Abstract
Cell-cell adhesion is a dynamic process that can activate multiple signaling pathways. These signaling pathways can be regulated through reversible tyrosine phosphorylation events. The level of tyrosine phosphorylation of junctional proteins reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. The receptor-tyrosine phosphatase DEP-1 (CD148/PTP-eta) has been implicated in cell growth and differentiation as well as in regulating phosphorylation of junctional proteins. However, the role of DEP-1 in regulating tight junction phosphorylation and the integrity of cell-cell junctions is still under investigation. In this study, we used a catalytically dead substrate-trapping mutant of DEP-1 to identify potential substrates at cell-cell junctions. We have shown that in epithelial cells the trapping mutant of DEP-1 interacts with the tight junction proteins occludin and ZO-1 in a tyrosine phosphorylation-dependent manner. In contrast, PTP-PEST, Shp2, and PTPmu did not interact with these proteins, suggesting that the interaction of DEP-1 with occludin and ZO-1 is specific. In addition, occludin and ZO-1 were dephosphorylated by DEP-1 but not these other phosphatases in vitro. Overexpression of DEP-1 increased barrier function as measured by transepithelial electrical resistance and also reduced paracellular flux of fluorescein isothiocyanate-dextran following a calcium switch. Reduced DEP-1 expression by small interfering RNA had a small but significant increase in junction permeability. These data suggest that DEP-1 can modify the phosphorylation state of tight junction proteins and play a role in regulating permeability.
Highlights
Din, and JAMs), which interact with cytoplasmic proteins (AF-6 and ZO-1, -2, -3), regulating assembly and maintenance of tight junctions
These studies suggest that maintenance of junctional integrity for both the TJ and adherens junction (AJ) is regulated in part by reversible tyrosine phosphorylation that results from a competing balance of protein-tyrosine kinase and protein-tyrosine
Together these results indicate that ZO-1 and occludin are substrates of DEP-1 and imply a role for DEP-1 in influencing the phosphorylation state of tight junction proteins and junction permeability
Summary
MCF10A cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 medium supplemented with 5% horse serum, 20 ng/ml epidermal growth factor (EGF), 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, 100 ng/ml cholera toxin and antibiotics. MDCK II, A431, and HEK 293 cells were cultured in Dulbecco’s modified Eagle’s medium-high glucose enriched with 10% fetal bovine serum and antibiotics. Cells were transfected with Lipofectamine 2000 (Invitrogen) or FuGENE 6
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