Endothelial Nitric Oxide Synthase Dimerization Is Regulated by Heat Shock Protein 90 Rather than by Phosphorylation

Abstract

Endothelial nitric oxide synthase (eNOS) is a multifunctional enzyme with roles in diverse cellular processes including angiogenesis, tissue remodeling, and the maintenance of vascular tone. Monomeric and dimeric forms of eNOS exist in various tissues. The dimeric form of eNOS is considered the active form and the monomeric form is considered inactive. The activity of eNOS is also regulated by many other mechanisms, including amino acid phosphorylation and interactions with other proteins. However, the precise mechanisms regulating eNOS dimerization, phosphorylation, and activity remain incompletely characterized. We utilized purified eNOS and bovine aorta endothelial cells (BAECs) to investigate the mechanisms regulating eNOS degradation. Both eNOS monomer and dimer existed in purified bovine eNOS. Incubation of purified bovine eNOS with protein phosphatase 2A (PP2A) resulted in dephosphorylation at Serine 1179 (Ser1179) in both dimer and monomer and decrease in eNOS activity. However, the eNOS dimer∶monomer ratio was unchanged. Similarly, protein phosphatase 1 (PP1) induced dephosphorylation of eNOS at Threonine 497 (Thr497), without altering the eNOS dimer∶monomer ratio. Different from purified eNOS, in cultured BAECs eNOS existed predominantly as dimers. However, eNOS monomers accumulated following treatment with the proteasome inhibitor lactacystin. Additionally, treatment of BAECs with vascular endothelial growth factor (VEGF) resulted in phosphorylation of Ser1179 in eNOS dimers without altering the phosphorylation status of Thr497 in either form. Inhibition of heat shock protein 90 (Hsp90) or Hsp90 silencing destabilized eNOS dimers and was accompanied by dephosphorylation both of Ser1179 and Thr497. In conclusion, our study demonstrates that eNOS monomers, but not eNOS dimers, are degraded by ubiquitination. Additionally, the dimeric eNOS structure is the predominant condition for eNOS amino acid modification and activity regulation. Finally, destabilization of eNOS dimers not only results in eNOS degradation, but also causes changes in eNOS amino acid modifications that further affect eNOS activity.

Enhancing eNOS activity with simultaneous inhibition of IKKβ restores vascular function in Ins2Akita+/− type-1 diabetic mice

“…4 Recent evidence also demonstrated that Ser1179 phosphorylation exclusively exists in eNOS dimer at both resting and stimulated condition. 5 As shown in Figure 4a, our results demonstrated a significant downregulation of both total and phospho eNOS expression in Ins2…”

“…3 In vitro studies from our laboratory and reports from other investigators have shown that the regulation of eNOS activity is mediated by heat shock protein-90 (Hsp-90). 4,5 The interaction of eNOS with Hsp-90 favors the phosphorylation of eNOS at Ser1177 by Akt kinase. This phosphorylation process is a key event which augments eNOS activity leading to NO production.…”

“…This phosphorylation process is a key event which augments eNOS activity leading to NO production. 5 Our earlier studies have shown negative regulation of eNOSHsp-90 axis by inhibitor kappa B kinase beta (IKKβ), an upstream mediator of NF-κB signaling pathway in endothelial cells under the influence of high glucose (HG). We have also demonstrated that blocking IKKβ restores eNOS-Hsp-90 interaction and NO production.…”

Compartmentalized nitric oxide signaling in the resistance vasculature

“…This interaction increases calmodulin binding, thus sensitizing the system to rises in calcium levels [58]. Recent work has shown that hsp90 is required for dimerization, and it is this dimerization that is the predominant condition for eNOS phosphorylation and activation rather than the modifications being the permissive event for dimerization [59]. Hsp90 promotes these phosphorylation events by potentially increasing the binding affinity for eNOS and Akt and causing a conformational shift that unmasks sites for modification [60, 61].…”

Differential effects of heat shock protein 90 and serine 1179 phosphorylation on endothelial nitric oxide synthase activity and on its cofactors

“…The expressed wild-type eNOS and S1179D eNOS proteins were harvested and purified according to the description of previous publications [17] [11]. …”