Vascular Endothelial Growth Factor Stimulates Tyrosine Phosphorylation and Recruitment to New Focal Adhesions of Focal Adhesion Kinase and Paxillin in Endothelial Cells

doi:10.1074/jbc.272.24.15442

Vascular Endothelial Growth Factor Stimulates Tyrosine Phosphorylation and Recruitment to New Focal Adhesions of Focal Adhesion Kinase and Paxillin in Endothelial Cells*

Husna Abedi and
Ian Zachary‡

From the Cruciform Project and Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, United Kingdom

Abstract

Vascular endothelial growth factor (VEGF) stimulated the tyrosine phosphorylation of multiple components in confluent human umbilical vein endothelial cells (HUVECs) including bands of M _r 205,000, corresponding to the VEGF receptors Flt-1 and KDR, and M _r 145,000, 120,000, 97,000, and 65,000–70,000. VEGF caused a striking and transient increase in mitogen-activated protein (MAP) kinase activity and stimulated phospholipase C-γ tyrosine phosphorylation, but it had no effect on phosphatidylinositol 3′-kinase activity. VEGF caused a marked increase in tyrosine phosphorylation of p125 focal adhesion kinase (p125^FAK), which was both rapid and concentration-dependent. VEGF produced similar effects on p125^FAK in the endothelial cell line ECV.304. VEGF stimulated tyrosine phosphorylation of the 68-kDa focal adhesion-associated component, paxillin, with similar kinetics and concentration dependence to that for p125^FAK. Thrombin and the phorbol ester, phorbol 12-myristate 13-acetate, also increased p125^FAK tyrosine phosphorylation in HUVECs. The effect of VEGF on p125^FAK tyrosine phosphorylation was completely inhibited by the actin filament-disrupting agent cytochalasin D and was partially inhibited by the protein kinase C inhibitor GF109203X. Inhibition of the MAP kinase pathway using a specific inhibitor of MAP kinase kinase had no effect on p125^FAK tyrosine phosphorylation. VEGF stimulated migration and actin stress fiber formation in confluent HUVEC, and VEGF-induced p125^FAK/paxillin tyrosine phosphorylation was accompanied by increased immunofluorescent staining of p125^FAK, paxillin, and phosphotyrosine in focal adhesions in confluent cultures of HUVECs. These findings identify p125^FAK and paxillin as components in a VEGF-stimulated signaling pathway and suggest a novel mechanism for VEGF regulation of endothelial cell functions.

Footnotes

↵* This work was supported by the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Supported by the British Heart Foundation. To whom correspondence should be addressed. Tel.: 0171 209 6620; Fax: 0171 813 2846; E-mail:I.Zachary{at}ucl.ac.uk.
↵1 The abbreviations used are: VEGF, vascular endothelial growth factor; anti-Tyr(P), anti-phosphotyrosine monoclonal antibody; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HUVEC, human umbilical vein endothelial cell; MAP, mitogen-activated protein; mAb, monoclonal antibody; PI phosphatidylinositol; p85α, p85α subunit of PI 3-kinase; p120GAP, p120 GTPase-activating protein; p125^FAK, p125 focal adhesion kinase; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PKC, protein kinase C; PLC-γ, phospholipase C-γ; PAGE, polyacrylamide gel electrophoresis; VSMC, vascular smooth muscle cells.
↵2 H. Abedi and I. Zachary, unpublished results.
- Received March 11, 1997.
- Revision received March 28, 1997.