Originally published In Press as doi:10.1074/jbc.M405493200 on July 6, 2004
J. Biol. Chem., Vol. 279, Issue 37, 39175-39185, September 10, 2004
Regulation of Vascular Endothelial Growth Factor Receptor 2-mediated Phosphorylation of Focal Adhesion Kinase by Heat Shock Protein 90 and Src Kinase Activities*
Fabrice Le Boeuf
,
François Houle, and
Jacques Huot
From the
Le Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec G1R 2J6, Canada
Exposure of endothelial cells to vascular endothelial growth factor (VEGF) induced tyrosine phosphorylation of focal adhesion kinase (FAK) on site Tyr407, 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 Tyr407. 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 Tyr407 within FAK. In contrast to phosphorylation of Tyr861, the phosphorylation of site Tyr407 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 
130VEGFR2, 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/Tyr407 pathway in transducing the VEGF signal that leads to the assembly of focal adhesions and endothelial cell migration.
Received for publication, May 17, 2004
, and in revised form, June 24, 2004.
* This work was supported by The Canadian Institutes for Health Research Grant MOP 15402. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Held a studentship from "La Fondation de Transplantation d'Organes de France."
To whom correspondence should be addressed: Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, 9 rue McMahon, Québec G1R 2J6, Canada. Tel.: 418-525-4444 (ext. 15553); Fax: 418-691-5439; E-mail: Jacques.Huot{at}phc.ulaval.ca.
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
