| First Author | Butt E | Year | 2000 |
| Journal | J Biol Chem | Volume | 275 |
| Issue | 7 | Pages | 5179-87 |
| PubMed ID | 10671564 | Mgi Jnum | J:60681 |
| Mgi Id | MGI:1353792 | Doi | 10.1074/jbc.275.7.5179 |
| Citation | Butt E, et al. (2000) 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 |
| abstractText | 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. |
