Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

Abstract

The heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Aktmediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane-and Golgi complex-targeted constructs, Ser 1179 is critical for NO production. This study provides clear evidence for functional roles of plasma membrane-and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes.Within the vascular endothelium, nitric oxide (NO) is generated via the enzyme endothelial nitric-oxide synthase (eNOS). 1

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

“…* P ＜ 0.05. activation should require the translocation of the free PKA catalytic subunit to the nucleus (Sastri et al, 2005). Furthermore, membrane targeting of eNOS to the plasma membrane or Golgi appears to be critical for the phosphorylation by protein kinases (Gonzalez et al, 2002;Fulton et al, 2004). Although the preparations used in this experiment are not fully informative in terms of organelle composition, it is conceivable that shear stress may stimulate PKA interaction with its substrates, i.e.…”

Altered Angiogenesis in Caveolin-1 Gene–Deficient Mice Is Restored by Ablation of Endothelial Nitric Oxide Synthase

“…Other factors may include the circumstances in which eNOS is able to produce superoxide, and the subcellular localization of eNOS, which in the absence of caveolae cannot be compartmentalized with upstream and downstream partners of the pathways leading to NO production. Targeting of eNOS to different cellular compartments has been shown to regulate the mechanism and degree of its activation 51,52 ; and, given than NO is short lived, the subcellular localization of its production is known to affect its actions, including protein S-nitrosylation. 53 Another way of interpreting our results is that ablation of caveolin-1 rescues the altered angiogenesis seen in the absence of eNOS.…”

Age‐related changes in endothelial nitric oxide synthase phosphorylation and nitric oxide dependent vasodilation: evidence for a novel mechanism involving sphingomyelinase and ceramide‐activated phosphatase 2A

“…eNOS activity, and hence NO production, is regulated in a complex and multilayered process. This includes regulation at the transcriptional and post-translational levels, availability of cosubstrates/cofactors, subcellular location, and proteinprotein interactions (Alderton et al ., 2001;Fulton et al ., 2004). Thus, eNOS in the aging vasculature may be vulnerable to dysregulation at multiple points that would limit NO synthesis and consequently, NO availability.…”