title: Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A.
abstract: Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Yong Chool
authors.initials: YC
authors.lastname: Boo
authors.forename: George
authors.initials: G
authors.lastname: Sorescu
authors.forename: Nolan
authors.initials: N
authors.lastname: Boyd
authors.forename: Ichiro
authors.initials: I
authors.lastname: Shiojima
authors.forename: Kenneth
authors.initials: K
authors.lastname: Walsh
authors.forename: Jie
authors.initials: J
authors.lastname: Du
authors.forename: Hanjoong
authors.initials: H
authors.lastname: Jo
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic; D002417:Cattle; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D013314:Stress, Mechanical; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004791:Enzyme Inhibitors; D009566:Nitrates; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D010768:Phosphoserine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin