Anal Bioanal Chem (2014) 406:1387–1396 DOI 10.1007/s00216-013-7535-4

RESEARCH PAPER

Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry Jingshan Lu & Ikuko Yao & Masahito Shimojo & Tayo Katano & Hitoshi Uchida & Mitsutoshi Setou & Seiji Ito

Received: 1 September 2013 / Revised: 29 October 2013 / Accepted: 22 November 2013 / Published online: 23 January 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract 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 PKGIα, 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 PKGIα; 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.

Published in the topical collection Biomedical Mass Spectrometry with guest editors Mitsutoshi Setou, Toshimitsu Niwa, and Akira Ishii.

L -arginine

Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7535-4) contains supplementary material, which is available to authorized users. J. Lu : I. Yao : M. Shimojo : T. Katano : H. Uchida : S. Ito (*) Department of Medical Chemistry, Kansai Medical University, Hirakata, Osaka 573-1010, Japan e-mail: [email protected] I. Yao Department of Optical Imaging, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan M. Setou Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan

Keywords MALDI-TOF MS . NanoLC–MS/MS . 3-Nitrotyrosine . Peroxynitrite . Protein kinase G-Iα Abbreviations 3-NT 3-Nitrotyrosine cGMP Guanosine 3′,5′-cyclic monophosphate NO Nitric oxide PKG-I cGMP-dependent protein kinase-I

Introduction Nitric oxide (NO), a labile and free-radical gas, is produced from and oxygen and plays important roles in a wide variety of physiological and pathophysiological processes such as neurotransmission, regulation of vascular tone, and mediation of immune responses [1–3]. NO activates soluble guanylate cyclase and increases the generation of guanosine 3′,5′-cyclic monophosphate (cGMP) [4]. Although cGMP modifies several intracellular processes including activation of protein kinases, ion channels, and phosphodiesterases, a primary action of elevated cGMP levels is the stimulation of cGMP-dependent protein kinase-I (PKG-I) in the nervous system as well as in the vascular system [5, 6]. PKG-I exists in two isoforms, designated α and β [5]. The highest levels of PKG-Iα activity and expression have been detected in cerebellar Purkinje cells [7, 8]. High levels of PKG-Iα are also present in nociceptive neurons of the dorsal root ganglia and in the dorsal horn of the spinal cord [9, 10]. Many

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studies have demonstrated that the NO/cGMP signaling pathway is present in neurons of the spinal cord and contributes to the development of hyperalgesia in models of acute and chronic pain [11–13] and to spinal motor neuron death, which is prevented by NO-stimulated cGMP synthesis [14]. Since target proteins of PKG-I have not been assigned in the spinal cord [9], the mechanisms through which NO mediates its hyperalgesic effects and neuron death are not completely understood. In addition to the classical cGMP/PKG pathway, NO participate in non-classical pathway, post-translational modification called nitration and S -nitrosylation. In nitration, tyrosine residues of protein are modified thorough the incorporation of a nitro group (− NO2) to the phenolic ring of tyrosine residues to form a 3-nitrotyrosine residue (3-NT), which represents a common consequence of oxidative stress associated with various pathological conditions and natural aging [15–18]. NO also directly modifies sulfhydryl residues of protein through S -nitrosylation, which has emerged as an important posttranslational protein modification [19–22]. The presence of nitration and S-nitrosylation can compromise the function and structure of proteins including PKG-Iα itself and affect their biological half-life. In fact, increased production of NO results in the impairment of PKG kinase activity through its tyrosine nitration in caveolin-knockout mice [23]. Exposure of cultured cells and recombinant PKGIα to an NO donor and peroxynitrite produces PKG-Iα nitration and reduces kinase activity [24]. Peroxynitrite is an oxidant and nitrating agent formed through the interaction between free-radical precursor molecules NO and superoxide, the latter of which is a potent oxidant involved in tissue damage. A recent study using mutated PKG-Iα and immunoprecipitation of PKG-α expressed in cultured cells suggested that peroxynitrite attenuates PKG-Iα activity via the nitration of Y345 and Y549 of this enzyme [23]. However, confirmation by mass spectrometry is needed for interpreting the mutation data, because it was unclear how the replacement of tyrosine residues with phenylalanine may have altered the overall structure of PKG-Iα [24]. Although attempts at purifying PKG-Iα in E. coli have been unsuccessful due to insolubility and proteolysis [25], the active enzyme has been successfully purified by the use of a baculovirus-mediated expression system in Sf9 insect cells [26]. In the present study, we nitrated purified recombinant PKG-Iα expressed in Sf9 cells by peroxynitrite and employed a proteomic approach to determine the sites of nitration in relation to PKG-Iα activity.

PKG-Iα/β (H-100) antibodies were obtained from Upstate Biotech (Lake Placid, NY, USA) and Santa Cruz Biotech (Santa Cruz, CA, USA), respectively. Histone IIA and peroxynitrite were purchased from Sigma-Aldrich (St. Louis, MO, USA). (±)-(E)-4-Methyl-2-[(E )-hydroxyimino]-5-nitro6-methoxy-3-hexenamide (NOR1) was supplied by Dojindo (Kumamoto, Japan). Other reagents were of reagent grade.

Material and methods

Detection of nitration and S-nitrosylation of PKG-Iα

Materials

Purified wild-type and mutated PKG-Iα were incubated with or without peroxynitrite or NOR1 for 10 min at 30 °C. The reaction mixtures were subjected to 7.5 % SDS-PAGE, and proteins were transferred electrophoretically to a PVDF

[γ- 32 P]ATP (>3,000 Ci/mmol) was purchased from PerkinElmer NEN (Boston, MA, USA). Anti-3-NT and anti-

Construction of PKG-Iα cDNA plasmid (pTriEX-4-PKG-Iα) The construct containing PKG-Iα fused to a His-tag at the N-terminus was generated by PCR amplification using PKG cDNA (RIKEN Fantom clone, D130073L22) as a template. PCR was performed by using KOD Plus Ver. 2 (TOYOBO, Shiga, Japan), and the PCR product was cloned into the BamHI/ HindIII sites of the pTriEX-4 plasmid (Novagen, Darmstadt, Germany). Tyrosine residues at 71, 141, 212, 336, and 345 in the plasmid pTriEX-4-PKG-Iα were mutated to phenylalanine by site-directed mutagenesis according to the manufacturer's instructions. Four mutations, at Y71, Y141, Y212, and Y336 (Y4F), were simultaneously introduced into the plasmid in the same manner as used for the single mutation. The plasmids were confirmed by DNA sequencing. Expression and purification of wild-type and mutant PKG-Iα For production of recombinant baculovirus, Sf9 insect cells were co-transfected with the plasmid pTriEX-4-PKG-Iα and BacMagic™ DNA (Novagen) according to the manufacturer's protocol. The lysates of Sf9 cells prepared at 72 h postinfection were purified on His-Trap HP column (1 ml) equilibrated with the homogenization buffer (20 mM Tris–HCl, pH 7.5, containing 1 mM DTT and 50 mM NaCl) and eluted with a linear gradient of imidazole from 0 to 0.5 M. The fractions containing recombinant PKG-Iα were pooled and applied to a HiTrap Q FF column (1 ml) equilibrated with 50 mM HEPES buffer (pH 6.5) containing 2 mM EDTA and 1 mM DTT and eluted with a linear gradient of NaCl from 0 to 1 M. PKG-Iα purity in these samples was analyzed by 7.5 % SDS-PAGE and Coomassie Blue staining. More than 1 mg of purified enzyme was obtained by this two-step chromatography, and the purity of the recombinant enzyme was more than 95 %. The recombinant enzyme was maximally activated by 100 nM cGMP. Mutant PKG-Iα proteins were also expressed in Sf9 cells and purified in the same manner as were the wildtype PKG-Iα.

Nitrated tyrosine residues of protein kinase G-Iα

membrane. The membrane was incubated at 4 °C overnight with rabbit anti-3-NT (1 μg/ml; 1:1,000) or anti-PKG-Iα/β (1:1,000) antibody and then for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Zymed) or goat anti-mouse IgG (1:10,000; GE Healthcare UK, Little Chalfont, Buckinghamshire, UK). The immunoreactivity was detected by using Enhanced Chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). The intensity of nitration was quantified by use of imageJ and normalized by that of PKG-Iα. S-Nitrosylation was detected by use of the biotin-switch method, as described previously using antibiotin antibody conjugated HRP (1:1,000) [27]. MALDI-TOF MS and nanoLC-ESI-QTOF MS/MS Samples treated with or without 3 mM peroxynitrite were applied to a 7.5 % gel for SDS-PAGE. After the gel had been stained by 2D-SILVER STAIN II (Cosmo Bio, Tokyo, Japan), PKG-Iα bands with Mr of 75 kDa were excised from the gel, destained, and in-gel digested as described previously [27, 28]. For single mass analyses, digested peptides were analyzed by use of a Shimadzu mass microscope (Kyoto, Japan) equipped with a matrix-assisted laser desorption/ionization chamber, a QIT, and a TOF instrument [29]. Mass peaks were picked up from the spectrum, and the finger mass printing analyses were processed by using the Mascot search engine (Matrix science, http://www.matrixscience.com/). For the tandem mass analyses, the hybrid liquid chromatography-tandem mass spectrometry system, QSTAR XL (AB SCIEX, Framingham, MA, USA), was used. The mobile phases used in this experiment were water/AcCN/ formic acid (98:2:0.1, v /v /v ) as solution A, and water/ AcCN/formic acid (10:90:0.1, v/v/v) as solution B. A linear gradient was carried out from 5 to 50 % of solvent B at a flow rate of 0.5 μl/min. The MS/MS analyses were performed automatically, and the peptide sequences were processed by using Mascot search engine and ProteinPilot™ (AB SCIEX). Determination of PKG-Iα activity Kinase activities of wild-type and mutant PKG-Iα were determined by measuring the incorporation of 32 P from [γ-32P]ATP into histone IIA. The standard reaction mixture (20 μl) contained 0.4–1.0 ng of purified recombinant PKG-Iα, 0.5 μg histone IIA, 10 μM cGMP, 20 mM MgCl2, and 1 mM 3-isobutyl-1-methylxanthine in 20 mM Tris–HCl (pH 7.5). The reaction was initiated by the addition of 1 μM ATP (1 μCi), and incubated for 5 min at 30 °C. The reaction was terminated by the addition of 5 μl of 5× SDS buffer, after which the reaction mixture was subjected to 17.5 % SDSPAGE. The [32P] incorporated into histone IIA was counted by using a Packard Scintillation counter model 5300.

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Statistics Data were presented as the mean±SD and analyzed by oneway ANOVA. Statistical significance was further examined by performing Student's t test using a Statview software program. P

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 target...
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