Abstract

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

Highlights

  • 7758 JOURNAL OF BIOLOGICAL CHEMISTRY and excessive neovascularization, contributes to the development of many pathologies, including retinopathies, rheumatoid arthritis, and tumor growth [2, 5,6,7]

  • Consistent with this, tyrosine phosphorylation of the Gab1⌬Grb2 mutant in response to stimulation was greatly attenuated, and this mutant was unable to associate with phosphorylated SHP2 and to activate ERK1/2 as WT Gab1 (Fig. 3A). These results suggest that the association of Gab1 with VEGFR2 complexes via Grb2 is required for optimal Gab1 tyrosine phosphorylation, Gab1 association with phosphorylated SHP2, and the promotion of Gab1-dependent ERK1/2 activation

  • Since Gab1 is associated with phosphatidylinositol 3-kinase (PI3K) and SHP2 in Vascular endothelial growth factor (VEGF)-stimulated endothelial cells (Fig. 1C) and optimal activation of PLC␥, ERK1/2, Src, and Akt requires Gab1 expression (Fig. 5), we investigated whether Gab1-associated PI3K and SHP2 were involved in VEGF-dependent signaling and migration

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Summary

Introduction

7758 JOURNAL OF BIOLOGICAL CHEMISTRY and excessive neovascularization, contributes to the development of many pathologies, including retinopathies, rheumatoid arthritis, and tumor growth [2, 5,6,7]. Gab1 depletion experiments demonstrate that endogenous Gab1 is required for the optimal VEGF-dependent signaling to PLC␥, ERK1/2, Src, and Akt, endothelial cell migration, and capillary formation.

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