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

Oncogene. 2007 Feb 15;26(7):1067-77. doi: 10.1038/sj.onc.1209855. Epub 2006 Aug 14.

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 RPSLY(685)AQVQ. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, CD / genetics
  • Antigens, CD / metabolism*
  • CHO Cells
  • Cadherins / genetics
  • Cadherins / metabolism*
  • Cell Movement / physiology*
  • Cells, Cultured
  • Cricetinae
  • Cricetulus
  • Endothelium, Vascular / cytology*
  • Endothelium, Vascular / enzymology*
  • Endothelium, Vascular / metabolism
  • Humans
  • Phosphorylation
  • Tyrosine / genetics
  • Tyrosine / metabolism*
  • Vascular Endothelial Growth Factor A / physiology*
  • src-Family Kinases / physiology*

Substances

  • Antigens, CD
  • Cadherins
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • cadherin 5
  • Tyrosine
  • src-Family Kinases