Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent proteinkinases

Endothelial nitric-oxide synthase (NOS-III) is defined as being strictly de pendent on Ca2+/calmodulin (CaM) for activity, although NO release from end othelial cells has been reported to also occur at intracellular free Ca2+ l evels 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 pr otein kinase II (cGK II) and the catalytic subunit of cAMP-dependent protei n kinase (cAK) in vitro. Phosphopeptide analysis by mass spectrometry ident ified Ser(1177), as well as Ser(633) which is situated in a recently define d CaM autoinhibitory domain within the flavin-binding region of human NOS-I II, Phosphoamino acid analysis identified a putative phosphorylation site a t Thr(495) in the CaM-binding domain. Importantly, both cAK and cGK phospho rylation of NOS-III in vitro caused a highly reproducible partial (10-20%) NOS-III activation which was independent of Ca2+/CaM, and as much as a 4-fo ld increase in V-max in the presence of Ca2+/CaM. cAK stimulation in intact endothelial cells also increased both Ca2+/CaM-independent and -dependent activation of NOS-III. These data collectively provide new evidence for cAK and cGK stimulation of both Ca2+/CaM-independent and -dependent NOS-III ac tivity, and suggest possible cross-talk between the NO and prostaglandin I, pathways and a positive feedback mechanism for NO/cGMP signaling.