Intracellular location regulates calcium-calmodulin-dependent activation of organelle-restricted eNOS.
Mislocalization of endothelial nitric oxide (NO) synthase (eNOS) in response to oxidized low-density lipoprotein, cholesterol depletion, elevated blood pressure, and bound eNOS interacting protein/NOS traffic inducer is associated with reduced NO release via unknown mechanisms. The proper targeting of eNOS to the plasma membrane or intracellular organelles is an important regulatory step controlling enzyme activity. Previous studies have shown that plasma membrane eNOS is constitutively phosphorylated on serine 1179 and highly active. In contrast, the activity of eNOS targeted to intracellular organelles is more complex. The cis-Golgi eNOS is fully activated by Akt-dependent phosphorylation. However, eNOS targeted to the trans-Golgi is decidedly less active in response to all modes of activation, including mutation to the phosphomimetic aspartic acid. In this study, we establish that when expressed within other intracellular organelles, such as the mitochondria and nucleus, the activity of eNOS is also greatly reduced. To address the mechanisms underlying the impaired catalytic activity of eNOS within these locations, we generated subcellular-targeted constructs that express a calcium-independent NOS isoform, iNOS. With the use of organelle specific (plasma membrane, cis- vs. trans-Golgi, plasma membrane, and Golgi, nucleus, and mitochondria) targeting motifs fused to the wild-type iNOS, we measured NO release from intact cells. With the exception of the Golgi lumen, our results showed no impairment in the ability of targeted iNOS to synthesize NO. Confirmation of correct targeting was obtained through confocal microscopy using identical constructs fused to the green fluorescent protein. We conclude that the reduced activation of eNOS within discrete cytoplasmic regions of the Golgi, the mitochondria and the nucleus is primarily due to insufficient access to calcium-calmodulin.
Jagnandan
Davin
D
Vascular Biology Center and Department of Pharmacology, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta Georgia 30912, USA.
Sessa
William C
WC
Fulton
David
D
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D002522:Chlorocebus aethiops; D003067:Coenzymes; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D015388:Organelles / Q000201:enzymology*; D013379:Substrate Specificity
D002147:Calmodulin; D003067:Coenzymes; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D002118:Calcium