Nitric oxide prevents phosphorylation of neuronal nitric oxide synthase at Serine1412 by inhibiting the Akt/PKB and CaM-K�II signaling pathways

Abstract

Abstract. Neuronal nitric oxide synthase (nNOS) is an important regulatory enzyme in the central nervous system catalyzing the production of NO, which regulates multiple biological processes in the central nervous system. However, the mechanisms by which nNOS activity is regulated are not completely understood. In the present study, the effects of protein kinases on the phosphorylation of nNOS in GH3 rat pituitary tumor cells were evaluated. We show that phosphorylation of nNOS at Ser1412 could be induced by the phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) agonist insulin, the calcium/calmodulin-dependent protein kinase II (CaM-K II) agonist A23187 or the cAMP-dependent protein kinase A (PKA) agonist IBMX, respectively. The phosphorylation levels of nNOS at Ser1412, induced by activation of Akt/PKB or CaM-K II, but not by PKA signaling, were reduced by pre-treatment with the NO donor diethylamineNONOate. This inhibitory effect could be reversed by addition of a reducing reagent, dithiothreitol. Furthermore, the levels of phosphorylation of nNOS at Ser1412, induced by Akt/PKB or CaM-K II but not by PKA signaling, were enhanced by inhibition of nNOS activity with 7-nitroindazole. These findings suggest that the activation of nNOS can be catalyzed by at least three protein kinases, Akt/PKB, CaM-K II or PKA. NO generated from nNOS feedback prevents the activation of nNOS by inhibiting either Akt/PKB or CaM-K II but not PKA signaling. IntroductionNitric oxide (NO), generated by the family of NO synthases (NOS), is a signaling molecule that participates in multiple biological processes in the central nervous system, including neurotransmitter release, plasticity and apoptosis (1). NO exerts its effects by regulating soluble guanylyl cyclase (sGC) to mediate cyclic guanosine monophosphate (cGMP) synthesis (2). In addition to the activation of sGC, NO also exerts cGMP-independent signaling, albeit at much higher levels than needed for sGC activation (2). These include modification of protein function by S-nitrosylation, now recognized as a prototype redox-dependent signaling component that mediates a variety of actions of endogenous NO (3). Under pathological circumstances, overproduction of NO can cause the formation of peroxynitrite, which is associated with excitotoxicity after stroke (4). S-nitrosylation and nitration of proteins by peroxynitrite also exacerbate central nervous system disorders such as dementia (5), Parkinson's disease (6), and Alzheimer's disease (7).Neuronal NOS (nNOS) is a critical regulatory enzyme in the central nervous system catalyzing the production of NO from arginine. It has been reported that the control of NO production by the NMDA receptor at neural synapses is associated with regulatory phosphorylations of nNOS (8). However, the mechanisms by which these phosphorylations are induced in neurons and by which they regulate nNOS activity are not known. We and others reported that activity of nNOS was regulated by Ca 2+ -dependent binding of calmodulin (CaM) and multiple protein...

cAMP‐dependent post‐translational modification of neuronal nitric oxide synthase neuroprotects penile erection in rats

“…nNOS phosphorylation is one of the major mechanisms regulating nNOS bioactivity . nNOS activation by phosphorylation can be induced by several protein kinases, including PKA, serine/threonine kinase/protein kinase B (Akt [or PKB]), AMP‐activated protein kinase, and Ca2+ /calmodulin‐dependent protein kinase II . Phosphorylation of nNOS on Ser‐1412 activates the enzyme, while phosphorylation of nNOS on Ser‐847 inhibits the enzyme.…”

“…nNOS phosphorylation is one of the major mechanisms regulating nNOS bioactivity [19,42]. nNOS activation by [44].…”

Molecular determinants of acute kidney injury

“… 74 This release of vesicular Ca 2+ can activate calcineurin (PP2B), which is able to block calcium/calmodulin-dependent protein kinase type II (CamKII) indirectly via protein phosphatases 1 (PP1). 75 , 76 CamKII is a downstream target of NMDA-R activation, 77 and is coupled to this receptor via scaffolders belonging to the disks large family DLGs, 78 that also bind to the Ca 2 +/Na+-importer channel AMPA-R. 79 Nitric oxide synthase (NOS) is also bound to DLGs 80 and activated by CamKII, 81 thereby linking NO-production directly to NMDA-R activity. CamKII is also involved in protein tyrosine kinase 2 (Pyk2) 82 and mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) 83 cascade activation, and both of those events can lead to a substantial number of diverse signaling end-points through various other cascades and molecules involved.…”

Coordination between Calcium/Calmodulin-Dependent Protein Kinase II and Neuronal Nitric Oxide Synthase in Neurons

“…Pathological high glutamate (100-500 µM) activates PPase and decreases the level of the phosphorylation of nNOS at Ser847, leading the overproduction of NO during pathogenesis in rat hippocampal neurons [49]. We reported that CaMKII phosphorylates not only Ser847 but also Ser1412 in cultured cells [51], which induce activation of nNOS by reducing the enzyme's Ca 2+ requirement and facilitating electron transfer [52]. The study of NO and superoxide generation activity of nNOS phosphorylated at not only Ser847 but also Ser1412 needs to be determined.…”