Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.
J Biol Chem
; 275(7): 5179-87, 2000 Feb 18.
Article
in En
| MEDLINE
| ID: mdl-10671564
ABSTRACT
Endothelial nitric-oxide synthase (NOS-III) is defined as being strictly dependent on Ca(2+)/calmodulin (CaM) for activity, although NO release from endothelial cells has been reported to also occur at intracellular free Ca(2+) levels that are substimulatory for the purified enzyme. We demonstrate here that NOS-III, but neither NOS-I nor -II, is rapidly and strongly activated and phosphorylated on both Ser and Thr in the presence of cGMP-dependent protein kinase II (cGK II) and the catalytic subunit of cAMP-dependent protein kinase (cAK) in vitro. Phosphopeptide analysis by mass spectrometry identified Ser(1177), as well as Ser(633) which is situated in a recently defined CaM autoinhibitory domain within the flavin-binding region of human NOS-III. Phosphoamino acid analysis identified a putative phosphorylation site at Thr(495) in the CaM-binding domain. Importantly, both cAK and cGK phosphorylation of NOS-III in vitro caused a highly reproducible partial (10-20%) NOS-III activation which was independent of Ca(2+)/CaM, and as much as a 4-fold increase in V(max) in the presence of Ca(2+)/CaM. cAK stimulation in intact endothelial cells also increased both Ca(2+/)CaM-independent and -dependent activation of NOS-III. These data collectively provide new evidence for cAK and cGK stimulation of both Ca(2+)/CaM-independent and -dependent NOS-III activity, and suggest possible cross-talk between the NO and prostaglandin I(2) pathways and a positive feedback mechanism for NO/cGMP signaling.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Calcium
/
Cyclic GMP-Dependent Protein Kinases
/
Cyclic AMP-Dependent Protein Kinases
/
Nitric Oxide Synthase
Type of study:
Prognostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
J Biol Chem
Year:
2000
Document type:
Article