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Persistent Activation of cGMP-dependent Protein Kinase by a Nitrated Cyclic Nucleotide Via Site-specific Protein S-Guanylation Soichiro Akashi, Khandaker Ahtesham Ahmed, Tomohiro Sawa, Katsuhiko Ono, Hiroyasu Tsutsuki, Joseph R Burgoyne, Tomoaki Ida, Eiji Horio, Oleksandra Prysyazhna, Yuichi Oike, Mizanur Md. Rahaman, Philip Eaton, Shigemoto Fujii, and Takaaki Akaike Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00774 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 24, 2016
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Biochemistry
Biochemistry: Article
Persistent Activation of cGMP-dependent Protein Kinase by a Nitrated Cyclic Nucleotide Via Site-specific Protein S-Guanylation
Soichiro Akashi§1, Khandaker Ahtesham Ahmed‡1,2, Tomohiro Sawa‡¶*, Katsuhiko Ono‡, Hiroyasu Tsutsuki‡, Joseph R. Burgoyne†, Tomoaki Ida§, Eiji Horio‖, Oleksandra Prysyazhna†, Yuichi Oike‖, Mizanur Md. Rahaman§, Philip Eaton†, Shigemoto Fujii§, and Takaaki Akaike§
From the §Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan ‡
Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University,
1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan ¶
Precursory Research for Embryonic Science and Technology, Japan Science and
Technology Agency, Kawaguchi, Saitama 332-0012, Japan †
Department of Cardiology, Cardiovascular Division, King’s College London, The Rayne
Institute, St Thomas’ Hospital, London, SE1 7EH, UK ‖
Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto
University, Honjo 1-1-1, Kumamoto 860-8556, Japan
*To whom correspondence should be addressed: Professor Tomohiro Sawa, Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University,, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan.
E mail:
[email protected] 1 ACS Paragon Plus Environment
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1
These authors contributed equally to this work.
2
Present address: Institute for Research and Medical Consultation, University of Dammam,
Dammam 1982-31441, Kingdom of Saudi Arabia.
Funding This work was supported in part by Grants-in-Aid for Scientific Research and Grants-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Area) from the Ministry of Education, Sciences, Sports and Technology, Japan; a grant from the Japan Science and Technology Agency Precursory Research for Embryonic Science and Technology program; and a Grants-in-Aid from the Japan Society for the Promotion of Science Fellows; The Naito Foundation.
ABBREVIATIONS 8-AET-cGMP, 8-(2-aminoethyl)thioguanosine-cGMP; 8-bromo-cGMP, 8-bromoguanosine 3′,5′-cyclic monophosphate; 8-nitro-cGMP, 8-nitroguanosine 3′,5′-cyclic monophosphate; DTT, dithiothreitol; ESI, electrospray ionization; iNOS, inducible isoform of NO synthase; LC, liquid chromatography; MLC, myosin light chain; MS/MS, tandem mass spectrometry; PDE, phosphodiesterase; PKG, cGMP-dependent protein kinase; RNOS, reactive nitrogen oxide species; ROS, reactive oxygen species; Rp-8-bromo-cGMPS, 8-bromoguanosine 3′,5′-cyclic monophosphorothioate, Rp-isomer; RP, reverse phase; sGC, soluble guanylate cyclase; TOF, time-of-flight; VASP, vasodilator-stimulated phosphoprotein.
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ABSTRACT 8-Nitroguanosine 3′ ′,5′ ′-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of guanosine 3′ ′,5′ ′-cyclic monophosphate (cGMP) formed endogenously under conditions associated with production of both reactive oxygen species and nitric oxide. It acts as an electrophilic second messenger in regulation of cellular signaling by inducing a post-translational modification of redox-sensitive protein thiols via covalent adduction of cGMP moieties to protein thiols (protein S-guanylation).
Here, we
demonstrate that 8-nitro-cGMP potentially S-guanylates thiol groups of cGMP-dependent protein kinase (PKG), the enzyme that serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses. S-Guanylation of PKG was found to occur in site specific manner; Cys42 and Cys195 were the susceptible residues among 11 Cys residues.
Importantly, S-guanylation at
Cys195, which locates in high affinity cGMP binding domain of PKG, causes persistent enzyme activation as determined by in vitro kinase assay as well as by organ bath assay. In vivo, S-guanylation of PKG was demonstrated to occur in mice without any specific treatment, and was significantly enhanced by lipopolysaccharide administration. These findings warrant further investigation for physiological and pathophysiological roles of S-guanylation-dependent persistent PKG activation.
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Accumulating evidence has emerged that ROS act as signaling molecules to regulate diverse cellular events such as cell proliferation, apoptosis, and senescence.1-6
ROS can directly
react with redox-sensitive protein thiols to cause oxidative modifications including the formation of sulfenic acid (-SOH) and disulfide (-S-S-) bonding in proteins.5,7,8
Those
redox-dependent post-translational modifications (PTMs) of protein thiols are involved in regulation of ROS signaling by affecting protein structures and functions via multiple mechanisms such as activation/inactivation of enzymes, modulation of protein-protein interactions, alteration of protein localization, and so forth.7,8 ROS react with cellular components to produce chemically reactive electrophilic metabolites such as lipid peroxidation products and nitrated fatty acids.4 Those electrophiles act as second messengers for ROS signaling via induction of alkylation of redox-sensitive protein thiols.4,6,8-11
Environmental as well as food-derived electrophiles also cause alkylation of
redox-sensitive proteins, hence, resulting in modifications of endogenous ROS signaling.6 8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is the first identified endogenous derivative of guanosine 3′,5′-cyclic monophosphate (cGMP) formed under production of both ROS and nitric oxide (NO).9,12-15
Owing to its electron withdrawing
nature of aromatic nitro-compounds, 8-nitro-cGMP functions as an electrophile and causes alkylation of redox-sensitive protein thiols.9,12,13 This PTM is called “protein S-guanylation”.9,12,13 In the reaction of S-guanylation, the nitro group of 8-nitro-cGMP is replaced by a thiol group of cysteine residues, with the release of nitrite ion, resulting in the formation of irreversible cGMP adducts.
Our previous studies revealed that 8-nitro-cGMP
modulates diverse ROS signaling pathways by acting as a ROS second messenger, depending on the S-guanylation targets.
For instance, 8-nitro-cGMP is capable of inducing antioxidant
gene expression, cellular senescence, and mitochondrial permeability transition pore opening, via S-guanylation of Keap112,14, H-Ras16, and mitochondrial heat shock proteins17. cGMP-dependent protein kinases, also commonly called protein kinase G (PKG), are kinases which phosphorylate a variety of substrate proteins such as PKGs themselves, myosin phosphatase small regulatory subunit, phosphodiesterases (PDEs), and vasodilator-stimulated phosphoprotein (VASP).18
Phosphorylation of those substrates induces diverse cellular
signaling, resulting in regulation of a variety of tissue responses ranging from smooth muscle relaxation, neuronal synaptic plasticity, to platelet functions.
In the resting state, catalytic
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site of PKG is covered by autoinhibitory domain that acts as pseudosubstrate to limit the access of substrate proteins.19
The binding of cGMP to PKG results in a conformational
change in structure that exposes the catalytic site, leading to increased substrate phosphorylation.
This type of PKG activation is referred to as a cGMP-dependent
mechanism for PKG activation.
Alternatively, thiol oxidation caused by oxidants such as
hydrogen peroxide and transition metals leads to enzyme activation even in the absence of cGMP.20,21 In this mechanism, oxidizing agents induce inter- or intra-subunit disulfide formation in PKG, resulting in conformational change leading increased catalytic activity independently of cGMP.
For hydrogen peroxide-induced PKG1α activation, Cys42
participates to inter-subunit disulfide formation.21
These notions suggest that PKG
activation may be controlled in stimuli-dependent manner. We previously found that 8-nitro-cGMP activated PKG in vitro.12,16
Because
8-nitro-cGMP can behave as a cGMP derivative, it may activate PKG via cGMP-dependent mechanism. activation.
Alternatively, thiol modifications of PKG by 8-nitro-cGMP may affect enzyme To clarify molecular mechanisms how 8-nitro-cGMP activate PKG, we
investigated the interaction between 8-nitro-cGMP and PKG during enzyme activation, particularly focusing on the occurrence of PKG S-guanylation.
Roles of PKG S-guanylation
on enzyme activation were determined by means of in vitro kinase assay.
Effects of
8-nitro-cGMP on vascular response, one of major biological responses associated with PKG activation, was examined by means of organ bath assay.
We also tried to verify whether
PKG could be the target for S-guanylation in vivo.
MATERIALS AND METHODS Materials. 8-Nitro-cGMP was prepared according to the method previously reported.12,14 8-Bromoguanosine 3′,5′-cyclic monophosphate (8-bromo-cGMP), cGMP, and LPS from Escherichia coli 0111:B4 were obtained from Sigma-Aldrich (Saint Louis, MO).
8-Bromoguanosine 3′,5′-cyclic monophosphorothioate, Rp-isomer
(Rp-8-bromo-cGMPS), was purchased from BioLog (Bremen, Germany). was from Nacalai Tesque (Kyoto, Japan).
Phenylephrine
Recombinant human PKG1α and PKG1β
(N-terminal 6His-tagged, recombinant, full-length PKG1α and PKG1β, expressed by baculovirus in the Sf21 insect cell line) was from Merck Millipore (Billerica, MA).
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Isotope-labeled 8-nitro-cGMP (8-15NO2-cGMP) and cGMP (c[15N5]GMP) were prepared as reported previously.12,14
Other reagents were of the highest grade commercially available
and were used without further purification.
Rabbit polyclonal antibody specific for protein
S-guanylation was generated as described previously.12,14 Preparation of Recombinant PKG. Recombinant human PKG1α was expressed by using the BacPAK Baculovirus Expression System (Clontech, Mountain View, CA). The plasmid pBacPAK9-FLAG PKG1α with N-terminal FLAG was constructed by subcloning the PCR fragment containing the FLAG PKG1α region amplified from pAdTrack-CMV-FLAG PKG1α.21
The Cys-to-Ser mutants of PKG1α (C42S, C195S,
C42S/C195S) were constructed via PCR mutagenesis from pBacPAK9-FLAG PKG1α. The sf21 cells were co-infected with mutant FLAG-tagged PKG1α plasmids and BacPAK6 viral DNA, and these cells were cultured in BacPAK complete medium (Clontech) at 27 °C for 5 h. Fresh BacPAK complete medium was added at 5 h, and incubation continued for another 72 h at 27 °C. The culture supernatant, which contained recombinant PKG1α-expressing virus, was then harvested.
After isolation of the recombinant PKG1α-expressing virus by means
of the plaque assay, sf21 cells were infected with isolated recombinant PKG1α-expressing virus and were cultured for 60 h. were sonicated.
Virus-infected sf21 cells were collected with PBS and
Cell lysates were mixed with anti-FLAG M2 affinity gel (Sigma-Aldrich)
and were then washed with PBS three times.
Recombinant PKG1α proteins were eluted by
using 3X FLAG peptide solution (Sigma-Aldrich). In Vitro S-Guanylation of PKG1α α by 8-Nitro-cGMP. PKG1α (100 ng) was reacted with 8-nitro-cGMP in 100 mM Tris-HCl (pH 7.4) at 37 °C.
In separate experiments,
PKG1α was preincubated with 100 µM Rp-8-bromo-cGMPS for 30 min, followed by reaction with 200 µM 8-nitro-cGMP.
The resultant protein preparations were subjected to
Western blotting and liquid chromatography (LC)-time-of-flight (TOF)-tandem mass spectrometry (MS/MS) analyses, as described below.
To identify the S-guanylation sites,
wild-type and Cys mutants of recombinant PKG1α were reacted with 200 µM 8-nitro-cGMP in 100 mM Tris-HCl buffer (pH 8.4) at 37 °C for 180 min.
In separate experiments, PKG1α
was reacted with 8-nitro-cGMP in the presence of 10 mM dithiothreitol (DTT) in 100 mM Tris-HCl (pH 7.4) at 37 °C.
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Western Blotting. Proteins were separated by using SDS-PAGE and were then transferred to a PVDF membrane.
Blots were blocked with 5% nonfat skim milk followed
by incubation with one of the following antibodies: anti-S-guanylation12,14, anti-PKG (Santa Cruz Biotechnology, Dallas, TX), anti-iNOS (BD Biosciences, San Jose, CA), anti-β-actin (Santa Cruz Biotechnology), anti-phospho-VASP (Cell Signaling Technology), anti-VASP (Alexis Biochemicals), or anti-GAPDH (Santa Cruz Biotechnology).
After incubation of
the samples with adequate second antibodies conjugated with HRP, immunoreactive bands were detected by using a chemiluminescence reagent (ECL Plus; GE Healthcare Bio-Sciences Corp., Piscataway, NJ) with a luminescent image analyzer (LAS1000UV; Fujifilm, Tokyo, Japan.).
For quantification of immunoreactive bands, densitometric
analyses were performed with the signal intensity of Western blotting images measured by using Image J software (National Institute of Health, USA). In Vitro Kinase Assay. PKG kinase activity was measured according to the literature.16 Briefly, 1 µg/ml PKG1α (Millipore) or the Cys mutants C42S or C195S or the C42S/C195S double mutant was incubated with cGMP or 8-nitro-cGMP at different concentrations in 20 mM Tris-HCl (pH 7.4) containing 16 mM MgCl2, 10 mM DTT, 0.1 mM ATP, and 60 µM Glasstide (Merck Millipore) at 37 °C for 30 min, followed by kinase activity determination with the ADP-Glo Kinase assay kit (Promega, Madison, WI) according to the manufacturer’s protocol.
Non-linear regression isotherm and Hill plot analyses were performed to
determine Hill coefficient using GraphPad Prism 6.0. In separate experiments, unbound free cGMP derivatives were separated from active PKG1α mixtures by using ultracentrifugation tubes (Ultra Centricon, cutoff of 30 kDa, Millipore).
In brief, after reacting PKG1α solutions with cGMP derivatives, they were
added to the ultracentrifugation tubes with 0.1% bovine serum albumin in 100 mM Tris-HCl (pH 7.4) to avoid nonspecific absorption of the enzyme to the tubes.
PKG1α solutions were
then subjected to ultrafiltration with repeated washing (six times) with 0.1% bovine serum albumin in 100 mM Tris-HCl (pH 7.4) to remove unbound cGMP derivatives (10,000 × g for 10 min, 4 °C).
Finally, PKG1α was recovered from the filter membranes by adding 50 µl of
100 mM Tris-HCl (pH 7.4) and was then used for the kinase assay.
Protein recovery after
desaltation was confirmed by means of Western blotting against PKG.
Amounts of PKG
subjected for enzyme assay were then adjusted based on the band intensities.
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the error range of the determined kinase activities, these assays were performed in the same day. Animal Study. C57BL/6 mice (male, 8 weeks old) were purchased from Kyudo Co. Ltd., (Tosu, Japan).
The animals were maintained and the experiments were conducted according
to the guidelines in the Laboratory Protocol of Animal Handling, Kumamoto University Graduate School of Medical Sciences.
For the endotoxin shock model, mice were injected
i.p. with E. coli LPS at a dose of 32 mg/kg. Myography. Vascular rings (5 mm) were isolated from the thoracic aorta of the mice and were mounted in a tension myograph (Model MTOB-2Z, Lab Support, Suita, Japan), stretched to the optimal pretension conditions of 1.0 g.
The aortic rings were bathed in
Krebs-Henseleit buffer, pH 7.6 (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 25 mM NaHCO3, 10 mM glucose), maintained in 37 °C, and gassed with 95% O2 and 5% CO2.
Vasotone measurements of the aortic rings were made
essentially as described previously21,22, by determining the responses of phenylephrine-contracted vessels to a number of agents.
The aortic vessels were treated
with 8-nitro-cGMP (200 µM) or 8-bromo-cGMP (200 µM) for 2 h followed by monitoring of the vasoconstrictive response to phenylephrine with or without washing the organ bath with Krebs-Henseleit buffer.
These vasoconstrictive responses were then recorded by using a
force transducer data acquisition system (PowerLab, Chart v5, ADInstruments, Colorado Springs, CO). LC-Electrospray Ionization (ESI)-QQQ-MS/MS Quantitation of 8-Nitro-cGMP. The human monocytic cell line, THP-1 cells (4.0 × 106 cells) were incubated with 10 µM 8-nitro-cGMP in DMEM at 37°C for 30 min.
Cells were then washed with PBS and
collected and homogenized in 1 ml of ice-cold methanol containing 2% acetic acid.
After
the homogenate samples were centrifuged at 10,000 × g at 4 °C for 10 min, the resultant supernatants were subjected to solid-phase purification of cGMP derivatives with the Oasis WAX cartridge (Waters, Milford, MA).
After the cartridge was washed with methanol,
cGMP derivatives were collected in the eluate with 1 ml of methanol containing 15% aqueous ammonia.
Eluted samples were dried in vacuo and then redissolved in distilled
water (0.2 ml). cGMP derivatives extracted from THP-1 cells were quantitated by means of
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LC-ESI-QQQ-MS/MS.
LC-ESI-QQQ-MS/MS was performed with an Agilent 6430 Triple
Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA), after reverse-phase (RP)-HPLC on a YMC-Triart C18 column (50 × 2.0 mm inner diameter; YMC Co., Ltd., Kyoto, Japan), with a linear 1-60% methanol gradient for 12 min in 0.1% formic acid at 40 °C. The total flow rate was 0.2 ml/min, and the injection volume was 50 µl.
Ionization
was achieved by using electrospray in the positive mode, with the spray voltage set at 4000 V. Nitrogen was the nebulizer gas, and the nebulizer pressure was set at 50 psi.
The
desolvation gas (nitrogen) was heated to 350 °C and was delivered at a flow rate of 10 L/min. For collision-induced dissociation, high-purity nitrogen was the collision gas at a pressure of 0.05 MPa. cGMP derivatives were quantified by using the multiple-reaction monitoring mode as reported previously.19
The peak widths of precursors and product ions were
maintained at 0.7 atomic mass unit at unit-unit resolution in the multiple-reaction monitoring mode.
Isotope-labeled 8-15NO2-cGMP and c[15N5]GMP (final concentration of each: 200
nM) were used as internal standards. Intracellular concentrations of 8-nitro-cGMP were calculated by dividing total amount of 8-nitro-cGMP detected with free cytoplasmic volume as mentioned below.
According to the
literature, up to half the volume of the cell is occupied by membrane-bounded organelles including nucleus, so that remaining 50% corresponds to free volume available to reactants diffusing in the cytoplasm.23
For 4.0 × 106 of THP-1 cells, free cytoplasmic volume was
estimated to be 6 µl. Determination of Partition Coefficient for cGMP Derivatives. The octanol/water partition coefficients (logPo/w) of cGMP derivatives were determined by shake-flask method.25 octanol.
Aqueous media (20 mM Tris-HCl [pH7.4] or 0.1 M HCl) were pre-saturated with Similarly, octanol was pre-saturated with the aqueous media.
Aqueous media
containing 100 µM cGMP derivatives were mixed with equal volume of octanol, then shake vigorously by using tube shaker (Bio Shaker M-BR-022UP; Taitec, Saitama, Japan) at 25 °C for 1 h.
After centrifuged at 10,000 × g at 25 °C for 2 min, octanol phase was collected and
diluted 100 times by 0.1 % formic acid.
The concentrations of 8-nitro-cGMP in octanol
phase (Co) were determined by means of LC-MS/MS as mentioned above. calculated using the following equation:
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LogPo/w were
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logPo/w = log Co/Cw = log Co/(100 µM – Co)
Isolation of PKG from Mouse Heart Tissues by Using cGMP-immobilized Agarose. To study the endogenous S-guanylation of PKG1α, we purified PKG1α from heart tissues of control and LPS-treated mice via the cGMP-agarose pulldown method with 8-(2-aminoethyl)thioguanosine-cGMP-agarose (8-AET-cGMP-Agarose) (BioLog) performed before Western blot analysis.26
Mouse heart homogenates (5 mg) were incubated with
8-AET-cGMP-Agarose for 2 h at 4 °C under rotation.
Nonspecifically bound proteins were
removed by rinsing three times with a wash buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, 5 mM sodium pyrophosphate, 1% Triton X-100), followed by boiling in SDS sample buffer at 95 °C for 5 min. cGMP-binding proteins were then separated by using SDS-PAGE followed by Western blot or proteomic analysis as mentioned below. Proteomic Analysis to Identify Proteins and Determine S-Guanylation Sites. Coomassie Brilliant Blue- or silver-stained gels were subjected to in-gel digestion, followed by peptide extraction and then proteomic analysis by means of LC-TOF-MS/MS. digestion and peptide extraction were performed according to the literature.27
In-gel
For
identification of S-guanylation sites, S-guanylated peptides were enriched by using immunoaffinity columns as described previously.16,17
These enriched peptide samples were
analyzed in an ESI-Q-TOF tandem mass spectrometer (6510; Agilent Technologies) with an HPLC chip-MS system comprising a nano pump (G2226; Agilent Technologies) with a four-channel microvacuum degasser (G1379B; Agilent Technologies), a microfluidic chip cube (G4240; Agilent Technologies), a capillary pump (G1376A; Agilent Technologies) with degasser (G1379B; Agilent Technologies), and an autosampler with thermostat (G1377A; Agilent Technologies).
MassHunter software (version B.02.00; Agilent Technologies) was
used to control all modules.
A microfluidic RP-HPLC chip (Zorbax 300SB-C18; 5 µm
particle size, 75 mm inner diameter, and 43 mm length) was used for peptide separation. Gradient nano flow at 600 nl/min was generated via the nano pump, with the mobile phase of 0.1% formic acid in MS-grade water (solvent A) and 0.1% formic acid in acetonitrile (solvent B).
The gradient was 5-75% solvent B in 9 min.
A capillary pump was used for loading
samples, at 4 µl/min, with a mobile phase of 0.1% formic acid.
The Agilent ESI-Q-TOF
instrument was operated in the positive ionization mode (ESI+) with the ionization voltage
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being 1850 V and the fragmentor voltage being 175 V at 300 °C.
Protonated molecular ions
were fragmented in the auto-MS/MS mode, starting with a collision energy voltage of 3 V that was increased by 3.7 V per 100 Da.
The m/z ranges selected were 300-2400 Da in the
MS mode and 59-3000 Da in the MS/MS mode.
The data output was one full mass
spectrum, with three fragmentation patterns per mass spectrum, every 250 ms.
The three
highest peaks of an MS spectrum were chosen for fragmentation. Mass lists as Mascot generic files were created and used as the input for Mascot MS/MS ion searches of the National Center for Biotechnology Information nonredundant (NCBI nr) database via the Matrix Science Web server Mascot version 2.2.
The default search
parameters used were the following: enzyme, trypsin; maximum missed cleavage, 1; variable modifications, carbamidomethyl (Cys) and cGMP (Cys); peptide tolerance ± 1.2 Da; MS/MS tolerance ± 1.2 Da; peptide charge, 2+ and 3+; instrument, ESI-Q-TOF.
For positive
identification, the resultant score of [-10 × log(P)] had to be lower than the significance threshold level (p < 0.05). Phosphorylation of VASP in Mouse Heart Tissues. Phosphorylation of VASP was analyzed as an index of PKG activation.
At 12 h after the LPS injection, mouse heart
tissues were collected and homogenized in 15 volumes of buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail (cOmplete, Mini, EDTA-free; Roche).
After incubation for 30 min on ice, the resulting homogenate was centrifuged at
10,000 × g at 4 °C for 30 min to allow sedimentation of tissue debris.
Supernatants (10 µg
of protein each) were subjected to Western blotting for phosphorylation of VASP. The same blots were analyzed with the antibodies anti-VASP to determine the total VASP. Statistical Analysis. All data are given as means ± S.D.
Data for each experimental
condition were acquired from at least three separate experiments.
Student’s t test was used
for statistical analyses.
RESULTS Analysis of PKG S-Guanylation in vitro. We first examined whether S-guanylation of PKG occurs or not during the reaction between 8-nitro-cGMP and PKG in vitro by means of Western blotting.
As shown in Fig. 1A and B, 8-nitro-cGMP treatment clearly induced
S-guanylation of PKG1α in a time- and dose-dependent manner. 11 ACS Paragon Plus Environment
S-Guanylation of PKG1α
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was clearly detected in the presence of excess amount of reducing agent 10 mM DTT (Fig. 1C).
On the other hand, PKG1α S-guanylation was partly inhibited in the presence of a
cGMP analogue Rp-8-bromo-cGMPS (Fig. 1D).
PKG1β, another isoform of PKG1, was
also effectively S-guanylated by 8-nitro-cGMP (Fig. 1E). The sites of S-guanylation in PKG1α was then analyzed. PKG1α was subjected for trypsin digestion.
8-Nitro-cGMP-treated
Peptides bearing S-guanylated cysteine
residues were enriched by affinity chromatography, followed by analyzing their amino acid sequences by tandem mass spectrometry combined with MASCOT database search.
As Fig.
2A shows, two peptides bearing the Cys residues Cys42 and Cys195 were determined to be S-guanylated.
Mass fragmentation patters for those peptides were shown in Supplemental
Tables S1 and S2.
Although PKG1α contains 11 Cys residues, no S-guanylated peptides
except for Cys42 and Cys195 were detected. The S-guanylation sites of PKG1α were further verified by using Cys to Ser mutants positioned at Cys42 (C42S), at Cys195 (C195S), and at both Cys42 and Cys195 (C42S/C195S).
As Fig. 2B clearly illustrates, S-guanylation of PKG1α was markedly
suppressed in the single mutants (C42S, C195S), and S-guanylation signal was decreased to undetectable level in the double mutant C42S/C195S.
Collectively, these data indicate that
S-guanylation occurs selectively at positions of Cys42 and Cys195 among the 11 Cys residues in PKG1α (Fig. 2C). Effects of S-guanylation on PKG Activity. We next performed in vitro kinase assay using recombinant PKGs with or without cysteine residue mutations to clarify the effects of S-guanylation on enzyme activity.
In consistent with our previous findings, 8-nitro-cGMP
was capable of activating wild-type PKG to a similar extent to that caused by native cGMP (Fig. 3A and B).
Apparent kinase activation constants (Ka) were determined to be 110 ± 5
nM and 119 ± 3 nM for native cGMP and 8-nitro-cGMP, respectively.
Non-linear
regression isotherm analyses suggest that both cGMP and 8-nitro-cGMP had Hill coefficient larger than 1, indicating positively cooperative binding towards PKG (Supplementary Fig. S3). It is noteworthy that significant difference between native cGMP and 8-nitro-cGMP was observed when enzyme activity was determined after removal of unbound cGMP and 8-nitro-cGMP from reaction mixtures.
As seen in Fig. 3C, upper panel, 8-nitro-cGMP
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activated PKG1α in a similar extent to that by native cGMP.
However, significantly higher
enzyme activation was determined for 8-nitro-cGMP-treated PKG after desaltation (Fig. 3C, lower panel).
This result suggests that interaction between PKG1α and cGMP depended on
the binding equilibrium, and thus cGMP would dissociate reversibly from PKG1α during desaltation.
In contrast, interaction between PKG1α and 8-nitro-cGMP was not
significantly affected by desaltation possibly due to the formation of stable S-guanylation modification.
In order to clarify whether irreversible activation of PKG by 8-nitro-cGMP is
attributable to S-guanylation or not, enzyme activation was analyzed with wild-type as well as Cys mutants of PKG1α.
As Fig. 3D shows, activation of the C42S mutant by
8-nitro-cGMP remained intact even after desaltation, similar to the effect observed for wild-type PKG1α.
In contrast, the C195S mutant as well as the C42S/C195S double mutant
became sensitive to desaltation in 8-nitro-cGMP-mediated enzyme activation.
These data
strongly suggest that S-guanylation occurring at Cys195 leads to irreversible PKG1α activation caused by 8-nitro-cGMP. Effects of 8-Nitro-cGMP on Vascular Responses against Vasoconstrictor Treatment. It has been well recognized that cGMP-PKG signal plays an important role in regulation of vascular tone via induction of smooth muscle relaxation.
Thus, we hypothesized that
8-nitro-cGMP can induce different vascular responses than cGMP does because of S-guanylation-dependent persistent activation of PKG.
To test this hypothesis, we
investigated the effects of 8-nitro-cGMP on the vascular response to the vasoconstrictor phenylephrine by means of an organ bath assay and mouse aortic rings ex vivo. 8-Bromo-cGMP was used as a membrane-permeable and PDE-resistant cGMP analogue instead of native cGMP. Because of hydrophobic nature of 8-nitro-cGMP as suggested by its logPo/w value (Supplemental Table S3), we first measured whether 8-nitro-cGMP penetrate cell membrane upon extracellular addition of 8-nitro-cGMP using cultured cells.
We found that, when
human monocyte-like THP-1 cells were treated with 10 µM 8-nitro-cGMP for 30 min, intracellular 8-nitro-cGMP was determined to be 104.3 ± 39.8 nM. abundant cellular thiol that affect 8-nitro-cGMP stability in cells.
Glutathione is the most Our in vitro study
suggested that even after incubation of 1 µM 8-nitro-cGMP with 1,000 times excess glutathione (1 mM) in neutral pH buffer at 37 °C for 2 h, approximately 50% of
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8-nitro-cGMP was remained in the reaction solution (Supplemental Fig. S5).
We further
examined whether 8-nitro-cGMP was stably detected in aortic rings upon exposure to exogenously added 8-nitro-cGMP. 8-nitro-cGMP in aortic rings.
As shown in new Supplemental Fig. S5, we could detect
8-Nitro-cGMP in aortic rings S-guanylates tissue proteins as
suggested by Western blotting (Supplemental Fig. S6). We then investigated the effects of 8-nitro-cGMP treatment on vascular responses. Under the condition, 8-nitro-cGMP treatment induced a marked reduction of phenylephrine-dependent vasocontraction of the mouse aorta (Fig. 4A, upper panel). Vascular hyporesponsiveness induced by 8-nitro-cGMP was clearly detected even after removal of 8-nitro-cGMP from the organ bath by washing the aorta with Krebs-Henseleit buffer (Fig. 4A, lower panel).
As seen in Fig. 4B (upper panel), 8-bromo-cGMP treatment
also reduced phenylephrine-dependent vasocontraction of the aorta when 8-bromo-cGMP was added during the assay.
However, in sharp contrast to 8-nitro-cGMP, the effects of
8-bromo-cGMP were completely nullified by removal of 8-bromo-cGMP from the assay medium (Fig. 4B, lower panel).
These data suggest that 8-nitro-cGMP shows prolonged
biological responses against vasculature, possibly via persistent activation of PKG by S-guanylation. Occurrence of PKG S-guanylation in vivo and their augmentation with LPS treatment. We finally investigated the occurrence of PKG S-guanylation in vivo.
Our
previous findings showed that formation of 8-nitro-cGMP requires production of both ROS and NO. route.
To induce in vivo production of ROS and NO, mice were administered LPS via i.p.
With the current LPS treatment protocol, significant iNOS expression in mouse heart
appeared 6 h after LPS treatment, and the iNOS expression level gradually increased until 12 h (Fig. 5A). Western blotting analyses revealed that LPS treatment increased the levels of protein S-guanylation in heart tissues (Fig. 5B). We therefore investigated whether S-guanylation of PKG was also enhanced by LPS treatment.
cGMP-immobilized-agarose gels were used to
extract cGMP-binding proteins including PKG from mouse heart homogenates.
Silver
staining of the electrophoresed gel indicated that several proteins were extracted from these homogenates according to cGMP-binding affinity (Fig. 5C).
Three major bands were
identified as PKG1 (band 1), cAMP-dependent protein kinase type I-alpha regulatory subunit (band 2), and actin (band 3), respectively (Table 1).
Western blotting with anti-PKG
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antibody verified the identity of band 1 as PKG1 (Fig. 5D).
An important result was that
Western blotting clearly showed S-guanylation of PKG1 (Fig. 5E) even without LPS treatment.
Densitometric analyses indicated that LPS treatment significantly enhanced the
extent of PKG1 S-guanylation; the ratios of band intensities for S-guanylated PKG1 to total PKG1 were 0.23 and 0.57 for PBS-treated control and LPS-treated samples, respectively. These data suggest that S-guanylation of PKG indeed occurs in vivo, particularly under excess production of NO and ROS. We finally investigated the phosphorylation of VASP as an index of PKG activation in mice with or without LPS treatment.
As shown in the Fig. 5F,
phosphorylation of VASP was enhanced by LPS treatment compared with PBS-treated control.
Relative intensity for phosphorylated versus total VASP in LPS-treated mice hearts
was approximately 1.8 times higher than that of PBS-treated control, which shows good correlation to the increment of PKG S-guanylation upon LPS treatment as mentioned above.
DISCUSSION In this study, we demonstrated that 8-nitro-cGMP potentially S-guanylates PKG1 in vitro. Mass spectrometric analyses as well as Western blotting with Cys mutants revealed that Cys42 and Cys195 were susceptible residues for S-guanylation.
In vitro kinase assay using
Cys mutants clearly indicated that S-guanylation at Cys195 contributed to the persistent activation of PKG.
As mentioned earlier, PKG1α consists of several functionally different
domains in its structure (Fig. 2C).
At N-terminal, coiled coils promote a parallel
homodimeric configuration, followed by an autoinhibitory (AI) domain and two tandem cGMP binding sites, which cooperatively regulate C-terminal catalytic activity.18,28,29
The
AI domain is flexible hinge segment having a pseudosubstrate sequence and is believed to autoinhibit the C-terminal catalytic center in the enzyme’s dormant state.18,28,29 localized in high affinity cGMP binding site (Fig. 2C).
Cys195 is
It was reported that cGMP binding
to both the high and low affinity domains induces the conformational change necessary for full kinase activity.18,28,29
Our study showed that S-guanylation of PKG1α was partially
inhibited in the presence of Rp-8-bromo-cGMP (Fig. 1).
This may suggest that binding of
8-nitro-cGMP to high affinity domain initially occurs, followed by nucleophilic attack by Cys195 to complete S-guanylation at Cys195, resulting in fixation of cGMP moiety in cGMP high affinity domain to cause persistent full enzyme activation.
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It should be noted that oxidation of Cys195 has been implicated in the cGMP-independent activation of PKG1α.
Landgraf et al. reported that exposure of PKG1α
to divalent cations with positive redox potentials promotes enzyme activation in the absence of cGMP.20
Disulfide bonds formed between Cys117-Cys195 and/or Cys312-Cys518 has
been suggested to involve in such cGMP-independent enzyme activation.20
They also
reported that alkylation by iodoacetamide of Cys residues of PKG1α including Cys195 did not cause enzyme activation, suggesting that simple alkylation including carboxymethylation occurring at Cys195 is not sufficient to activate PKG1α.20
Detailed structural analyses by
Osborne et al. suggested that disulfide bond formation between Cys117-Cys195 may disrupt the interaction between the AI and catalytic center thereby releasing the kinase from a state of autoinhibition.19
In contrast, it seems unlikely that Cys312 is capable of forming a disulfide
bridge with Cys518 from the activation loop in the catalytic domain.
It is thus suggested
that Cys195 plays different roles in PKG1α activation, i.e., disulfide bridge formation with Cys312 or S-guanylation in response to oxidants or 8-nitro-cGMP, respectively. The molecular docking analysis fo PKG1α with 8-nitro-cGMP suggested that 8-nitro-cGMP binds to the high affinity binding pocket identical to that for native cGMP (Supplemental Fig. S8).
From docking analysis, distance between Cys195 and C-8 of
non-covalently attached 8-nitro-cGMP was predicted to be ~21Å.
This suggests that, after
S-guanylation, cGMP moiety derived from 8-nitro-cGMP retained in the high affinity binding pocket, which may contribute to persistent activation of the enzyme.
This speculation
should be supported by more precise structural analysis in future. Cys42, an another site for S-guanylation, is present between coiled coil dimerization domain and AI domain.
It has been suggested that H2O2 treatment induced inter-subunit
disulfide formation between Cys42 of each subunit, which activates PKG1α via induction of conformational change independently cGMP binding.21
Our data showed that S-guanylation
occurring at Cys42, however, did not affect enzyme activation as suggested by the finding that C42S mutant could be activated by 8-nitro-cGMP to the similar extent to that of wild type enzyme. We demonstrated in this study that 8-nitro-cGMP induced S-guanylation not only of PKG1α but also of PKG1β (Fig. 3).
PKG1α and PKG1β are isoforms of PKG1 generated
by the prkg1 gene via alternative splicing.30
These two isoforms differ only in the
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amino-terminal leucine zipper domain.31
With regard to protein S-guanylation, PKG1β
lacks the amino-terminal Cys residue that corresponds to Cys42 of PKG1α, but the other Cys residues are exactly the same as those in PKG1α (Supplemental Fig. S9). This finding suggests that Cys210, which corresponds to the Cys195 of PKG1α, is the site susceptible to 8-nitro-cGMP-mediated S-guanylation. We previously found that 8-nitro-cGMP shows a biphasic effect on vasculature.12
In
aortic rings with intact endothelium, 8-nitro-cGMP at concentrations higher than 10 µM induced relaxation of precontracted vascular strip, whereas 8-nitro-cGMP at concentrations less than 10 µM substantially enhanced vasoconstriction.
8-Nitro-cGMP-dependent
vasoconstriction could be attributable to the redox active property of this molecule: 8-nitro-cGMP can accelerate superoxide production from endothelial NOS via electron uncoupling reactions.
Superoxide thus formed nullified bioavailability of NO.
other hand, 8-nitro-cGMP showed vasorelaxation effect.
On the
This suggests that 8-nitro-cGMP
directly induces vasorelaxation possibly via PKG activation.
In this study, we showed that
vascular relaxation caused by 8-nitro-cGMP was persistently observed suggesting that irreversible activation of PKG1α via S-guanylation may contribute to such persistent vascular relaxation. proteins.
Activated PKG1α would induce vascular relaxation by phosphorylating target Myosin light chain (MLC) phosphorylation is known to be a key determinant of
smooth muscle contractility.32,33
Activated PKG1α promotes MLC dephosphorylation by
activating MLC phosphatase, which results in smooth muscle relaxation.32,33
PKG1β,
however, regulates smooth muscle relaxation by alternative mechanisms.32,33
For instance,
PKG1β activation leads to inhibition of MLC kinase via suppression of calcium release from sarcoplasmic reticulum.32,33 We previously demonstrated that 8-nitro-cGMP S-guanylates the intracellular envelop of invaded bacteria and initiates thereby ubiquitination and autophagy.34
Dey et al. reported
that cGMP down-regulates PKG1 singal, at least in part, via degradation of the enzyme by activating ubiquitin-proteasome pathway.35
Thus, stability and degradation of S-guanylated
PKG1α needs be clarified for further understanding of the action of 8-nitro-cGMP on vascular regulation. As shown in Fig. 5B and Supplemental Fig. S6, multiple proteins were targets for protein S-guanylation during 8-nitro-cGMP stimuli.
Roles of protein S-guanylation
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occurring other than PKG should be investigated, because certain electrophiles are known to induce vasodilation.
For example, curcumin, a naturally occurring phenolic compound
isolated from turmeric, has electrophilic nature, and was found to show relaxant effect on porcine coronary arterial ring segments.36
Such vasodilating effect of curcumin was
suggested to involve activation of endothelium-dependent NO formation.
On the other hand,
endothelium independent vasodilating action of curcumin was also reported, suggesting that vasodilating action of curcumin might be context dependent manner.37
It was also reported
that 4-hydroxynonenal, an electrophile formed during lipid peroxidation, induces relaxation of human cerebral arteries.38
The vasodilating effect of 4-hydroxynonenal was endothelium
dependent, possibly via modulation of cellular signaling in endothelium.
Further study is
warranted to identify the target proteins as well as their functions for vascular tone regulation. Biological levels of 8-nitro-cGMP are regulated by multiple factors.
In cells and
tissues, abundant substrate GTP is first nitrated by biologically relevant nitrating agents.14 Peroxynitrite is a strong nitrating agent formed from the reaction of NO and superoxide radical.39
At physiological pH, peroxynitrous acid, a protonated form of peroxynitrite,
decomposes via hemolysis to give the hydroxyl radical and nitrogen dioxide.
Hydroxy
radical thus formed oxidizes guanine moiety resulting in the formation of guanine cation radical, which secondary reacts with nitrogen dioxide to form 8-nitroguanine. Nitrite-myeloperoxidase-hydrogen peroxide system is another important system to form 8-nitroguanine.
In this system, nitrite is oxidized by myeloperoxidase in the presence of
hydrogen peroxide to form nitrogen dioxide.
Reaction of myeloperoxidase with hydrogen
peroxide also form myeloperoxidase complex I that can oxidize guanine moiety to form guanine cation radical. 8-nitroguanine. guanylyl cyclase.
Finally, guanine radical reacts with nitrogen dioxide to form
8-Nitro-GTP is then converted to 8-nitro-cGMP by the catalytic action of In this study, we found that 8-nitro-cGMP was present in mice tissue
without any specific treatment.
This suggests that basal level production of nitrating agents
may occur under physiological conditions.
This is in consistent with our previous data that
basal level formation of 8-nitro-cGMP was determined even in unstimulated mammalian cultured cells and in mouse tissues.14-16
It is interesting to note that PKG S-guanylation was
also evident under physiological condition (Fig. 5), suggesting that PKG is sensitive target for endogenous protein S-guanylation. Metabolic conversion of 8-nitro-cGMP is important to determine the regulation of 18 ACS Paragon Plus Environment
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S-guanylation-dependent ROS signaling.
We recently identified that reactive cysteine
persulfides potentially react with 8-nitro-cGMP, resulting in conversion of 8-nitro-cGMP to 8-SH-cGMP.40
Because of highly nucleophilic nature of cysteine persulfides, they react
effectively with 8-nitro-cGMP to form unstable cysteine persulfide-cGMP adducts that are easily reduced to form 8-SH-cGMP.
Transsulfuration pathway enzymes cystathione
β-synthase and cystathione γ-lyase contribute, at least in part, to the endogenous formation of cysteine persulfides by catalyzing CS bond cleavage in cystine.40
8-SH-cGMP thus formed
is further metabolized to native cGMP via oxidative desulfuration mediated by biologically relevant oxidants such as H2O2 and peroxynitrite of which formation is pronounced under oxidative stress conditions.16
Finally cGMP is degraded by phosphodiesterases.
In conclusion, this study demonstrated that S-guanylation of PKG at Cys195 leads to persistent activation of the enzyme.
We also showed the in vivo occurrence of PKG
S-guanylation under basal condition as well as endotoxin shock condition.
These findings
warrant further investigation for physiological and pathophysiological roles of S-guanylation-dependent persistent activation of PKG and its downstream signaling cascade.
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Xu, P. H., Long, Y., Dai, F., and Liu, Z. L. (2007) The relaxant effect of curcumin on porcine coronary arterial ring segments, Vascul. Pharmacol. 47, 25-30.
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Dash, J. R., and Parija, S. C. (2013) Spasmolytic effect of curcumin on goat ruminal artery is endothelium independent and by activation of sGC, Res. Vet. Sci. 95, 588-593. 22 ACS Paragon Plus Environment
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Acknowledgments. We thank J. B. Gandy for her editing of the manuscript.
Thanks are
also due to Drs S. Khan, M.H.A. Rahman, F.Y. Wei, and K. Miyata for technical assistance.
Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx.
Figures S1-S9 and Tables S1-S3.
FIGURE LEGENDS
Figure 1.
Western blot analysis of PKG1 S-guanylation in vitro. Dependence on time (A)
and dose (B) of PKG1α S-guanylation by 8-nitro-cGMP.
(A) Recombinant human PKG1α
was reacted with 200 µM 8-nitro-cGMP at 37 °C for the indicated time periods.
PKG1α
was reacted with different concentrations of 8-nitro-cGMP at 37 °C for 180 min in the absence of additive (B), or in the presence of 10 mM DTT (C).
(D) Effect of a cGMP
analogue on PKG1α S-guanylation by 8-nitro-cGMP. PKG1α was reacted with 200 µM 8-nitro-cGMP for 180 min in the presence of various concentrations of Rp-8-bromo-cGMPS. (E) Time-dependent S-guanylation of PKG1β by 8-nitro-cGMP.
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Biochemistry
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Page 24 of 32
PKG1β was reacted with 200 µM 8-nitro-cGMP at 37 °C for the indicated time periods. 100 ng of PKG1 per lane were loaded.
Figure 2.
Full length blot images are in Supplemental Fig. S1.
Site-specific S-guanylation of PKG1α by 8-nitro-cGMP.
(A) LC-TOF-MS/MS
analysis of S-guanylated peptides. S-Guanylation at Cys42 (left panel) and at Cys195 (right panel) of human PKG1α was found.
*C indicates the S-guanylated Cys residue.
mass fragmentation patterns are shown in Supplemental Tables S1 and S2. analysis of PKG1α S-guanylation.
Peptide
(B) Western blot
Wild-type PKG1α and Cys mutant PKG1α were reacted
with 200 µM 8-nitro-cGMP for 180 min, followed by Western blotting. Western blotting confirmed the loading control of PKG1α. S2.
Full length blot images are in Supplemental Fig.
(C) Domain structure of human PKG1α.
Arrows indicate Cys residues.
AI,
autoinhibitory domain.
Figure 3.
Irreversible activation of PKG1α by 8-nitro-cGMP in vitro. Recombinant human
PKG1α was incubated with different concentrations of cGMP (A) or 8-nitro-cGMP (B) for 30 min.
Enzyme activity was then determined by using an in vitro kinase assay with the
PKG substrate peptide Glasstide and [32P]ATP, as described in the text. S.D. (n = 3).
Data are means ±
Results for non-linear regression isotherm analysis and Hill slopes for both
cGMP and 8-nitro-cGMP are shown in Supplemental Fig. S3. PKG1α by 8-nitro-cGMP or cGMP with or without desaltation.
(C) Activation of human Enzyme activity was
determined by means of an in vitro kinase assay. PKG1α was reacted with 200 µM 8-nitro-cGMP or cGMP for 5, 10 and 30 min.
Reaction mixtures were then subjected to
ultracentrifugation to remove free (not covalently bound) cGMP derivatives (desaltation). Data are means ± S.D. (n = 3).
**, p < 0.01 versus non-desaltation.
confirmed recovery of PKG1α after desaltation (inset). Supplemental Fig. S4.
Western blotting
Full length blot images are in
(D) Effects of desaltation on activation by 8-nitro-cGMP of
wild-type PKG1α or Cys mutant recombinant PKG1α.
Data are means ± S.D. (n = 3).
**,
p < 0.01 versus non-desaltation. WT, wild type.
Figure 4.
Induction by 8-nitro-cGMP of sustained hyporesponsiveness of mouse aorta to
phenylephrine.
Mouse aortic rings with intact endothelium were treated with 200 µM
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Biochemistry
8-nitro-cGMP (A) or 8-bromo-cGMP (B) or were untreated (PBS control) in Krebs-Henseleit buffer at 37 °C for 2 h. Vasocontraction induced by different concentrations of phenylephrine was then measured.
In separate experiments, aortic rings were washed with
Krebs-Henseleit buffer before measurement of vasocontraction.
Figure 5.
Identification of endogenous PKG1α S-guanylation in mouse heart.
(A) Time
course of iNOS expression in mouse heart tissues after LPS treatment. C57BL/6 mice received LPS (32 mg/kg) via i.p. injection. S7.
Full length blot images are in Supplemental Fig.
(B) Western blotting of endogenous protein S-guanylation in mouse heart tissues after
LPS treatment for 12 h (left panel).
Relative band intensity to that of GAPDH was
determined by densitometric analysis on three major bands indicated by arrow heads (right panel). (C) silver staining of protein samples of mouse heart homogenates eluted from cGMP-immobilized-agarose.
Three major bands (1-3) noted in the gel were subjected to
in-gel digestion followed by LC-TOF-MS/MS proteomic analyses. the same protein samples as in B, of PKG (D) and S-guanylation (E).
Western blotting, with Arrowheads point to
the molecular size identical to that of PKG1α. (F) Western blotting of phosphorylated levels of VASP in mouse heart tissues after LPS treatment (left panel).
Band intensities of
phosphorylated VASP relative to total VASP were determined by densitometric analysis (right panel).
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Page 26 of 32
TABLE 1 Proteomic identification of proteins in mouse heart homogenates after immunoaffinity purification with cGMP-agarose Sample
Band
Protein name
Score
Protein sequence
Nominal
coverage (%)
mass
620
25
76,302
633
39
43,158
Actin, alpha cardiac muscle 1
190
24
41,992
Actin, cytoplasmic 1
141
21
41,710
cGMP-dependent protein
264
14
76,302
no. PBS control 1
cGMP-dependent protein kinase 1
2
cAMP-dependent protein kinase type I-alpha regulatory subunit
3
LPS treated
1
kinase 1 2
cAMP-dependent protein
685
43
43,158
kinase type I-alpha regulatory subunit 3
Actin, alpha cardiac muscle 1
347
36
41,992
Actin, cytoplasmic 1
283
36
41,710
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Biochemistry
Fig. 1. Akashi et al.
A
0
Time (min)
60
15
180
S-Guanylation
B 8-Nitro-cGMP (µM)
10
50
100
200
!
! ! S-Guanylation
C
8-Nitro-cGMP (µM)
0
0.1
1
10
100 !
S-Guanylation
D Rp-8-Br-cGMPS (µM) 8-Nitro-cGMP (200 µM)
+
1 +
0
15
10 +
100 +
S-Guanylation PKG
E
Time (min) S-Guanylation
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60
180
Biochemistry
Fig. 2. Akashi et al.
A
y10!y9!y8!y7!y6! y1!
y3!
y1! y7!
*C Q S V L P V P S T H I G P R! b2! y2!
y6! b2! y3!
y4! y7!
y10! y9!
1000! (m/z)!
600!
C195S
Wild type
B
y7!
C42S/195S
200!
1000! (m/z)!
600!
y5!
y3!
C42S
200!
y5!y4! y3!y2!
Q *C F Q T I M M R!
y8!
S-Guanylation!
PKG!
C
REGULATORY DOMAIN! 58-72! 1! 40! N!
Dimer!
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
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42!
111! AI!
CATALYTIC DOMAIN!
228! cGMP! high!
344! cGMP! low!
118! 129!158!175! 195!
313!
474!
ATP-! binding!
441! 467!
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670! Substrate-binding!
519!
595!
C!
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Fig. 3. Akashi et al.
C 12000! 10000! 8000! 6000! 4000! 2000!
70000!
PKG!
60000! 50000! 40000!
25000!
30000! 20000! 10000! 5!
Log[cGMP] (M)
6000! 4000! 2000! 0! 0.0001! 0.001!0.01! 1! 10! -9 -8 0.1! -7 -8 -5 100! -4 Control Log[8-Nitro-cGMP] (M)
PKG activity! (counts per minute)
12000!
8000!
10! 30! Time (min)
Desaltation (+)! cGMP! 8-Nitro-cGMP!
14000!
10000!
D
cGMP! 8-Nitro-cGMP!
0!
0! -9 -8 0.1! -7 -6 -5 100! -4 Control 0.0001! 0.001!0.01! 1! 10!
B
No desaltation! cGMP! 8-Nitro-cGMP!
70000!
40000!
cGMP! 8-Nitro-cGMP!
**
30000! 20000! 10000!
Desaltation! –! +!
20000! 15000! **
10000!
**
5000!
PKG!
60000! 50000!
PKG activity! (counts per minute)
14000! PKG activity! (counts per minute)
PKG activity! (counts per minute)
A
PKG activity! (counts per minute)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Biochemistry
**
**
0! 5!
10! 30! Time (min)
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0! WT wt
C42S C195S C42S/! 42 195 195/42 C195S PKG1α
Biochemistry
Fig. 4. Akashi et al.
B 140!
Contraction (% of control)
Contraction (% of control)
A Without washing
120! 100!
PBS 8-Nitro-cGMP
80! 60! 40! 20!
120! 100!
Contraction (% of control)
140!
With washing PBS 8-Nitro-cGMP
80! 60! 40! 20! 0! -9 0.01 -8 -7 -6 Control 0.0001 0.001 0.1 1 Log[Phenylephrine] (M)
-5 10
140! 120! 100!
Without washing PBS 8-Bromo-cGMP
80! 60! 40! 20! 0! -9 0.01 -8 -7 -6 Control 0.0001 0.001 0.1 1 Log[Phenylephrine] (M)
0! -9 0.01 -8 -7 -6 -5 Control 0.0001 0.001 0.1 1 10 Log[Phenylephrine] (M) Contraction (% of control)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
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160! 140! 120! 100!
-5 10
With washing PBS 8-Bromo-cGMP
80! 60! 40! 20! 0! -9 0.01 -8 -7 -6 Control 0.0001 0.001 0.1 1 Log[Phenylephrine] (M)
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-5 10
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Fig. 5. Akashi et al.
B
C
LPS
kDa
iNOS!
β-Actin!
100 75
1 2
50 37
3
25 20
GAPDH
D kDa
0.4!
50 37
1
1
2 3
2 3
0.2!
0! band 1! band 2! band 3!
kDa
150
150
100
100
75
250 150 100 75
0.6!
37
F
E PBS LPS
M PBS M LPS kDa
Relative band intensity! (Normalized to GAPDH)
Time after LPS treatment (h) 0 6 12
PBS! LPS!
0.8!
PBS LPS kDa 75
PBS
50 Phospho-! VASP 37
75
50
50
37
37
75 Total-! 50 VASP 37
1.4!
LPS Relative intensity! (phospho-VASP/total-VASP)
A
PBS
S-guanylation
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Biochemistry
1.2! 1! 0.8! 0.6! 0.4! 0.2! 0! PBS! LPS!
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O
Substrate-binding site
O 2N
O
AI
NH 2 NH 2
COO
N
NH N
Substrate
NH 2
O
O
Cys195
N
P O
OH
OH
S
cGMP NH 2 NH 2
Irreversible activation
S cGMP H
PKG1α
Graphic for Table of Contents
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