Abstract
The heterogeneous localization of endothelial nitric-oxide 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 Akt-mediated 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, Ser1179 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 30349-30357 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 279 |
| Issue number | 29 |
| DOIs | |
| State | Published - Jul 16 2004 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
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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, David J; Babbitt, Roger; Zoellner, Stefan; Fontana, Jason; Acevedo, Lisette; McCabe, Timothy J.; Iwakiri, Yasuko; Sessa, William C.
In: Journal of Biological Chemistry, Vol. 279, No. 29, 16.07.2004, p. 30349-30357.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - 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
AU - Fulton, David J
AU - Babbitt, Roger
AU - Zoellner, Stefan
AU - Fontana, Jason
AU - Acevedo, Lisette
AU - McCabe, Timothy J.
AU - Iwakiri, Yasuko
AU - Sessa, William C.
PY - 2004/7/16
Y1 - 2004/7/16
N2 - The heterogeneous localization of endothelial nitric-oxide 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 Akt-mediated 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, Ser1179 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.
AB - The heterogeneous localization of endothelial nitric-oxide 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 Akt-mediated 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, Ser1179 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=3142705856&partnerID=8YFLogxK
U2 - 10.1074/jbc.M402155200
DO - 10.1074/jbc.M402155200
M3 - Article
C2 - 15136572
AN - SCOPUS:3142705856
VL - 279
SP - 30349
EP - 30357
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 29
ER -