Quantitative Phosphoproteomic Analysis of Early Alterations in

Article pubs.acs.org/jpr

Quantitative Phosphoproteomic Analysis of Early Alterations in Protein Phosphorylation by 2,3,7,8-Tetrachlorodibenzo‑p‑dioxin Melanie Schulz,†,‡ Stefanie Brandner,† Carola Eberhagen,† Friederike Eckardt-Schupp,§ Martin R. Larsen,‡ and Ulrich Andrae*,† †

Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany ‡ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark § Institute of Radiation Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany S Supporting Information *

ABSTRACT: A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥1.5 in one of the experiments per time point. The vast majority of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers and heterodimers to different enhancers suggesting that this phosphorylation represents a novel mechanism contributing to the alteration of gene expression by TCDD. Other proteins with altered phosphorylation included, among others, various transcriptional coregulators previously unknown to participate in TCDD-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity. KEYWORDS: TCDD, dioxin, protein phosphorylation, phosphoproteomics, SILAC, SIMAC, ARNT, transcriptional regulation, GTPases, 5L cells

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regulatory regions of aryl hydrocarbon-responsive genes.6−8 Binding to the DREs initiates chromatin remodeling and recruitment of coregulators9 which enables the transcription of a set of target genes comprising the so-called “AhR gene battery”. The induced gene products include several xenobioticmetabolizing enzymes such as cytochrome P4501A1 (CYP1A1). During the past few years, evidence has been accumulated that TCDD can also cause biochemical alterations that are easily detectable within a few minutes after addition of the compound to cells in vitro. These effects cannot be the consequence of AhR/ARNT-mediated gene activation as this is dependent on a stimulation of de novo transcription and translation which require a certain minimal time in the range of a few hours to become apparent. For these responses, which in the majority of the cases have been claimed to be dependent on a functional AhR, but not ARNT10 and which appear to be

INTRODUCTION 2,3,7,8-Tetrachlorodibeno-p-dioxin (TCDD) is a ubiquitous and persistent environmental contaminant with the potential to cause a broad spectrum of adverse health effects including a variety of developmental defects, cardiovascular disease, diabetes, porphyria, hormonal disturbances and cancer in humans.1,2 In spite of enormous efforts to uncover the mechanisms of action underlying the various deleterious effects of TCDD, the precise molecular events mediating toxicity are still largely unknown. TCDD is a ligand of the so-called aryl hydrocarbon receptor (AhR), a primarily cytosolic receptor which plays important roles in normal cell physiology3 and which mediates the toxic effects of TCDD.4,5 Binding of TCDD or a wide variety of other AhR agonists to the receptor results in the translocation of the ligand−receptor complex into the cell nucleus and the dimerization with a related protein, the AhR nuclear translocator (ARNT). This heterodimer can act as a ligand-activated transcription factor by binding to specific DNA sequences known as xenobiotic response elements or dioxin response elements (DREs), enhancers located in © 2013 American Chemical Society

Received: October 8, 2012 Published: January 8, 2013 866

dx.doi.org/10.1021/pr3009429 | J. Proteome Res. 2013, 12, 866−882

Journal of Proteome Research

Article

kinase (MAPK) in an AhR-dependent, but, importantly, transcription-independent manner, suggesting a novel crosstalk between AhR and MAPK signaling. p38 activation was observed as early as 1 h after addition of TCDD and resulted in subsequent transcriptional induction of the proto-oncogene cjun. The signaling pathway resulting in p38 phosphorylation and the kinase involved remained unclear. In the present phosphoproteomic analysis of the effects of TCDD in 5L cells, short exposure periods of 0.5, 1, and 2 h were used, that is, treatments that were unlikely to result in significant alterations in the abundance of proteins with TCDD-regulated expression, which requires some time because of its dependence on de novo transcription and translation. The study was performed with the expectation to uncover proteins not previously known to exhibit rapid alterations in phosphorylation as a consequence of TCDD exposure and which might give clues to pathways involved in nongenomic signaling preceding alterations in gene expression. Moreover, it was anticipated to identify proteins with altered phosphorylation as a consequence of their involvement in the regulation of transcription induced by the DRE-bound liganded AhR/ ARNT heterodimer. Finally, the study was expected to provide novel information on the phosphoproteome of rat cells with respect to the identification of novel phosphorylation sites of known phosphoproteins and the discovery of proteins previously unknown to be phosphorylated.

largely mediated by rapid phosphorylation events, the term “protein phosphorylation pathway”11 or, more recently, “nongenomic” pathway was coined to discriminate them from the classical “genomic” pathway involving Ahr/ARNT-mediated induction of gene expression. Whereas the details of the phosphorylation-mediated pathways may vary between different types of cells, a salient feature in all cells examined is that they appear to be initiated by a rapid increase in intracellular Ca2+ concentration which has been consistently demonstrated in organ cultures of TCDD-exposed animals,12 primary cell cultures13−17 and mammalian cell lines.18−20 This increase in intracellular calcium, which takes place within a few minutes, is the earliest effect detectable in TCDD-exposed cells and has been suggested to constitute the trigger of the nongenomic action of TCDD.10 For certain cell lines, the elevation of the intracellular Ca2+ concentration by TCDD has been reported to result in the activation of the ubiquitous Ca2+-dependent cytosolic phospholipase A2 (cPLA2) resulting in the cleavage of arachidonic acid (AA) from glycerophospholipids.21−23 TCDDinduced cPLA2 activation was also detected via an increase in the activating Ser505 phosphorylation of the enzyme.21,24 The AA formed has been implicated in the rapid elevation of Src kinase activity that occurs in several cell types and which takes place in MCF10A cells within 30 min.21,25 Quite recently, a novel branch of the nongenomic pathway linking the TCDD-induced rapid elevation of intracellular Ca2+ concentration to the up-regulation of AhR target genes, that is, to the genomic response, via phosphorylation events has been identified.20,26,27 For MCF-7 cells it was shown that the increase in intracellular Ca2+ concentration results in a transient activation of the ubiquitous calmodulin (CaM)-dependent protein kinase Iα (CaMKI α) which turned out to be a necessary prerequisite for the TCDD-triggered nuclear import of the ligand-activated AhR and promoter activation of AhR target genes. Confirmatory data were presented for primary human macrophages.20 Evidence was obtained that exposure to TCDD was accompanied by an increased Thr177 phosphorylation of CaMKI α,20 which has been associated with full CaMKI α activation.28 The protein kinase mediating CaMKI α activation remained unclear.20 Whereas the genomic response to TCDD has been analyzed in numerous large-scale studies at the level of the transcriptome and the proteome both in vitro and in vivo, a systematic study of the proteins involved in the early nongenomic action of TCDD has not been performed previously. Therefore, the present study was conducted with the aim to identify TCDDinduced alterations in protein phosphorylation occurring early after the onset of TCDD exposure to achieve a better knowledge of the alterations in signal transduction potentially involved in the nongenomic effects of TCDD. The rat hepatoma cell line 5L was used as a model system. 5L cells are epithelial-like cells which express both the AhR and the ARNT proteins and which are highly responsive to TCDDinduced gene activation. They have been previously used in a variety of studies dealing with the role of AhR in cell cycle control and TCDD toxicity,29−37 the regulation of the expression of xenobiotic metabolizing enzymes,38−40 and the effects of AhR and TCDD on microRNA levels.41 In addition, they have been successfully employed in studies aiming at the detection of novel target genes of the AhR.42,43 With respect to nongenomic actions of TCDD in this cell line, Weiss et al.32 observed that TCDD exposure of the cells is associated with the rapid phosphorylation of the p38 Mitogen-activated protein

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EXPERIMENTAL PROCEDURES

Cells

The 5L cell line is a descendant of the cell line H4IIEC3 established by Pitot et al.44 from the Reuber H35 rat hepatoma.45 Cells were grown as monolayers in 10-cm culture dishes containing RPMI 1640 medium supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. SILAC Labeling

For stable isotope labeling of the 5L cells using the SILAC (stable isotope labeling by amino acids in cell culture) approach, 13C6-lysine (Lys6) (Invitrogen, Carlsbad, U.S.A.), 15 N4-arginine (Arg4) (Cambridge Isotope Laboratories, Andover, U.S.A.) and 13C615N4-arginine (Arg10) (Invitrogen) were used according to the labeling scheme of van Hoof et al.46 in order to circumvent potential peptide quantitation problems associated with the conversion of isotopically labeled arginine into labeled proline. The amino acids were dissolved in RPMI medium without arginine and lysine supplemented with 10% dialyzed fetal bovine serum, 100 μg/mL streptomycin, and 100 U/ml penicillin. For the “light” condition, the medium was supplemented with Lys0 and Arg4, and for the “heavy” condition with Lys6 and Arg10. The concentrations of arginine and lysine were 75 mg/L. The cells were grown for about 7 cell cycles (7 days) with intermediate splitting after 3 days and replating in labeling media at a density of 7.5 × 105 cells/10-cm dish. Treatment of the Cells with TCDD

During the last 16 h before the initiation of the TCDD exposure, the cells were starved in fresh labeling medium without serum. The “heavy” cells were treated with 1 nM TCDD (purity ≥99%, Ö kometric, Bayreuth, Germany) dissolved in DMSO and the “light” cells with DMSO alone as a control for 0.5, 1, and 2 h. The DMSO concentration in the 867



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Hydrophilic Interaction Chromatography (HILIC)

medium was 0.1%. Safety considerations: Skin contact with TCDD-containing solutions and contamination of laboratory benches and laminar flow hoods with TCDD must be avoided.

The monophosphorylated and nonphosphorylated peptides from the SIMAC enrichment were fractionated by HILIC. The dried fraction was resuspended in B-solvent (80% ACN, 0.1% TFA) and loaded onto a 2 mm TSKGel Amide-80 HILIC column (Tosoh Bioscience, Stuttgart, Germany) connected to an Ä KTA Basic FPLC system (GE Healthcare, Hillerod, Denmark). Loading was performed in B-solvent at a flow rate of 200 μL/min for 12 min. The peptides were separated by an inverse gradient (100%−70% B-solvent within 28 min and 70%−0% B-solvent within 5 min) with an increasing amount of A-solvent (0.1% TFA) at a flow rate of 200 μL/min. Sixteen fractions were collected and dried down in a vacuum centrifuge.

Cell Lysis and Protein Precipitation

Following exposure of the cells the medium was removed and cells were washed twice with 10 mL ice-cold PBS. Then 1.5 mL of lysis buffer containing 1 μL/mL benzonase, 1 mM MgCl2, 25 mM Tris HCl, pH 7.4, 120 mM NaCl, 1% Triton X-100, one protease inhibitor tablet (Complete, Roche, Mannheim, Germany) per 50 mL buffer, 0.5 mL of phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) per 50 mL, and 1 mM sodium pervanadate were added to each 10-cm dish. The sodium pervanadate solution was prepared by treating a solution of 100 mM sodium orthovanadate, pH 10, with an equal volume of a 0.36% hydrogen peroxide solution immediately before use. The lysates were transferred to Eppendorf tubes, shaken for 30 min at 4 °C and centrifuged at 15000g for 10 min at 4 °C. Following determination of the protein concentrations with the Bradford assay, the supernatants from the “light” and the “heavy” cells were mixed 1:1. The proteins (800 μg) were precipitated with four volumes of ice-cold ethanol at −20 °C. After 15 min, four volumes of icecold acetone were added and the mixture was incubated at −20 °C overnight. The precipitated proteins were centrifuged at 15 000g for 20 min at 4 °C. The pellet was washed 3 times with ethanol/acetone/water 2:2:1.

Phosphopeptide Enrichment by Titanium Dioxide (TiO2) Chromatography

To enrich the mainly monophosphorylated peptides from the individual HILIC fractions, the TiO2 method was used essentially as described by Larsen et al.49 with slight modifications.50 TiO2 beads (GL Sciences, Tokyo, Japan) were washed with water, 100% ACN and loading buffer (1 M glycolic acid in 80% ACN, 5% TFA). Each HILIC fraction was dissolved in 5 μL 1% SDS, 200 μL loading buffer, and the appropriate amount of TiO2 was added. For 200 μg of peptides 0.6 mg TiO2 particles were used. The peptides and the TiO2 resin were incubated for 15 min under vigorous shaking. The beads were spun down by centrifugation at 2000g for 30 s. The supernatant was collected in a fresh tube and the TiO2 beads were washed with loading buffer (80% ACN/5% TFA), then 30% ACN/0.1% TFA and, finally, distilled water. The phosphopeptides were eluted from the beads with 50 μL 1% NH4OH using an incubation time of 10 min under vigorous shaking. The supernatant was acidified with 5 μL 100% FA. The phosphopeptides were desalted using Poros R3 P10 micro columns and analyzed by LC-MS/MS. The collected supernatant from the first TiO2 incubation with loading buffer was again treated with TiO2 as described above.

Proteolysis

The precipitated proteins were dissolved in 6 M urea, 2 M thiourea and 50 mM NH4HCO3. The proteins were reduced for 2 h at 30 °C with 20 mM DTT and alkylated with 40 mM iodoacetamide for 45 min in the dark at room temperature. Then they were digested with endoproteinase Lys-C (Wako, Richmond, U.S.A.) (1/100 w/w) for 3 h. For digestion with trypsin, the solutions were diluted in 50 mM NH4HCO3 to a urea concentration of less than 1 M and incubated with sequencing grade modified trypsin (Promega, Madison, U.S.A.) (1/50 w/w) at 30 °C for 16 h. The digestion was quenched by adding trifluoroacetic acid (TFA) to reach a pH of ≤3.

StageTip Peptide Purification

Peptides were purified using modified StageTips. StageTips were prepared by stamping out a small plug of a 3 M Empore C8 disk (3 M Bioanalytical Technologies, St. Paul, U.S.A.) and placing it at the end of a P10 tip. Poros R3 material in ACN was packed in the pipet tip onto the top of the C8 disk. The microcolumn was washed with 100% ACN and equilibrated with 0.1% TFA. Phosphopeptide samples were loaded onto the columns using a 1 mL disposable syringe. The columns were washed once with 0.1% TFA in water and the peptides were eluted with 50% ACN and 0.1% TFA. Samples were dried down in a vacuum centrifuge and redissolved in 0.5 μL 100% FA and 4.5 μL 0.1% FA prior to analysis by LC-MS/MS.

Phosphopeptide Enrichment by Sequential Elution from IMAC (SIMAC)

The phosphorylated peptides were enriched using SIMAC essentially as described.47 Briefly, PHOS-Select Iron Affinity Gel beads (Sigma-Aldrich) were washed twice with 0.1% TFA and once with loading buffer (50% acetonitrile (ACN), 0.1% TFA) to remove the glycerol. The beads were resuspended in loading buffer and the sample was added. For 200 μg of peptides, 50 μL gel beads were used. After incubation by endover-end rotation for 1 h, the peptide-bead mixture was transferred to a constricted P200 gel loading tip. The flowthrough and the first wash with loading buffer (100 μL) were collected and combined. The monophosphorylated peptides were eluted with 20% ACN, 1% TFA (100 μL) and combined with the flow-through. This solution was dried down in a vacuum centrifuge. The multiphosphorylated peptides were eluted off the IMAC material with 1% NH4OH (70 μL) and this solution was subsequently acidified with 100% formic acid (FA) to a final FA concentration of 1%. The multiphosphorylated peptides were desalted using Poros Oligo R3 reversed phase material (PerSeptive Biosystems, Framingham, U.S.A.) packed in P10 micropipet tips as previously described48 and analyzed by LC-MS/MS.

Phosphopeptide Identification and Quantitation

LC-MS/MS Analysis. The enriched and purified phosphopeptides were analyzed on a LTQ Orbitrap XL Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany). The samples were applied onto an EASY nano-HPLC system (Thermo Fisher Scientific, Odense, Denmark) comprising a self-packed 16-cm analytical column (100 μm I.D., 360 μm O.D., ReproSil-Pur C18 AQ 3 μm (Dr. Maisch, AmmerbuchEntringen, Germany). The peptides were loaded with a flow rate of 550 nL/min in 0.1% FA (100% phase A) as solvent and eluted at a flow rate of 250 nL/min with a gradient from 100% phase A to 34% phase B (95% ACN, 0.1% FA) for 100 min. 868



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peptide with the highest ion score was kept. If one peptide was detected with different IPI accessions, only the peptide belonging to the IPI identifier with the highest number of different peptides observed was retained.54 Then, a normalization of the calculated log2 peptide ratios was performed. A global normalization method involving the adjustment of the log median of the peptide ratios to zero was used. Statistical Analysis. Three different, independent biological replicates were analyzed for the data sets obtained for the 30-min and 1-h exposures to TCDD and two biological replicates were analyzed for the 2-h data set. Only peptides found at least twice for a given time point were used for the following statistical test. The normalized ratios were fitted to a linear model and an empirical Bayes moderated t test was conducted using the R/Bioconductor package “limma”. The resulting p-values were used to calculate the q-values36 with the R-package “q-value”. Peptides with a q-value ≤0.05 were accepted as statistically significantly regulated. Volcano plots were generated with the limma package. Annotation. Following statistical analysis, the IPI identifier of every unique phosphorylated peptide was mapped to a Uniprot, GO, KEGG, REFSEQ, and ENSEMBL identifier. Mapping was done using the software R in combination with the packages BioIDMapper and biomaRt. Phosphoset Analysis. The identification of known and hitherto unknown phosphorylation sites was performed with an in-house written Perl script. For this analysis, the Swiss-Prot accession numbers were used and searched against the UniProt database.

The nano-HPLC system was connected online to the mass spectrometer with a nano ES ion source and PicoTip emitter needles (New Objective, Berlin, Germany). The mass spectrometer was operated in positive ion mode and a datadependent acquisition with an automatic switch between MS and MS/MS was employed. Each MS scan was followed by 5 MS/MS scans. The full scan was acquired in the Orbitrap with an automatic gain control (AGC) target value of 1 × 106 ions and a maximum fill time of 500 ms. The top 5 most intense ions were selected for fragmentation in the linear trap quadrupole (LTQ). Fragmentation was induced by CID. For the LTQ, the AGC target value was set to 3 × 104 ions and a maximum fill time of 300 ms. For an improved fragmentation of phosphopeptides, the multistage algorithm51 was enabled for each MS/MS spectrum, which showed a loss of phosphoric acid from the parent ion (neutral loss of 97.97, 48.99, or 32.66 Thomson). Ions selected for MS/MS were dynamically excluded for a duration of 45 s. Database Searching. The acquired multistage activation spectra were converted into Mascot generic format (mgf) using the software DTASuperCharge, version 1.37 (http://msquant. sourceforge.net/). Deisotoping was performed using the default settings from DTASuperCharge. The processed spectra were searched against the rat IPI protein database version 3.60 which contains 39863 protein sequences. The search was performed using an in-house Mascot server (version 2.2) with carbamidomethyl (C) as a fixed modification and acetylation (protein N-terminal), oxidation (M), phosphorylation (ST and Y) and the SILAC labeling (K-6, R-4, R-10) as variable modifications. Trypsin was chosen as the enzyme and up to two miss-cleavages were allowed. For the parent ion a mass accuracy of 10 ppm was used and for the fragment ions a mass accuracy of 0.8 Da. A concatenated decoy database search was performed derived from the IPI rat database. Proteins with a false discovery rate (FDR) of ≤1% and with peptides having an ion score of ≥20 (MudPit scoring) were accepted. This ion score cutoff was slightly lower than that of ≥22 recently employed by Engholm-Keller et al.52 and selected because an evaluation of a representative part of low-scoring MS/MS spectra of the peptides identified in the present study had indicated that an ion score cutoff of ≥22 would have been too conservative as an ion score of ≥20 yielded highly reliable results (not shown). Quantitation and Phosphosite Localization. Relative quantitation of phosphopeptides and nonphosphorylated peptides was performed using the software MSQuant (version 1.0a70). To enable the processing of the data generated with the labeling scheme of van Hoof et al.,46 the source code of MSQuant was changed. MSQuant quantifies the peptides based on the first isotopic peak of the isotopically labeled peptides by averaging the ion intensities over multiple MS scans. As a measure of the reliability of the phosphorylation site assignment, the posttranslational modification (PTM) score for every phosphosite was calculated using MSQuant.53 Thereafter, every MS and MS/MS spectrum was manually validated. Peptides with a low signal-to-noise ratio, ion score 1.5. bThe asterisk (*) in the sequence indicates the position of the phosphorylation. cThe respective phosphorylation site of the protein had previously been observed for species other than the rat (known by similarity) or not been observed for any species (novel). dThe normalized SILAC ratio TCDD/DMSO is the normalized SILAC ratio “heavy/light” (H/L). eThe indicated two alternative positions of the phosphorylation site correspond to two splice isoforms that had been observed for the rat at the mRNA level.

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Table 2. Phosphopeptides Identified As Regulated (q-Value 6500 different phosphopeptides revealed that only a tiny fraction, less than 0.5%, exhibited evidence for a regulation of their phosphorylation status by TCDD. This finding supports the notion that early alterations in protein phosphorylation by dioxins are likely 878



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(4) Fernandez-Salguero, P. M.; Hilbert, D. M.; Rudikoff, S.; Ward, J. M.; Gonzalez, F. J. Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol. Appl. Pharmacol. 1996, 140, 173−9. (5) Bunger, M. K.; Glover, E.; Moran, S. M.; Walisser, J. A.; Lahvis, G. P.; Hsu, E. L.; Bradfield, C. A. Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor. Toxicol. Sci. 2008, 106, 83−92. (6) Safe, S. Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol. Lett. 2001, 120, 1−7. (7) Sogawa, K.; Numayama-Tsuruta, K.; Takahashi, T.; Matsushita, N.; Miura, C.; Nikawa, J.-i.; Gotoh, O.; Kikuchi, Y.; Fujii-Kuriyama, Y. A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer. Biochem. Biophys. Res. Commun. 2004, 318, 746−55. (8) Boutros, P. C.; Moffat, I. D.; Franc, M. A.; Tijet, N.; Tuomisto, J.; Pohjanvirta, R.; Okey, A. B. Dioxin-responsive AHRE-II gene battery: identification by phylogenetic footprinting. Biochem. Biophys. Res. Commun. 2004, 321, 707−15. (9) Beischlag, T. V.; Luis Morales, J.; Hollingshead, B. D.; Perdew, G. H. The aryl hydrocarbon receptor complex and the control of gene expression. Crit. Rev. Eukaryot. Gene Expr. 2008, 18, 207−50. (10) Matsumura, F. The significance of the nongenomic pathway in mediating inflammatory signaling of the dioxin-activated Ah receptor to cause toxic effects. Biochem. Pharmacol. 2009, 77, 608−26. (11) Enan, E.; Matsumura, F. Evidence for a second pathway in the action mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Significance of Ah-receptor mediated activation of protein kinase under cell-free conditions. Biochem. Pharmacol. 1995, 49, 249−61. (12) Canga, L.; Levi, R.; Rifkind, A. B. Heart as a target organ in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity: decreased beta-adrenergic responsiveness and evidence of increased intracellular calcium. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 905−9. (13) McConkey, D. J.; Hartzell, P.; Duddy, S. K.; Håkansson, H.; Orrenius, S. 2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation. Science 1988, 242, 256−9. (14) Canga, L.; Paroli, L.; Blanck, T. J.; Silver, R. B.; Rifkind, A. B. 2,3,7,8-tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts. Mol. Pharmacol. 1993, 44, 1142−51. (15) Hanneman, W. H.; Legare, M. E.; Barhoumi, R.; Burghardt, R. C.; Safe, S.; Tiffany-Castiglioni, E. Stimulation of calcium uptake in cultured rat hippocampal neurons by 2,3,7,8-tetrachlorodibenzo-pdioxin. Toxicology 1996, 112, 19−28. (16) Tannheimer, S. L.; Barton, S. L.; Ethier, S. P.; Burchiel, S. W. Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca2+ and cell proliferation in primary human mammary epithelial cells. Carcinogenesis 1997, 18, 1177−82. (17) Lin, C.-H.; Juan, S.-H.; Wang, C. Y.; Sun, Y.-Y.; Chou, C.-M.; Chang, S.-F.; Hu, S.-Y.; Lee, W.-S.; Lee, Y.-H. Neuronal activity enhances aryl hydrocarbon receptor-mediated gene expression and dioxin neurotoxicity in cortical neurons. J. Neurochem. 2008, 104, 1415−29. (18) Puga, A.; Nebert, D. W.; Carrier, F. Dioxin induces expression of c-fos and c-jun proto-oncogenes and a large increase in transcription factor AP-1. DNA Cell Biol. 1992, 11, 269−81. (19) Puga, A.; Hoffer, A.; Zhou, S.; Bohm, J. M.; Leikauf, G. D.; Shertzer, H. G. Sustained increase in intracellular free calcium and activation of cyclooxygenase-2 expression in mouse hepatoma cells treated with dioxin. Biochem. Pharmacol. 1997, 54, 1287−96. (20) Monteiro, P.; Gilot, D.; Le Ferrec, E.; Rauch, C.; LagadicGossmann, D.; Fardel, O. Dioxin-mediated up-regulation of aryl hydrocarbon receptor target genes is dependent on the calcium/ calmodulin/CaMKIalpha pathway. Mol. Pharmacol. 2008, 73, 769−77. (21) Dong, B.; Matsumura, F. Roles of cytosolic phospholipase A2 and Src kinase in the early action of 2,3,7,8-tetrachlorodibenzo-p-

restricted to few signaling pathways. The observation that transcriptional regulators comprised the largest functional group of proteins with altered phosphorylation corroborates the suitability of the experimental approach applied as it is fully in line with the well-known activity of TCDD as a modulator of transcription. By contrast, the discovery that regulators of small GTPases constituted the second largest group was completely unexpected and sheds light on hitherto unknown activities of TCDD in cells. The importance of these alterations, like that of the phosphorylation changes of the other proteins identified in the present study, for the toxicity of TCDD can now be scrutinized in future investigations. It should also be noted that the identification of thousands of phosphorylation sites clearly unaffected by TCDD during short exposures has provided an invaluable source of information on regulatory pathways not subject to phosphorylation changes. These data could significantly aid in the selection of further experimental approaches dealing with the clarification of the mechanisms of TCDD toxicity. And, finally, the present study has identified phosphorylation changes which exhibited q-values just above the set threshold value and were therefore disregarded according to the stringent significance criteria applied. It appears very likely, however, that this group of phosphopeptides comprises further phosphorylation sites which are actually subject to regulation by TCDD. A closer look at these peptides might yield further stimuli for future targeted investigations on phosphorylation-mediated early alterations in cellular signaling by dioxins.

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ASSOCIATED CONTENT

* Supporting Information S

Tables S1−S9 and Figures S1−S5. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

The MS/MS mass spectra of all individual phosphopeptides identified have been deposited with the ProteomeCommons.org Tranche network and may be accessed using the following URL: https://proteomecommons.org/data set.jsp?i= 7eOKZmaZM61cU58heX9amEydDZy7FjmVv Qi1JWnbHZznP8FRtlceK66QYGbrs7XBhg0As2ZJbp9JUZ2fP CyNogaozncAAAAAAABi2A%3D%3D

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +49 89 3187 2221. Fax: +49 89 3187 3449. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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