Regulation of vascular endothelial growth factor receptor 2-mediated phosphorylation of focal adhesion kinase by heat shock protein 90 and Src kinase activities.
Exposure of endothelial cells to vascular endothelial growth factor (VEGF) induced tyrosine phosphorylation of focal adhesion kinase (FAK) on site Tyr(407), an effect that required the association of VEGF receptor 2 (VEGFR2) with HSP90. The association of VEGFR2 with HSP90 involved the last 130 amino acids of VEGFR2 and was blocked by geldanamycin, a specific inhibitor of HSP90. Moreover, geldanamycin inhibited the VEGF-induced activation of the small GTPase RhoA, which resulted in an inhibition of phosphorylation of FAK on site Tyr(407). In this context, the inhibition of RhoA kinase (ROCK) with Y27632 or by expression of dominant negative forms of RhoA or ROCK impaired the VEGF-induced phosphorylation of Tyr(407) within FAK. In contrast to phosphorylation of Tyr(861), the phosphorylation of site Tyr(407) was insensitive to Src kinase inhibition by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2). We also found that the recruitment of paxillin to FAK was inhibited by geldanamycin but not by PP2, whereas both geldanamycin and PP2 inhibited the recruitment of vinculin to FAK. In accordance, the recruitment of paxillin and vinculin to FAK was inhibited in cells that express the mutant FAK-Y407F, whereas the expression of the mutant Y861F inhibited the recruitment of paxillin but not of vinculin. Importantly, cell migration was abolished in cells in which the signal from the VEGFR2-HSP90 pathway was blocked by the expression of Delta130VEGFR2, a deletant of VEGFR2 that does not associate with HSP90. Our findings underscore for the first time the key role played by the VEGFR2-HSP90-RhoA-ROCK-FAK/Tyr(407) pathway in transducing the VEGF signal that leads to the assembly of focal adhesions and endothelial cell migration.
Le Boeuf
Fabrice
F
Le Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec G1R 2J6, Canada.
Houle
François
F
Huot
Jacques
J
D000577:Amides / Q000494:pharmacology; D016227:Benzoquinones; D018821:CD18 Antigens / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic; D008164:Luminescent Proteins / Q000378:metabolism; D009154:Mutation; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D000577:Amides; D016227:Benzoquinones; D018821:CD18 Antigens; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D008164:Luminescent Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011725:Pyridines; D011809:Quinones; D016596:Vinculin; C108830:Y 27632; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019061:src-Family Kinases; D020742:rhoA GTP-Binding Protein; C001277:geldanamycin