Phosphoproteomic Profiling of NSCLC Cells Reveals that Ephrin B3

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Phosphoproteomic Profiling of NSCLC Cells Reveals that Ephrin B3 Regulates Pro-survival Signaling through Akt1-Mediated Phosphorylation of the EphA2 Receptor † Sara Stahl, Rui Mm Branca,† Ghazal Efazat,† Maria Ruzzene,‡ Boris Zhivotovsky,§ Rolf Lewensohn,† Kristina Viktorsson,† and Janne Lehti€o*,†,|| †

Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, SE-171 76 Stockholm, Sweden Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden ‡ Department of Biological Chemistry, Venetian Institute of Molecular Medicine (VIMM), University of Padova, Padova, Italy Science for Life Laboratory, Stockholm, Box 1031, 17121 Solna, Sweden

)

§

bS Supporting Information ABSTRACT: The ephrin and Eph signaling circuit has been reported as deregulated in a number of tumor types including nonsmall cell lung cancer (NSCLC). Here we show that suppression of the ephrin-familly member ephrin B3 decreases NSCLC cell proliferation and has profound effects on cell morphology. To reveal which signaling networks ephrin B3 utilize to regulate such effects on growth and morphology, differential regulation of phosphorylated proteins was analyzed in the NSCLC cell line U-1810. Using strong cat ion exchange (SCX) and TiO2-based fractionation followed by nano-LC and mass spectrometry analysis, we identified 1083 unique phosphorylated proteins. Out of these, 150 proteins were found only when ephrin B3 is expressed, whereas 66 proteins were found exclusively in U-1810 cells with silenced ephrin B3. Network analysis of changes in the phosphoproteome with regard to the presence or absence of ephrin B3 expression generated a hypothesis that the site specific phosphorylation on Ser-897 detected on the erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2) is critical for the survival of NSCLC cells. Upstream of the EphA2 phosphorylation, activation of Akt1 on Ser 129 was also revealed as part of the ephrin B3-mediated signaling pathway. Phosphorylation of these sites was further confirmed by immune-based strategies in combination with mass spectrometry. Moreover, by further stepwise pathway walking, annotating the phosphorylated sites and their corresponding kinases upstream, our data support the process in which a Heat shock protein 90 isoform (HSP90AA1) acts as a protector of EphA2, thereby saving it from degradation. In addition, protein kinase CK2 (CK2) is suggested as a dominant kinase, activating downstream substrates to generate the effects on NSCLC proliferation and morphology. KEYWORDS: phosphoproteomics, ephrin B3, EphA2, Akt1, lung cancer

’ INTRODUCTION Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for more than 1 million deaths each year.1 Nonsmall cell lung cancer (NSCLC) is the most common type and accounts for at least 85% of all diagnosed lung cancer cases.1 Treatment options for NSCLC depend on the stage of disease but include surgery, radiation and platinum-based doublet chemotherapy. Unfortunately, several NSCLC patients are diagnosed with advanced and or metastatic disease, a stage at which efficacy of therapy is modest. Hence, in current clinical practice, there is an urgent need for targeted and individualized therapies that impact the tumor driving pathways from multiple angles, as well as to reduce the side effects compared to current NSCLC r 2011 American Chemical Society

treatments. Indeed, analysis of signaling aberrations in NSCLC has generated targeted therapies such as those targeting the EGFR-signaling pathway, which now are in clinical practice. Unfortunately, not all NSCLC cases respond to such treatment, which emphasizes the need for further discovery of signaling networks that may be the Achilles heels of NSCLCs and thereby constitute novel therapeutic targets. In our previous studies, we have shown that the staurosporine analog PKC 412 can sensitize the highly radio-resistant NSCLC cell line U-1810 to γ-radiation by significantly increasing the Received: January 14, 2011 Published: March 17, 2011 2566

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Journal of Proteome Research levels of mitotic catastrophe and apoptosis,2,3 and moreover, using gene array analysis, we found that down regulation of the ephrin family member, ephrin B3, in part was responsible for this radio-sensitizing effect (Stahl et al., Manuscript submitted). The ephrin B3 ligand belongs to the family of ephrins which consist of the ephrin ligands and their corresponding receptors, the Ephs, the latter of which form the largest class of receptor tyrosine kinases (RTKs).4 A feature of the ephrin/EphR-interaction is the bidirectional signaling activity where Eph kinase acts as a transducer in the Eph receptor expressing cell, whereas reverse signaling occurs in the ligand expressing cell.5 In normal cells, the Eph-ephrin signaling circuit acts to control the positioning of cells by modulating adhesive or repulsive properties.6 The Ephrin-signaling not only accurately guides the path of migrating cells but also facilitates contact and communication between neighboring cell populations, particularly at epithelial mesenchymal boundaries.7 For example, during somite morphogenesis, cells undergo a mesenchymal to epithelial transition that involves changes in cell shape and cell adhesive interactions wherein Eph-ephrin signaling is reported to play a key role.8 With respect to tumors, depending on ephrin and eph receptor subtype as well as on tumor origin, it has been demonstrated that the ephrin and eph receptors signaling circuit often is deregulated as compared to normal nontumorus tissue resulting in either decreased or increased signaling. In studies pertinent to lung cancer, differential gene expression in normal lung vs tumor tissue showed that ephrin A3 was up-regulated 26 times in the tumor.9 Ephrin A2 was present only in the tumor and not detected in normal lung. Significant differences were also observed in tumors of other organs. Another study showed that levels of EphA2 expression in NSCLC can provide information about clinical outcome.10 The highest levels of EphA2 expression were found in those patients who went on to develop brain metastases, whereas low levels were expressed in patients who did not relapse or who developed contralateral disease. These findings show the clinical importance and support the relevance to further study this family of proteins. Detection of circulating EphA2 levels could serve as a diagnostic marker for metastatic disease, as well as serve as a guide to clinical management of metastatic disease. Also, the absence of EphA2 in normal lung tissue makes it an ideal receptor for antibody targeting, which can specifically target tumor tissue, while minimizing adverse effects to normal cells.1113 Importantly, to our knowledge, no studies have so far addressed how the ligand ephrin B3 influences NSCLC signaling and thereby contribute to its malignant phenotype. In the past decade, MS-based shotgun proteomics has undergone an enormous development, enabling the detection of a very large number of proteins in comparable samples at different conditions.14 However, the identifications of the proteins in a sample set, even when provided with relative quantities, are usually difficult to interpret in terms of cellular signaling and biologic relevance. The relevant information for understanding the communication, both intracellular and between cells, often lies in the fine-tuned regulations of proteins mediated through protein phosphorylations. However, the big challenge when it comes to phosphoproteomics of a complex sample, such as of a total cell lysate, is the low abundance of the phosphorylated proteins compared to nonphosphorylated proteins. To tackle this problem, the enrichment of phosphorylated peptides from complex samples through combinations of SCX-chromatography and IMAC (immobilized metal affinity chromatography)15 or TiO216 has become standard procedures. Nevertheless, these

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experiments often still require milligrams of starting material and should preferably be analyzed by a high resolution mass spectrometer.17 In the present study, we aimed to understand more about active cellular signals affected by ephrin B3 in U-1810 cells. We used a phosphoproteomic approach to perform a global and unsupervised screen for overall changes in the phosphoproteome between U-1810 cells in the presence or absence of ephrin B3. This strategy enabled examination of the in vivo on/off phosphorylation status for substrates directly or indirectly regulated by ephrin B3 which resulted in generation of novel and biologically relevant information regarding the ephrin B3/EphA2 signaling circuit.

’ MATERIAL AND METHODS Cell Culture, Treatments, and Protein Extraction

A previously described NSCLC cell line, U-181018 was used in these studies. Cells were seeded in 15 cm dishes (4 dishes/ treatment) and cultured in DMEM medium supplemented with 2 mM L-Glutamine and 10% heat-inactivated fetal calf serum (FCS) until 60% confluence. The NSCLC-cells were then transfected with either custom-made siRNA targeting EphrinB3 (50 -CCAGGAGTATAGCCCTAAT-3, Qiagen) or Nontarget siRNA (Dharmacon) as previously described.19 To assess proliferation capacity of the U-1810 cells in presence or absence of ephrin B3, cells were diluted 1:1 in 0.4% Trypan blue solution (Sigma Aldrich) and the number of viable cells were counted using phase contrast microscopy. Cell morphology was analyzed using a Nikon Eclipse TS100 light microscope (20 magnification) and photos were further processed with Adobe Photoshop CS3. For phosphoproteomic analysis, the U-1810 cells were harvested after 48 h, when a reduced proliferation and an elongated phenotype were observed (Figure 1C) by using Cell dissociation solution (CDS) (Sigma Aldrich). In samples intended for mass spectrometry analysis, protein extraction was carried out by resuspending and incubating the harvested cells in 1 mL of lysis buffer (8 M Urea, 75 mM NaCl, 50 mM Tris-acetate pH 8.2, protease and phosphatase- inhibitor tablets) for 10 min. Samples intended for immunoprecipitation (IP) were resuspended and incubated in 1 mL of the IP lysis buffer included in the Pierce Direct IP kit (prod #26148 Pierce/Thermo) before centrifugation at 10 000 g for 10 min. Protein concentrations were determined using the Bradford DC assay (Bio-Rad) according to the manufacturer’s instructions. Reduction, Alkylation, and Tryptic Digestion

Reduction, alkylation and digestion were carried out essentially as described in ref 15. Briefly, 15 mg of protein from cell lysate was reduced in 5 mM dithiothreitol (DTT) for 25 min at 56 °C and alkylated in 14 mM Iodoacetamide (IAA) for 30 min in darkness at room temperature, followed by another addition of 5 mM DTT and incubation for 15 min at room temperature. Cell lysates were diluted four times in 50 mM Tris-acetate pH 8.2, and 1 mM CaCl2 was added. Proteins were digested overnight at 37 °C using Trypsin 1:100 (Promega). Samples were then acidified with 0.4% Triflour acetic acid (TFA) (final concentration) and centrifuged for 10 min at 2500 g. The supernatant was kept for further analyses. Cleanups I and II

Cleanups I (before SCX chromatography) and II (before TiO2 enrichment) were performed using Sep-PAK vac tC18 2567

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Figure 1. Silencing of ephrin B3 causes profound effects on NSCLC U-1810 cell proliferation and morphology. U-1810 cells were either transfected with nontargeting siRNA or siRNA against ephrin B3 and analyzed for their ephrin B3 protein expression using Western blot (A, left). The proliferation capacities of U-1810 cells in the presence or absence of ephrin B3 expression were analyzed by trypan blue counting (A, right). Data is presented as % of non targeting siRNA treated cells and are the mean ( SD out of three separate siRNA transfections. U-1810 cell morphology in the presence of ephrin B3 (left) and in absence of ephrin B3 (right) was examined (B).

cartridge 6 cm3/500 mg (I) and Sep-PAK vac tC18 cartridge 1 cm3/100 mg (II) according to the manufacturer’s instructions and as described elsewhere.15 SCX Chromatography

SCX chromatography was performed as in ref 15, with minor changes: each sample was resuspended in 200 μL of buffer A (7 mM KH2PO4, pH 2.65, 30% ACN (v/v)) for manual injection into the loop; an Agilent 1200 LC system (Agilent Technologies, Santa Clara, CA) was used, complete with binary pump, diode array detector (DAD SL) and fraction collector (Analyt FC). Three runs of 5 mg of peptides were done for each condition. Acetonitrile (ACN) was allowed to evaporate from the collected fractions. Identical fractions from the three runs per sample (15 mg peptides in total) were then pooled before cleanup II. Enrichment of Phosphorylated Peptides

After clean up II, each fraction from SCX fractionation was resuspended in 120 μL binding buffer (80% ACN, 5% TFA and 1 M Glucolic Acid). Two-hundred forty microliters of TiO2 magnetic beads TiO2 (Mag Sepharose, GE Healthcare) were equilibrated in 500 μL binding buffer using a magnetic tube rack. Each sample was then incubated with 30 μL of bead slurry for 40 min at room temperature with continuous shaking. The eppendorf tubes containing the mix of magnetic beads and sample were then placed in the magnetic tube rack where beads were allowed to congregate at the magnet for one minute before collecting the supernatant, which was added to new beads for further enrichment. The beads were taken off the magnet rack, washed with

binding buffer, and then again placed in the rack. This washing step was repeated three times and finally the beads were rinsed once with wash buffer (80% ACN, 1% TFA). Phosphorylated peptides were eluted by incubating the beads for 30 min in 5% NH4OH. The elution step was repeated once and eluates were pooled before lyophilization and further mass spectrometry analysis. Nano-LC and Mass Spectrometry Analysis

Each fraction was resuspended into 8 μL 5% ACN, 4% Formic acid (FA), of which 0.8 μL was injected into online HPLCMS performed on a hybrid LTQ-Orbitrap Velos mass spectrometer (Thermo). An Agilent HPLC 1200 system was used to provide the gradient for online reversed-phase nano-LC at a flow of 0.4 μL/min. Solvent A was 97% water (milli-Q, Millipore Corporation, Billerica, MA), 3% acetonitrile (ACN), 0.1% formic acid (FA); solvent B was 5% water, 95% ACN, 0.1% FA. The linear gradient went from 2% B up to 30% B in 51 min, followed by a steep increase to 100% B in 3 min. The sample was injected into a C18 guard desalting column (Agilent) prior to a 15 cm long C18 picofrit column (100 μm internal diameter, 5 μm bead size, Nikkyo Technos Co., Tokyo, Japan) installed on to the nano electrospray ionization (NSI) source of the Orbitrap Velos instrument. Acquisition proceeded in ∼3.5 s scan cycles, starting by a single full scan MS at 30 000 resolution (profile mode), followed by two stages of data-dependent tandem MS (centroid mode): the top 5 ions from the full scan MS were selected first for collision induced dissociation (CID, at 29% energy) with MS/MS detection in the ion trap (ITMS), and finally for higher energy collision dissociation (HCD, at 45% energy) with

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Journal of Proteome Research MS/MS detection in the orbitrap (FTMS). Precursors were isolated with a 2 m/z width and dynamic exclusion was used with 60 s duration. Additional experiments were performed using electron transfer dissociation (ETD), as an alternative fragmentation method for peptide ions. Both samples were run in three replicates. Data Analysis

All MS data was searched by Mascot2.2 (Matrix Science Limited, London, U.K.) under the software platform Proteome Discoverer 1.1 (Thermo) against the IPIhuman target decoy (20100120) protein sequence database with a 5% False Discovery Rate (FDR) cut off limit. A precursor mass tolerance of 10 ppm, and product mass tolerances of 0.02 Da for HCD-FTMS and 0.8 Da for CID-ITMS were used. The algorithm considered tryptic peptides with a maximum of 4 missed cleavages; carbamidomethylation of cysteine as the fixed modification; and oxidation of methionine, and phosphorylation of serine, threonine and tyrosine as the variable modifications. Peptides fulfilling 95% confidence (i.e., < 5% FDR) were exported to Protein center (PROXEON) for further analyses. Here, proteins found phosphorylated in three out of three replicates were listed and passed on to computer based network analysis using the Ingenuity Pathway Analysis software (Ingenuity Systems, Inc., Redwood City, CA) concertedly with manual evaluation of data assisted by Protein Center (PROXEON/Thermo Fisher Scientific). In addition, High energy collision induced dissociation (HCD) spectra of peptides from selected active signaling candidates, that is, EphA2, FAK1, Akt1 and HSP90AA1, were carefully analyzed by manual inspection. Scores and E-values for these proteins Western Blot and Immunoprecipitation

Immunoprecipitations (IP) of 1 mg of protein extract using 510 μg of immobilized antibody were carried out using the Pierce Direct IP kit (prod #26148 Pierce/Thermo), according to Manufacturer’s instructions with one modification; instead of using the provided elution buffer, the antigen was eluted from the antibody using the same denaturing buffer (8 M Urea, 75 mM NaCl, 50 mM Tris pH 8.2, protease and phosphatase inhibitor tablets) as was used for cell lysis prior to mass spectrometry. IP experiments; both blotted and silver stained were performed in two independent experiments. SDSPAGE were run, by loading either 30 μg of total protein or half of IP elution volume on NU PAGE 412% gradient Bis-Tris gels (Invitrogen), in MES running buffer, and then blotted onto a PVDF membrane as described elsewhere.18 Antibodies used for IP and Western Blot were: anti-EphA2 (sc-10746), from Santa Crus Biotechnology, and anti-Phospho-Ser/Thr/Tyr (ab15556), Antiephrin B3 (ab53063) and anti-Ephrin A1(ab65072) from Abcam .To assess phosphorylation of Akt1 on Ser 129 and expression of CK2R, antibodies which previously have been described in ref 20 was used. Silver staining of gels was performed using SilverQuest Silver Staining Kit (Cat. No. LC6070, Invitrogen) according to the manufactures instructions. SDS-PAGE experiments were performed in triplicates showing one representative blot.

’ RESULTS AND DISCUSSION The ephrin and Eph receptor signaling circuits are reported to be deregulated in a number of tumor types including Non small cell lung cancer21,22 where several mutations in the Eph receptors are reported.23,24 The effect of ephrin B3 on NSCLC signaling

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has not been analyzed so far and was in focus of the current study in which phosphoproteomics and pathway analysis were used to reveal key signaling components regulated by ephrin B3 in NSCLC. Silencing of Ephrin B3 Causes Profound Effects on NSCLC U-1810 Cell Proliferation and Morphology

Upon silencing of ephrin B3 using siRNA (Figure 1A), U-1810 cells decrease proliferation (Figure 1B) and dramatically change their morphology from a small round appearance to a flattened and elongated structure (Figure 1C). To understand the cellular signals behind these dramatic effects, we set up a global study of the active cellular signals regulated by ephrin B3. Identifying Changes in the Phosphoproteome Induced in Presence or in Absence of Ephrin B3

In order to reveal which signaling networks ephrin B3 utilizes to achieve its effect (Figure 1B and C), we analyzed the differential regulation of phosphorylated proteins in the NSCLC cell line U-1810, in the presence or absence of ephrin B3 expression. For this purpose, U-1810 cells were transfected with either non target siRNA or siRNA for ephrin B3 and were cultured for 48 h until cells with silenced ephrin B3 achieved a clear change in morphology (Figure 1C). Cells were then harvested, proteins extracted and digested to peptides before pre fractionation using SCX-chromatography. Each SCX-fraction was then further enriched for phosphorylated peptides using TiO2 magnetic beads (TiO2 Mag Sepharose, GE Healtcare). Finally, high resolution mass spectrometry was used for identification of phosphorylated proteins which subsequently were submitted for data analysis and further evaluation. A schematic illustration of the experimental work flow is described in Figure 2. Using this strategy, from a total of 28047 MS/MS spectra, we identified 1788 unique phosphorylated peptides along with 630 supportive unique nonphosphorylated peptides (Figure 3A left and Supplementary Table 1, Supporting Information) corresponding to a total of 1083 phosphorylated proteins (Figure 3A right and Supplementary Table 1). 1105 (Figure 3B left) of all peptides were found in all three replicates in at least one condition (in presence or in absence of ephrin B3, or regardless of ephrin B3 status). Out of these 1105, 806 were phosphorylated peptides (Supplementary Table 2, Supporting Information). 546 peptides (Figure 3B left and Supplementary Table 2) corresponding to 150 phosphorylated proteins (Figure 3B right and Supplementary Table 1) were specific for cells expressing ephrin B3, 285 peptides (Figure 3B left and Supplementary Table 2) corresponding to 66 phosphorylated proteins (Figure 3B right and Supplementary Table 2) were specific for cells with silenced ephrin B3, and 274 peptides (Figure 3B left and Supplementary Table 3, Supporting Information) were phosphorylated regardless of ephrin B3 status. Those phosphoproteins that were found in three out of three replicates (Figure 3B right and Supplementary Table 2) were submitted to further bioinformatic analyses. Analysis of active networks through mapping of active signaling molecules generates a hypothesis of active EphA2 and Focal adhesion kinase 1 (FAK1) as involved in ephrinB3-mediated cell survival signaling. Presently, there is no standard software that can adequately build networks or pathways based primarily on status and changes of protein phosphorylation. Nevertheless, Ingenuity pathway analysis (IPA) is based on transcription data and connects proteins in networks based on protein relations as reported in literature. In order to sort out key signaling molecules regulated by ephrin B3, our lists of phosphorylated proteins were 2569



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Figure 2. Schematic picture showing the experimental set up and work flow of the study. Total protein fraction from NSCLC cells either expressing or not expressing ephrin B3 was extracted and further digested into peptides. Peptides were fractionated using SCX-chromatography and each fraction was enriched for phosphorylated peptides using magnetic beads covered in Titaniumdioxide. Protein IDs from the corresponding phosphorylated peptides was submitted to bioinformatic analyses and additional upstream signaling events was pin pointed from the candidates identified from these analyses. Finally, confirmation of key signaling events was done using complementary methods, that is, immuno-based strategies.

submitted to IPA. This resulted in a plethora of networks (data not shown), most of which describe relations between phosphorylated proteins in cells expressing ephrin B3, pointing at a generally higher signal activity in these cells. These results were not unexpected as the cells expressing ephrin B3 are highly proliferative aggressive tumor cells whereas the silencing of ephrin B3 instead decreased growth (Figure 1). When generating our data set of phosphorylated proteins, we have not pursued a quantification of the occupancy rate of the phosphorylation sites. Rather, we used a simple phosphorylated vs nonphosphorylated approach as a strategy to generate data sets describing on/off cellular signaling networks. In parallel to the bioinformatic network analyses by IPA, we also overviewed lists of specific phosphorylated proteins manually. The manual evaluation was assisted using Protein center (PROXEON/Thermo Fisher Scientific) and the brief information generated for each protein by this program. Using this combination of the computerized

network analysis and manual annotation, the EphA2 and its downstream target FAK12527 was highlighted as potential candidates to play critical roles for the ongoing survival-signaling cascade supported by ephrin B3. The two proteins appeared in the top scoring (score 58) network generated by IPA analysis from the input of the 150 phosphoproteins specifically found in NSCLC cells harboring expression of ephrin B3 (Supplementary Figure 1, Supporting Information). Functions associated with this network as postulated by the IPA knowledgebase are described to control cellular assembly and organization, cell morphology and cellular development much in line with the observed difference in cell shape and growth when changing the levels of ephrin B3 in U-1810 cells (Figure 1). The identified sequences and the sites of phosphorylation as well as the corresponding mass spectra of EphA2 and FAK1 are shown in Figure 4A and B and Figure 5A and B. 2570



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Figure 3. Wenn diagram showing differentially phosphorylated proteins and corresponding supporting peptides in NSCLC cells in presence or in absence of ephrin B3 expression. Wenn diagram showing unique peptides (both phosphorylated and nonphosphorylated) and corresponding phosphoproteins in presence or absence of ephrin B3 as indicated in the figure. (A) Left, all peptides identified in the study. Right, all phosphoproteins identified in the study (for specific information, see Supplementary Table 1, Supporting Information). (B) Left, only peptides identified in all three experimental replicates. Right, only phosphoproteins identified in all three experimental replicates (for specific information, see Supplementary Table 2, Supporting Information).

FAK1 encodes a cytoplasmic protein tyrosine kinase, which is found concentrated in focal adhesions between growing cells.28 Activation of this protein is suggested as an important early step in cell growth and in intracellular signal transduction pathways triggered in response to cell interactions with the extracellular matrix.29,30 EphA2 is like ephrin B3 also belonging to the family of ephrins. EphA2 is a receptor which indeed has been connected to lung cancer. Hence, EphA2 directed therapeutics against NSCLC through inhibition of EphA2 has already been addressed in some studies.31 The physiologic role of Eph receptors is crucial in several processes such as cell migration, vascular development and tissue-border formation. In addition, it has been shown that EphA2 is overexpressed in several cancer forms including NSCLC and promotes cell proliferation, motility, invasion, metastasis and angiogenesis in malignant tumors. Also, overexpression of this receptor is associated with a poor prognosis in different tumor types including lung.32 However dual roles of EphA2 have been described; thus, depending on the circumstances, EphA2 may express both anti- and pro-survival properties.33 Manual Verification of Phospho-MS/MS Spectra, Securing Phosphorylation of Ser 897 on EphA2

The combination of higher energy collision induced dissociation (HCD) and the high-resolution, high-accuracy of the Orbitrap instrument allows for much improved localization of

phosphosites in peptides.34 The high accuracy fragment ions of the orbitrap analyzer bring an obvious boost to confidence in annotation of tandem mass spectra, and HCD suffers less from phospho loss than lower energy collision induced dissociation (CID). Therefore, even though the extremely fast scan rate with which CID spectra can be produced by the ion trap which makes this acquisition mode extremely useful to provide phosphopeptide identifications, it is the HCD spectrum that contains reliable information concerning the exact location of the phosphorylation within the peptide sequence. As a side note, it should be mentioned that electron transfer dissociation (ETD) spectra contain even better information on the internal phospho site because ETD does not suffer from phospho loss. However, the lack of coverage power (i.e., ETD yields much less phosphopeptide identifications because it requires high positive charge densities in precursor peptide ions to provide useful tandem mass spectra—a condition rarely compatible with the phosphorylation modification) limits the usefulness of this fragmentation method. HCD on the orbitrap has been used before to locate phosphosites in peptides.35,36 Despite recent successful efforts to improve the automation of phosphosite localization within each peptide,34 we here carried out manual verification of the annotations of the HCD spectra to secure the precise position of phosphorylation. 2571



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Figure 4. Total protein sequences and identified peptide sequences of proteins phosphorylated only in presence of ephrin B3. Total protein sequences with the sequence of the identified phospho-peptides as well as in existing cases also other identified peptides are highlighted as indicated in the figure showing candidate phosphorylation sites of importance for ephrin B3-mediated survival-signaling in NSCLC cells. (A) EphA2R P-Ser 897, (B) FAK1 P-Ser 538, (C) Akt1 P-Ser 129 (P-Ser 124 both in presence and absence of ephrin B3), and (D) HSP90AA1 P-Ser 263 (34% sequence coverage only in presence of ephrin B3).

In Figure 5A, the HCD FTMS (Fourier transform mass spectrum obtained in the orbitrap) of a phosphopeptide (LPSTSGSEGVPFR) unique to the EphA2 receptor protein is shown. Given that there are four residues amenable to phosphorylation (S3, T4, S5, and S7), we provide the four possible annotations in table format under the spectrum. When a theoretical peak from any given annotation is observed in the spectrum, the Δmass (i.e., difference between theoretical and observed m/z’s) is given in the respective table position. The annotation that considers S3 to be the phospho site is the one that explains the largest number of fragment ions in the observed spectrum. The spectrum itself is annotated in this way (with S3 phospho). Because HCD FTMS is high accuracy, we can take

great confidence in the presence and also in the absence of theoretical fragment ions. The fact that the unmodified y7, y8, y9 and y10 ions (in accordance to the correct S3phospho annotation) are present alongside their respective water loss ions (-H2O) is sufficient to prove that the phospho modification could not have been on S7, S5 or T4. Had it been a case of phosphorylation on T4, for example, the unmodified y10 ion could not have been present, but only its phoshorylated and phospho loss versions. In addition, the absence of an unmodified b3 ion (according to the three incorrect annotations) in the spectrum, conjugated with the presence of a phosphorylated b3 ion (as considered by the correct annotation) leaves no room for doubt. Particularly 2572



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Figure 5. Continued 2573



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Figure 5. Annotated high resolution tandem mass spectra of relevant phosphopeptides. Peaks colored blue correspond to C-terminal ions, whereas peaks colored red correspond to N-terminal ions. Below each spectrum a table is given with Δmass values (i.e., the difference, in ppm, between the theoretical and the observed m/z of a fragment ion) for observed peaks. In the table, “” implies that the theoretical fragment ion was not found in the spectrum using a 10 ppm error tolerance. The annotated spectrum of a phosphopeptide from (A) EphA2R was used to illustrate the method of determining phosphorylation sites within the peptide, and therefore all candidate annotations are shown. For the phosphopeptides of (B) FAK1, (C) AKT1, and (D) HSP90AA1, only the correct annotation table is displayed.

because HCD does suffer from phospho loss, and therefore phosphorylated fragment ions are expected to be much weaker than their unmodified or phospho loss versions; and here we observe a phospho b3 ion in detriment of an unmodified b3 ion. All together, we are confident when we conclude that the top annotation, proposing a phosphorylation on Ser-897 is the only possibility of a correct annotation. For the other phosphopeptides discussed in this study, only the correct annotation is displayed (figures 5B-D). Nonetheless, these spectra have been manually evaluated similarly as described above and the complete sets of alternative annotations are supplied in the Supporting Information (Supplementary Figure 2AC). In addition, the scores and E-values for main role proteins of this study are presented in Supplementary Table 3. No Alteration in the EphA2 Ligand, Ephrin A1 Expression in Ephrin B3 Silenced NSCLC U-1810 Cells

In the current study we found that ephrin B3 mediates phosphorylation of Ser 897 on the EPHA2 receptor (Figure 4A and 5A).

Recently, Miao et al. proved that circumstances that declare anti- or pro-survival properties of EphA2 may be coupled to the presence of a specific ligand. Hence, in the absence of its ligand ephrin A1, EphA2 loses its catalytical (kinase) function and instead acts as a substrate to Akt1.33 Thus, Miao et al proved that the pro-survival kinase, Akt1 is generating the phosphorylation at Ser 897 on EphA2 and thereby drives survival signaling in tumor cells and in contrast, when EphA2 is ligated by ephrin A1, migration and invasion are inhibited.33 In our case, we detect the phosphorylation on Ser 897 of EphA2 only when ephrin B3 is present. Taking this into consideration, it indicates that the presence of ephrin B3 prevents ephrin A1 from ligating EphA2 in this NSCLC- cell line. To test if the effect of ephrin B3 on EphA2 Ser 897 phosphorylation in U-1810 cells is mediated by alteration of ephrin A1 expression, we compared ephrin A1 levels in these cells with our without ephrin B3 expression (Figure 6A). A similar expression of ephrin A1 was however found in cells irrespectively of Ephrin B3 expression, suggesting that ephrin B3 inhibition does not alter ephrin A1 expression levels. 2574



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Figure 6. Immuno-based confirmation of selected candidates from hypothesis generating mass spectrometry data. The NSCLC cell line, U-1810 was used for all experiments and samples were treated as indicated. For westen blots (wb) 30 μg of total protein from total cell lysates were loaded and GAPDH was used as loading control. For immunoprecipitations (IP); 1 mg of total protein were used as starting amount. (A) Wb of Ephrin A1 (MW 24 kDa). (B) IP of EphA2R (MW 130 kDa) probed with pan-phospho antibody, showing two independent experiments. (C) 1D-gel, well 1 and 2, Crude lysates from U-1810 cells ( ephrin B3; well 4 and 5, IP of EphA2R (MW 130 kDa) from U-1810 cells ( ephrin B3. The gel was stained using Silver stain. (D) Wb of Akt1 P-Ser 129 (MW 60 kDa) in the presence or absence of ephrin B3 expression. (E) Wb of CK2 alpha (MW 45 kDa) the presence or absence of ephrin B3 expression.

EphA2 is Not Expressed in NSCLC U-1810 Cells if Ephrin B3 is Absent

To further validate the differential phosphorylation of EpHA2, we carried out immunoprecipitation (IP) of EphA2. As there are no phospho-antibodies available for the Ser-897 site of EphA2 we used a pan-phospho antibody on the blot of the immunoprecipitated sample and a unique band at 130 kDa pointing at phosphorylation on EphA2 (Figure 6B) was observed only in presence of ephrin B3. When imunoprecipitating EphA2 in cells

with silenced and non silenced ephrinB3 followed by SDS-PAGE and silver staining to visualize total protein levels, it appeared that EphA2 was not only phosphorylated in presence of ephrin B3, but the protein itself actually only existed in presence of ephrinB3 (Figure 6C). In gel digestion and mass spectrometric analysis of the 130 kDa band (Figure 6C) confirmed identification of EphA2 (data not shown). Differential CK2-mediated phosphorylation of Akt1 on Ser 129 contributes to the downstream activation of EphA2 in 2575



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Figure 7. Hypothetical illustration describing ephrin B3-mediated tumor driving signaling in NSCLC cells. The phosphorylation of EphA2R on Ser 897 is only possible in presence of ephrinB3 expression. Hypothetically expression of ephrin B3 by some means hinder the ligation of EphA2R by ephrin A1, either by binding ephrin A1 or by direct ligation of the EphA2 receptor. The phosphorylation of EphA2R is likely generated by Akt1 which in turn is activated by CK2. Moreover, CK2 is also phosphorylating HSP90AA1 on Ser 263 which then use its chaperon properties to protect the EphA2R from proteosomal degradation. Thereby, downstream signaling through activation of FAK1 on Ser 538 is regulating a continuous proliferation.

Ephrin B3 expressing U-1810 cells. Based on the fact, that in presence of ephrinB3; EphA2 is phosphorylated on Ser 897, a site previously shown to be phosphorylated by Akt1 in human astrocytoma,33 we generated the hypothesis that Akt1 is active (and thereby phosphorylated) in the presence of ephrin B3 also in U-1810 cells. However, Akt1 did not appear in our list of proteins only phosphorylated in presence of ephrin B3 (Supplementary Table 2, Supporting Information) but was indeed found in the original list containing all phosphorylated proteins generated in this study (Supplementary Table 1, Supporting Information). Interestingly, it appears that Akt1 was phosphorylated on Ser 124 (Figures 4C and 5C) regardless of the level of ephrinB3 expression, and for that reason was not initially listed as differentially phosphorylated. However, Akt1 did show differential phosphorylation on a second phophorylation site. Hence, in cells expressing ephrin B3, Akt1 was phosphorylated also on site, Ser 129 (Figures 4C and 5C) which was not the case in cells with silenced ephrin B3. This was also confirmed using Western blot (Figure 6D) whereas no alteration in total levels of Akt1 was detected (data not shown). Thus our data support a mechanism in which Ephrin B3 expression specifically results in Akt Ser 129 phosphorylation and which suggest a potential role for this kinase in phosphorylating EphA2 on Ser 897. In addition, we have previously proved that protein kinase CK2 (CK2) is directly responsible for hyper activation of Akt1 through phosphorylation on Ser 129 in other tumor cells notably Jurkat cells.20 Thus, we hypothesized that CK2 may exert a similar function also in NSCLC cells when ephrin B3 is expressed but not in U-1810 cells devoid of ephrin B3 expression. CK2 r is Highly Expressed in U-1810 NSCLC cells

The serine/threonine kinase CK2 is invariably elevated in tumors as compared to normal tissues. By itself, CK2 does not appear to be an oncogene, since mutations causing gain of function similar to those determining the oncogenic potential of many protein kinases, have never been reported in the case of CK2.37 Moreover, CK2 has been described to harbor an

extraordinary pleiotropy which was further proven in a weblogo phosphoproteome analysis published by Salvi and co-workers 2008.38 In fact, this study indicates that CK2 alone could contribute to the generation of a substantial amount of the human phosphoproteome, potentially as much as 20% also at basal conditions. Thus, the hypothesis; that a common denominator of diverse tumor cells is a nononcogenic dependency to elevated levels of CK2, has recently been raised.37 As a next step we therefore examined CK2 R levels in U-1810 cells using Western blot (Figure 6E). Indeed we found this protein to be highly expressed both in presence and absence of ephrin B3 expression. This is also expected with regard to the multitasking role described for this kinase, and therefore, its activity is probably not specific only for ephrin B3 regulated signaling. HSP90AA1 is activated by CK2 and is potentially saving EphA2 from proteasomal degradation in presence of ephrin B3 in NSCLC cells. As Akt1 and CK2 had gained our attention, we searched the public domain data together with our phospho-data for additional signaling molecules that could participate in this pathway. One possible candidate turned out to be heat shock protein HSP90AA1.37 Interestingly, we found HSP90AA1 to be highly expressed in U-1810 cells in presence of ephrin B3 as it was identified with sequence coverage of 34% compared to 0% in absence of ephrin B3 expression (Figure 4D). Moreover, HSP90AA1 was activated by phosphorylation on Ser 263 only in presence of ephrin B3 expression (Figure 4D and 5D).The role of heat shock proteins is to act as chaperons and protect its clients from denaturation and proteosomal digestion. Interestingly, EphA2 has recently been reported as such a client to HSP90 which in turn is reported to be required for EphA2 stability.39 Moreover, another observation further corroborating our model is that CK2 generates the phosphorylation on Ser 263 of HSP90AA1 which is detected in our data set in presence of ephrin B3 expression but not in its absence (Figure 4D and 5D). As described above, IP of EphA2 in U-1810 cells with silenced and nonsilenced ephrinB3 showed that the EphA2 actually only existed in presence of ephrinB3 expression (Figure 6C). This 2576


Journal of Proteome Research finding strongly correlates with our theory of CK2 acting as a dominating kinase in presence of ephrin B3 expression in U-1810 cells, since our data strongly suggest CK2 to also regulate HSP90AA1 which in turn protects EphA2 from proteasomal degradation. Hence, CK2 is allowing EphA2 to stay active and drive survival signaling as long as ephrin B3 is present.

’ CONCLUSIONS In this study we have used the information from differentially phosphorylated proteins in a NSCLC cell line in presence and absence of ephrin B3. We were able to back track the upstream kinases from the differentially phosphorylated substrates and thereby perform upstream pathway walking; a work which led to increased understanding of the role of ephrin B3/EphA2 mediated survival signaling in NSCLC cells. Given the previous observed role of EphA2 in NSCLC tumor genesis (i.e., in invasion and metastasis) and given its observed upregulation in NSCLC patient derived material our novel finding of its role in ephrin B3 signaling is timely and intriguing. Our data suggests that, in NSCLC cells EphA2 act as a substrate to Akt1 in the presence of high levels of both ephrin B3 and ephrin A1, tempting us to speculate that the role of ephrinB3 in NSCLC is to block the binding of ligand ephrin A1 to EphA2 and thereby promoting cell survival. At present, we can not rule out ephrin B3 directly acting as a binding partner of ephrin A1 thereby preventing it from binding the receptor. Yet another plausible explanation is that ephrin B3 itself acts as ligand to EphA2 thereby making EphA2 to work as a dependence receptor of ephrin B3, similarly to what has been previously described for the relationship of ephrin B3 and another Eph receptor EphA4.40 In addition, overexpression of EphA2 is known to result in increased levels of focal adhesions,31 and interestingly, our data show that FAK1 is phosphorylated on Ser 538, in line with the upstream increase of total levels and phosphorylation of EphA2 in presence of ephrin B3. In summary, a potential signal scenario in these highly malignant NSCLC cells might be that ephrin B3 drives survival by blocking the interaction between ephrin A1 and EphA2, thereby allowing CK2 to phosphorylate Akt1 on Ser 129 which subsequently enable Akt1-mediated phosphorylation of EphA2 on Ser-897. We also suspect that CK2 in parallel is activating HSP90AA1 (via phosphorylation of Ser 263) which then chaperones EphA2 and protects it from proteosomal degradation. In turn, downstream of EphA2, FAK1 is activated through phosphorylation of Ser 538. This hypothesis (and phospho-proteomics data supporting it), is illustrated in Figure 7. Given that CK2, HSP90 and EphA2 all have been considered as targets for cancer therapy, our data strongly suggest that such a therapy might be of relevance for NSCLC in which ephrin B3 is present. Further studies are therefore warranted as it might give rise to novel treatment approaches for NSCLC, a tumor disease with poor outcome and where novel treatment strategies are urgently needed. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary Figure 1. Top scoring network (score 58) generated from IPA analysis. Highlighting ephrin B3-mediated activation of the EphA2 receptor and PTK2/FAK1 as potential important factors for survival of NSCLC cells. Supplementary

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Figure 2. Phospho-MS/MS spectra. All possible annotations of spectra from phosphorylated peptides corresponding to AKT1 double and single phosphorylation, FAK1 and HSP90AA1. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Janne Lehti€o, Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, SE-171 76 Stockholm, Sweden. Phone: þ46 8 5177 639. Fax: þ46 8 5177 1000. E-mail: Janne.Lehti€[email protected].

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Phosphoproteomic Profiling of NSCLC Cells Reveals that Ephrin B3

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