Enhanced Electron Flux and Reduced Calmodulin Dissociation May Explain “Calcium-independent” eNOS Activation by Phosphorylation

Abstract

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K m /EC 50 for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.

Endothelial nitric oxide synthase induces heat shock protein HSPA6 (HSP70B′) in human arterial smooth muscle cells

“…Furthermore, Ad-eNOS and Ad-eNOS S1179D induced HSPA6 by 23.75 and 53.2-fold respectively above Ad-Null (Fig.2C). S1179D eNOS activated HSPA6 by 2.24 fold compared to wild-type eNOS, which parallels the two-fold increase in the rate of NO production from this mutant compared to wild-type eNOS [15]. In contrast, HSP70A and HSP70B were not further elevated by mutant eNOS S1179D and remained at similar levels to that induced by wild-type eNOS ( Supplementary Fig.…”

“…The fold induction of HSPA6 closely parallels the known NO production from these two different eNOS transgenes with the constitutively active eNOS mutant producing double the amount of NO compared to wild-type eNOS [15]. Therefore, we would infer that HSPA6 is responding in a dose dependent manner to the NO generation by these differing transgenes.…”

“…Viral stocks were stored at -80 o C and the viral titre determined by plaque assay. All other adenoviruses packaged with bovine eNOS (Ad-eNOS), a constitutively active form of human eNOS (Ad-eNOS S1179D) [15,16] or without a transgene (Ad-Null) were amplified and purified as above from previously used viral stocks [12].…”

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells

“…Phosphorylation enhances the electron flux through the protein domains of eNOS, resulting in increased nitric oxide production [33]. In human aortic ECs, Ser1177 of eNOS is phosphorylated by the serine/threonine kinase Akt [34,35].…”

Sphingosine-1-phosphate and modulation of vascular tone

“…eNOS regulation by S1P involves another set of signalling pathways that modulate enzyme's phosphorylation at the serine 1177 residue; this phosphorylation of eNOS at its C terminus 'sensitizes' the enzyme to activation at lower levels of calcium in vitro. 55 Full activation of eNOS by S1P requires a pathway comprising G-protein-coupled S1P receptors; pertussis toxin-sensitive G-proteins, especially G-protein b-g subunits that modulate the b-isoform of phosphoinositide 3-kinase (PI3-K); and protein kinase Akt, which phosphorylates eNOS at the serine 1177 residue. 23,27 Subsequent studies, using siRNA-mediated knock down of selected signalling proteins in ECs, established that the AMP-activated protein kinase and the small G-protein Rac1 represent a key upstream regulatory pathway that couples S1P receptor activation and stimulation of PI3-Kb/Akt.…”