Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells.
Vascular endothelial growth factor (VEGF) stimulates the tyrosine phosphorylation of focal adhesion kinase (FAK), increases focal adhesion formation and is chemotactic for human umbilical-vein endothelial cells (HUVECs). In the present study we identified the major sites of VEGF-induced FAK tyrosine phosphorylation and investigated the mechanism mediating this pathway in the action of VEGF. VEGF increased the focal adhesion localization of FAK phosphorylated at Tyr-397 (Y397) and Y861 but stimulated a marked increase in phosphorylation at Y861 without significantly affecting the total level of phospho-Y397 FAK. Inhibition of Src with the specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) completely blocked VEGF-induced Y861 phosphorylation without decreasing the level of phospho-Y397 FAK. We also examined the role of Src in mediating endothelial functions of VEGF in which FAK has been implicated as having a role. PP2 markedly inhibited VEGF-induced chemotaxis and wound-healing cell migration. The Src inhibitor also decreased the anti-apoptotic effect of VEGF determined by surface staining of annexin V but did not increase FAK proteolysis or prevent the VEGF-dependent inhibition of FAK proteolysis. In contrast, the specific PtdIns 3-kinase inhibitor LY294002 induced apoptosis and markedly decreased p125(FAK) expression and increased FAK proteolysis but had little effect on Y861 phosphorylation. These findings identify Src-dependent FAK phosphorylation at Y861 as a novel VEGF-induced signalling pathway in endothelial cells and suggest that this pathway might be involved in the mechanisms mediating VEGF-induced endothelial cell migration and anti-apoptosis.
Abu-Ghazaleh
R
R
Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK.
Kabir
J
J
Jia
H
H
Lobo
M
M
Zachary
I
I
D017209:Apoptosis; D015153:Blotting, Western; D002448:Cell Adhesion; D002455:Cell Division; D002465:Cell Movement; D002470:Cell Survival; D002478:Cells, Cultured; D002633:Chemotaxis; D002867:Chromones / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D015151:Immunoblotting; D008222:Lymphokines / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014945:Wound Healing; D019061:src-Family Kinases / Q000378:metabolism*
D002867:Chromones; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D008222:Lymphokines; D009025:Morpholines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019061:src-Family Kinases