Tyrosine phosphorylation of VE‐Cadherin following activation of Src‐family kinases is not sufficient to decrease endothelial barrier function.

Abstract

Src has been implicated as a major mediator leading to the loss of endothelial cell (EC) barrier function following exposure to inflammatory mediators and growth factors. One mechanism proposed for Src induced loss of endothelial barrier function is via phosphorylation of adherens junction proteins including VE‐cadherin. To assess the role of Src in EC barrier function independent of other signaling pathways, we activated Src directly either by expressing a constitutive active Src (caSrc) or by expressing dominant negative c‐Src kinase (CSK‐). Both strategies resulted in similar levels of Src activation as assessed by an increase in Src auto‐phosphorylation at Y419 and by phosphorylation of known Src substrates. Surprisingly, caSrc, but not CSK‐ expression, resulted in a loss of EC barrier function. Both caSrc and CSK‐ expression resulted in the phosphorylation of VE‐cadherin at Y658 and Y731. However, only CSK‐ induced the phosphorylation of Y685. Co‐IP and IF studies demonstrated that phosphorylation of VE‐cadherin did not impair VE‐Cadherin membrane localization nor its ability to bind p120 and β‐Catenin. CaSrc, but not of CSK‐, induced drastic changes in the cellular morphology, the actin cytoskeleton, focal adhesions and an increased MLC phosphorylation. Our data suggest that Src‐dependent phosphorylation of VE‐cadherin is not sufficient to decrease endothelial barrier function. Support R01HL77870