title: Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism.
abstract: Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, GA 30322, USA.
authors.forename: Yong Chool
authors.initials: YC
authors.lastname: Boo
authors.forename: Jinah
authors.initials: J
authors.lastname: Hwang
authors.forename: Michelle
authors.initials: M
authors.lastname: Sykes
authors.forename: Belinda J
authors.initials: BJ
authors.lastname: Michell
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Kemp
authors.forename: Hazel
authors.initials: H
authors.lastname: Lum
authors.forename: Hanjoong
authors.initials: H
authors.lastname: Jo
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008222:Lymphokines / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism; D013314:Stress, Mechanical; D013912:Threonine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013912:Threonine; D012694:Serine; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases