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DOI 10.1002/pmic.200600176
Proteomics 2006, 6, 4838–4844
RESEARCH ARTICLE
Peroxynitrite treatment in vitro disables catalytic activity of recombinant p38 MAPK Rose P. Webster1, Stephen Macha2, Diane Brockman1 and Leslie Myatt1, 3 1
Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Cincinnati, OH, USA Rieveschl Laboratories of Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA 3 Department of Molecular and Cell Physiology, University of Cincinnati, College of Medicine, Cincinnati, OH, USA 2
Protein tyrosine nitration is a post-translational modification occurring under conditions of oxidative stress in a number of diseases. The causative agent of tyrosine nitration is the potent prooxidant peroxynitrite that results from the interaction of nitric oxide and superoxide. We have previously demonstrated existence of nitrotyrosine in placenta from pregnancies complicated by preeclampsia, which suggested the possibility of the existence of nitrated proteins. Nitration of various proteins has been demonstrated to more commonly result in loss of protein function. Potential nitration of p38 MAPK, a critical signaling molecule has been suggested and also tentatively identified in certain in vivo systems. In this study we demonstrate for the first time nitration of recombinant p38 MAPK in vitro and an associated loss of its catalytic activity. LC-MS data identified tyrosine residues Y132, Y245 and Y258 to be nitrated. Nitration of these specific residues was deduced from the 45.0-Da change in mass that these residues exhibited that was consistent with the loss of a proton and addition of the nitro group.
Received: March 8, 2006 Revised: April 18, 2006 Accepted: May 21, 2006
Keywords: p38 MAPK / Nitrotyrosine / Peroxynitrite / Preeclampsia
1
Introduction
Tyrosine residues in proteins can be nitrated by peroxynitrite resulting in the formation of nitrotyrosine residues, which are markers of both oxidative and nitrative stress [1]. Our laboratory has earlier demonstrated nitrotyrosine residues in the villous vessels and stroma of placentae from pregnancies complicated by either preeclampsia or pregestational diabetes [2–5]. Peroxynitrite treatment of the normal placental vasculature in vitro leads to the formation of nitrotyrosine residues and alters vascular reactivity of the
Correspondence: Dr. Rose P. Webster, Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, PO Box 670526, Cincinnati, OH 45267-0526, USA E-mail:
[email protected] Fax: 11-513-5586138 Abbreviations: ATF2, activating transcription factor 2; FA, formic acid; IAA, iodoacetamide; MAPK, mitogen activated protein kinase; MKK6, MAPK6
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
placenta to resemble that observed in placentae from pregnancies complicated by either preeclampsia or pregestational diabetes [6]. Nitration of several proteins including manganese superoxide dismutase [7], p53 [7–9], cytochrome P450 [10], Ca-ATPase of the sarcoplasmic reticulum [11], mitochondrial creatine kinase [12], tyrosine hydroxylase [13], and prostacyclin synthase [14–16] have been reported before. Protein nitration has been observed to have three different effects, the most common, being inhibition of protein function, which has been observed in case of manganese superoxide dismutase [17], p53 [8], nuclear factor kappa B [18] and tyrosine hydoxylase [19]. At times, nitration either has no effect, e.g. protein kinase epsilon [20], or it can activate protein function as observed with cyclooxygenase-2 [21], poly-ADPribose polymerase [22] and fibrinogen [23]. Nitration of cytoskeletal proteins such as actin and tubulin [19] has been suggested to have a physiological role in vivo. In this study, we hypothesized that nitration of p38 mitogen activated protein kinase (MAPK) would lead to inhibition of its catalytic activity. p38 MAPK, a member of the highly www.proteomics-journal.com
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conserved MAPK superfamily regulates diverse cellular processes in response to a number of extracellular stimuli [24]. It is activated directly by an upstream kinase MAPK6 (MKK6) [25]. Our hypothesis was based on reports in the literature that either suggested [26] or tentatively [27] identified nitration of p38 MAPK, and our own (unpublished) observations of increased nitration of phospho-p38 MAPK in the preeclamptic placenta. We also speculated that we would be able to identify specific tyrosines that were nitrated in p38 MAPK. We have used Western blots, p38 MAPK activity assays and LC-MS to investigate our hypothesis.
2
Materials and methods
2.1 Materials Peroxynitrite, degraded peroxynitrite, mouse monoclonal anti-nitrotyrosine antibody and MAPK6 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Nonradioactive p38 MAP kinase assay kit, polyclonal antibodies to phospho-p38, p38 MAP kinase, phospho-activating transcription factor 2 (ATF2) and total ATP2 antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Western blot chemiluminescence reagent was purchased from Perkin Elmer (Boston, MA, USA). Precast 8–16% gradient gels and kaleidoscopic molecular weight marker were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Horseradish peroxidase linked molecular weight standards from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) were used for Western blotting. The recombinant proteins, p38 MAPK and phospho p38 MAPK were purchased from BioSource International Inc. (Camarillo, CA, USA). Protein estimation was carried out with BCA kit (Pierce, Rockford, IL, USA).
2.2 Western blotting Proteins were separated on 8–16% gradient precast gels, transferred onto nitrocellulose membranes, and blocked with 5% milk in 20 mM Tris-buffered saline (pH 7.5) containing 0.1% Tween (TTBS) for 1 h. Blots were probed with either anti-nitrotyrosine (1:2000), anti-p38 (1:500), or antiphospho p38 (1:500) antibodies as required in 5% BSA/ TTBS overnight, and were detected using horseradish peroxidase-conjugated, secondary antibody (1:10 000) in 5% milk/TTBS for 1 h. In the case of anti-nitrotyrosine, blots were blocked with 1% BSA, and both primary and secondary antibodies were prepared in the same solution. Products were visualized using chemiluminescence (Perkin Elmer). Band intensity was measured using AlphaImager software from Alpha Innotech Corporation (San Leandro, CA, USA). Equal protein loading was confirmed by Ponceau S staining. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.3 p38 MAPK activity assays The assay was carried out essentially according to the manufacturer’s protocol. Reactions were carried out with kinase buffer containing 25 mM Tris (pH 7.5), 5 mM b-glycerophosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, 10 mM MgCl2, 200 mM ATP and 2 mg ATF2 at 307C for 30 min. Reactions were stopped with 25 mL 36 Laemmli buffer and boiled at 957C for 5 min. Reaction mixtures were separated on 12% SDS-PAGE gels and Western blots for phospho-ATF2 (1:1000) were performed. The amount of phospho-ATF2 formed was used as a measure of p38 MAPK activity. Recombinant proteins used in the assay are described below. 2.4 In vitro nitration of recombinant p38 MAPK The method was adapted from Zhang et al. [28]. Recombinant p38 MAPK (active/inactive as required, 0.5 mg) was diluted in assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM b-glycerol phosphate, 5 mM EGTA, 1 mM sodium othovanadate and 1 mM DTT) supplemented with a magnesium/ATP mixture (75 mM magnesium chloride and 500 mM ATP). After addition of peroxynitrite (1 mM), the solution was incubated at 307C for 30 min. This concentration was chosen because it has been reported [29] that peroxynitrite formation could reach up to 1 mM/min in a inflamed organ in vivo. The reaction was either stopped by the addition of Laemmli’s buffer or further used to measure activity of p38 MAPK. The reaction mixture was then separated on an SDSPAGE gel and Western blotted for anti-nitrotyrosine detection. In experiments performed to measure the effect of nitration on catalytic activity, two different conditions were tested. In one, peroxynitrite was added to recombinant phospho-p38 (active form) purchased commercially. This was done in parallel with controls, which had no peroxynitrite added or had degraded peroxynitrite added. In the second, peroxynitrite was added simultaneously with recombinant p38 MAPK (inactive form) and recombinant MKK6. In this case, samples without addition of peroxynitrite were used as controls (data not shown). The effect of nitration on activity of both preactivated phospho-p38 and activated phospho-p38 were thus compared. 2.5 MS identification of nitrated residues Recombinant p38 MAPK (0.5 mg) that had been nitrated as described above was separated by SDS-PAGE, stained with CBB and the band corresponding to p38 MAPK was excised for MS. For comparison, non-nitrated recombinant p38 MAPK was similarly processed and subjected to LC-MS analysis. The excised gel pieces were destained for 10 min, washing three times with 200 mL 25 mM NH4HCO3/50% ACN. The gel pieces were treated with a freshly prepared 10 mM DTT, vortexed briefly and incubated at 567C for 45 min. After removing the DTT solution, 55 mM iodoacetamide (IAA) in 25 mM NH4HCO3 was added. The IAA solution was removed and diswww.proteomics-journal.com
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carded; samples were treated with 25 mM NH4HCO3 for 10 min, then twice with 25 mM NH4HCO3/50% ACN. Samples were dried for 20 min in a SpeedVac and trypsin solution added. The gel pieces were re-hydrated in 0.01 mg/mL trypsin solution, and then incubated at 47C for 45 min. A small amount of 25 mM NH4HCO3 solution was added to keep the gels hydrated during digestion. The digestion was performed overnight at 377C in a water bath. The peptides were extracted two times, once with 5% formic acid (FA) and then with 50% ACN/ 25 mM NH4HCO3 solution. To perform LC-MS runs, 10 mL of the sample was loaded on a 350675 mm C18 capillary column (made in-house) using Waters Cap-LC system. This system has an auto-sampler to facilitate the sample loading. The peptides were separated and eluted from the column using the Cap-LC gradient composed of solvent A (95% water, 5% ACN, 0.1% FA), and solvent B (95% ACN, 5% water and 0.1% FA). A linear gradient was run starting at 15% solvent B:85% solvent A to 80% solvent B in 70 min. The flow rate was set at 7 mL/min but split into about 50:50 before passing through the column. The Cap-LC system was directly connected to the Waters Q-TOF 2 mass spectrometer, in which the eluted peptides were directly detected (LCMS), fragmented (LC-MS/MS) and data collected accordingly. The collected data were processed using MassLynx 4.0 software to construct peak list files. The peak list files were used for the protein database search using licensed MASCOT 2.1 software from Matrix Science Ltd. (London, UK). The peptide sequences were obtained from the MASCOTprotein database report; such sequences are the same ones used to identify the protein. All experiments using immunoblots and LC-MS were repeated to confirm the data obtained.
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Figure 1. (A) Lane 1 shows p38 MAPK treated with degraded (deg.) peroxynitrite (indicated as ONOO in all the figures). p38 MAPK (0.5 mg) (lanes 2 and 3) was reacted with 1 mM peroxynitrite and probed on immunoblots with anti-nitrotyrosine antibody. Lanes 4 and 5 shows recombinant p38 MAPK not treated with either peroxynitrite or degraded peroxynitrite. In the last lane (M), the molecular weight marker was loaded and the corresponding weights are indicated. (B) Immunoblot was then stripped and probed with antibody to total p38 MAPK (labeled as T.p38) to verify protein loading. The signs (1) and (2) have been used in this figure and in Fig. 2 to indicate the presence or absence of a particular reagent tested.
Results
3.1 In vitro nitration and effect on catalytic activity of recombinant p38 MAPK Experiments were performed to establish whether recombinant p38 MAPK can be nitrated in vitro (Fig. 1A). Western blot analyses clearly showed extensive nitration of the recombinant p38 MAPK (lanes 2 and 3). There was no band detected (lanes 4 and 5) with non-nitrated peroxynitrite. The blot was then stripped and probed for total p38 MAPK (Fig. 1B), which shows that there were equal amounts of protein loaded in each lane. We then determined the effect of nitration on p38 MAPK activity. As seen in Fig. 2A, there was complete inhibition of p38 MAPK activity after nitration. This inhibition was irrespective of whether the active phospho-p38 MAPK was preformed or formed at the time of nitration. This blot was stripped and probed for total p38 MAPK (Fig. 2B) to verify protein loading and for total ATF2 (Fig. 2C) to confirm that a comparable amount of the substrate ATF2 was used in each reaction. The presence of the nitrating agent either concurrently with or after the phosphorylating event does not seem to affect the loss in catalytic © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Active recombinant phospho-p38 was reacted (A) with either degraded peroxynitrite (lane 1), or peroxynitrite (lanes 2 and 3). A control reaction was also carried out in the absence of both (lanes 4 and 5). p38 MAPK was reacted with MKK6 and peroxynitrite (lanes 6 and 7), or MKK6 alone (lanes 8 and 9) or peroxynitrite alone (lanes 10 and 11). All the different reactions were evaluated for their ability to phosphorylate ATF2. (B) Immunoblot was stripped and probed for total p38 (labeled as t.p38 MAPK in B) and total ATF2 (labeled as t.ATF2 in C) to verify protein loading.
activity of p38 MAPK. These experiments show for the first time that p38 MAPK can be nitrated in vitro, and this leads to a complete loss of p38 MAPK catalytic activity. 3.2 Identification of nitrated tyrosine residues in p38 MAPK by LC-MS Analysis of the LC-MS/MS data using a MASCOT protein database search software identified three well-sequenced nitrated peptides (Table 1). All the three nitrated peptides www.proteomics-journal.com
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Table 1. Identification of the nitrated tyrosine residues in recombinant p38 MAPK by LC-MS. Three peptides were identified to be nitrated by LC-MS data. Each of the peptides identified was found to be present both in the 45.0-Da heavier and lighter forms in the sample that was subjected to nitration. Y 132, 245 and 258 were nitrated
Peptide Sequence
Start–End
Mass (m/z)
Sample Type
LTDDHVQFLIYQILR
122–136 122–136 122–136
1917.96 (0.03) 1873.15 (0.14) 1872.95 (0.06)
Nitrated Nitrated Non-nitrated
LTGTPPAYLINR
238–249 238–249 238–249
1359.66 (0.06) 1314.72 (0.01) 1314.66 (0.07)
Nitrated Nitrated Non-nitrated
NYIQSLTQMPK
257–267 257–267 257–267
138 2 689 (0.03) 1337.62 (0.04) 1337.66 (0.00)
Nitrated Nitrated Non-nitrated
Figure 4. LC-MS/MS spectra of the peptide LTDDHVQFLIYQR from non-nitrated (A) and nitrated (B) p38 MAPK sample is shown. In (A), y5 (indicated with an asterisk in the figure) fragment appears at m/z 692.41 Da, and in (B) y5 fragment appears at m/z 737.39 Da. The y5 (indicated with an asterisk) fragment in (B) has 44.98 mass units more than y5 in (A) due to nitration on tyrosine (Y), as shown in Table 2.
Figure 3. Electrospray mass spectra of p38 MAPK sample showing a doubly charged tryptic peptide LTDDHVQFLIYQR. (A, B) Spectrum from the non-nitrated and nitrated samples, respectively. The mass difference between the nitrated and nonnitrated m/z peaks is 22.5 Da. The peptide in (B) is nitrated. As shown in (B), there is a small peak of the non-nitrated peptide as indicated by the asterisk.
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
were doubly charged. The first one is LTDDHVQFLIYQILR (122–136, Table 1) with m/z 1917.96 (6 0.03 Da). It is seen in Fig. 3 that this particular peptide is doubly charged (i.e., m/z 937.5 and 960.0) and, therefore, the difference in the masses between the nitrated and non-nitrated is 22.5 Da. The y5 (Table 2A) ions from the nitrated sample had a mass of 737.39, whereas the mass of the corresponding ion from the non-nitrated peptide was 692.41. Peak b11 (Table 2B) also show a difference in their masses of 44.98 Da. The fragmentation of this particular peptide and the resulting y and b ions are clearly seen in Fig. 4. This difference in molecular weights between the two ions is consistent with the addition of a nitro group to, and loss of a hydrogen atom from, these peptides. Another peptide with the same sequence but different mass was also identified in the nitrated sample. This had an m/z 1872.95 (6 0.06 Da). The presence of this nonnitrated peptide in the same sample might be an indication that the nitration process was incomplete or the formed nitro-peptide is unstable under certain experimental procedures to which the sample was subjected. The second identified nitrated peptide was LTGTPPAYLINR (238–249, Table 2) with m/z 1359.66 (6 0.06 Da). Peaks y5 and b8 (data not shown), resulting from the fragmentation of the peptide bearing the nitrated tyrosine, show a difference of approxiwww.proteomics-journal.com
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Table 2. Series of b and y ions generated from the fragmentation of peptide LTDDHVQFLIYQR (122–136). The fragmentation of the peptide LTDDHVQFLIYQILR (122–136) (1873.15 Da) from the non-nitrated sample and the nitrated sample provided the sequence series of b and y ions shown in A and B, respectively. The peaks b11 and y5 (B) from nitrated sample exhibit a shift of 44.98 Da relative to those of non-nitrated sample b11 and y5 (B). Fragments (y6, y7, y8, y9, y10, y11, y12, y13 and y14 from one end and b12, b13 and b14 from the other end) generated by progressive digestion of subsequent amino acids show the same approximately 45 Da difference in their masses between the nitrated and non-nitrated sample
B
A No.
b
Seq.
y
No.
No.
b
Seq.
y
No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
114.09 215.14 330.17 445.19 582.25 681.32 809.38 956.45 1069.53 1182.62 1345.68 1473.74 1586.82 1699.91
L T D D H V Q F L I Y Q I L R
1760.93 1659.89 1544.86 1429.83 1292.77 1193.70 1065.65 918.58 805.49 692.41 529.35 401.29 288.20 175.12
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
114.09 215.14 330.17 445.19 582.25 681.32 809.38 956.45 1069.53 1182.62 1390.66 1518.72 1631.81 1744.89
L T D D H V Q F L I Y(NO2) Q I L R
1805.92 1704.87 1589.84 1474.82 1337.76 1238.69 1110.63 963.56 850.48 737.39 529.35 401.29 288.20 175.12
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
mately 45 Da that is consistent with nitration. In this case also, there was a peptide with the same sequence but an m/z value of 1314.73 (6 0.01 Da), which was the non-nitrated peptide. Similarly, amino acid mapping of the peptide corresponding to amino acids 257–267 with the sequence NYIQSLTQMPK (257–267) was carried out. This peptide has a mass of 1382.68 (6 0.03 Da, data not shown). Peaks y10 and b2 obtained from the modified and unmodified samples also showed a difference in mass of approximately 45 Da, corresponding to nitration. Like the other two peptides discussed above, there was a peptide with the same sequence in the nitrated sample with m/z 1337.66 (6 0.00 Da, data not shown). The mass difference between the two peptides is 45.02 Da. This peptide in both the nitrated and non-nitrated sample demonstrated oxidation of methionine. It was observed that peptides generated from the progressive digestion of residues subsequent to the one nitrated also exhibited the difference in mass that was consistent with nitration. This was observed in all three peptides, when the sequence was considered from the N-terminal or C-terminal end, and this helped confirm the nitration at that particular residue.
4
Discussion
To our knowledge in vitro nitration of p38 MAPK has not been demonstrated before. LC-MS data has identified three tyrosine residues Y132, Y245 and Y258. Interestingly, the tyrosine in the catalytic site at position 182 was not among those that were nitrated. It is significant though that all of the three tyr© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
osines nitrated are from the C terminus of the protein, which contains the catalytic site, the magnesium binding site and the phosphorylation lip [30]. Our data also conclusively establishes that in vitro nitration of recombinant p38 MAPK completely inhibits its activity. This inhibition may not be solely due to tyrosine nitration. Peroxynitrite can also bring about oxidation of cysteine, methionine and tryptophan [31] and this could contribute to the total inhibition observed in vitro. We have observed (unpublished) an association between the reduced activity of phospho-p38 MAPK and increasing levels of nitration in placenta from pregnancies complicated by preeclampsia. Data presented here shows a direct inhibitory effect of nitration on p38 MAPK catalytic activity. On the basis of our observations both in vitro and in vivo of the effect of nitration on p38 MAPK activity, it is reasonable to state that nitration is associated with inhibition of p38 MAPK protein activity. It is highly likely that nitration of p38 MAPK interferes with its interaction with other proteins in vivo. Several reports [32–34] exist concerning interaction of active p38 MAP kinase with other proteins, and also the ability of p38 MAPK to regulate various gene functions [35]. Nitration of p38 MAPK could potentially impair several critical functions at both cellular and genetic levels. Interaction of reactive nitrogen species with biomolecules leads to the nitration of protein tyrosine residues. This well-established PTM caused by peroxynitrite results in very low yields of biological nitration for several reasons. One of the main reasons is that precursors of peroxynitrite, i.e., superoxide, hydrogen peroxide and the nitrosyl radical undergo multiple processes including reactions among www.proteomics-journal.com
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dues and result in 3-nitrotyrosine to tyrosine ratios in the range of 0.1–1.0. Therefore, it seems a loss of function is a rare event. Yet, there are several reports [5, 10, 11] that have shown loss of function due to nitration of proteins. Our data also very clearly demonstrate that nitrated p38 MAPK has no catalytic activity. Further studies directed at mutating the tyrosine residues that have been identified to be nitrated are in progress to demonstrate the effect of nitration on p38 MAPK function in an in vivo cell culture model.
This work was supported by NIH RO1 grant no. HL07529.
5
References
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Figure 5. The structure of p38 MAPK isoform CSBP1 (Database Swiss-Prot: acc. no. Q16539-2) was created by the SWISS MODEL protein modeling server by homology modeling with six very closely related (96% homology) protein structures in the protein database (http://www.rcsb.org/pdb/). Request for the structure was made according to the instructions in the Swiss PDB viewer (http://www.expasy.org/spdbv/). This model is used here only to show the relative positions of the nitrated residues with respect to the residues in the catalytic site threonine 180 and tyrosine 182.
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