1. ¹Laboratoire Développement et Vieillissement de l'Endothélium, Département Recherche et Dynamique Cellulaires, Université Joseph Fourier, Grenoble, Inserm, Grenoble, France
2. ²Institut de Recherches SERVIER, Centre de Croissy, Chemin de Ronde, Croissy sur Seine, France
3. ³Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden

Correspondence: Dr I Vilgrain, Inserm EMI 0219, Laboratoire de Développement et Vieillissement de l'Endothélium, Département Recherche et Dynamique Cellulaires, Université Joseph Fourier, CEA Grenoble, 17, rue des Martyrs, ISERE, 38054 Grenoble Cedex 9, France. E-mail: ivilgrain @cea.fr

Received 3 January 2006; Revised 15 May 2006; Accepted 23 June 2006; Published online 14 August 2006.

Abstract

Src-family tyrosine kinases are regulatory proteins that play a pivotal role in the disorganization of cadherin-dependent cell–cell contacts. We previously showed that Src was associated with vascular endothelial (VE)-cadherin and that tyrosine phosphorylation level of VE-cadherin was dramatically increased in angiogenic tissues as compared to quiescent tissues. Here, we examined whether VE-cadherin was a direct substrate for Src in vascular endothelial growth factor (VEGF)-induced VE-cadherin phosphorylation, and we identified the target tyrosine sites. Co-transfections of Chinese hamster ovary cells (CHO) cells with VE-cadherin and constitutively active Src (Y530F) resulted in a robust tyrosine phosphorylation of VE-cadherin that was not detected with kinase-dead Src (K298M). In an in vitro Src assay, the VE-cadherin cytoplasmic domain is directly phosphorylated by purified Src as well as the tyrosine residue 685 (Tyr)685-containing peptide RPSLY⁶⁸⁵AQVQ. VE-cadherin peptide mapping from human umbilical vein endothelial cells stimulated by VEGF and VE-cadherin-CHO cells transfected with active Src revealed that Y685 was the unique phosphorylated site. The presence of PhosphoY685 was confirmed by its ability to bind to C-terminal Src kinase-SH2 domain in a pull-down assay. Finally, we found that in a VEGF-induced wound-healing assay, cadherin adhesive activity was impaired by Src kinase inhibitors. These data identify that VEGF-induced-VE-cadherin tyrosine phosphorylation is mediated by Src on Y685, a process that appears to be critical for VEGF-induced endothelial cell migration.