title: Endomembrane H-Ras controls vascular endothelial growth factor-induced nitric-oxide synthase-mediated endothelial cell migration.
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.
authors.affiliation: Vascular Biology Section, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: Dagmar J
authors.initials: DJ
authors.lastname: Haeussler
authors.forename: David R
authors.initials: DR
authors.lastname: Pimentel
authors.forename: Xiuyun
authors.initials: X
authors.lastname: Hou
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Burgoyne
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
authors.forename: Markus M
authors.initials: MM
authors.lastname: Bachschmid
mesh_terms: D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004790:Enzyme Induction / Q000502:physiology; D006801:Humans; D018919:Neovascularization, Physiologic / Q000502:physiology; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Angiogenesis; Cell Migration; Endomembrane; H-Ras; Nitric Oxide; Nitric-oxide Synthase; Protein Palmitoylation; Vascular Endothelial Growth Factor (VEGF)
references.citation: Science. 2005 Mar 18;307(5716):1746-52
references.pmid: 15705808
references.citation: Nature. 2006 Dec 7;444(7120):724-9
references.pmid: 17086201
references.citation: Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19777-82
references.pmid: 17170139
references.citation: J Cell Biol. 2008 Apr 7;181(1):131-41
references.pmid: 18391074
references.citation: Biochem Soc Trans. 2003 Dec;31(Pt 6):1171-7
references.pmid: 14641020
references.citation: Mol Biol Cell. 2008 Apr;19(4):1404-14
references.pmid: 18234837
references.citation: Mol Cell Biol. 2001 Mar;21(5):1444-52
references.pmid: 11238881
references.citation: PLoS One. 2010 Aug 05;5(8):e11991
references.pmid: 20700538
references.citation: Nature. 2003 Aug 7;424(6949):694-8
references.pmid: 12845332
references.citation: PLoS One. 2011;6(8):e23160
references.pmid: 21826235
references.citation: J Biol Chem. 2000 Feb 18;275(7):5096-103
references.pmid: 10671553
references.citation: Antioxid Redox Signal. 2012 Oct 15;17(8):1099-108
references.pmid: 22472004
references.citation: J Biol Chem. 2011 Feb 18;286(7):5319-27
references.pmid: 21062747
references.citation: Cell Res. 2012 Apr;22(4):661-76
references.pmid: 21968647
references.citation: Nature. 1999 Jun 10;399(6736):601-5
references.pmid: 10376603
references.citation: Circ Res. 2008 Jun 6;102(11):1350-8
references.pmid: 18467631
references.citation: J Mol Cell Cardiol. 2007 Feb;42(2):271-9
references.pmid: 16839566
references.citation: Cell. 2010 Apr 30;141(3):458-71
references.pmid: 20416930
references.citation: Nat Rev Mol Cell Biol. 2002 May;3(5):339-48
references.pmid: 11988768
references.citation: Vascul Pharmacol. 2010 Nov-Dec;53(5-6):215-22
references.pmid: 20813203
references.citation: Br J Pharmacol. 2002 Dec;137(7):1021-30
references.pmid: 12429574
references.citation: Cancer Res. 2010 May 1;70(9):3803-12
references.pmid: 20388785
references.citation: J Biol Chem. 2004 Jul 16;279(29):30349-57
references.pmid: 15136572
references.citation: Oncogene. 1999 Apr 1;18(13):2221-30
references.pmid: 10327068
references.citation: Oncogene. 2004 Jan 8;23(1):192-200
references.pmid: 14712224
references.citation: Mol Cell Biol. 2003 Aug;23(16):5726-37
references.pmid: 12897144
references.citation: Nat Cell Biol. 2002 May;4(5):343-50
references.pmid: 11988737
references.citation: Nat Rev Mol Cell Biol. 2003 May;4(5):373-84
references.pmid: 12728271
references.citation: Mol Endocrinol. 1999 May;13(5):692-704
references.pmid: 10319320
references.citation: Cell. 2007 Jun 1;129(5):957-68
references.pmid: 17540175
references.citation: J Biol Chem. 2005 Sep 2;280(35):31141-8
references.pmid: 16000296
references.citation: Nat Cell Biol. 2005 Aug;7(8):837-43
references.pmid: 16041367
references.citation: Genes Cancer. 2011 Mar;2(3):261-74
references.pmid: 21779497
references.citation: Mol Pharmacol. 2010 Jul;78(1):142-50
references.pmid: 20406854
references.citation: Biochem Biophys Res Commun. 1999 Feb 16;255(2):545-8
references.pmid: 10049745
references.citation: J Cell Biol. 2003 Sep 1;162(5):933-43
references.pmid: 12952943
references.citation: Nat Rev Mol Cell Biol. 2011 Dec 22;13(1):39-51
references.pmid: 22189424
references.citation: Arterioscler Thromb Vasc Biol. 2003 May 1;23(5):802-8
references.pmid: 12637339
references.citation: Nat Chem Biol. 2010 Jun;6(6):449-56
references.pmid: 20418879
references.citation: Cell. 1980 Apr;19(4):1005-14
references.pmid: 6247068
references.citation: J Biol Chem. 2002 Oct 18;277(42):39554-60
references.pmid: 12189156
references.citation: Science. 1996 Dec 20;274(5295):2086-9
references.pmid: 8953040
references.citation: Mol Cell Biol. 2011 Mar;31(5):983-97
references.pmid: 21189290
references.citation: Mol Cell. 2011 Jan 21;41(2):173-85
references.pmid: 21255728
references.citation: Mol Membr Biol. 2009 Jan;26(1):80-92
references.pmid: 19115142
references.citation: Biochemistry. 2003 Oct 14;42(40):11716-25
references.pmid: 14529282
references.citation: Anal Biochem. 1999 Apr 10;269(1):17-20
references.pmid: 10094770