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Time-Resolved Quantitative Phosphoproteomics: New Insights into Angiotensin-(1−7) Signaling Networks in Human Endothelial Cells Thiago Verano-Braga,† Veit Schwam ̈ mle,† Marc Sylvester,† Danielle G. Passos-Silva,§ § Antonio A. B. Peluso, Gisele M. Etelvino,§ Robson A. S. Santos,*,§ and Peter Roepstorff*,† †

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark National Institute of Science and Technology in Nanobiopharmaceutics, Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Brazil

§

S Supporting Information *

ABSTRACT: Angiotensin-(1−7) [Ang-(1−7)] is an endogenous ligand of the Mas receptor and induces vasodilation, positive regulation of insulin, and antiproliferative and antitumorigenic activities. However, little is known about the molecular mechanisms behind these biological properties. Aiming to identify proteins involved in the Ang-(1−7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1−7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1−7). Our data suggest that the antiproliferative activity of Ang-(1−7) is due to the activation or inactivation of several target phosphoproteins, such as forkhead box protein O1 (FOXO1), mitogen-activated protein kinase 1 (MAPK), proline-rich AKT1 substrate 1 (AKT1S1), among others. In addition, the antitumorigenic activity of Ang-(1−7) is at least partially due to FOXO1 activation, since we show that this transcriptional factor is activated and accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1−7). Moreover, Ang-(1−7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1−7) in glucose homeostasis. In summary, this study provides new concepts and new understanding of the Ang-(1−7) signal transduction, shedding light on the mechanisms underlying Mas activation. KEYWORDS: ACE2/Ang-(1−7)/Mas axis, angiotensin-(1−7) signaling, Mas receptor, mass spectrometry, phosphoproteomics, renin-angiotensin system, TiO2−SIMAC

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INTRODUCTION The renin angiotensin system (RAS) plays a key role in cardiovascular physiology and pathophysiology by regulating vascular, kidney, and heart function. In the classical view, the RAS is considered to be a circulating endocrine system with the octapeptide angiotensin (Ang) II as an effector hormone and Ang-(1−7) as an inactive fragment of this system. Angconverting enzyme (ACE) is a crucial player in this classical view since it produces Ang II from the decapeptide Ang I. Recently, however, there were major changes to this general view of RAS. The discovery of Mas as the Ang-(1−7) receptor1 and ACE22 as an isoform of the ACE family that predominantly metabolizes Ang II to form Ang-(1−7) focused attention onto this once neglected peptide. While Ang II binds preferentially to the AT1 receptor and promotes vasoconstriction, cell proliferation, and hypertrophy, Ang-(1−7), via Mas activation, induces the opposite effects of vasodilatation, antiproliferation, and antihypertrophy. In addition, Ang-(1−7) has an antitumorigenic activity, which seems to be related to its antiproliferative and antiangiogenesis actions.3,4 Mas is a G protein-coupled receptor and is the endogenous receptor of Ang-(1−7).1 One of the main sites for the formation and action of Ang-(1−7) is the vascular endothelium. © 2012 American Chemical Society

Human aortic endothelial cells (HAEC) constitutively express this receptor5 as do certain tumor cell lines, for example, SKLU-1, SK-MES-1, and A549.6 It is already known that Mas activation leads to vasodilatation due to nitric oxide (NO) production through phosphatidylinositol 3-kinase (PIK3)/ protein kinase B (AKT)-dependent pathways.5 Moreover, Ang-(1−7) negatively regulates downstream effectors of Ang II signaling as the mitogen-activated protein kinase 1 (MAPK1) and NAD(P)H oxidase.7 Furthermore, in recent years, a series of studies revealed a clear connection between RAS and insulin pathways wherein Ang II negatively modulates insulin signaling. In contrast, Ang-(1−7) seems to be an enhancer of insulin actions.8,9 For instance, Mas-knockout animals developed a metabolic syndrome-like state which is characterized by dramatic changes in glucose and lipid metabolisms.9 Overall, therefore, it is clear that the impact of Ang-(1−7) signaling on physiologically important processes is likely to be significant; however, the molecular mechanisms behind the actions of Ang-(1−7) as well as the details of its associated signaling cascade are still elusive. This dearth of Ang-(1−7) Received: February 22, 2012 Published: April 12, 2012 3370

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1:100 ratio with trypsin. The reaction was quenched by adding trifluoroacetic acid (TFA; 0.1% final concentration) followed by centrifugation at 13 000g for 5 min.

data is partly because, thus far, only Western blot-based studies have been used to interrogate Ang-(1−7) signal transduction. This strategy has several advantages, such as high sensitivity and specificity if good antibodies are available. However, the number of simultaneously analyzed proteins is limited using this approach, and only relatively few phospho-specific antibodies with proven performance exist. In addition, discovery of unknown components of a regulatory network by Western blots alone is not possible. In response to this, mass spectrometry-based shotgun phosphoproteomics has emerged as a powerful approach to study signal transduction systems and has already been applied for comprehensive studies of insulin,10 Ang II,11,12 growth factor,13 and other signaling pathways. In this study, we characterized the Ang-(1−7) signaling network using a time-resolved quantitative phosphoproteomic approach. The vast majority of phosphoproteins identified in our study have not previously been associated with Ang-(1−7) signaling. Moreover, the transcriptional factor forkhead box protein O1 FOXO1 was found to be accumulated in the nucleus of Ang-(1−7) treated A549 tumor cells, probably triggering an antiproliferative cascade. In addition, known insulin downstream effectors were found to be regulated by Ang-(1−7), indicating an overlap of these two pathways.

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Peptide Labeling and Phosphopeptide Enrichment

Peptides (100 μg) were labeled with iTRAQ reagent according to manufacturer’s specifications. Samples were combined in 1:1:1:1 ratio and lyophilized. A new method recently developed by Engholm-Keller and collaborators14 that combines TiO2 chromatography15 and SIMAC16 was used to enrich for phosphopeptides. Samples were resuspended in 500 μL loading solution (80% acetonitrile (ACN), 5% TFA, 1 M glycolic acid), transferred to a low-binding sample tube and incubated for 30 min with approximately 3.6 mg of TiO2 beads previously equilibrated with loading solution. Eventual phosphopeptide losses were minimized by repeating this step twice. Briefly, after the first incubation, the loading solution containing unbound phosphopeptides was transferred to an additional low-binding tube and incubated with 1.8 mg of TiO2 and then with 0.9 mg of TiO2 for 30 min. After incubation, the beads were washed with (i) 100 μL of loading solution, (ii) 100 μL of 80% ACN, 2% TFA, and then (iii) 20% ACN, 0.1% TFA. Phosphopeptides were eluted with 70 μL of 1.5% NH3 and lyophilized. Afterward, sample was subjected to SIMAC. Briefly, samples were resuspended in 500 μL of loading solution (0.1% TFA, 50% ACN) and transferred to a low-binding tube containing IMAC beads previously equilibrated with the loading solution. After 45 min incubation at RT, beads were washed with the loading solution and then 50 μL of an acidic solution (1% TFA, 20% ACN) was added to elute mostly monophosphorylated peptides; this acidic fraction and the flow through were combined, dried down in a vacuum centrifuge and subjected to another TiO2 chromatography step. IMAC beads were then incubated with 70 μL of a basic solution (0.5% NH3) to elute mainly multiply phosphorylated peptides.

EXPERIMENTAL SECTION

Materials and Reagents

Human Aortic Endothelial Cells (HAEC) and the cell culture medium (Clonetics EGM-2 BulletKit) were purchased from Lonza (Basel, Switzerland). Trypsin was from Promega (Fitchburg, MI). Poros Oligo R3 reversed-phase material was from PerSeptive Biosystems (Framingham, MA). TiO2 beads were obtained from GL Science (Tokyo, Japan). Iron-coated PHOS-select metal chelate beads were from Sigma (Steinheim, Germany). Empore C8 extraction disk was from 3M Bioanalytical Technologies (St. Paul, MN). Ammonia solution (25%) was from Merck (Darmstadt, Germany). Protease and phosphatase inhibitor cocktails were from Roche (Mannheim, Germany). RapiGest SF Surfactant was from Waters (Milford, MA). The iTRAQ reagents were from Applied Biosystems (Foster City, CA). All other chemicals were obtained from Sigma (Steinheim, Germany).

Sample Desalting Steps

Samples were desalted using self-made microcolumns packed with Poros Oligo R3 reversed-phase resin prior to iTRAQlabeling and after phosphopeptide enrichment. The microcolumns were prepared by stamping out a small plug of C8 material from an Empore C8 extraction disk and placing the plug in the constricted end of a P10 or P200 tips in order to prevent leakage of the reversed phase resin. R3 beads (suspended in 100% ACN) were packed in the tip by the application of air pressure. Each acidified sample was loaded onto an R3 microcolumn and washed three times with 0.1% TFA. Peptides were eluted first with 50% ACN, 0.1% TFA and then 70% ACN, 0.1% TFA. Samples were then lyophilized.

HAEC Stimulation with Ang-(1−7) and In-Solution Digestion

HAEC were grown in four 100 mm dishes at 37 °C according to manufacturer’s specifications until they reached 65% confluence. Cells were then incubated with 10−7 M final concentration of Ang-(1−7) for 3, 5, and 20 min. This peptide concentration was used based on our previous study where we showed that this is the most effective concentration of Ang-(1− 7) to stimulate HAEC.5 Untreated cells were used as control (time = 0 min). After washing with 5 mL of ice-cold HEPES buffer, 400 μL of lysis solution (7 M urea, 2 M thiourea, 200 mM triethylammonium bicarbonate, 0.05% RapiGest, 0.5 mM Na-pervanadate, protease and phosphatase inhibitor cocktails) was added and the cells were then harvested on ice. Cell lysis was enhanced and DNA sheared by tip sonication on ice. Samples were incubated at room temperature (RT) with 10 mM dithiothreitol for 1 h. After reduction, free thiols were alkylated with 40 mM iodoacetamide in the dark for 40 min (RT). Samples were diluted 7-fold with 20 mM triethylammonium bicarbonate and proteins digested overnight (RT) in

Reversed-Phase NanoLC−ESI MS/MS analysis

Enriched phosphopeptides were resuspended with 0.1% formic acid (FA) and separated by reversed-phase liquid chromatography on an in-house packed Reprosil-Pur C18-AQ column (3 μm; Dr. Maisch GmbH, Ammerbuch, Germany) (length, 17 cm; inner diameter, 100 μm; outer diameter, 360 μm) using an Easy-LC nanoHPLC (Thermo Fisher, Waltham, MA). The chromatography gradient was 0−34% solvent B (90% ACN, 0.1% FA) for 213 min at a flow rate of 250 nL/min. The LTQOrbitrap XL instrument (Thermo Fisher, Waltham, MA) was operated in a data-dependent MS/MS scan mode using multistage activation (MSA).17 After a survey scan (400− 1500 m/z; 30 000 resolution at 400 m/z), the top three most intense ions were selected for both low-resolution CID-MSA3371

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medium (Sigma, Steinheim, Germany) supplemented with 10% fetal bovine serum, 100 μg/mL penicillin, and 100 units/mL streptomycin. A549 cells were allowed to grow in poly-lysine treated glass slides for 2 days. The cells were incubated with DMEM-F12 medium without fetal bovine serum for 1 h before the experiment. HAEC were grown as previously described in this Experimental Section. HAEC and A549 were treated with 10−7 M Ang-(1−7) for 3 min. Cells incubated only with medium were used as control. The cells were fixed with 4% pformaldehyde in PBS for 15 min. The slides were washed three times with PBS, treated with 0.5% Triton X-100 in PBS for 10 min and incubated for 1 h with 5% bovine serum albumin diluted in PBS. The slides were then incubated overnight with the polyclonal anti-FOXO1 antiserum (Cell Signaling Technology, Danvers, MA) diluted in PBS. The slides were washed three times, and staining was detected with an Alexa Fluor 488conjugated anti-rabbit antiserum (Invitrogen, Grand Island, NY). Vectashield (Vector Laboratories, Burlingame, CA) containing 4′,6-diamidino-2-phenylindole (DAPI) was used for mounting, and the slides were visualized with a 60× oil immersion objective on a ZEISS Meta confocal microscope (Zeiss, Oberkochen, Germany). Fluorescence quantification was done by using the ImageJ software (http://rsb.info.nih. gov/ij/). For untreated and Ang-(1−7) treated cells, roughly 30 cells were analyzed. Nuclei were individually examined, and the mean label intensity under subsaturating conditions was determined. Unpaired t test with Welch’s correction was performed to compare measured nuclei fluorescence values of Ang-(1−7) treated and untreated in A549 and HAEC cells.

MS/MS (normalized collision energy = 35; activation time 10 ms) and high-resolution HCD-MS/MS (normalized collision energy = 55; activation time 5 ms; 7500 resolution at 400 m/z). Raw data were viewed in Xcalibur v2.0.7 (Thermo Fisher, Waltham, MA). Data Process and Database Search

The MS/MS spectra were processed using Proteome Discoverer (Version 1.2, Thermo Fisher, Waltham, MA) and submitted to database searching against the human sequence library in the Swiss-Prot protein sequence database (Sprot 2010_12 version: 523 151 sequences; 184 678 199 residues) using an in-house Mascot server (version 2.2.04, Matrix Science Ltd., London, U.K.). Trypsin was chosen as the enzyme allowing up to two missed cleavages. S-carbamidomethylcysteine was defined as a fixed modification. Oxidation (Met), Nacetylation (protein N-terminus), phosphorylation (Ser, Thr, and Tyr) and iTRAQ reagents (protein N-terminus and Lys side chain) were defined as dynamic modifications. A decoy database search was performed using a concatenated decoy derived from the Swiss-Prot human database. Only peptides with up to 1% false discovery rate (FDR) were considered for further analysis. All annotated spectra are available from the Tranche data repository under the project name: HAEC phosphoproteome; https://proteomecommons.org/groupssearch.jsp. Data Normalization and Significance Analysis

The two biological replicates with two technical replicates each were considered four experiments in the statistical analysis. The quantitative analysis was carried out on the log2-values of the measured intensities and the data was normalized using the median. Multiple measurements of the phosphopeptides were merged with the R Rollup function from the DanteR package (http://www.omics.pnl.com) allowing for one-hit-wonders and using mean instead of the default median. The common quantitative approach consists in comparison of the ratios of the four replicates. In an equivalent way, we subtracted the mean over the four experimental conditions for each peptide in each replicate, decreasing the influence of measurement errors of the first condition. Significant up/down-regulations between experimental stages were determined by means of p-values. The values were calculated using the ANOVA procedure of the DanteR software and corrected for multiple testing with Benjamini-Hochberg.18 All peptides, independently of their specific p-values, were used to detect common trends in the phosphorylation profiles. Therefore, we carried out fuzzy cmeans cluster analysis that allows the identification of these trends even in noisy data providing that parameter adjustment is carried out carefully. We set the fuzzifier, the parameter adapted to the noise in the system, equal to 2. The other parameter for the clustering, the cluster number, was taken by evaluating two validation indices, namely, the Xie-Beni index19 and the minimum centroid distance.20 These indices provide a measure for the quality of the procedure comparing clustering runs carried out with different cluster numbers. As a result, each peptide obtains a membership value estimating the degree with which it belongs to the most similar cluster. The power of the analysis was increased by inspecting both cluster membership and p-values in the final analysis.

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RESULTS

Quantitative Phosphoproteome Analysis of Ang-(1−7) Signaling

To study the Ang-(1−7) signaling pathway and to identify novel downstream effectors, we used an MS-based quantitative phosphoproteomic approach in Ang-(1−7) treated cells. HAEC were treated with 10−7 M Ang-(1−7) for 3, 5, or 20 min. Untreated cells were used as a control. Following protein digestion and iTRAQ peptide labeling, samples were subjected to the newly developed TiO2−SIMAC (TiO2−IMAC−TiO2) phosphopeptide enrichment method14 (Figure 1). This approach allowed highly specific phosphopeptide enrichment. Around 99% of all peptides identified in this work were phosphorylated (Supplementary Figure S1). Furthermore, more than 50% of all identified phosphopeptides were found in at least two replicates, indicating a reasonable phosphopeptide overlap that could then be subjected to significance analysis (Supplementary Figure S2). Data Normalization and Variance Analysis

The distributions of phosphopeptide log-intensities were slightly asymmetric (Figure 2A), and therefore, normalization to the median was performed. Principal component analysis provides a method to assess sample quality when comparing different experimental conditions. In our results, the scoring plot showed that samples of different experimental conditions clearly formed distinguishable clusters. In addition, the system showed a strong and rapid response after 3 min of Ang-(1−7) stimulation (Figure 2B). The detection of significantly regulated phosphopeptides was carried out applying analysis of variance (ANOVA). Figure 2C depicts volcano plots for the p-values, corrected for multiple testing, versus log-ratios. The ANOVA tests resulted in a substantial number of significantly

FOXO1 Immunolocalization

A549 human lung adenocarcinoma cells (Rio de Janeiro Cell Bank, Rio de Janeiro, Brazil) were grown in DMEM-F12 3372

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Figure 1. Experimental setup. HAEC were grown until they reached 65% confluence and then stimulated with 10−7 M Ang-(1−7) for 3, 5, and 20 min. Untreated cells were used as a reference time point. Proteins were digested overnight with trypsin (1:50) at room temperature, desalted, labeled with iTRAQ, and mixed with the same peptide amount. Phosphorylated peptides were enriched combining TiO2 and SIMAC. Samples were then subjected to LC−MS/MS analysis using CID-MSA fragmentation.

Supplementary Tables 2 and 3). In this manuscript, we use the term up-phosphorylation to describe a specific phosphosite showing an increase in phosphorylation level following Ang(1−7) stimulation, while we use down-phosphorylation to describe sites exhibiting a decrease in phosphorylation level. Figure 3A depicts over-represented categories detected in the regulated phosphoproteins set based on the Gene Ontology annotation of biological processes. As expected, several proteins involved in gene expression and signal transduction were identified. It is worth specifically mentioning that downstream effectors in the insulin and the vascular endothelial growth factor (VEGF) signaling networks were found over-represented in our data set. Proteins involved in apoptosis regulation were also found to be over-represented. It is important to note that these biological processes are not over-represented in the entire data set (regulated and nonregulated proteins; Supplementary Figure S3) indicating that gene expression, apoptosis/ antiapoptosis, and signal transduction are biological processes over-represented only in the regulated data set. We also used the motif-X algorithm25 to determine overrepresented sequence motifs for the regulated phosphorylation sites, using the Swiss-Prot Homo sapiens database as back-

changing phosphopeptides. These data also show that confident detections only slightly correlate to the observed fold-change. Phosphosite Localization

We used the MD-score21 and the Ascore algorithm22 to assign phosphorylation sites. We decided to combine these two methods because Ascore outperforms the MD-score for pS/pT sites while the MD-score is more reliable for pY.21 We used MD-score ≥9 or Ascore ≥19 as threshold. An in-house developed Perl-based program was used to overlay peptide sequences and remove redundancy from phosphorylation site identifications.23 A total of 1288 unique phosphosites with 99% certainty that the sites were assigned correctly (1% FLR) were identified. These sites were found on 699 different proteins and their distributions were 88.9%, 9.4%, and 1.7% for pS, pT, and pY, respectively, which is comparable with previous observations.11,24 All validated phosphosites with their MD-score and Ascore values are listed in Supplementary Table 1. Ang-(1−7) Triggers Phosphorylation Changes on Different Effectors

Stimulation of HAEC with Ang-(1−7) statistically changed the phosphorylation level of 121 sites on 79 phosphoproteins (see 3373

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Figure 2. Data normalization and significance analysis. (A) Boxplots for each of the four replicates. The distributions are skewed to values with high intensities due to missing measurements of low-abundance phosphopeptides beyond the detection limits of the experimental setup. Therefore, the data was normalized to the median. (B) The two largest principal components for all replicates and times after normalization and merging of redundancies. Experimental time-points exhibit similar PCA coordinates (114 = 0 min, 115 = 3 min, 116 = 5 min and 117 = 20 min.). The system shows a strong and immediate response after 3 min of Ang-(1−7) stimulation. (C) P-values versus intensity changes for each time-point following Ang-(1−7) stimulation. The p-values were corrected for multiple testing. Points above the red lines are considered as significant, having p-values below 0.05.

ground. We used a ±6 amino acid residue sequence window surrounding the phosphorylated residues (S, T, or Y). Only motifs with p < 10−6 were accepted. The WebLogo software (http://weblogo.berkeley.edu)26 was used to visualize enriched sequence motifs as logo plots. We used the NetworKIN algorithm27 to predict kinases involved in the phosphorylation of a specific motif as well as their substrates. Figure 3B depicts the two phosphorylation motifs over-represented in our data set. Kinases from the CMGC group recognize the prolinedirected pS/T-P motif, while members of the AGC and CAMK groups phosphorylate the serine and threonine residues in the basophilic R/K-X-X-pS/T consensus sequence. It is worth mentioning that we were able to identify two regulated kinases from the CMGC group, MAPK1 and DYRK1B, and one from the AGC group, AKT1, in our data set (Supplementary Table 3).

Regulation patterns in the time-dependent up/downphosphorylation profile could be identified using the fuzzy cmeans algorithm. All phosphosites identified in our study were grouped in 10 different clusters based on their phosphorylation kinetics. Sites with membership value higher than 0.3 and p ≤ 0.05 are depicted bellow each cluster (Figure 4). Several phosphosites were earlier regulated after Ang-(1−7) stimulation. For example, S124 on AKT1, S338 on PIK3C2A, and S331 on RASIP1 were significantly up-phosphorylated after 3 min (Figure 4 and Supplementary Table 2). The phosphorylation of S124 on AKT1 partially activates this kinase.28 However, only phosphorylation of both T308 and S473 can fully activate the kinase, leading to its translocation to the nucleus where it phosphorylates FOXO1 at T24, S256, and S319, inactivating this transcriptional factor.29−31 In our data, however, the S256 on FOXO1 was down-phosphorylated, which 3374

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Figure 3. Phosphoproteins annotation. (A) Over-represented biological processes of regulated phosphoproteins. (B) The regulated phosphosites are within the R-X-X-pS/T and pS/T-P phosphorylation motifs. Kinase families that recognize these consensus sequences as well as kinase-substrate networks were predicted using the NetworKIN algorithm (http://networkin.info/search.php).

is known to lead to FOXO1 activation. This transcriptional factor plays an important role in many cellular processes, orchestrating programs of gene expression involved in apoptosis and cell cycle arrest, stress resistance, repair of damaged DNA, glucose metabolism, and tumor suppression.29−31 Similarly to FOXO1, the Y187 site on MAPK1 was downphosphorylated after 3 min of Ang-(1−7) treatment. To be fully activated, MAPK1 needs to be phosphorylated at T185 and Y187 by upstream kinases.32 Thus, this pro-proliferative kinase seems to become inactivated by Ang-(1−7). It is worth mentioning that two other kinases, PAK2 and DYRK1B, had their phosphosites rapidly affected by Ang-(1−7). DYRK1B, a member of the dual-specificity family of protein kinases, is predominantly localized in the nucleus and is activated by Y273 autophosphorylation. Interestingly, Ang-(1−7) induced a longlasting up-phosphorylation of Y273, which may suggest a positive feedback effect. Once activated, DYRK1B can phosphorylate its

substrates at serine and threonine residues to induce cell cycle arrest, cellular differentiation, and survival. Moreover, DYRK1B can enhance FOXO1 transcriptional activity independent of its kinase action.33 In addition, this kinase is highly expressed in solid tumors, where it seems to act as a tumor survival factor.34 Conversely, MOBKL1A was up-phosphorylated at T35 only after 20 min of Ang-(1−7) stimulation (Figure 4 and Supplementary Table 2). Once phosphorylated at T35 and T12, this kinase inhibits cells proliferation.35 These data suggest that AKT1, MAPK1, PAK2, and DYRK1B are more upstream kinases of the Ang-(1−7) signaling machinery compared to MOBKL1A. HDAC1 is a nuclear enzyme that deacetylates lysine residues on the N-terminal part of the core histones H2A, H2B, H3, and H4, inducing cell proliferation and reducing gene expression. Phosphorylation of S421 and S423 leads to its activation.36 However, Ang-(1−7) induced a rapid and long-lasting downphosphorylation of these sites which decreases HDAC1 activity 3375

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Figure 4. Clustering of time-dependent up/down-phosphorylation profiles using fuzzy c-means, Phosphosites with similar phosphorylation profile were grouped into 10 different clusters. Proteins containing phosphosites with membership values higher than 0.3 and p ≤ 0.05 are shown below each cluster.

Interacting Genes” or STRING.37 Figure 5 depicts the interaction network of proteins showing an Ang-(1−7)-induced change in phosphorylation. Only high confidence (score = 0.700) interactions were allowed. Stronger interactions are indicated by thicker lines. AKT1 and MAPK1 seem to be critical kinases in this network, interacting with several proteins. PGM2 and PGM2L1, enzymes involved in carbohydrate

(Figure 4 and Supplementary Table 2). As a consequence, overall histone acetylation levels are expected to increase, tending, in most cases, to favor gene expression and cell cycle arrest.36 To better understand the Ang-(1−7) signaling networks, we decided to build a protein−protein interaction map using the online database resource “Search Tool for the Retrieval of 3376

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needs to be performed in order to avoid common pitfalls in quantitative phosphoproteomics; other factors than protein phosphorylation and/or dephosphorylation can influence the relative abundance of the phosphopeptides leading to data misinterpretation. To date, protein expression can influence the up-phosphorylated data set. However, as we used a short-term treatment, significant changes in the protein expression due to the Ang-(1−7) treatment are not expected to occur at such a short time scale. It is worth mentioning that 84% of the regulated phosphopeptides were differentially regulated within 5 min. Only 16 phosphopeptides (16%) were regulated within 20 min. Among them, 11 phosphopeptides belong to proteins that contain other phosphopeptides that are either not regulated or regulated earlier (3 or 5 min), indicating that it is the phosphosite itself, and not the entire protein that was regulated by Ang-(1−7). Unfortunately, for 5 phosphoproteins (ANP32B, CYBRD1, MOBKL1A, OSBPL10, YTHDC1), we could not find other phosphopeptides to support this argument, and therefore, further validation methods should be performed in order to confirm these phosphorylation events. However, it is important to stress that even 20 min can be considered a short time for protein expression.39,40 Conversely to biosynthesis, protein degradation can interfere in the downphosphorylated data set. In our study, we validated the downphosphorylation of FOXO1 with immunohistochemistry since this protein seems to play an important role in the Ang-(1−7) signaling. Moreover, the presence of modified amino acids (e.g., oxidized Met, and spontaneous deamidation of Asn and Gln), as well as tryptic missed cleavages due to steric hindrance of phospho-groups nearby the cleavage site can affect the phosphopeptide relative abundance and therefore should be taken into account during database search and data interpretation. To overcome these issues, we defined as dynamic modifications the oxidation of Met, and allowed up to 2 missed cleavages in the database search. In our experience, spontaneous deamidation is not frequent under the experimental conditions used in this study, and therefore, we did not include this modification.

metabolism, are not linked with the main network, suggesting that we were not able to detect their upstream partners in this study. Ang-(1−7) Induces FOXO1 Accumulation in the Nucleus

FOXO1 plays a pivotal role in the regulation of cell proliferation, apoptosis, glucose metabolism, and others.38 In addition, FOXO1 has a strikingly anticarcinogenic activity.29 These effects are in accordance with some of the observed Ang(1−7) actions.3,4,6,9 Therefore, we believe that FOXO1 is an important node for the Ang-(1−7) signaling although this relationship has not been reported so far. Thus, we decided to investigate in more details whether FOXO1 is indeed activated by Ang-(1−7). As already mentioned in this manuscript, FOXO1 is downphosphorylated at S256 following 3 min of Ang-(1−7) treatment. The down-phosphorylation of this regulatory site induces the translocation of this transcriptional factor from the cytoplasm to the nucleus where it becomes transcriptionally active and regulates a number of target genes.31 Following 3 min of Ang-(1−7) treatment, FOXO1 was accumulated in the HAEC nucleus which is in accordance with the downphosphorylation of S256 (Figure 6). Aiming to investigate whether the anticarcinogenic activity of Ang-(1−7)3,6 could be at least partially explained by FOXO1 activation, we performed the same immunocytochemical assay using A549 human lung adenocarcinoma cells. Cells treated with Ang-(1−7) statistically changed the FOXO1 localization (Figure 6). Thus, Ang-(1−7) may well also induce downphosphorylation of S256 at FOXO1 in A549 cells which leads to the migration of this transcriptional factor to the nucleus. This molecular event suggests that the Ang-(1−7) anticarcinogenic activity involves FOXO1 activation, as well as the inactivation of proliferative proteins such as MAPK1, and HDAC1.

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DISCUSSION

Challenges in Quantitative Phosphoproteomics

Quantitative phosphoproteomics is now a routine in laboratories worldwide. However, a careful data validation

New Insights into the PIK3/AKT-Dependent Ang-(1−7) Signaling Pathway

Ang-(1−7) was regarded as an inactive component of RAS for many years. However, this scenario changed in recent decades, and today, this heptapeptide is known to play a pivotal role in cardiovascular homeostasis. Specifically, Ang-(1−7) counterregulates many of the Ang II effects, such as vascular contraction and growth. Western blot-based studies revealed that class I phosphatidylinositol 3-kinase (PIK3) as well as the serine/threonine protein kinase B (AKT1) are components of the Ang-(1−7)/Mas signaling and involved in vasodilatation through NO production via endothelial nitric oxide synthase (eNOS) activation.5,8 However, in our phosphoproteomic study, we identified a class II isoform of PIK3 (PIK3C2A) which is not an upstream kinase of the AKT1 signaling branch.41,42 This protein contains a C2 domain that acts as a calcium-dependent phospholipid binding motif mediating translocation of proteins to membranes. Although the full cellular effects of PIK3C2A are yet to be deciphered, this kinase induces translocation of GLUT4 to the plasma membrane in response to insulin stimulation.42 AKT1 is the main isoform found in endothelial cells and modulates angiogenesis as well as endothelial cell migration, survival, and growth.43,44 It is fully activated through

Figure 5. Protein−protein interaction networks, Functional interaction network of phosphoproteins regulated by Ang-(1−7) created by the STRING web application. Stronger interactions are represented by thicker lines. 3377

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Figure 6. FOXO1 immunolocalization. The FOXO1 immunolocalization was performed in HAEC and A549 cells untreated (control) and treated with 10−7 M Ang-(1−7) for 3 min. The cells were stained with DAPI, a DNA-specific stain. The anti-FOXO1 was detected by using the Rabbit antiIgG conjugated with Alexa 488 green-fluorescent dye. DAPI, 4′,6-diamidino-2-phenylindole; *p ≤ 0.05; **p ≤ 0.01.

phosphorylation of T308 and S473 residues. 45,46 The phosphoproteome of Ang-(1−7) treated endothelial cells showed an increase in phosphorylation of S124 on AKT1 in comparison to control cells. It has already been postulated that S124 phosphorylation may stimulate AKT1 activation through facilitating the phosphorylation of T308 and S473, thereby modulating the above-mentioned endothelial cell functions as already postulated.28 Even though we could not detect pS473 in this study, it is known that Ang-(1−7) induces phosphorylation of this site.5 Activated AKT1 inhibits the transcription factor FOXO1 through phosphorylation of S256, S319, and T24.30 However, in this work, we have demonstrated that Ang-(1−7) induces activation of this transcriptional factor through S256 down-phosphorylation. The differing inferences that can be drawn from these observations suggest a highly controlled system, possibly with more regulatory inputs than have thus far been described.

The phosphoproteome of Ang-(1−7) treated cells also showed a negative regulation of other proteins involved in cell proliferation as well as in tumorigenesis, such as MAPK1 and HDAC1. Sampaio et al.7 demonstrated that Ang-(1−7) negatively modulates MAP kinases activated by Ang II, thereby inhibiting cell proliferation. Our findings are in agreement with this study since Ang-(1−7) led to down-phosphorylation of Y187 on MAPK1. This event suggests MAPK1 inactivation since phosphorylation of this site is crucial for its activity.32 Ang-(1− 7) also seems to induce a long-lasting inactivation of HDAC1, via down-phosphorylation of the regulatory sites S421 and S423.36 This nuclear enzyme plays a pivotal role in tumorigenesis. Multiple types of solid tumor, such as prostate, gastric, colorectal, breast, and brain tumors, all overexpress HDAC1. Moreover, substrates of this enzyme are proteins involved in tumorigenesis and cancer progression [to review the role of HDAC1 in cancer, see ref 49]. On the other hand, Ang-(1−7) induced activation of the antiproliferative and antitumorigenic FOXO1 that is implicated in the activation of target genes that orchestrate apoptosis, cell cycle arrest, and oxidative stress resistance.38 In addition, we demonstrated that Ang-(1−7) induced FOXO1 accumulation in the nucleus of tumor cells, where it can increase the expression of target genes. Thus, FOXO1 seems to be an important effector of Ang-(1−7) signaling. Besides FOXO1, RASIP1 is also a protein involved in angiongenesis which had its phosphorylation profile differentially regulated following Ang-(1−7) treatment. RASIP1 is an essential protein for blood vessel morphogenesis.50 However, it is yet to be established whether the phosphorylation of this protein stimulates or

Ang-(1−7) Antiproliferative and Anticarcinogenic Activities

Cell growth promotion is one of the key functions of AKT. The predominant downstream effector of the cellular proliferative pathway appears to be the mTOR complex 1 (mTORC1).47 The 40 kDa proline-rich AKT substrate PRAS40 (or AKT1S1) is an mTOR binding partner, acting to inhibit mTOR activity upon binding. AKT phosphorylates AKT1S1 on T246, resulting in dissociation of AKT1S1 from mTORC1 and its binding to 14-3-3.48 Cells treated with Ang-(1−7) showed down-phosphorylation of T246 on AKT1S1. This result suggests that Ang-(1−7) induces the binding of AKT1S1 to mTOR, leading to mTOR inhibition. 3378

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Figure 7. Ang-(1−7)/Mas signaling time-resolved model. Represented in this figure are some phosphorylation events of the Mas receptor activation and their cellular consequences. Proteins in blue have been identified and statistically quantified in this study. Proteins in gray have not been identified in this study but they were included to facilitate the signaling interpretation. This figure was created using Servier Medical Art (www. servier.com). *p ≤ 0.05.

approach.10 It is worth mentioning that two enzymes involved in carbohydrate metabolism, phosphoglucomutase-2 (PGM-2) and glucose 1,6-bisphosphate synthase (PGM2L1), had also their phosphorylation levels regulated by Ang-(1−7) in our study. Finally, PIK3C2A, which induces GLUT4 translocation and then glucose uptake,42 was significantly up-phosphorylated following Ang-(1−7) treatment. Thus, we believe that our data corroborates previous observations and sheds light on the important role of the RAS in glucose homeostasis.

inhibits angiogenesis. In summary, our data suggest that the antiproliferative and antitumorigenic effects of Ang-(1−7) are at least partially due to FOXO1 activation and MAPK1, mTOR, HDAC1 inactivation. Insulin and Ang-(1−7) Share Common Downstream Effectors

The RAS plays an important role in glucose metabolism and homeostasis. Ang II negatively modulates insulin signaling at multiple levels, for example, inhibiting insulin-activated PIK3/ AKT signaling, and inhibiting vasodilator and glucose transport properties of insulin.51 Conversely, there is evidence suggesting that Ang-(1−7) can improve insulin sensitivity. Mas-knockout mice present dyslipidemia, increased levels of insulin and leptin, and approximately 50% increase in abdominal fat mass. Furthermore, Mas deletion leads to glucose intolerance and reduce insulin sensitivity as well as a decrease in insulinstimulated glucose uptake.9 These results clearly show that Mas deficiency induces a metabolic syndrome-like state. Furthermore, Ang-(1−7) positively regulates the insulin signaling effectors JAK2, IRS-1, and AKT1 in rat heart in vivo.8 In our phosphoproteome study, the known downstream effectors of the insulin signaling network AKT1, FOXO1, and CAV1 had their phosphorylation profile differentially regulated by Ang(1−7). In addition, phosphorylation levels of vimentin (VIM) and paxillin (PXM) were also regulated by Ang-(1−7). These two proteins were recently reported as downstream targets of insulin signaling by using a MS-based phosphoproteomic

Time-Resolved Ang-(1−7) Signaling

On the basis of our phosphoproteome study and from the available literature, we proposed a model for the Ang-(1−7)/ Mas signal transduction (Figure 7). In this model, we tried to correlate the known Ang-(1−7) biological effects with some important phosphorylation events that we have identified in this study. Following 3 min of Ang-(1−7) stimulation, several sites were statistically up/down-phosphorylated, including important regulatory phosphosites on MAPK1, DYRK1B, AKT1, FOXO1, and HDAC1. As discussed above, up-phosphorylation of S124 on AKT1 may activate this kinase. Following S256 dephosphorylation, FOXO1 becomes active and then translocates to the nucleus where it can increase transcription of genes involved in apoptosis, cell arrestment, glucose metabolism, oxidative protection, and tumor suppression.29 The accessibility of these genes is probably improved by HDAC1 inactivation. DYRK1B was also activated after 3 min. Once activated, this 3379

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(CEMEL - ICB/UFMG, Belo Horizonte, Brazil). This project was partially financed by CAPES.

kinase can induce cell cycle arrest. Moreover, DYRK1B can interact with FOXO1 and then increases its transcriptional activity.33 Ang-(1−7) inhibits the activation of the proproliferative proteins MAPK1, HDAC1, and MOBKL1A which is regulated only after 20 min of Mas activation. In addition, T246 on AKT1S1 was rapidly down-phosphorylated (3 min after stimulation), which may induce its binding to the mTOR complex, inactivating this cell proliferation agent. The protein kinases PAK2 and PIK3C2A were also earlier downand up-phosphorylated, respectively. However, it is still unclear whether these sites regulate the kinases activity. PAK2 is activated during caspase-mediated apoptosis and PIK3C2A induces GLUT4 translocation to the plasma membrane in response to insulin stimulus. In summary, we performed for the first time a large-scale MSbased phosphoproteomics approach to interrogate the Ang-(1− 7)/Mas signaling in human endothelial cells. In total, 79 out of 699 identified proteins had their phosphorylation sites differentially regulated by Ang-(1−7). We performed extensive literature mining to assign biological relevance of the identified phosphorylation sites; based on this, we proposed a comprehensive signaling model that expands our understanding of Ang-(1−7) signaling networks. Furthermore, the identification of insulin downstream effectors implies a degree of cross-talk between these two signaling pathways, consistent with other reports. Finally, we showed for the first time that Ang-(1−7) anticarcinogenic activity can be at least partially explained by the activation and translocation of the transcriptional factor Forkhead box protein O1 (FOXO1) to the nucleus, where it can regulate expression of target genes.

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(1) Santos, R. A.; Simoes e Silva, A. C.; Maric, C.; Silva, D. M.; Machado, R. P.; de Buhr, I.; Heringer-Walther, S.; Pinheiro, S. V.; Lopes, M. T.; Bader, M.; Mendes, E. P.; Lemos, V. S.; CampagnoleSantos, M. J.; Schultheiss, H. P.; Speth, R.; Walther, T. Angiotensin(1−7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (14), 8258−63. (2) Oudit, G. Y.; Crackower, M. A.; Backx, P. H.; Penninger, J. M. The role of ACE2 in cardiovascular physiology. Trends Cardiovasc. Med. 2003, 13 (3), 93−101. (3) Soto-Pantoja, D. R.; Menon, J.; Gallagher, P. E.; Tallant, E. A. Angiotensin-(1−7) inhibits tumor angiogenesis in human lung cancer xenografts with a reduction in vascular endothelial growth factor. Mol. Cancer Ther. 2009, 8 (6), 1676−83. (4) Machado, R. D.; Santos, R. A.; Andrade, S. P. Mechanisms of angiotensin-(1−7)-induced inhibition of angiogenesis. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 2001, 280 (4), R994−R1000. (5) Sampaio, W. O.; Souza dos Santos, R. A.; Faria-Silva, R.; da Mata Machado, L. T.; Schiffrin, E. L.; Touyz, R. M. Angiotensin-(1−7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways. Hypertension 2007, 49 (1), 185−92. (6) Gallagher, P. E.; Tallant, E. A. Inhibition of human lung cancer cell growth by angiotensin-(1−7). Carcinogenesis 2004, 25 (11), 2045−52. (7) Sampaio, W. O.; Henrique de Castro, C.; Santos, R. A.; Schiffrin, E. L.; Touyz, R. M. Angiotensin-(1−7) counterregulates angiotensin II signaling in human endothelial cells. Hypertension 2007, 50 (6), 1093− 8. (8) Giani, J. F.; Gironacci, M. M.; Munoz, M. C.; Pena, C.; Turyn, D.; Dominici, F. P. Angiotensin-(1 7) stimulates the phosphorylation of JAK2, IRS-1 and Akt in rat heart in vivo: role of the AT1 and Mas receptors. Am. J. Physiol.: Heart Circ. Physiol. 2007, 293 (2), H1154− 63. (9) Santos, S. H.; Fernandes, L. R.; Mario, E. G.; Ferreira, A. V.; Porto, L. C.; Alvarez-Leite, J. I.; Botion, L. M.; Bader, M.; Alenina, N.; Santos, R. A. Mas deficiency in FVB/N mice produces marked changes in lipid and glycemic metabolism. Diabetes 2008, 57 (2), 340−7. (10) Kruger, M.; Kratchmarova, I.; Blagoev, B.; Tseng, Y. H.; Kahn, C. R.; Mann, M. Dissection of the insulin signaling pathway via quantitative phosphoproteomics. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (7), 2451−6. (11) Christensen, G. L.; Kelstrup, C. D.; Lyngso, C.; Sarwar, U.; Bogebo, R.; Sheikh, S. P.; Gammeltoft, S.; Olsen, J. V.; Hansen, J. L. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists. Mol. Cell. Proteomics 2010, 9 (7), 1540−53. (12) Xiao, K.; Sun, J.; Kim, J.; Rajagopal, S.; Zhai, B.; Villen, J.; Haas, W.; Kovacs, J. J.; Shukla, A. K.; Hara, M. R.; Hernandez, M.; Lachmann, A.; Zhao, S.; Lin, Y.; Cheng, Y.; Mizuno, K.; Ma’ayan, A.; Gygi, S. P.; Lefkowitz, R. J. Global phosphorylation analysis of betaarrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR). Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (34), 15299− 304. (13) Blagoev, B.; Ong, S. E.; Kratchmarova, I.; Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 2004, 22 (9), 1139−45. (14) Engholm-Keller, K.; Birck, P.; Størling, J.; Pociot, F.; MandrupPoulsen, T.; Larsen, M. R. TiSH − a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. J. Proteomics, Submitted for publication. (15) Graves, J. D.; Krebs, E. G. Protein phosphorylation and signal transduction. Pharmacol. Ther. 1999, 82 (2−3), 111−21. (16) Thingholm, T. E.; Jensen, O. N.; Robinson, P. J.; Larsen, M. R. SIMAC (sequential elution from IMAC), a phosphoproteomics

ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure S1, number of identified peptides, phosphopeptides, unique phosphopeptides, and nonphosphopeptides using theTiO2−SIMAC approach; Supplementary Figure S2, (A) Venn diagram depicting the overlap of the phosphopeptides identified in each replicate, (B) percentage of high-confidence phosphopeptides identified only in one (blue bar) or in more than one (red bar) replicate; Supplementary Figure S3, over-represented biological processes of all identified phosphoproteins; Supplementary Table 1, high-confidence unique phosphopeptides identified in the study; Supplementary Table 2, list of all phosphopeptides with significant changes in their phosphorylation level; Supplementary Table 3, phosphorylated proteins statistically up- or down-phosphorylated after Ang-(1−7) stimulation. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*R.A.S.S.: phone, (55) 31-3409-2956; fax, (55) 31-3409-2924; e-mail, [email protected]. P.R.: phone, (45) 6550-2404; fax, (45) 6593-2661; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Alistair Edwards for proof reading this manuscript. The immunocytochemical data was obtained using microscopes from the Center of Electron Microscopy 3380

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