization, although some staining at cell-cell borders
could still be observed (Fig. 1A, d). This observation was
further confirmed by subjecting A431 cells to sequential detergent
extraction to release cytosolic (saponin-soluble) proteins,
membrane-bound (Triton-soluble) proteins, and (Triton-insoluble)
presumably junction or cytoskeleton-associated proteins after 0, 12, or
24 h of EGF treatment. Western blot analysis of the different
pools revealed a modest, reproducible increase in the cytosolic pool
and a concomitant decrease in the detergent-insoluble pool of Pg (Fig.
1, B and C). The Western blot shown in Fig.
1B is representative of five different experiments, and
results of ECL detection were confirmed by reprobing with an
125I-conjugated secondary antibody (data not shown). Data
from three experiments were quantified using the Molecular Analyst
software and plotted as percentage of total plakoglobin (Fig. 1C,
upper graph). Results were also expressed as the difference
between the percentage of total plakoglobin present in the
Triton-insoluble pool and the cytosolic pool. This difference in the
percentage of total Pg significantly decreases (p < 0.05) over the time course of EGF treatment (Fig. 1C, lower
graph), thus illustrating the shift in Pg solubility upon long
term EGF treatment. In addition to these alterations seen when
normalizing to total Pg, a decrease in total Pg levels was frequently
observed, possibly due to increased turnover of protein in the soluble
non-junctional cell compartment (not shown). When this decrease is
taken into account, alterations in each subcellular fraction are even
more pronounced.
Plakoglobin Is Rapidly Phosphorylated in Response to EGF and
Tyrosine-phosphorylated Plakoglobin Is Predominantly in a
Triton-soluble Pool--
Although Pg has been shown to be
tyrosine-phosphorylated in response to growth factors such as EGF and
hepatocyte growth factor/Scatter Factor (13, 27), the effect of
tyrosine phosphorylation on Pg subcellular distribution and interaction
with junctional binding partners is unknown. To begin to address this
question, we analyzed Pg phosphorylation status and subcellular
distribution following short term EGF treatment. Different Pg
constructs represented in Fig. 2 were
established for these studies.
First, A431 cells were treated with EGF for 1, 2, and 5 min and
subjected to a sequential detergent extraction. By using the Pg-specific antibody 11E4, endogenous Pg was immunoprecipitated from
each cell fraction. The subcellular distribution of Pg in the absence
of EGF was similar to its previously described (22) distribution under
normal growth conditions, that is a small proportion of Pg was
cytosolic but the majority of the protein was distributed between the
Triton-soluble and Triton-insoluble pools (Fig.
3B, EGF 0 min). Treatment of
A431 cells with EGF for 2 or 5 min did not detectably alter the
subcellular distribution of total Pg (Fig. 3B, EGF 2 and
5 min). After EGF treatment, Pg was rapidly phosphorylated
on tyrosine residues (Fig. 3A), consistent with previous
reports (13). Although Pg protein was detected in all three subcellular
pools using the Pg-specific antibody, tyrosine-phosphorylated Pg was
found predominantly in the membrane-associated Triton-soluble pool of
proteins and not in the Triton-insoluble, junction-associated pool
(Fig. 3A).
The Plakoglobin C Terminus Is Required for EGF-induced Tyrosine
Phosphorylation--
By using a Pg antibody that recognizes both endogenous and ectopic Pg,
endogenous Pg and Pg
In the absence of growth factor, the majority of Pg Tyrosine-phosphorylated Desmoglein 2 Is in a Complex with
Tyrosine-phosphorylated Plakoglobin in the Membrane-associated
Triton-soluble Pool of Proteins--
After sequential detergent
extraction, two higher molecular weight proteins (~160-180 kDa)
co-immunoprecipitating with Pg were also detected by the
phosphotyrosine antibody (Fig. 3, A, C, and E).
At ~160 kDa, desmogleins, the transmembrane desmosomal cadherins, are
well characterized binding partners for Pg (for review see Ref. 29).
Therefore, we tested whether the most abundant desmoglein in A431
cells, desmoglein 2, represented one of these high molecular weight
phosphorylated proteins found in the 11E4 immunoprecipitates.
Triton-soluble lysates of A431 cells treated for 0, 5, or 20 min with
EGF were processed for Pg immunoprecipitation. The immunoprecipitates
were subjected to immunoblot analysis for phosphotyrosine confirming
the presence of the two high molecular weight phosphorylated proteins
at both time points of EGFR activation (Fig.
4A). The membranes were
stripped and reprobed to confirm the identity of the ~80-kDa
phosphorylated band as Pg. In addition, the lowest of the two high
molecular weight bands detected on the phosphotyrosine blots was
identified as desmoglein 2 (Dsg2). To our knowledge these data
represent the first report of desmoglein tyrosine phosphorylation
in response to EGFR activation.
Since EGFR is reported to interact with
To extend this analysis, A431 cells were serum-starved overnight and
treated with EGF for 0, 5, or 20 min to reproduce conditions under
which the Specific Tyrosine Residues of the C-terminal Domain of Pg Are
Targeted by EGFR Activity--
COS-7 cells transiently expressing N-
or C-terminal truncations of Pg were analyzed to confirm the
requirement of the C terminus of Pg for EGF-induced tyrosine
phosphorylation. COS-7 cells were transfected with EGFR, ECad-Dsg1, and
Pg
Plakoglobin tyrosine point mutant constructs were used in order to
identify the C-terminal tyrosine residues of Pg targeted by
EGF-dependent phosphorylation. COS-7 cells were transfected with EGFR, Ecad-Dsg1, and either Pg F660 in which tyrosine residue Tyr-660 was substituted for a phenylalanine or with Pg Phe-693, Phe-724, Phe-729 in which the three C-terminal tyrosine residues Tyr-693, Tyr-724, and Tyr-729 were mutated into phenylalanines (Fig.
2). After overnight serum starvation, cells were treated with EGF for 0 or 5 min and processed for immunoprecipitations with the 9E10 antibody.
Although Myc-tagged Pg point mutants were both efficiently
immunoprecipitated (Fig. 5B, Blot: Pg), Pg
Phe-660 was phosphorylated in response to the 5-min EGF treatment,
whereas Pg Phe-693, Phe-724, and Phe-729 showed no sign of
EGF-dependent phosphorylation (compare lanes 2 and 5, Fig. 5B, Blot: PY). The lack of
phosphorylation of Pg Phe-693, Phe-724, and Phe-729 was also confirmed
after 24-h activation of the EGFR (data not shown). This indicated that
Pg C-terminal residues Tyr-693, Tyr-724, and/or Tyr-729 are specific
targets for EGF-dependent phosphorylation of Pg.
Pg Phosphorylated upon EGFR Activation Is Associated with Dsg2 but
Not with DP N-terminal Domain--
A reported consequence for
EGF-dependent phosphorylation of EGF-dependent Phosphorylation of Plakoglobin Prevents
the Formation of DP-NTP and Plakoglobin Complexes--
The results
described in Fig. 6 indicate that in transiently transfected COS-7
cells, the Pg associated with DP-NTP after EGF treatment is not
tyrosine-phosphorylated. These results, however, do not rule out that
the Pg associated with DP-NTP is not available for phosphorylation by
EGFR. To take a more direct approach to determine whether
phosphorylation of Pg prevents its association in a complex with
DP-NTP, in vitro techniques using recombinant Pg and DP-NTP
were initiated. However, efforts were hampered due to technical
problems including the inefficiency of in vitro
phosphorylation of Pg by purified EGFR kinase. Consequently, we took a
different approach, generating an A431 stable cell line inducible for
DP-NTP. Our goal was to create a system in which Pg becomes
tyrosine-phosphorylated prior to the synthesis of its DP binding
partner. This would rule out the possibility that phosphorylated Pg is
not associated with DP-NTP simply because this interaction makes Pg
inaccessible for tyrosine phosphorylation. A time course of induction
demonstrated that DP-NTP was detectable in the cell lysates only after
Plakoglobin and In the present study, we demonstrate that, like Intriguingly, in a short time frame of growth factor-induced tyrosine
phosphorylation, a transient but reproducible decrease in the
Triton-insoluble pool of the deletion mutant Pg In A431.Pg The transmembrane receptor tyrosine kinase EGFR was reported previously
to associate both with its substrate Several mechanisms have been proposed to explain the adhesive changes
associated with cadherin-catenin tyrosine phosphorylation (41). One
model suggests that tyrosine phosphorylation of the cadherin-catenin
complex leads to a decreased association of the complex with the
cytoskeleton. Linkage of the classical cadherins to the cytoskeleton
has been shown to be required for classical cadherin-mediated adhesion
in epithelial cells (42, 43), and a decreased association with the
cytoskeleton is thought to lead to decreased adhesion. It has also been
suggested that phosphorylation of junctional components other than
In addition to its ability to interact with desmosomal cadherins (21,
48-52), Pg has been shown to interact with the desmosomal plaque
protein desmoplakin (20, 53, 54). Experiments conducted in transiently
transfected COS-7 cells showed that upon short term EGFR activation, no
interaction between tyrosine-phosphorylated Pg and the N-terminal
domain of desmoplakin was detected (Fig. 6B). This finding
raised the possibilities that the association of Pg in a complex with
DP prevents its phosphorylation and/or that the phosphorylation of Pg
inhibits its association with DP. These hypotheses were tested first by
activating the EGFR kinase for 24 h, a time frame during which
exchange between the different pools of proteins is expected to occur.
The experiments still showed no DP associated with phosphorylated Pg in
the membrane-associated Triton-soluble pool of proteins, consistent
with the idea that phosphorylation of the catenin inhibits its
interaction with the N-terminal domain of desmoplakin (Fig.
6B). Also supporting this interpretation are results from
experiments carried out using A431 cells inducible for DP-NTP
expression (Fig. 7). In these experiments pre-phosphorylated Pg was
unable to participate in de novo interactions with the DP N terminus.
Together, these findings suggest that desmosomal protein interactions
are affected by EGFR-dependent tyrosine phosphorylation in
a manner comparable to that reported previously (10) for adherens
junctions, that is the EGF-dependent phosphorylation of
catenins affects the linkage of the actin or intermediate filament cytoskeleton with the adherens junction or the desmosome components, respectively. As in adherens junctions, where the
EGF-dependent phosphorylation of -catenin and loss
of classical cadherin-mediated adhesion, the effects of tyrosine
phosphorylation on the function of the adherens junction and
desmosome-associated protein plakoglobin is unknown. In the
present study, we investigated the effects of epidermal growth factor
receptor (EGFR) tyrosine kinase activation on the subcellular
distribution of plakoglobin and its association with its junctional
binding partners. Long term epidermal growth factor (EGF) treatment of
A431 cells revealed a modest decrease in the cytoskeleton-associated
pool of plakoglobin (Pg) and a corresponding increase in the cytosolic
pool of Pg. After short term EGF treatment, plakoglobin was rapidly
phosphorylated, and tyrosine-phosphorylated Pg was distributed
predominantly in a membrane-associated Triton X-100-soluble pool, along
with a co-precipitating high molecular weight tyrosine-phosphorylated
protein identified as desmoglein 2. Analysis of deletion and point
mutants defined the primary EGFR-dependent targets as one
or more of three C-terminal tyrosine residues. Whereas phosphorylated
Pg remained associated with the desmoglein tail after both short and
long term EGFR activation, no phosphorylated Pg was found associated
with the N-terminal Pg-binding domain (DPNTP) of the intermediate
filament-associated protein, desmoplakin. Together these results are
consistent with the possibility that EGF-dependent tyrosine
phosphorylation of Pg may modulate cell-cell adhesion by compromising
the link between desmosomal cadherins and the intermediate filament cytoskeleton.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin have
been reported to associate with the tyrosine kinases c-ErbB-2, the
epidermal growth factor receptor (EGFR) (12-13), and protein-tyrosine
phosphatases PTP
(14) and LAR (15). These data suggest that these
members of the armadillo family of proteins may function as modulatable
components within complex adhesive structures allowing for rapid
responses to intracellular or extracellular signals.
-catenin has been
correlated with decreased classical cadherin-mediated adhesion (7, 9).
In ras-transformed MCF-10A human breast epithelial
cells, tyrosine phosphorylation of
-catenin correlated with an
increased detergent solubility of both
-catenin and E-cadherin (8).
The increased detergent solubility was interpreted as a decreased
association with the cytoskeleton, as protein complexes associated with
the cytoskeleton are generally thought to be Triton X-100-insoluble.
Tyrosine phosphorylation of
-catenin in EGF-treated HSC-1 squamous
carcinoma cells also correlated with an increased detergent solubility
of E-cadherin (6). EGF treatment of MDA-MB-468 human breast cancer
cells promoted dissociation of actin,
-actinin, and vinculin from
the E-cadherin-catenin complex (10). Treatment of K562 leukemia cells
with pervanadate was shown to cause a significant amount of
-catenin
to dissociate from the E-cadherin complex (16). In addition, both
-catenin and Pg were tyrosine-phosphorylated in response to
pervanadate or EGF treatment, but their association with E-cadherin was
not compromised (10, 16). However, a recent in vitro study
reported that Src-mediated phosphorylation of Tyr-654 of
-catenin
regulates the interaction between E-cadherin and
-catenin (11).
Taken together, these reports suggest that tyrosine phosphorylation of
-catenin and Pg may lead to a decreased association of these
proteins with the actin cytoskeleton.
-catenin may lead to a decreased
association with the cytoskeleton, less is known about the effects of
tyrosine phosphorylation on Pg and its association with cytoskeletal
components. We sought to examine the effects of EGF treatment on Pg
with the ultimate goal of understanding the possible contribution of Pg
phosphorylation on the regulation of junctional interactions during
EGF-triggered cell migration. We investigated the effect of long term
and short term EGF treatment on the subcellular distribution of Pg and
Pg truncation mutants using biochemical approaches in epithelial cells
that assemble both desmosomes and adherens junctions. We detected
modest shifts in the subcellular distribution of Pg during cell
migration occurring after 24 h of EGF treatment. We confirmed that
endogenous Pg in A431 cells was rapidly phosphorylated on one or more
of three C-terminal tyrosine residues in response to EGF and was
distributed exclusively in the membrane-soluble, non-junctional pool of
proteins. Furthermore, while the interaction between Pg and the
desmoglein tail was maintained, no interaction was detected between
phosphorylated Pg and the N-terminal domain of desmoplakin (DP), which
provides a link between the desmosomal plaque and the intermediate
filament cytoskeleton (19, 20). Collectively, these results are
consistent with the idea that tyrosine phosphorylation of the Pg C
terminus affects the ability of Pg to assemble into functional
junctions by compromising anchorage of the intermediate filament
cytoskeleton through DP, thus contributing to the remodeling of
epithelia in response to growth factors during wound healing and invasion.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin promoter (21). N- and C-terminal deletions of Pg were
generated and subcloned into the LK 444 vector as described previously
(22). Pg point mutants were generated using an overlap extension
strategy. The mutant Pg Phe-660 was generated by PCR amplification
using the following primers and p240 which is identical to the
previously described p236 Pg construct as template (21): LNPg 140, 5'GCATGGAGATCTCC3' (AGATCT,
BglII restriction site); LNPg 141, 5'TCCGGAAGTCTGGGTTCT3' (GAA, Phe-660); LNPg
142, 5'AGAACCCAGACTTCCGGA3' (TTC, Phe-660);
LN77, 5'GACGTAAGCTTCTAGGCCAGCATGTGGTCT3'
(AAGCTT, HindIII restriction site).
N(A7) and Pg
C (10) were used for the analyses
reported here; however, similar results were seen for multiple clones
expressing Pg
N or Pg
C. An A431 stable cell line,
tetracycline-inducible for the expression of DP-NTP, was established as
described (23).2 DP-NTP
expression was induced using 2 µg/ml doxycycline. For transient
transfections, COS-7 cells were cultured in DMEM containing 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin and
transfected using the calcium phosphate precipitation method described
previously (21).
-catenin antibody used for immunoprecipitations was
purchased from Transduction Laboratories, Lexington, KY. The 5H10 mouse
monoclonal
-catenin antibody and the desmoglein 2 monoclonal
antibody 6D8 were kind gifts from Dr. M. Wheelock. The 795 rabbit
polyclonal anti-E cadherin antibody was a kind gift from Dr. R. Marsh.
The 1407 polyclonal antibody was raised in chickens using 400 µg of
recombinant full-length Myc-tagged human Pg to immunize each animal
(immunizations performed by Aves Labs, Tigard, OR). The
anti-phosphotyrosine mouse monoclonal antibody 4G10 and the anti-Shc
rabbit polyclonal antibody were purchased from Upstate Biotechnology
Inc., Lake Placid, NY), and the anti-phosphotyrosine HRP-conjugated
PY99 was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit and
mouse IgG (clone MOPC 31C) were obtained from Sigma. The anti-FLAG
epitope M2 monoclonal antibody coupled to agarose beads was purchased
from Sigma. EGFR monoclonal antibodies Ab12 and Ab13 were purchased
from Neomarkers (Fremont, CA). For immunoblotting, the following
dilutions were used: concentrated 11E4 supernatant (1:2000), NW161
(1:2500), 1407 (1:5000), 9E10 (1:2), Ab12, Shc antibody and 6D8 ascites
(1:1000), 5H10 hybridoma supernatant (1:100), 4G10 (1:1000), and PY99
(1:200). Primary antibodies were diluted in PBS, 5% milk. Secondary
antibodies, goat anti-mouse peroxidase, goat anti-rabbit peroxidase,
and goat anti-chicken peroxidase (Kirkegaard & Perry Laboratories,
Gaithersburg, MD), were used at a dilution of 1:5000 in PBS, 5% milk.
Antibodies were detected using enhanced chemiluminescence (Amersham
Pharmacia Biotech). For immunoprecipitations, 2-5 µl of concentrated
11E4, 1 µl of the rabbit polyclonal antibody directed against the C terminus of plakoglobin, 3 µg of rabbit or mouse IgG, 2 µl of the
E-cadherin 795 rabbit polyclonal antibody, 6 µl of 9E10 ascites, 3 µg of monoclonal
-catenin antibody, 5 µl of 6D8 ascites, 5 µl
of EGFR Ab13 antibody, or 40 µl of M2 agarose were used per reaction.
8 M EGF
(Biomedical Technologies Inc., Stoughton, MA), prepared in serum-free
DMEM containing 0.1% bovine serum albumin, for designated times and
then rinsed with cold complete PBS. Cells were then processed for
sequential detergent extraction or immunoprecipitation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of long term EGF treatment on A431
epithelial cell morphology and plakoglobin distribution.
A, after 24 h of serum starvation cells were treated
with 10 nM EGF for 0 or 24 h. In the absence of EGF,
A431 cells remain in tight colonies (a). After long term EGF
treatment A431 cells exhibit a more fibroblastic morphology accompanied
by cell scattering (b). Indirect immunofluorescence using
the Pg N-terminal antibody 11E4 was performed to detect Pg before
(c) and after (d) long term EGF treatment. Pg
shifts from cell borders to a more diffuse, cytoplasmic distribution
after 24 h of EGF treatment. B, after 24 h of
serum starvation cells were treated with 10 nM EGF for 0, 12, or 24 h followed by sequential detergent extraction. Detergent
extracts were run on SDS-PAGE gels transferred to nitrocellulose and
immunoblotted with the Pg N-terminal antibody 11E4. The Western blot
shown is representative of five different experiments. C,
the data obtained by Western blot analysis as presented in B
was scanned using Molecular Analyst, and the percentage of plakoglobin
present in each detergent extract was graphically represented as a
proportion of total Pg at each time point. The cytosolic pool of Pg
showed a modest increase while the Triton-insoluble pool decreased. The
average difference between these two detergent pools is represented in
the 2nd bar graph. *, the decrease in the difference of
total plakoglobin between the Triton-insoluble pool and the cytosolic
pool of proteins from time 0 to 24 h is statistically significant
as assessed by a two-tailed t test (p = 0.03).
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Fig. 2.
Schematic representation of tyrosine residues
and their substitutions in the N and C termini of plakoglobin.
There are a total of 22 tyrosine residues in Pg, 11 of which are in the
N and C termini of Pg (5 in the N terminus and 6 in the C terminus).
The Pg N deletion removes the 5 tyrosine residues in the N terminus
(Tyr-20, Tyr-22, Tyr-53, Tyr-61, and Tyr-74), whereas the Pg
C
deletion removes the 6 tyrosine residues in the C terminus and 1 tyrosine in the 13th armadillo repeat (Tyr-660, Tyr-693, Tyr-701,
Tyr-705, Tyr-724, Tyr-729, and Tyr-737). The Tyr-660 of the Pg Phe-660
point mutant has been substituted for a phenylalanine. The C-terminal
tyrosine residues, Tyr-693, Tyr-724, and Tyr-729, were substituted for
phenylalanines to generate the Pg Phe-693, Phe-724, Phe-729 point
mutant construct. The triple lines represent the armadillo
repeats found in Pg and its family members (48). All deletion and point
mutant Pg constructs were C-terminally Myc epitope-tagged.
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Fig. 3.
Subcellular distribution and tyrosine
phosphorylation status of plakoglobin and plakoglobin deletion
mutants. After 24 h of serum starvation, A431, A431.Pg C,
and A431.Pg
N cells were treated with 10 nM EGF for 0, 2, or 5 min followed by sequential detergent extraction and
immunoprecipitation with the Pg antibody 11E4 or the polyclonal Pg
C-terminal antibody for the Pg
N samples. Immunoprecipitates were
run, in duplicate, on SDS-PAGE gels, transferred to nitrocellulose, and
immunoblotted with the 4G10 anti-phosphotyrosine antibody (A,
C, and E) or the Pg N-terminal antibody 11E4
(B) or the Myc monoclonal antibody 9E10 (D and
F). Although Pg could be detected in all three pools
(B), tyrosine-phosphorylated Pg was predominantly in the
Triton-soluble pool (A). Upper bands (~180 kDa)
are discussed in the text and addressed in Fig. 4. Pg
C could be
detected in all three pools using the Myc antibody
(arrowhead in D) and was not
tyrosine-phosphorylated in response to EGF treatment
(arrowhead in C). Endogenous Pg from A431.Pg
C
cells was still phosphorylated in response to EGF (asterisk
in C). As described previously (22), the majority of Pg
N
was detected in the cytosolic and Triton-soluble pools (F).
Tyrosine-phosphorylated Pg
N (arrowhead in E)
and endogenous Pg (asterisk in E) could be
detected in both these pools. After 2 min of EGF treatment a transient
decrease in the amount of Pg
N in the Triton-insoluble pool could be
detected (F). The fold decrease in the Triton-insoluble pool
of Pg
N, established by scanning densitometry, was 3.7 ± 1.8 (from three different experiments).
-Catenin has been shown to be phosphorylated
directly by the EGF receptor in vitro (13). Interestingly,
the armadillo repeats of
-catenin were not phosphorylated,
suggesting that tyrosine residues in the N and/or C termini of
-catenin were targets for EGF receptor phosphorylation. Plakoglobin,
which has high structural identity to
-catenin (18), has a total of
22 tyrosine residues, 5 in the N terminus, 11 in the armadillo repeats,
and 6 in the C terminus. To determine whether the Pg end domains are
required for EGF-dependent tyrosine phosphorylation
in vivo, A-431.Pg
C and A431.Pg
N cells lines stably
expressing C- or N-terminal Pg truncations were established and
subjected to EGF stimulation and sequential detergent extraction as
above for endogenous Pg. The Pg
N polypeptide lacks the 5 tyrosine
residues in the N terminus, whereas the Pg
C polypeptide lacks the 6 tyrosine residues in the C terminus and 1 tyrosine residue from the
13th armadillo repeat (Fig. 2).
C were immunoprecipitated from each subcellular
fraction. Pg
C was detected in all three pools with the majority of
Pg
C in the Triton-soluble and Triton-insoluble pools (Fig.
3D). Similar to Pg from control cells, EGF treatment did not
detectably alter the subcellular distribution of the Triton-insoluble or -soluble pools; however, a small increase in the cytosolic pool of
the Pg
C deletion was observed. After EGF treatment
tyrosine-phosphorylated endogenous Pg was detected (Fig. 3C,
asterisk), but phosphorylation of Pg
C was not detectable (Fig.
3C, arrowhead), suggesting that the Pg C terminus is
required for EGF-induced tyrosine phosphorylation (Fig. 3C
and Fig. 5A). A small amount of tyrosine-phosphorylated endogenous Pg was present in the cytosolic pool, consistent with its
previously reported association with Pg
C (22).
N was distributed
between the cytosolic and Triton-soluble pools, and only a small
proportion was detected in the Triton-insoluble pool (Fig. 3F,
EGF 0 min). Interestingly, a transient decrease of ~4-fold was
observed in the Triton-insoluble pool of Pg
N after 2 min of EGF
treatment in three independent experiments, reflecting an increase in
the detergent solubility of Pg
N. Similar to endogenous Pg from
control cells and in contrast to Pg
C, Pg
N was rapidly phosphorylated on tyrosine residues in response to EGF (Fig. 3E, EGF 2 and 5 min). However, unlike
tyrosine-phosphorylated Pg from control cells, tyrosine-phosphorylated
Pg
N was detected in both the cytosolic and Triton-soluble pools,
with the majority of the phosphorylated protein found in the cytosolic
pool. Tyrosine-phosphorylated endogenous Pg from A431.Pg
N cells was
detected mainly in the Triton-soluble pool, but some was also present
in the cytosolic pool, again consistent with its reported interaction
with N-terminally deleted Pg (22).
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Fig. 4.
Identification of high molecular weight
phosphorylated proteins that co-immunoprecipitate with Pg.
A, A431 cells were subjected to overnight serum starvation
prior to 0, 5, or 20 min of EGF treatment. The cells were lysed for
immunoprecipitation (IP) using the 11E4 Pg antibody.
Immunoprecipitates were run on SDS-PAGE gels and transferred to
nitrocellulose and immunoblotted with the HRP-conjugated PY99
anti-phosphotyrosine antibody (left blot). Membranes were
stripped and reblotted with the anti-desmoglein 2 (Dsg2) 6D8
antibody and the 1407 anti-plakoglobin antibody. The Pg IPs revealed
two high molecular mass phosphorylated bands at ~160-180 kDa, the
lowest one of which appears to be the known Pg-binding partner Dsg2
(Dsg2 blot). B, A431 cells were processed for
immunoprecipitation as described above. The cell lysates were subjected
to immunoprecipitation using Sepharose beads controls, Ab13 EGFR
antibody, mouse and rabbit immunoglobulins, 5H10 -catenin,
E-cadherin, Pg, and Dsg2 antibodies. The immunoprecipitates were
subjected to Western blot analysis using Ab12, an EGFR antibody and
PY99, a phosphotyrosine antibody. A tyrosine-phosphorylated band
migrating approximately at the molecular weight of EGFR is consistently
observed nonspecifically associated with the beads. However, EGFR was
not detected co-immunoprecipitating with any of the tested proteins
under these particular experimental conditions, even though a robust
signal for EGFR was detected in the EGFR immunoprecipitates. In
addition, other expected co-immunoprecipitating partners were observed,
including Dsg2 with Pg (see tyrosine-phosphorylated band representing
Dsg2 in Pg lane),
-catenin with E-cadherin, and
E-cadherin with
-catenin (not shown). C, A431 cells were
processed for immunoprecipitation as described in A.
Sepharose beads were used as a control for the Ab13 EGFR
immunoprecipitation reactions, which were performed in glycerol
containing HEPES buffer (1% Triton, 50 mM HEPES, 150 mM NaCl, 1 mM NaVO3, 10% glycerol,
pH 7.4). The immunoprecipitates were run on SDS-PAGE gels and analyzed
by Western blot using Ab12 EGFR antibody or 5H10
-catenin antibody,
1407 Pg antibody, or Shc antibody. The previously characterized
interaction between the adaptor protein Shc and EGFR was detected in
these conditions, whereas
-catenin and Pg interactions with EGFR
were not detected.
-catenin (13) and we
repeatedly detected a phosphorylated protein of a similar molecular
weight to EGFR in Pg immunoprecipitation experiments (Fig. 3, A,
C, and E), we hypothesized that the receptor associates with Pg to trigger its phosphorylation. To address this question, we
carried out immunoprecipitations using antibodies directed against
-catenin or Pg from cells treated with EGF for 0 and 30 min. In
addition, because Dsg2 was shown to co-immunoprecipitate with Pg (Fig.
4A), we tested whether immunoprecipitating Dsg2 would
co-immunoprecipitate EGFR in a manner similar to the reported co-immunoprecipitations of EGFR with E-cadherin (30). Analysis of the
resulting immunoprecipitates by blotting with an EGFR antibody showed
that under the conditions used for these experiments, EGFR was not
associated with any of the immunoprecipitated proteins. However, it was
easily detected by direct EGFR immunoprecipitation (Fig.
4B). The membrane was stripped and reprobed for
tyrosine-phosphorylated proteins revealing a high molecular weight band
of similar molecular weight to EGFR in all EGF-activated
immunoprecipitates, including beads only and IgG controls. In the lane
corresponding to the specific EGFR immunoprecipitation after EGF
treatment, the signal was much stronger due to the presence of
activated EGFR as shown in the EGFR blot (Fig. 4B).
Therefore, we conclude that the higher of the two phosphorylated high
molecular weight bands detected under these conditions in the Pg
immunoprecipitations (Fig. 4A) was due to a nonspecific
interaction of the Sepharose beads or IgG with a
tyrosine-phosphorylated protein of a similar molecular weight to EGFR.
Note that the phosphorylated specific band corresponding to Dsg2 is
detected in both Pg and Dsg2 immunoprecipitates (Fig. 4B).
In addition, co-precipitation of E-cadherin with
-catenin and
-catenin with E-cadherin was confirmed following stripping of the
blot (data not shown).
-catenin-EGFR interaction was detected previously (13).
Cells were harvested in 0.5% Triton buffer, glycerol containing HEPES
buffer to facilitate the maintenance of protein complexes during the
immunoprecipitation procedures. The cell lysates were subjected to EGFR
immunoprecipitation using Sepharose beads as a control. The
immunoprecipitates were probed with an EGFR antibody first, and the
membranes were subsequently stripped and reprobed for
-catenin, Pg,
or the well characterized EGFR-binding adaptor protein Shc (Fig.
4C). Under these experimental conditions, no
-catenin nor
Pg were detected in association with EGFR, although high amounts of
receptor were immunoprecipitated as shown by the EGFR blot and Shc
co-immunoprecipitated with EGFR (Fig. 4C).
C or Pg
N. The 9E10 anti-Myc antibody was used to
immunoprecipitate the C-terminally tagged Pg deletion mutants (Fig.
5A, lanes 1, 2, 6, and
7). Slight phosphorylation of Pg
C was detected in this
system as compared with the high level of phosphorylation observed for
Pg
N (compare lanes 2 and 7, Fig.
5A), confirming that the C terminus of Pg is required for
efficient EGF-induced tyrosine phosphorylation as indicated by the
results obtained in A431.Pg
C cells (Fig. 3).
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Fig. 5.
Requirement of plakoglobin C-terminal
tyrosine residues Tyr-693, Tyr-724, and Tyr-729 for EGF-induced
tyrosine phosphorylation of Pg. A, COS-7 cells
transiently transfected with Ecad-Dsg1, EGFR, and Myc-tagged Pg C or
Myc-tagged Pg
N were serum-starved overnight, treated with 10 nM EGF for 0 or 5 min, and harvested in 0.5% Triton
buffer. Cell lysates were subjected to 9E10 immunoprecipitation
(IP), and immunoprecipitates were run on SDS-PAGE gels,
transferred to nitrocellulose, and immunoblotted with the
HRP-conjugated PY99 anti-phosphotyrosine antibody (PY). The
PY99 antibody was stripped off the nitrocellulose membranes, and the
blots were reprobed with the 1407 Pg antibody. Phosphorylated Pg
C
was barely detectable (lane 2), whereas
tyrosine-phosphorylated Pg
N was easily detected following EGFR
activation (lane 7) as indicated by the
asterisks. The upper bands correspond to the
nonspecific high molecular weight protein co-migrating with EGFR.
B, COS-7 cells were transiently transfected with EGFR,
ECad-Dsg1, and Pg Phe-660 or Pg Phe-693, Phe-724, and Phe-729. And
24 h later, the cells were serum-starved overnight. Prior to lysis
and immunoprecipitation, cells were treated with 10 nM EGF
for 0 or 5 min. Immunoprecipitates were run on SDS-PAGE gels,
transferred to nitrocellulose, and immunoblotted with the
HRP-conjugated PY99 anti-phosphotyrosine antibody (PY). The
PY99 antibody was stripped off the nitrocellulose membranes, and the
blots were reprobed with the 1407 Pg antibody. The single Pg point
mutant Phe-660 was a substrate for EGF-dependent tyrosine
phosphorylation (lane 2). The substitution of Tyr-693,
Tyr-724, and Tyr-729 into phenylalanines prevented phosphorylation of
Pg Phe-693, Phe-724, and Phe-729 (lane 5).
-catenin is to modulate
negatively the interaction of E-cadherin with the actin cytoskeleton
(10). To explore whether the EGF-dependent phosphorylation
of Pg can similarly affect the interaction of desmosome components with
the intermediate filament cytoskeleton, we tested the effect of Pg
tyrosine phosphorylation on the interaction between Pg and its direct
binding partners Dsg and DP (20). DP links the desmosomal plaque to
intermediate filaments (19, 31-33). COS-7 cells were transiently
transfected with EGFR, Dsg2, and Pg, and in parallel with EGFR,
Ecad-Dsg1, Pg, and a FLAG epitope-tagged desmoplakin N-terminal
polypeptide (DP-NTP) (19). DP-NTP binds Pg and is recruited to
cell-cell borders in COS-7 cells in the presence of Pg and a desmosomal
cadherin tail (20). Cells were serum-starved overnight and treated with
EGF for 0 of 5 min or 24 h prior to 6D8 antibody or M2-agarose
immunoprecipitations. The 6D8 antibodies precipitated Dsg2, whereas the
M2 antibodies precipitated FLAG-tagged DP-NTP, as shown by the
immunoblot probed with 6D8 and NW161, which is directed against the DP
N terminus (Fig. 6, A and
B, Blot Dsg2 and Blot DPNT). Pg was
co-immunoprecipitated with Dsg2 independently of EGF treatment as shown
by the 1407 blot (Fig. 6A, right panel, Blot Pg)
and was efficiently phosphorylated as shown by the Pg
immunoprecipitations (Fig. 6A, left panel, Pg IP). Likewise,
Pg was co-immunoprecipitated with DP-NTP independently of EGF treatment
as assessed with the 1407 anti-Pg antibody (Fig. 6B, Blot
Pg) and was efficiently phosphorylated as shown by the Pg
immunoprecipitations (Fig. 6B, Pg IP). However, no
tyrosine-phosphorylated Pg was detected in association with DP-NTP
after either short or long term EGF treatment (Fig. 6B, Blot
PY), whereas phosphorylated Pg was detected in the Dsg2
immunoprecipitations (Fig. 6A, middle panel, blot PY).
Interestingly, a high level of phosphorylation of Dsg2 was detected in
these samples as shown by the upper band in the PY blot (Fig. 6A,
middle panel). These findings suggest that EGFR activation
promotes tyrosine phosphorylation of Pg, which remains associated with
its transmembrane binding partner Dsg2, while it does not associate, in
the cytosolic or membrane associated pools of proteins, with the
N-terminal domain of its desmosomal plaque binding partner DP.
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Fig. 6.
Phosphorylated Pg is found in a complex with
Dsg2 but not DP-NTP after EGFR activation. A, COS-7
cells were transiently transfected with EGFR, Dsg2, and Pg. After
24 h incubation at 37 °C, the cells were serum-starved
overnight. Prior to immunoprecipitation (IP), cells were
treated with 10 nM EGF for 0 or 24 h. 11E4 Pg
immunoprecipitates were run on SDS-PAGE gels and transferred to
nitrocellulose. The nitrocellulose blots were probed with the
HRP-conjugated PY99 anti-phosphotyrosine antibody (PY),
stripped, and reprobed with the 1407 Pg antibody (Pg).
Significant phosphorylation of total Pg was observed following EGF
treatment (left panels). Anti-Dsg2 6D8 immunoprecipitates,
generated from the same cell lysates used for Pg immunoprecipitations,
were run on SDS-PAGE gels, transferred to nitrocellulose, and
immunoblotted with the HRP-conjugated PY99 anti-phosphotyrosine
antibody (PY). The PY99 antibody was stripped off the
nitrocellulose membrane, and the blots were reprobed with the 1407 Pg
antibody (Pg). Phosphorylated Pg was easily detected
co-immunoprecipitating with its well characterized binding partner
Dsg2. The band denoted ns is nonspecific (middle
panels). The strength of the phosphorylation signal obtained for
Dsg2 (see middle panel) hindered the stripping of the PY99
antibody to efficiently reprobe the PY blot with Dsg2 Ab. Therefore,
parallel 6D8 immunoprecipitates were run on SDS-PAGE gels and
transferred to nitrocellulose. The top of the membrane was
immunoblotted with anti-Dsg2 6D8 antibody, and the bottom was probed
with 1407 Pg antibody. The ability of Dsg2 to associate with Pg was not
altered by the tyrosine phosphorylation of both Pg and Dsg2 resulting
from the 24-h EGF treatment (right panel). B,
COS-7 cells were transiently transfected with EGFR, ECad-Dsg1, Pg, and
FLAG epitope-tagged DP-NTP. After 24 h incubation at 37 °C, the
cells were serum-starved overnight. Prior to immunoprecipitation, cells
were treated with 10 nM EGF for 0 or 5 min or 24 h. Pg
immunoprecipitations using the 9E10 Myc antibody were run on SDS-PAGE
gels and transferred to nitrocellulose. The nitrocellulose blots were
probed with the HRP-conjugated PY99 anti-phosphotyrosine antibody
(PY), stripped, and reprobed with the 1407 Pg antibody
(Pg). Significant phosphorylation of total Pg was observed
following 5 min or 24 h of EGF treatment (left panels).
Anti-FLAG M2-conjugated agarose beads immunoprecipitates generated from
the same cell lysates used for Pg immunoprecipitations were run in
duplicate, on SDS-PAGE gels, transferred to nitrocellulose, and
immunoblotted with the HRP-conjugated PY99 anti-phosphotyrosine
antibody (PY) and the NW161 DP N-terminal antibody
(DPNT). The PY99 antibody was stripped off the
nitrocellulose membranes, and the blots were reprobed with the 1407 Pg
antibody (Pg). Although phosphorylated Pg was readily
detected in the control experiment shown on the left, no
phosphorylated Pg was ever found associated with the DP N-terminal
polypeptide in the EGF-treated samples both after 5 min and 24 h
of activation of the receptor tyrosine kinase (right
panels). The blots presented in this figure are
representative of five independent experiments.
12 h of induction with doxycycline (Fig.
7A), whereas we showed
previously that Pg is phosphorylated in A431 cells as early as 2 min
after EGF treatment (Fig. 3). Therefore, cells were treated with
doxycycline and EGF for 24 h and subjected to parallel Pg and
M2-agarose immunoprecipitations. Phosphorylated Pg was detected
independently of doxycycline treatment (Fig. 7B, Pg IP).
DP-NTP was only detected in the M2-immunoprecipitates derived from
doxycycline-induced cells as shown by the DPNT blot (Fig.
7B). As expected, Pg was co-immunoprecipitated with the induced DP-NTP (Fig. 7B, blot Pg). However, no
phosphorylated Pg was detected in the M2 immunoprecipitates (Fig.
7B, blot PY). These results indicate that newly synthesized
DP-NTP is able to form new complexes with non-phosphorylated Pg,
whereas it is unable to associate with tyrosine-phosphorylated Pg.
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Fig. 7.
Lack of phosphorylated Pg in newly formed
Pg·DP-NTP complexes. A, stably transfected A431 cells
inducible for FLAG epitope-tagged DP-NTP expression, in response to
doxycycline, were subjected to a time course of induction. Cells were
treated with 2 µg/ml doxycycline for 0, 1, 2, 6, or 12 h and
scraped in Laemmli buffer prior to separation on SDS-PAGE gels. As
shown in this representative time course of doxycycline induction,
expression of DP-NTP was detected after 12 h of induction by
immunoblot analysis using the DP N-terminal antibody NW161.
B, A431 cells inducible for FLAG-tagged DP-NTP were treated
with 2 µg/ml doxycycline and 10 nM EGF for 24 h. The
cells were then processed for immunoprecipitation with M2-conjugated
agarose beads directed against the FLAG tag and the Pg antibody 11E4.
Immunoprecipitates were run on SDS-PAGE gels and transferred to
nitrocellulose. Membranes with 11E4 immunoprecipitates were probed with
the PY99 antibody. After 24 h of EGF treatment, phosphorylated Pg
was present regardless of doxycycline induction. The membranes with
transferred M2 immunoprecipitates were probed with poly-FLAG and PY99
phosphotyrosine antibody. The PY99 antibody was stripped off the
membrane which was subsequently reprobed with 1407 Pg antibody. The
exposure shown for the PY99 blot is twice as long as for the poly-FLAG
or 1407 blots. No phosphorylated Pg was found associated to
DP-NTP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin are substrates for tyrosine kinases
and associate with tyrosine phosphatases suggesting that
phosphorylation and dephosphorylation of members of the armadillo
family of proteins may modulate cell-cell adhesion (12-14, 34-36).
Results from several laboratories (6, 8, 10, 37) suggest that tyrosine
phosphorylation of cadherin-catenin complexes increases the detergent
solubility of cadherins and catenins, possibly leading to a decreased
association of the cadherin-catenin complex with the cytoskeleton and
decreased cell adhesion. Although tyrosine phosphorylation of the
adherens junction protein
-catenin has been correlated with a
decrease in classical cadherin mediated adhesion in certain cells (5, 7, 9), much less is known about the effects of tyrosine phosphorylation
on Pg, which is a component of both adherens junctions and desmosomes
(17). Furthermore, the role of tyrosine phosphorylation in regulating
protein-protein interactions among desmosomal components is unknown.
-catenin, Pg becomes
more detergent-soluble in scattering cells in response to long term EGF
treatment (Fig. 1) suggesting a decrease in its association with the
cytoskeleton. As reported previously for
-catenin (13), Pg is
rapidly phosphorylated on tyrosine residues upon EGFR activation, and
this tyrosine phosphorylation is dependent on 1 or more of 3 residues
in its C terminus. Furthermore, tyrosine-phosphorylated Pg was
predominantly found in the membrane-associated Triton-soluble pool
(Fig. 3), indicating a similar localization to the receptor tyrosine
kinase EGFR. We did not detect any tyrosine-phosphorylated Pg in the
Triton-insoluble pool of proteins in either short (Fig. 3) or long (not
shown) term EGF treatment, suggesting the possibilities that
phosphorylated Pg is unable to integrate into junctions and/or that
junctional Pg is not a substrate for EGFR tyrosine kinase activity.
However, we cannot rule out the possibility that Pg may rapidly
exchange out of the Triton-insoluble pool upon phosphorylation, thereby
preventing its detection in the junctional pool.
Tyrosine-phosphorylated
-catenin was restricted to the
Triton-soluble pool of proteins in ras-transformed
cells (8) and was found associated to E-cadherin after growth factor
treatment (13). Consistent with these observations we found that the Pg
contained in the Triton-soluble pool is associated with Dsg2 after EGF
treatment (Fig. 4A). Moreover, tyrosine-phosphorylated Pg
remains associated with the desmosomal cadherin Dsg2 (Fig. 6). During
this study we also detected tyrosine-phosphorylated desmoglein 2 in
response to EGFR activation, and this may represent another regulatory
step in the EGF-dependent regulation of desmosomes.
N was observed after
short term EGF treatment (Fig. 3F). Our ability to detect a
decrease in the level of Pg
N, but not full-length Pg or Pg
C, in
the Triton-insoluble pool may be due to the fact that levels of Pg
N
found in this pool are already relatively low. Thus, it seems possible
that the higher levels of Pg found in the Triton-insoluble pool could
mask subtle changes in the distribution of a relatively minor
population of tyrosine-phosphorylated full-length Pg in control cells
after short term growth factor treatment. The rapid changes in the
solubility observed for Pg
N in response to EGF may represent early
events that normally occur during remodeling of intercellular junctions
and acquisition of a motile phenotype. Taken together, the rapid
phosphorylation of Pg and the reproducible detection of changes in its
subcellular distribution after long term EGF treatment may indicate
that the regulatory effects of EGFR kinase on full-length Pg are a
multistep process, which evolves from the initial phosphorylation of Pg
to eventually promote its subcellular redistribution, hence remodeling
of desmosomes. An increase in the cytosolic pool of Pg
C was also
observed, even though this truncated Pg molecule does not become
tyrosine-phosphorylated. This raises the possibility that interactions
with other partners that normally stabilize Pg
C in the
membrane-associated pool may be compromised in an
EGF-dependent manner (e.g. because they are EGFR
substrates) by deletion of the Pg C terminus.
N and A431.Pg
C cells, endogenous
tyrosine-phosphorylated Pg was not only detected in the membrane-bound
Triton-soluble pool but also somewhat unexpectedly in the cytosolic
pool (Fig. 3E). Furthermore, the majority of the
phosphorylated deletion mutant Pg
N was found in the cytosolic pool
(Fig. 3E). Possibly due to its increased metabolic stability
through the deletion of its GSK3
phosphorylation site, which targets
full length Pg for degradation (38, 39), Pg
N accumulates in the
cytosolic pool (22). Therefore, since catenins are thought to exchange between non-cadherin-associated and cadherin-associated pools (40), it
is possible that tyrosine-phosphorylated Pg
N exchanges from the
membrane-bound, Triton-soluble pool into the cytosolic pool where it is
easily detected upon accumulation. This exchange might also be enhanced
due to changes in stability in the membrane pool, as described above
for Pg
C. The presence of tyrosine-phosphorylated full-length Pg in
the cytosolic pool may be due to its increased ability to associate
with Pg
N and Pg
C (as reported previously (22)).
-catenin (13) as well as
E-cadherin (30). In our experimental conditions, we did not detect a
stable interaction between EGFR and components of either the classic or
desmosomal cadherin-catenin complex (Fig. 4). Although the reasons
underlying the apparent discrepancy between the current results and
those reported previously are unknown, it has been shown that the
interaction between E-cadherin and EGFR is regulated by
calcium-mediated cell contact and other factors that influence the
presence of the receptor in the cadherin-catenin complex (30). Along
with potential cell type-specific differences in the formation of these
signaling complexes, differences in culture conditions and cell density
could account for the variation in reported findings. The fact that
plakoglobin is nevertheless robustly phosphorylated in an
EGF-dependent manner in the present study is consistent
with the possibilities that (a) labile or transient
interactions between activated EGFR and the membrane-associated desmoglein-plakoglobin complex are sufficient to drive the observed tyrosine phosphorylation of these desmosomal proteins and/or
(b) that a downstream non-receptor tyrosine kinase is
responsible for the observed tyrosine phosphorylation.
-catenin may be involved in the modulation of classical
cadherin-based adhesion (44, 45). As Pg preferentially incorporates
into desmosomes in cells that assemble both desmosomes and adherens
junctions (17, 46, 47), Pg may play a particularly important role in
cytoskeletal attachment and adhesive function in these junctions.
-catenin, Pg, and p120
does not affect their interaction with E-cadherin (10), the
phosphorylation of Pg does not affect its interaction with the
desmosomal cadherin Dsg2. Moreover, phosphorylated Pg does not
associate with the desmosomal cytoskeletal linking protein DP. In this
way, tyrosine phosphorylation of Pg could represent an early regulatory
element of the EGF-dependent cascade of events leading to
the shift from an adhesive to a migratory phenotype necessary for
epithelial cell motility.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. P. Bertics for analyzing the plakoglobin sequence for potential EGF-dependent tyrosine phosphorylation sites. We also thank Drs. M. Wheelock, J. Papkoff, and R. Marsh for antibodies provided. We also thank J. K. Park and Dr. L. J. Bannon for helping in the establishment of the A431-inducible cell line. We especially thank Drs. L. J. Bannon, E. Bornslaeger, A. P. Kowalczyk, K. Ishii, and L. M. Godsel for critical reading of the manuscript and insightful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant R01AR42989 (to L. H.) and National Institutes of Health Grants R01AR41836 and P01DE12328 Subproject 4 (to K. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Warner Lambert Consumer Healthcare Research Fellowship from the Dermatology Foundation.
¶ Supported in part by National Institutes of Health Training Grant 5T32CA09560-10. Current address: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724.
** To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. Tel.: 312-503-5300; Fax: 312-503-8240; E-mail: kgreen@nwu.edu.
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M102731200
2 A. C. Huen, J. K. Park, L. J. Bannon, E. A. Bornslaeger, K. J. Green, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Pg, plakoglobin; EGF, epidermal growth factor; EGFR, EGF receptor; DP, desmoplakin; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; Dsg2, desmoglein 2; Ab, antibody.
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REFERENCES |
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