Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry
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The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.
KeywordsMALDI-TOF MS NanoLC–MS/MS 3-Nitrotyrosine Peroxynitrite Protein kinase G-Iα
Guanosine 3′,5′-cyclic monophosphate
cGMP-dependent protein kinase-I
We thank Dr. Hayasaka and Ms. Suzuki for their assistance with the MS/MS analyses. This work was supported in part by grants from the programs Grants-in-Aid for Scientific Research (B) and (C), a Grant-In-Aid for Young Scientists A, Grant-in-Aid for Scientific Research on Innovative Areas, and Challenging Exploratory Research from Japan Society for the Promotion of Science, by the Science Research Promotion Fund of the Japan Private School Promotion Foundation, Research Grants for PRESTO and SENTAN, and by Japan Foundation of Applied Enzymology.
Conflict of interest
The authors have declared no conflict of interest.
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