Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry.
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.
Department of Medical Chemistry, Kansai Medical University, Hirakata, Osaka, 573-1010, Japan.
Jingshan
J
Lu
Ikuko
I
Yao
Masahito
M
Shimojo
Tayo
T
Katano
Hitoshi
H
Uchida
Mitsutoshi
M
Setou
Seiji
S
Ito
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009566:Nitrates / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis*; D030421:Peroxynitrous Acid; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D061987:Sf9 Cells; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics
D009566:Nitrates; D010446:Peptide Fragments; D011994:Recombinant Proteins; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D014357:Trypsin