Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.
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.
Butt
E
E
Medical University Clinic, Institute of Clinical Biochemistry and Pathobiochemistry, Josef Schneider Strasse 2, D-97080 Würzburg, Germany. butt@klin-biochem.uni-wuerzburg.de
Bernhardt
M
M
Smolenski
A
A
Kotsonis
P
P
Fröhlich
L G
LG
Sickmann
A
A
Meyer
H E
HE
Lohmann
S M
SM
Schmidt
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005415:Flavins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002147:Calmodulin; D005415:Flavins; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D002118:Calcium