Involvement of cortactin and phosphotyrosine proteins in cell-cell contact formation in cultured bovine corneal endothelial cells

Phosphotyrosine proteins involvement, particularly cortactin, was studied in cell-cell contacts of cultured bovine corneal endothelial (BCE) cells. These proteins, including α-catenin, vinculin and cortactin, are localized at cell-cell contacts separate from the cortical actin ring. Approximately 50% of cortactin isoforms p80 and p85 were associated with the Triton-insoluble fraction while phosphotyrosine proteins were in the soluble fraction. Disruption of cell-cell contacts by EDTA treatment was associated with a decrease in cortactin isoforms p80 (26%) and p85 (57%). Cortactin isoform p85 was phosphorylated at Y⁴⁶⁶, expressed in reattaching cells and associated with the Triton-soluble fraction, whereas cortactin isoform p80 was phosphorylated at Y⁴²¹ and associated with the Triton-insoluble fraction. In sub-confluent cultures, pY⁴²¹-cortactin was localized at the leading edge and pY⁴⁶⁶-cortactin at a perinuclear area. In confluent cultures both pY⁴⁶⁶- and pY⁴²¹-cortactin isoforms were localized at the cell-cell contacts. In conclusion, in BCE cells, the most prominent appearance of cortactin was at the cell-cell contacts separate from the cortical actin ring. Isoform p80 was phosphorylated at Y ⁴²¹ and associated with the Triton-insoluble fraction and isoform p85 was phosphorylated at Y⁴⁶⁶ and associated with the Triton-insoluble fraction.