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Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site

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.

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Abbreviations

CHO:

Chinese hamster ovary cells

HUVECs:

humanumbilical vein endothelial cells

VEGF:

vascular endothelial growth factor

VEGFR-2:

vascular endothelial growth factor receptor 2

VE-cadherin:

vascular endothelial-cadherin

SDS:

sodium dodecyl sulfate

SDS–PAGE:

SDS–polyacrylamide gel electrophoresis

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Acknowledgements

This work was possible thanks to INSERM, the Commissariat à l’Energie Atomique, the Association pour la Recherche sur le Cancer (ARC no. 5588), the Fédération Nationale des Centres de Lutte contre le Cancer, the Fondation pour la Recherche Médicale and the Ligue Nationale contre le Cancer. We thank Michael Burbridge and Nicolas Cauquil to have provided the constructions for Src mutants. We are indebted to Daniel Dumont and Paul Van Slyke for scientific discussions.

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Correspondence to I Vilgrain.

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Wallez, Y., Cand, F., Cruzalegui, F. et al. Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site. Oncogene 26, 1067–1077 (2007). https://doi.org/10.1038/sj.onc.1209855

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