Distinct VE-cadherin serine and tyrosine phosphorylation sites and their role for inflammation-induced vascular permeability in vivo.

VE-cadherin is a major component of endothelial adherens junctions and pivotal to the regulation of vascular barrier integrity. Whereas two phosphorylation sites of VE-cadherin (Y685 and Y731) are known to be relevant for the regulation of endothelial junctions in vivo, several others were suggested to be relevant based on in vitro studies. Here, we analyze for two of these, serine 665 (S665) and tyrosine 658 (Y658), whether they are relevant for the induction of vascular permeability in vivo. To this end, we generated and characterized two point-mutated VE-cadherin knock-in mouse lines where either S665 was replaced by valine (S665V) or Y658 by phenylalanine (Y658F). We found that the induction of vascular permeability by histamine or VEGF in the skin was clearly reduced in S665V mice, whereas Y658F mice showed a normal increase of permeability. In line with this, we found that histamine-induced endocytosis was impaired for the VE-cadherin-S665V mutant, but not for the Y658F mutant. Comparing the regulation of VE-cadherin phosphorylation at S665, Y658 and Y685, we found that only phosphorylation of S665 and Y685 were strongly induced by inflammatory mediators, while phosphorylation of Y658 increased weakly. Interestingly, phosphorylation of S665 and Y685 occurred with different kinetics, but independent of each other. Collectively, our results demonstrate that Y658 is irrelevant for vascular leak formation in the context of several tested inflammatory mediators and establish S665 of VE-cadherin as an important phosphorylation site regulating the induction of endothelial permeability in vivo.