TY - JOUR
T1 - Asymmetric dimethylarginine induces endothelial nitric-oxide synthase mitochondrial redistribution through the nitration-mediated activation of Akt1
AU - Rafikov, Ruslan
AU - Rafikova, Olga
AU - Aggarwal, Saurabh
AU - Gross, Christine
AU - Sun, Xutong
AU - Desai, Julin
AU - Fulton, David
AU - Black, Stephen M.
PY - 2013/3/1
Y1 - 2013/3/1
N2 - 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 Ser617 and Ser1179. 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 (Tyr350) located within the client-binding domain. Replacement of Tyr350 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 Ser617 and Ser1179 by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr350.
AB - 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 Ser617 and Ser1179. 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 (Tyr350) located within the client-binding domain. Replacement of Tyr350 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 Ser617 and Ser1179 by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr350.
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U2 - 10.1074/jbc.M112.423269
DO - 10.1074/jbc.M112.423269
M3 - Article
C2 - 23255608
AN - SCOPUS:84874771073
SN - 0021-9258
VL - 288
SP - 6212
EP - 6226
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 9
ER -