Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Sakoda, Tsuyoshi; Hirata, Ken‐ichi; Kuroda, Ryo; Miki, Nobuo; Suematsu, Masakuni; Kawashima, Seinosuke; Yokoyama, Mitsuhiro

doi:10.1007/bf01076076

Cited by 49 publications

(32 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, there is ample evidence for a functional role of Golgi complex-localized eNOS. As originally described in transfected cells (33,37), eNOS is found in the peripheral aspects of the Golgi complex, and this targeting is required for optimal NO release from cells. More recently, using immunofluorescence microscopy to label eNOS phosphorylated at Ser 1179 and NO imaging with 4,5-diaminofluorescein diacetate, we found that, in response to the angiogenic vascular endothelial growth factor, Golgi complex-localized eNOS is phosphorylated at Ser 1179 , and we identified "bursts" of NO emanating from the perinuclear region of endothelial cells (30).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have shown that WT and G2A eNOS have similar catalytic abilities and cofactor requirements (38,39) despite the inability of mislocalized G2A eNOS to produce NO after stimulation of cells with calcium-mobilizing agonists (33,37). Therefore, we measured the catalytic competency of the newly generated eNOS fusion proteins in vitro using the conversion of L-[ 3 H]arginine to L-[ 3 H]citrulline as an index of NOS activity in detergent-solubilized COS cell extracts.…”

Section: Generation and Characterization Of Enos Targetingmentioning

confidence: 99%

“…1). Previous work has shown that G2A eNOS retains full catalytic ability in vitro; however, the stimulated release of NO from intact cells expressing this mutant is impaired because the enzyme fails to target the Golgi complex and plasmalemmal rafts/caveolae and is not efficiently phosphorylated by Akt at Ser 1179 (30,33,37). By exploiting these properties of G2A eNOS, we generated Golgi complex-and plasma membranetargeted eNOS to assess the functional importance of subcellular targeting (Fig.…”

Section: Generation and Characterization Of Enos Targetingmentioning

confidence: 99%

See 2 more Smart Citations

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

Fulton

Babbitt

Zoellner

et al. 2004

Journal of Biological Chemistry

126

125

View full text Add to dashboard Cite

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

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Generation and Characterization Of Enos Targetingmentioning

confidence: 99%

Section: Generation and Characterization Of Enos Targetingmentioning

confidence: 99%

See 1 more Smart Citation

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

Fulton

Babbitt

Zoellner

et al. 2004

Journal of Biological Chemistry

126

125

View full text Add to dashboard Cite

show abstract

“…Myristoylation and dual palmitoylation at its extreme N terminus target eNOS to the cytoplasmic face of the Golgi complex and to the plasma membrane (7), where eNOS is thought to be fully capable of activation (8,9). Misrouting of acylation-deficient eNOS impairs NO production (10,11), indicating that correct subcellular targeting is critical for stimulus-dependent activation of the enzyme (8). Posttranslational modifications are efficiently complemented by multiple proteinprotein interactions that help regulate eNOS activity with respect to time and space.…”

mentioning

confidence: 99%

NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

Zimmermann

Opitz

Dedio

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

162

135

View full text Add to dashboard Cite

Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a Cterminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS-NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. N itric oxide (NO) is a potent mediator in biological processes such as neurotransmission, inflammatory response, and vascular homeostasis (1). The prime source of NO in the cardiovascular system is endothelial NO synthase (eNOS), which is tightly regulated with respect to activity and localization. For example, coordinated phosphorylation contributes to activity control of eNOS because of activating and inhibiting phosphorylation at S1179 and T495, respectively (2-6). Myristoylation and dual palmitoylation at its extreme N terminus target eNOS to the cytoplasmic face of the Golgi complex and to the plasma membrane (7), where eNOS is thought to be fully capable of activation (8, 9). Misrouting of acylation-deficient eNOS impairs NO production (10, 11), indicating that correct subcellular targeting is critical for stimulus-dependent activation of the enzyme (8). Posttranslational modifications are efficiently complemented by multiple proteinprotein interactions that help regulate eNOS activity with respect to time and space. For instance, chaperone hsp90 bound to eNOS may mediate vascular endothelial growth factor-induced eNOS phosphorylation by promoting the interaction between eNOS and Akt (12, 13). At the plasma membrane, eNOS is complexed to and inhibited by the master components of caveolae, i.e., caveolin-1 in endothelial cells (9, 14) and caveolin-3 in cardiac myocytes (15). After stimulus-induced [Ca 2ϩ ] i increase, the Ca 2ϩ -calmodulin complex displaces eNOS from caveolin (16), stimulates eNOS to produce NO, and subsequently leads to the redistribution of eNOS from plasma membrane caveolae (17). The complexity...

show abstract

“…Subcellular localization of eNOS: eNOS is largely membrane-associated as a result of Nterminal myristoylation and palmitoylation modifications (30,152,157 translocates remain unclear, but many include the nuclear membrane, the endoplasmic reticulum, or trans-golgi system (83).…”

Section: Endothelial Nitric Oxide Synthase (Enos)mentioning

confidence: 99%

Functional proteomic analysis of the myocardial PKCepsilon subproteome PKCepsilon-AKT-eNOS signaling modules and during.

Zhang¹

View full text Add to dashboard Cite

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Cited by 49 publications

References 25 publications

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

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

NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

Functional proteomic analysis of the myocardial PKCepsilon subproteome PKCepsilon-AKT-eNOS signaling modules and during.

Contact Info

Product

Resources

About