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J Biol Chem. 2013 May 24;288(21):15380-9. doi: 10.1074/jbc.M112.427765. Epub 2013 Apr 2.

Endomembrane H-Ras controls vascular endothelial growth factor-induced nitric-oxide synthase-mediated endothelial cell migration.

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1
Vascular Biology Section, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

Abstract

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling.

KEYWORDS:

Angiogenesis; Cell Migration; Endomembrane; H-Ras; Nitric Oxide; Nitric-oxide Synthase; Protein Palmitoylation; Vascular Endothelial Growth Factor (VEGF)

PMID:
23548900
PMCID:
PMC3663556
DOI:
10.1074/jbc.M112.427765
[Indexed for MEDLINE]
Free PMC Article
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