Activation of Src by inhibition of Csk does not decrease endothelial barrier function

Endothelial barrier function in the vasculature is known to be tightly regulated by the adherens junction (AJ). Recent studies have suggested that Src phosphorylation of AJ proteins contributes to a decrease in endothelial barrier function following cardiac ischemia and stroke. We sought to develop an in vitro model of Src activation in order to determine the mechanism of AJ phosphorylation, particularly p120, in Src‐induced loss of barrier function. In these studies we activated endogenous Src by expressing a dominant negative form of c‐terminal‐src kinase (DN‐Csk), a kinase known to inactivate Src by phosphorylation of tyrosine 527. Indeed, expression of DN‐Csk in confluent human lung or human dermal microvascular cells activated endogenous Src as shown by increases in the dephosphorylation of tyrosine 527 and phosphorylation of tyrosine 416. In addition expression of DN‐Csk resulted in a time dependent increase in global tyrosine phosphorylation including phosphorylation of paxillin, a protein found in cell‐matrix AJs. Surprisingly, we found that increased Src activation following expression of DN‐Csk is not sufficient to cause a loss of barrier function in endothelial cells. These results suggest that the context of Src activation is critical to modulating endothelial barrier function and suggests that Csk may play a greater role in regulation of cell‐matrix junctions, as shown by paxillin phosphorylation, than at cell‐cell junctions. K02‐HL‐04332; R01‐HL‐68079.