Abstract
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 beta-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.
Highlights
¶ Supported in part by National Institutes of Health Training Grant 5T32CA09560-10
In order to determine the subcellular distribution of the adherens junction and desmosome component Pg after prolonged epidermal growth factor (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, some staining at cell-cell borders could still be observed (Fig. 1A, d)
Summary
¶ 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. 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] 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, contributing to the remodeling of epithelia in response to growth factors during wound healing and invasion
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