Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase

Yong Chool Boo, George P. Sorescu, Philip M. Bauer, David J Fulton, Bruce E. Kemp, David G. Harrison, William C. Sessa, Hanjoong Jo

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

Shear stress stimulates NO production involving the Ca2+- independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+] i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+] i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.

Original languageEnglish (US)
Pages (from-to)729-741
Number of pages13
JournalFree Radical Biology and Medicine
Volume35
Issue number7
DOIs
StatePublished - Oct 1 2003
Externally publishedYes

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Phosphorylation
Nitric Oxide Synthase
Endothelial cells
Calcium
Endothelial Cells
HEK293 Cells
Cyclic AMP-Dependent Protein Kinases
Shear stress
Blood Vessels
Catalytic Domain
1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester

Keywords

  • Endothelial cells
  • Free radicals
  • eNOS mutants

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

Cite this

Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase. / Boo, Yong Chool; Sorescu, George P.; Bauer, Philip M.; Fulton, David J; Kemp, Bruce E.; Harrison, David G.; Sessa, William C.; Jo, Hanjoong.

In: Free Radical Biology and Medicine, Vol. 35, No. 7, 01.10.2003, p. 729-741.

Research output: Contribution to journalArticle

Boo, Yong Chool ; Sorescu, George P. ; Bauer, Philip M. ; Fulton, David J ; Kemp, Bruce E. ; Harrison, David G. ; Sessa, William C. ; Jo, Hanjoong. / Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase. In: Free Radical Biology and Medicine. 2003 ; Vol. 35, No. 7. pp. 729-741.
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AU - Boo, Yong Chool

AU - Sorescu, George P.

AU - Bauer, Philip M.

AU - Fulton, David J

AU - Kemp, Bruce E.

AU - Harrison, David G.

AU - Sessa, William C.

AU - Jo, Hanjoong

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N2 - Shear stress stimulates NO production involving the Ca2+- independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+] i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+] i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.

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