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.
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 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.
Fulton
David
D
Department of Pharmacology and the Program in Vascular Cell Signaling and Therapeutics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA.
Babbitt
Roger
R
Zoellner
Stefan
S
Fontana
Jason
J
Acevedo
Lisette
L
McCabe
Timothy J
TJ
Iwakiri
Yasuko
Y
Sessa
William C
WC
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D009227:Myristic Acids / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000737:chemistry; D052250:Nitric Oxide Synthase Type III; D010169:Palmitic Acids / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000737:chemistry; D014162:Transfection; D014471:Umbilical Veins / Q000378:metabolism
D002147:Calmodulin; D009227:Myristic Acids; D010169:Palmitic Acids; D011518:Proto-Oncogene Proteins; D009569:Nitric Oxide; D012694:Serine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D003545:Cysteine; D002118:Calcium