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Originally published In Press as doi:10.1074/jbc.M506692200 on September 23, 2005

J. Biol. Chem., Vol. 280, Issue 48, 40355-40363, December 2, 2005
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Mechanisms of Plakoglobin-dependent Adhesion

DESMOSOME-SPECIFIC FUNCTIONS IN ASSEMBLY AND REGULATION BY EPIDERMAL GROWTH FACTOR RECEPTOR*

Taofei Yin{ddagger}, Spiro Getsios{ddagger}, Reto Caldelari§, Lisa M. Godsel{ddagger}, Andrew P. Kowalczyk¶, Eliane J. Müller||, and Kathleen J. Green{ddagger}1

From the {ddagger}Departments of Pathology and Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, §CELLnTEC Advanced Cell Systems AG, 122 Länggass-Strasse, Postfach, Bern CH-3001, Switzerland, the Departments of Dermatology and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322, and the ||Institute of Animal Pathology, University of Bern, 122 Länggass-Strasse, Postfach, Bern CH-3001, Switzerland

Received for publication, June 20, 2005 , and in revised form, August 29, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related {beta}-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not {beta}-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adherens junctions and desmosomes are intercellular junctions that link the actin and intermediate filament (IF)2 cytoskeleton to sites of cadherin-based adhesion, respectively. These junctions both utilize members of the Armadillo family of signaling/adhesion molecules to connect the cadherin cytoplasmic domains to cytoskeleton linker proteins, {alpha}-catenin/vinculin in adherens junction and the plakin family member desmoplakin (DP) in desmosomes, as well as to regulate the stability and strength of these junctions (1-4). Two important Armadillo adapters are {beta}-catenin and plakoglobin. {beta}-Catenin normally binds exclusively to classic cadherins and is restricted to adherens junctions. However, plakoglobin (PG) is able to associate with classic and desmosomal cadherins and can be found in both adherens junctions and desmosomes (5). In light of the interdependence of adherens junctions and desmosomes during their formation and maturation (6, 7) and the fact that these junctions contribute synergistically to adhesive strength (8), it is possible that PG strengthens adhesion through its incorporation into both organelles. However, the molecular mechanism of PG-regulated adhesion remains poorly understood.

Intercellular adhesion is also constantly subject to modulation, especially during epithelial-to-mesenchymal transitions that occur during development and wound healing (9). During tumor metastasis, down-regulation of cell adhesion is a critical step, since it has been observed that tumor cells migrate from the tumor mass as single cells (10). Receptor tyrosine kinases, including members of the EGFR family, are critical participants in the process of epithelial-to-mesenchymal transitions (11). In addition to its mitogenic activity, epidermal growth factor (EGF) has been shown to promote motility in epithelial cells including keratinocytes (12-14). Both {beta}-catenin and PG have been identified as targets for EGFR-dependent phosphorylation, and it is thought that modulation of intercellular adhesion contributes to the acquisition of motility during epithelial-to-mesenchymal transitions (15). Consistent with this idea, EGF treatment of HSC-1 squamous carcinoma cells and MDA-MB-468 breast cancer cells resulted in tyrosine (Tyr) phosphorylation of {beta}-catenin and dissociation of adherens junction molecules from the adhesion complex (16, 17). In vitro assays confirmed that EGFR specifically phosphorylates {beta}-catenin at Tyr654, and this phosphorylation leads to significantly decreased binding to E-cadherin (18).

Less attention has been paid to the effects of EGF stimulation on the stability and assembly state of desmosomes. These adhesive organelles are crucially important in maintaining the mechanical integrity of epithelial and other stress-bearing tissues by tethering IF to the membrane through protein adhesion complexes containing DP and PG (19). The important role of PG in tissue integrity and function is demonstrated by skin and heart defects in PG knock-out mice (20, 21) and patients with autosomal mutation of the PG gene (22, 23). Loss of PG leads to decreased number and altered structure of desmosomes in the epidermis of mouse skin (20), and PG null keratinocytes exhibited weakened intercellular adhesion (24, 25). Likewise, the proper recruitment and distribution of the PG-associated protein DP to desmosomal plaques is required for IF attachment as well as strong intercellular adhesion and epithelial integrity in vitro and in vivo (7, 8).

Modulation of these junctions by altering desmosome protein-protein interactions is thus likely to make an important contribution to remodeling of tissues during wound healing and metastasis. This idea is supported by the observation that blocking EGFR signaling promoted desmosome assembly and increased cell-cell adhesion in oral squamous carcinoma cells (26). A variety of Tyr kinases have been found to phosphorylate PG at specific sites and induce distinct effects on its association with adhesion partners (27). EGFR activation results in PG phosphorylation at a three-Tyr cluster in its C-terminal domain (Tyr693, Tyr724, and Tyr729). This phosphorylation leads to the dissociation of PG from the N-terminal domain of DP (18, 28). However, a direct connection between EGFR-dependent phosphorylation of PG and potential alterations in adhesive strength between cells has not been demonstrated.

To determine the underlying mechanism by which PG strengthens adhesion and to examine how PG-dependent adhesion is regulated, we employed keratinocyte cultures established from the skin of PG knockout mice and an EGFR-dependent phosphorylation deficient PG mutant (24, 28). We show that the previously demonstrated decreased adhesive strength in PG-/- cells is accompanied by reduced steady state protein levels and junction incorporation of desmosomal components, whereas adherens junction assembly remains largely unaffected. Long-term EGF treatment decreased junction localization of DP as well as adhesive strength in control PG+/- cells and PG-/- cells expressing ectopic PG. These alterations were both attenuated in PG null cells expressing a PG mutant deficient in EGFR-dependent phosphorylation. Therefore, PG is not only an integral component of the desmosomal adhesive complex but also provides a mechanism for regulating desmosomal adhesion in response to growth factor stimulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Keratinocyte cultures established from the skin of PG+/- or PG-/- mice (24) were cultured in defined keratinocyte SFM (Invitrogen), supplemented with 10 ng/ml EGF and 10-10 M cholera toxin. In experiments where calcium concentration was varied, cells were incubated in calcium-free keratinocyte SFM (Invitrogen), supplemented with 10 ng/ml EGF and 10-10 M cholera toxin, with calcium level achieved by adding CaCl2. For starvation, cells were switched to unsupplemented defined keratinocyte SFM (Invitrogen) with calcium concentration adjusted to the appropriate level using 1 M CaCl2. In experiments examining the effects of EGF stimulation, cells were starved in 0.3 mM calcium (starvation medium) for 8 h and then rinsed and incubated in starvation medium containing Me2SO for 18 h as control or in Me2SO-containing starvation medium for 17 h and then treated with 10 nM EGF for 1 h or in starvation medium plus 10 nM EGF for 18 h. In some experiments, cells were treated with 10 µM PKI166 (Novartis), a small molecule tyrosine kinase inhibitor that blocks EGFR/ErbB2 activation (29). For these experiments, cultures were either incubated in starvation medium for 17 h in PKI prior to 1-h EGF treatment (PKI 18hrs EGF 1h) or in 10 µM PKI166 plus 10 nM of EGF for 18 h (PKI + EGF 18hrs).

Generation of Adenoviruses—The pAdeasy adenovirus-packaging system was kindly provided by Dr. W. G. Tourtellotte (Northwestern University Feinberg School of Medicine). Previously characterized Myc-tagged, full-length PG (PG) and PG mutants with Tyr693, Tyr724, and Tyr729 mutated to Phe (3YF) (28) were used to generate recombinant viruses according to published protocols (30). Infection rates were monitored using the GFP expressed in tandem with the construct of interest, and ~90% infection was achieved for all constructs in all experiments. The recombinant adenoviruses carrying the Myc-tagged human {beta}-catenin were characterized previously (31).

Antibodies—Full-length PG was detected with 1407 (28). For PG immunoprecipitation, a mouse monoclonal antibody {gamma}-catenin was used (BD Biosciences). DP was detected with a rabbit polyclonal antibody NW6 (32). A mouse monoclonal antibody 4B2 cross-reacting with the cytoplasmic domain of desmoglein (Dsg) 1 and Dsg 2 (Dsg1/2) was characterized by Dusek et al.3 The rabbit polyclonal Dsg3 antibody 3067 was a gift from Dr. J. R. Stanley (University of Pennsylvania). The rabbit polyclonal {beta}-catenin antibody c-2206 and rat polyclonal E-cadherin antibody DECMA were purchased from Sigma. The Myc-tagged proteins were detected with a rabbit polyclonal Myc antibody purchased from Bethyl Laboratories (Montgomery, TX). The anti-phosphotyrosine mouse monoclonal antibody clone 4G10 was from Upstate USA, Inc. The mouse monoclonal GFP antibody JL-8 was from BD Biosciences. The following dilutions were used for the above antibodies in Western blots: 1407 (1:5000), NW6 (1:2500), 4B2 (1:100), 3067 (1:5000), c-2206 (1:5000), DECMA (1:500), Myc (1:10,000), 4G10 (1:500), and JL-8 (1:2000). For immunoprecipitation, 3 µl of {gamma}-catenin antibody was used per 500 µl of lysate.

Immunofluorescence, Western Blot, and Triton Solubility Assay—Western blot and immunofluorescence were performed as described previously (8). For analysis of the Triton X-100-insoluble pool, cells were lysed in 0.5% Triton X-100 buffer (0.5% Triton X-100, 145 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 2 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) followed by centrifugation (16,000 x g, 10 min). Triton X-100-insoluble pellets were solubilized in urea sample buffer (33) and then subjected to Western blot analysis. Densitometry analysis was performed using Molecular Analyst software (Bio-Rad).

Mechanical Cell Dissociation Assay—Confluent keratinocyte cultures were incubated in dispase (1 unit/ml; Roche Applied Science) until detached from the culture dishes as previously described (8). Released intact monolayers were carefully washed twice with Dulbecco's phosphate-buffered saline and transferred to 15-ml conical tubes. Enough Dulbecco's phosphate-buffered saline was added for a final volume of 5 ml. Tubes were subjected to 30 inversion cycles. Fragments of the cell monolayers generated by shear stress were counted, and statistical analysis was performed on the average of three separate experiments. An increased number of fragments reflected weakened intercellular adhesion.

Immunoprecipitation—Immunoprecipitations were performed using appropriate antibodies as described previously (28). For immune complex retrieval, 40 µl of Gamma Bind Plus Sepharose beads (Amersham Biosciences) were used per reaction. Immune complexes were released by incubation in Laemmli sample buffer at 95 °C and were subjected to Western blot analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PG-/- Cells Exhibit Decreased Expression and Membrane Incorporation of Desmosomal Proteins—To determine the mechanism by which PG strengthens adhesion, we utilized keratinocyte cultures established from the skin of PG-/- mice, which we previously showed exhibit weakened intercellular adhesion (24). We first examined whether the protein levels of desmosomal adhesion molecules were altered in PG-/- cells cultured at varying calcium concentrations. PG+/- and PG-/- cells were switched to medium containing 0.05, 0.07, 0.2, 0.65, or 1.2 mM calcium for 3 days and then lysed and subjected to Western blot analysis using appropriate antibodies. Consistent with previous observations (24), PG-/- keratinocytes did not express PG. An antibody (4B2) cross-reacting with both Dsg1 and Dsg2 (Dsg1/2) detected a marked decrease in steady state levels in PG-/- cells compared with PG+/- cells at all calcium concentrations tested. Dsgs appeared as doublets that were most pronounced in the PG-/- cells. DP I/II (34) and Dsg3 were also down-regulated at lower calcium concentrations in PG-/- cells. Furthermore, Dsg1/2, and to a lesser extent DP I/II, levels were still somewhat depressed at 1.2 mM calcium in PG-/- cells compared with PG+/- cells (Fig. 1A).



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  		FIGURE 1.
PG-/- cells exhibit decreased protein expression level and membrane incorporation of desmosomal proteins. PG+/- and PG-/- cells were cultured in medium containing the indicated concentration of calcium for 3 days. Whole cell lysates (A) or Triton-soluble and -insoluble pools of cell lysates (B) were collected and subjected to Western blot analysis. The expression levels and Triton-insoluble pools of DP, Dsg1/2, and Dsg3 are decreased in PG-/- cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. Note that Dsgs are detected as doublets that are most pronounced in the PG-/- cells. Similar doublets were reported in another study using mouse keratinocytes (40). These doublets may represent differentially processed proteins or different isoforms of Dsg1 that are known to exist in mice (52). The lower PG band observed in the PG-/- cells expressing vPG is probably due to proteolytic degradation of the ectopic protein. A similar band of lower molecular weight endogenous PG can also be observed occasionally in PG+/- cells. C, PG+/- and PG-/- were cultured in medium containing the indicated concentration of calcium for 24 h and then subjected to immunofluorescence analysis. DP is labeled in red, and Dsg1/2 is labeled in green. Overlays of desmosomal protein DP and Dsg1/2 are shown. Membrane localization of Dsg1/2 in PG-/- cells was only detected at 1.2 mM calcium level.

 
To test whether loss of PG affects the proper junction incorporation of desmosomal proteins, we first examined the Triton solubility of adhesion proteins in these two cell lines. PG+/- and PG-/- cells cultured at different calcium levels were lysed in 0.5% Triton X-100 buffer and divided into Triton-soluble and Triton-insoluble pools, with the Triton-insoluble pool, most likely representing the protein that is associated with the IF network (35, 36). The ratios of Triton-insoluble/Triton-soluble pool of Dsg1/2, Dsg3, and DP were decreased in PG-/- cells compared with PG+/- cells at all calcium concentrations tested (Fig. 1B), with the most striking change exhibited by DP. These data suggest that desmosomal proteins are not as stably associated with the cytoskeleton and/or junctions in PG-/- cells.

We then examined the membrane localization of desmosomal proteins in response to increased calcium concentration in PG+/- and PG-/- cells using immunofluorescence. PG+/- and PG-/- cells were switched to medium containing the indicated concentration of calcium for 24 h prior to fixation and immunofluorescence analysis. At 0.05 mM calcium, neither DP nor Dsg1/2 was localized at cell-cell borders in either cell line. When the calcium level was increased to 0.2 mM, Dsg1/2 and DP readily co-localized at junctions in a dotty desmosomal staining pattern in PG+/- cells. On the other hand, although DP began to appear at PG-/- cell-cell borders in 0.2 mM calcium, Dsg1/2 was not detected at the plasma membrane until the calcium level was raised to 1.2 mM. In addition, DP staining often appeared more continuous in PG-/- cells in contrast with the dotty staining pattern in PG+/- cells (Fig. 1C). Taken together, PG-/- cells exhibited defects in both protein levels and membrane recruitment of desmosomal proteins.

Adherens Junctions Are Not Comparably Affected in PG-/- Cells—Because PG can be incorporated into adherens junctions, we also examined the protein levels and membrane association of E-cadherin and {beta}-catenin. Although some studies suggested that PG may regulate the levels of {beta}-catenin by competing for the binding of degradation machinery (37, 38), we did not observe changes in {beta}-catenin levels in PG-/- cells (Fig. 2A). The detergent solubility of {beta}-catenin was also comparable in both cell lines (Fig. 2B). The steady state level of the classical cadherin, E-cadherin, was moderately decreased in PG-/- cells compared with PG+/- cells (Fig. 2A). Nevertheless, the Triton solubility profiles for E-cadherin were comparable in both cell lines especially at the high calcium concentrations (Fig. 2B).

We then examined the localization of E-cadherin and {beta}-catenin in PG-/- and PG+/- cells at different calcium concentrations. In contrast to DP and Dsg1/2 staining, the localization of E-cadherin and {beta}-catenin as well as their recruitment to the borders in response increased calcium level were comparable in PG+/- and PG-/- to cells (Fig. 2C).

Reintroduction of PG into PG Null Cells Leads to an Accumulation of Junction-associated Desmosomal Proteins—We demonstrated previously that re-expression of PG, but not the closely related {beta}-catenin in PG-/- cells, restored adhesive strength (25). To test whether PG strengthens adhesion through the regulation of protein levels and/or junction incorporation of desmosomal proteins, we infected PG-/- cells with adenoviruses encoding Myc-tagged PG, {beta}-catenin, or GFP control. The cells were incubated in medium containing 0.65 mM calcium for 3 days and then lysed and subjected to immunoblot analysis. Note that infection of PG-/- cells with adenoviruses frequently resulted in a modest decrease in the expression level of DP and Dsg1/2 compared with uninfected PG-/- cells. However, compared with GFP or {beta}-catenin, expression of PG resulted in varying degrees of recovery of DP and Dsg1/2, bringing levels closer to those of the endogenous proteins in the PG+/- cells. Moreover, expression of PG in PG-/- cells altered the banding pattern of Dsg1/2 to that resembling PG+/- cells. The steady state levels of adherens junction proteins E-cadherin and {beta}-catenin were largely unaffected by expressing PG (Fig. 3A).



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  		FIGURE 2.
Adherens junctions are largely unaffected in PG-/- cells compared with PG+/- cells. PG+/- and PG-/- cells were cultured in medium containing the indicated concentration of calcium for 3 days. Whole cell lysates (A) or Triton-soluble and -insoluble pools of cell lysates (B) were collected and subjected to Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase controls are the same as shown in Fig. 1. C, PG+/- and PG-/- were cultured in medium containing the indicated concentration of calcium for 24 h and then subjected to immunofluorescence analysis. E-cadherin is labeled in red, and {beta}-catenin is labeled in green. Overlays of E-cadherin and {beta}-catenin were shown. Although the expression levels of E-cadherin are somewhat depressed, particularly at lower calcium concentrations, the Triton solubility profiles of E-cadherin and {beta}-catenin were comparable in PG+/- and PG-/- cells, as were membrane localization of E-cadherin and {beta}-catenin in response to increased calcium levels.

 
We then examined the effect of PG expression on solubility status of junctional proteins. PG-/- cells infected with adenoviruses encoding PG or {beta}-catenin or GFP were switched to medium containing 0.65 mM calcium for 3 days and then subjected to fractionation into Triton-soluble and -insoluble pools. The protein level of DP and Dsg1/2, but not E-cadherin, was increased in the Triton-insoluble pool of vPG versus vGFP cells (Fig. 3B), whereas the soluble pool was relatively unchanged. {beta}-Catenin expression also led to a somewhat more modest increase in the insoluble pool of DP but not Dsg1/2. Immunofluorescence analysis demonstrated that virally expressed PG efficiently recruited Dsg1/2 to the junctions in PG-/- cells, whereas {beta}-catenin and GFP failed to do so (Fig. 3C). These results demonstrate that PG primarily increases the steady state level and assembly of desmosome components without detectably affecting adherens junction status.

EGF Treatment Results in Tyr Phosphorylation of PG and Decreases Intercellular Adhesion in Keratinocytes—Toward addressing whether EGF treatment regulates desmosomal adhesion in normal keratinocytes, we examined desmosome protein distribution in PG+/- cells after growth factor treatment. We focused primarily on the protein interaction partners DP and PG, based on previous work demonstrating that tyrosine phosphorylation of PG compromised its interaction with DP without altering its association with Dsg (28). Because high calcium promotes the maturation of desmosomes, which may become resistant to destabilizing stimuli (39), we performed experiments at a calcium concentration of 0.3 mM, which allows desmosome formation while enhancing the effects of EGF stimulation. PG+/- cells were starved in 0.3 mM calcium for 8 h and then treated with 10 nM EGF for 1 or 18 h. The cells were then fixed, and the localization of DP and PG was examined by immunofluorescence. DP and PG localized at cell-cell borders in a dotty pattern in starved PG+/- cells. Upon1hofEGF treatment, DP staining was reduced at the borders and increased in the cytoplasm. Whereas intercellular PG staining was retained, the fluorescence pattern was less regular, and the borders appeared ruffled. After 18 h of EGF treatment, DP staining was largely absent from cell-cell borders, whereas PG staining remained (Fig. 4A). To confirm that the changes in DP and PG staining patterns in EGF-treated cells were the result of EGFR activation, we treated cells with 10 µM PKI166, a small molecule tyrosine kinase inhibitor that specifically blocks EGFR/ErbB2 activation (29) as described under "Materials and Methods." PKI166 treatment resulted in a dramatic recruitment of DP and PG to borders, consistent with our previous observations in SCC cells (26), and PG+/- colonies appeared more compact than Me2SO control cells (Fig. 4A).

Consistent with the immunofluorescence observations, detergent solubility analysis demonstrated that 1 h of EGF treatment induced an increase in the ratio of DP in the Triton-soluble/insoluble pool with a small but reproducible increase in the PG ratio, both of which were blocked by PKI166. 18 h of EGF treatment resulted in a further decrease in the DP-insoluble pool, with PKI166 partially preventing the changes induced by EGF treatment at this time point (Fig. 4B).

We then addressed whether these changes correlated with EGFR-dependent Tyr phosphorylation of PG. PG+/- cells treated as described above were subjected to immunoprecipitation with a monoclonal PG antibody ({gamma}-catenin), and the phosphorylation status of PG was examined using an antibody against phosphotyrosine.1hof treatment with 10 nM EGF induced an increase in tyrosine phosphorylation of PG. Somewhat surprisingly, based on the sustained effects of EGF treatment on desmosome assembly status, PG phosphorylation was attenuated after 18 h of EGF treatment. As expected, pretreatment with PKI166 blocked the phosphorylation of PG by1hofEGF stimulation (Fig. 4C). To test whether EGF treatment altered intercellular adhesive strength, we performed a mechanical dissociation assay using PG+/- cells treated as described above, as described under "Materials and Methods." Although 1 h of EGF treatment was sufficient to induce tyrosine phosphorylation of PG and a partial decrease in junctional DP and PG, it did not weaken the adhesive strength. In contrast, 18 h of treatment resulted in a 3-fold increase in the number of cell monolayer fragments compared with Me2SO-treated cells. Treating PG+/- cells with 10 nM EGF together with 10 µM PKI166 for 18 h completely blocked the effects of long term treatment of EGF on adhesive strength, confirming that the weakened adhesion was a result of EGFR activation (Fig. 4D).



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  		FIGURE 3.
Reintroduction of PG into PG null cells leads to an accumulation of desmosomal proteins, distributed to the junction-associated pool. PG+/-, PG-/-, and PG-/- cells infected with adenovirus encoding GFP, Myc-tagged {beta}-catenin, or Myc-tagged PG were cultured in medium containing 0.65 mM calcium for 3 days (A and B) or 24 h (C). At this calcium concentration, junctions form, but cells remain more flattened compared with higher calcium concentrations, thus facilitating microscopic evaluation. A, whole cell lysates were subjected to Western blot analysis. Densitometry determinations demonstrated ~3-fold increases in DPI/II and ~2-fold for Dsg1/2 in vPG versus vGFP control cells. B, Triton-soluble and -insoluble pools were collected and subjected to Western blot analysis. Re-expression of PG increased the protein expression level in the Triton-insoluble pool of desmosomal but not adherens junction proteins. C, cellular localization of Dsg1/2 (shown in green) and PG or Myc (shown in red) were examined by immunofluorescence and overlaid. Dsg1/2 accumulated at junctions in PG-/- cells infected with vPG but not v{beta}-catenin or GFP control viruses. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
3YF PG Is Not Phosphorylated by EGF Treatment—The difference in the temporal relationship between PG phosphorylation and changes in adhesive strength after EGF treatment raised the possibilities that 1) PG phosphorylation is not directly linked to the long term effects of EGF or that 2) PG phosphorylation is required for initiating a cascade of signaling or adhesion events that do not require its sustained phosphorylation. To distinguish between these possibilities, we generated a recombinant adenovirus using a previously characterized PG mutant in which Tyr693, Tyr724, and Tyr729 were altered to Phe (28) (3YF; Fig. 5A). When expressed in PG-/- cells under normal culture conditions (Fig. 5B), the 3YF mutant was capable of recruiting Dsg1/2 to the borders in response to increased calcium similar to the wild-type counterpart (Fig. 5D). Consistent with this observation, the protein level of Dsg1/2 was increased in both PG-/- cells expressing wild-type PG and 3YF PG, which mainly distributed to the Triton-insoluble pool. Likewise, the protein level of DP in the Triton-insoluble pool was also increased by the expression of PG and 3YF PG (Fig. 5C).

To confirm that the 3YF cannot be phosphorylated by EGF treatment in murine keratinocytes, we infected PG-/- cells with adenoviruses carrying either wild-type (WT) or 3YF PG and then treated the cells as described for PG+/- cells. Exogenous PG was then immunoprecipitated with an antibody against PG (11E4), and the phosphorylation state was examined using 4G10. As predicted,1hofEGF treatment induced tyrosine phosphorylation of wild-type PG but not the 3YF PG (Fig. 5E).

Expression of 3YF PG Prevents Loss of DP from Junctions Induced by Long Term EGF Treatment—Phosphorylation of PG at the tyrosine cluster Tyr693, Tyr724, and Tyr729 leads to its dissociation from the N-terminal domain of DP (18, 28). To test whether PG Tyr phosphorylation contributes to the loss of DP from the IF-associated pool in keratinocytes treated with EGF, we compared the detergent solubility of 3YF PG and WT PG upon EGF treatment. PG-/- cells expressing WT or 3YF PG were starved in 0.3 mM calcium for 8 h and then treated with EGF or with EGF and PKI166, as described above. Similar to PG+/- cells, in PG-/- cells expressing WT PG, 18 h of EGF treatment resulted in a significant increase in the ratio of Triton-soluble/insoluble DP and a slight increase for PG, where as 1 h of EGF treatment induced only a slight shift for both PG and DP. Loss of DP from the detergent-insoluble pool at 18 h was greatly attenuated in PG-/- cells expressing 3YF PG (Fig. 6A).

Consistent with the detergent solubility data, 18 h of EGF treatment resulted in a loss of DP border staining in PG-/- cells expressing WT PG but not 3YF PG (Fig. 6B). To quantify these changes, we measured the total fluorescence intensity along the border for PG and DP. The resulting average fluorescence intensities per unit length for DP and PG in the cells were plotted in Fig. 6C. The percentage change by 18 h of EGF treatment compared with control level was also calculated and plotted (Fig. 6D). 18 h of EGF treatment caused a similar modest decrease in PG fluorescence along the borders in both PG-/- cells expressing wild type and 3YF PG. On the other hand, border staining of DP decreased ~70% in EGF-treated PG-/- cells expressing WT PG but remained unaffected in PG-/- cells expressing 3YF PG. These results suggest that the initial phosphorylation of PG at Tyr693, Tyr724, and Tyr729 is important for long term EGF treatment-induced loss of DP from junctions.



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  		FIGURE 4.
EGF treatment results in Tyr phosphorylation of PG and decreased adhesive strength in keratinocytes. PG+/- cells were starved at 0.3 mM calcium level for 8 h and then treated with Me2SO (DMSO) as control or 10 nM EGF for 1 or 18 h or 10 µM PKI166 for 18 h prior to 1 h of incubation with 10 nM EGF or 10 µM PKI166 together with 10 nM EGF for 18 h, as described under "Materials and Methods." A, cellular localization of PG and DP were examined with immunofluorescence. B, Triton solubility of DP and PG were examined. Representative blots from three separate experiments are shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Densitometry analysis of the soluble pool and the insoluble pool was performed, and the ratio of soluble versus insoluble pool (S/I) was determined. EGF treatment results in decreased membrane incorporation of PG and DP, which is blocked by PKI166. C, PG was immunoprecipitated with an anti-{gamma}-catenin antibody and subjected to immunoblot using an antibody against phosphorylated Tyr (4G10). The same membrane was then stripped and reblotted with an antibody against PG. PG is Tyr-phosphorylated after 1 h of EGF treatment, which is blocked by PKI166. D, adhesive strength was examined using the dispase-based dissociation assay. 18 h of EGF treatment reduced adhesive strength in PG+/- cells.

 
Expression of 3YF PG Attenuates the Effects of Long Term EGF Treatment on Adhesive Strength—To test whether the initial phosphorylation of PG is essential for long term EGF treatment-induced weakening in adhesion, we expressed wild-type or 3YF PG in PG-/- cells and then examined adhesive strength using the mechanical dissociation assay. Compared with starved cells, long term EGF treatment induced an ~6.5-fold increase in fragment numbers generated in PG-/- cells expressing wild-type PG. On the other hand, the fragment number was only increased by 3-fold in PG-/- cells expressing 3YF PG (Fig. 7). Therefore, initial tyrosine phosphorylation of the three-Tyr cluster at the C terminus of PG by EGFR activation is important for long term EGF treatment-induced weakening in adhesion, even though PG was no longer phosphorylated at the end of this process. The fact that 3YF did not completely block the effect of EGF on adhesion suggests that PG may not be the sole target in long term EGF treatment, consistent with the observed increase in fragment numbers in PG-/- cells after long term EGF treatment (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PG is among a select subset of Armadillo proteins whose normal distribution is not restricted to a single junction type. The ability of PG to incorporate into both adherens junctions and desmosomes led us to ask whether its role in keratinocyte adhesion is manifested through one or both junctions. Here, we show that loss of PG largely affected desmosomes in PG-/- keratinocytes by reducing the steady state protein levels and recruitment of desmosome components to the plasma membrane. Whereas re-expression of PG substantially reversed these defects and restored intercellular adhesive strength, it did not detectably affect adherens junction assembly. {beta}-Catenin was unable to compensate for PG in restoring membrane localization of desmosomal molecules or to strengthen intercellular adhesion in PG null cells. Interestingly, previous studies showed that loss of {beta}-catenin did not impair adhesive strength in {beta}-catenin null keratinocytes, suggesting that PG can compensate for the loss of {beta}-catenin in these cells (40). Collectively, these results are consistent with the idea that PG plays an essential role in keratinocyte adhesion that cannot be replaced by {beta}-catenin and that it accomplishes this role primarily through regulation of desmosome assembly and function.

It is not known whether the observed decrease in protein expression, which was particularly evident in the case of Dsg1/2, occurs at a transcriptional or post-translational level, although it has been shown that Armadillo proteins are important for both the membrane stability and trafficking of classic and desmosomal cadherins to junctions (3, 41, 42). In this regard, it is tempting to speculate that the partial reversion of the Dsg1/2 banding pattern in vPG cells may reflect an effect on post-translational modification that could occur during Dsg trafficking to the plasma membrane. In any case, it seems likely that together the decrease of desmosomal cadherins and absence of PG results in a loss of membrane anchors available to recruit DP to the membrane, and thus both contribute to weakening of intercellular adhesion.



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  		FIGURE 5.
Three-tyrosine mutant (3YF) PG incorporates into junctions similarly to wild-type PG and is not phosphorylated by EGF treatment. A, a schematic diagram of selected Tyr residues in PG. PG+/-, PG-/-, and PG-/- cells infected with adenoviruses encoding GFP, {beta}-catenin, PG, or 3YF PG were incubated in medium containing 0.65 mM calcium for 3 days (B and C) or 24 h (D). B, whole cell lysates were subjected to Western blot analysis. C, solubility of DP, Dsg1/2, and PG were examined by Western blot. D, cellular localization of Dsg1/2 (shown in green) and Myc-tagged PG or 3YF PG (shown in red) were examined by immunofluorescence and overlaid. 3YF PG is able to increase the expression level, Triton-insoluble pool, and membrane localization of desmosomal proteins as well as wild-type PG. E, PG-/- cells infected with adenoviruses encoding PG or 3YF PG were starved at 0.3 mM calcium level for 8 h and then treated with Me2SO (DMSO) as control or 10 nM EGF for 1 h. PG was immunoprecipitated with an antibody against PG (11E4), and phosphorylation level was determined by blotting with an antibody against phosphotyrosine (4G10). The membrane was then stripped and blotted with an antibody against PG. 3YF PG is not Tyr-phosphorylated after 1 h of EGF stimulation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
It has been proposed that PG is required for the initiation of desmosome assembly, because in a squamous epithelial cell line that had lost classic cadherin expression, desmosomes did not assemble until both E-cadherin and PG or an E-cadherin/PG chimera was introduced (6). However, it has been reported that desmosome-like structures form in vivo in PG knock-out mice (20) and in vitro in PG null keratinocytes, albeit with altered structure and dimension of the cytoplasmic plaque (43). Furthermore, although its ability to do so is impaired, DP can still incorporate into the Triton-insoluble pool and be recruited to cell-cell borders in PG-/- keratinocytes, suggesting that other factors can compensate to promote desmosome assembly in keratinocytes. In PG-/- skin, {beta}-catenin localized to desmosomes and associated with Dsg1 (44), prompting the authors to propose that this Armadillo protein can compensate for loss of PG in vivo. This observation is consistent with our finding that overexpressing {beta}-catenin modestly increased the Triton-insoluble pool of DP. However, {beta}-catenin overexpression did not increase the membrane incorporation of Dsgs; nor did it restore adhesive strength (25), suggesting that {beta}-catenin cannot compensate for the adhesive function of PG.

It is also possible that recruitment of DP to junctions in the absence of PG is mediated through association with plakophilins, which have been shown to interact with both desmosomal cadherins and DP (45-47). However, this method of recruiting DP into borders may not be optimal, as evidenced by the continuous staining pattern of DP at the cell membrane, decreased pool of DP that is associated with cytoskeleton, and decreased adhesive strength in the PG-/- cells. Consistent with a role for plakophilins in this process, the linear staining pattern of DP in PG-/- cells resembles that observed in HT1080 cells expressing Dsg2, a tail-less DP, and plakophilin 2, but no PG (48). In this system, both plakophilin 2 and PG were required for segregating components into adherens junctions and desmosomes. Furthermore, the observation that plakophilins and PG were both necessary for forming structurally optimal desmosome plaques (47) is consistent with the idea that desmosomes assembled without PG are not efficient in maintaining adhesion.

Our results not only identify PG as a key factor in maintaining keratinocyte adhesive strength but also show that this adapter molecule can modulate the adhesive function of the complex in response to external cues such as the growth factor EGF. Previous studies demonstrated that the closest relative of PG, {beta}-catenin, is a target for phosphorylation by EGFR and its homologue ErbB2 (18, 49, 50) and that its phosphorylation correlated with EGF-induced dissociation of adherens junctions in cancer cell lines (16, 17). However, the physiological significance of these alterations in adherens junctions and whether they translated to changes in adhesive strength was not examined. Although less attention has been paid to desmosomes as targets for growth factor regulation, our previous findings showed that inhibition of EGFR signaling preferentially promoted desmosome assembly and enhanced adhesive strength in oral squamous cancer cells (26).

Here, we demonstrate that long term EGF treatment caused dissociation of DP from keratinocyte intercellular junctions and decreased intercellular adhesive strength as examined by a mechanical dissociation assay. The PG null keratinocytes and 3YF mutant deficient for EGFR-dependent phosphorylation allowed us to directly address the role of PG phosphorylation in these EGF-dependent processes. Our results are consistent with a model whereby the initial EGF-dependent phosphorylation of PG results in long term potentiation of DP uncoupling from junctions, correlated with weakening of intercellular adhesion that can be attenuated by expressing the 3YF PG mutant. Although the mechanism by which initial phosphorylation of PG by EGFR mediates these long term effects will require further investigation, several possibilities exist. Aside from EGFR, several other tyrosine kinases also target PG, and each has its own targeting sequence and unique effects (18). For example, phosphorylation at Tyr643 of PG by Src or phosphorylation of Tyr133, possibly by Fyn, resulted in decreased binding to {alpha}-catenin but increased binding to DP, whereas phosphorylation at Tyr549 by Fyn or Fer induced the opposite effects (18). In addition, recent studies demonstrated that the C terminus of PG plays an important role in regulating protein-protein interaction with the Armadillo domain and indicated that the C terminus may itself bind to the Armadillo domain (27, 51). It is possible that the initial phosphorylation of PG by EGFR at the C terminus alters its conformation, exposing or shielding other Tyr phosphorylation sites that may be phosphorylated by kinases activated downstream of EGFR signaling and subsequently contributing to the decreased adhesion in long term EGF-treated keratinocytes.



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  		FIGURE 6.
Expression of the 3YF PG decreases loss of DP from junctions induced by long term EGF treatment. A, PG-/- cells infected with adenoviruses encoding PG or 3YF PG were starved at 0.3 mM calcium level for 8 h and then treated with Me2SO (DMSO) or 10 nM EGF for 1 or 18 h or 10 µM PKI166 for 18 h prior to a 1-h incubation with 10 nM EGF as described under "Materials and Methods." Triton solubility of DP and PG were examined. Representative blots from three separate experiments are shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Densitometry analysis of the soluble pool and the insoluble pool was performed, and the ratio of soluble versus insoluble pool (S/I) was determined. Loss of DP from the Triton-insoluble pool was reduced at 18 h of EGF treatment in PG-/- cells expressing 3YF PG. B, PG-/- cells infected with adenoviruses encoding PG or 3YF PG were starved in 0.3 mM calcium for 8 h and then treated with 10 nM EGF for 18 h or no EGF as control. Cellular localization of PG and DP was examined by immunofluorescence. DP remains at borders after 18 h of EGF stimulation in PG-/- cells expressing 3YF PG. C, the fluorescence staining of PG or DP in B at each border was traced and measured using the Metamorph software. The total fluorescence intensity was then divided by the distance of the border. The y axis represents the average fluorescence per unit of distance. The average fluorescence per unit of distance after 18 h of EGF treatment is compared with that at control condition using Student's t test. The asterisk represents statistically significant difference (t < 0.001). D, change of fluorescence intensity per unit of PG and DP by 18 h of EGF incubation in B was determined by dividing each value of fluorescence intensity per unit by the value of its corresponding control. The y axis represents percentage change.

 



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  		FIGURE 7.
Expression of the 3YF PG attenuates the effects of long term EGF treatment on adhesive strength. PG-/- cells infected with adenoviruses encoding PG or 3YF PG were starved in 0.3 mM calcium for 8 h and then treated with or without 10 nM EGF for 18 h. Adhesive strength was examined using the dispase-based mechanical dissociation assay. 18-h EGF treatment induces less weakening of adhesion in PG-/- cells expressing 3YF PG compared with that expressing wild-type PG.

 
These data provide new insight into the multiple functions of the Armadillo protein PG in the assembly and adhesive function of desmosomes. In addition, our findings support the idea that PG acts as a rheostat that fine tunes the strength of connections between epidermal keratinocytes in response to environmental cues such as growth factors and thus may be an important regulator of wound healing and other epithelial remodeling processes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01AR41836, R01AR43380, and Project 4 of P01 DE12328 and the Joseph L. Mayberry endowment (to K. J. G.), National Institutes of Health Grant R01AR048266 (to A. P. K.), and Swiss National Science Foundation Grant 31-59456.99 (to E. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Feinberg School of Medicine, 303 E Chicago Ave., Chicago, IL 60611. Tel.: 312-503-5300; Fax: 312-503-8249; E-mail: kgreen{at}northwestern.edu.

2 The abbreviations used are: IF, intermediate filament; PG, plakoglobin; DP, desmoplakin; Dsg, desmoglein; EGF, epidermal growth factor; WT, wild type; EGFR, EGF receptor; PKI, protein kinase inhibitor; GFP, green fluorescent protein. Back

3 R. L. Dusek, S. Getsios, F. Chen, J. K. Park, E. V. Amargo, V. L. Cryns, and K. J. Green, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Dr. W. G. Tourtellotte (Northwestern University Feinberg School of Medicine, Chicago, IL) for providing the reagents and technical assistance in the adenoviral work and Dr. J. R. Stanley (University of Pennsylvania) for antibody reagents.



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