1. Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.
- Author
-
Butt E, Bernhardt M, Smolenski A, Kotsonis P, Fröhlich LG, Sickmann A, Meyer HE, Lohmann SM, and Schmidt HH
- Subjects
- Amino Acid Sequence, Animals, Calmodulin metabolism, Enzyme Activation, Flavins metabolism, Humans, Molecular Sequence Data, Nitric Oxide Synthase chemistry, Nitric Oxide Synthase Type III, Phosphorylation, Protein Conformation, Sequence Homology, Amino Acid, Substrate Specificity, Calcium metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Cyclic GMP-Dependent Protein Kinases metabolism, Nitric Oxide Synthase metabolism
- 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.
- Published
- 2000
- Full Text
- View/download PDF