title: Asymmetric dimethylarginine induces endothelial nitric-oxide synthase mitochondrial redistribution through the nitration-mediated activation of Akt1.
abstract: We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser(617) and Ser(1179). Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr(350)) located within the client-binding domain. Replacement of Tyr(350) with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser(617) and Ser(1179) by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr(350).
authors.affiliation: Pulmonary Disease Program, Vascular Biology Center, Georgia Health Sciences University, Augusta, Georgia 30912, USA.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Rafikov
authors.forename: Olga
authors.initials: O
authors.lastname: Rafikova
authors.forename: Saurabh
authors.initials: S
authors.lastname: Aggarwal
authors.forename: Christine
authors.initials: C
authors.lastname: Gross
authors.forename: Xutong
authors.initials: X
authors.lastname: Sun
authors.forename: Julin
authors.initials: J
authors.lastname: Desai
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D012756:Sheep
chemical_list: D004791:Enzyme Inhibitors; D024101:Mitochondrial Proteins; C018524:N,N-dimethylarginine; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C494918:AKT1 protein, human; C514862:Akt1 protein, mouse; D051057:Proto-Oncogene Proteins c-akt