Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site.
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.
Wallez
Y
Y
Laboratoire Développement et Vieillissement de l'Endothélium, Département Recherche et Dynamique Cellulaires, Université Joseph Fourier, Grenoble, Inserm, Grenoble, France.
Cand
F
F
Cruzalegui
F
F
Wernstedt
C
C
Souchelnytskyi
S
S
Vilgrain
I
I
Huber
P
P
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*; D019061:src-Family Kinases / Q000502:physiology*; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D014443:Tyrosine; D042461:Vascular Endothelial Growth Factor A; D019061:src-Family Kinases; D000099152:Cadherin 5; C467484:VEGFA protein, human