Phosphoproteome Analysis of HeLa Cells Using Stable Isotope

Phosphoproteome Analysis of HeLa Cells Using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Ramars Amanchy, Dario E. Kalume, Akiko Iwahori, Jun Zhong, and Akhilesh Pandey* McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry and Oncology, Johns Hopkins University, 733 N. Broadway, Baltimore, Maryland 21205 Received May 9, 2005

Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosinephosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes. Keywords: phosphoproteomics • tandem mass spectrometry • SILAC • EGF receptor signaling • tyrosine phosphorylation

Introduction Reversible phosphorylation of proteins is an important mechanism for modulating signal transduction pathways. Phosphorylation of proteins is known to regulate enzymatic activity, subcellular localization, protein-protein interaction, and degradation of proteins. Phosphorylation events relay signals from extracellular stimuli through interactions that are dependent on phosphorylated residues in specific contexts.1 In eukaryotes, the residues that undergo protein phosphorylation are serine and threonine, and to a lesser extent, tyrosine. However, the isolation of phosphoproteins from complex mixtures and the determination of phosphorylation sites still remain a challenge.2,3 Most traditional methods for characterizing the phosphoproteome are limited because of inadequate amounts of proteins and low stoichiometry of phosphorylation. Mass spectrometry has become the technique of choice for phosphoproteome profiling and analysis especially because of its sensitivity and high-throughput.4,5 Recent developments in mass spectrometry promise to provide novel insights into dynamics of protein activities regulated by post-translational modifications.6 A large majority of the available data on tyrosine phosphorylation events comes from in vitro studies. Although such * To whom correspondence should be addressed. Tel: (410) 502-6662. Fax: (410) 502-7544. E-mail: [email protected]. 10.1021/pr050134h CCC: $30.25

 2005 American Chemical Society

experiments help determine whether a molecule could be a potential substrate of a particular kinase, it is experimentally difficult to confirm if the phosphorylation indeed occurs in vivo. Thus, cell-based strategies are a prerequisite for characterization of the phosphoproteome to find true protein kinase substrates. Oda et al. used stable isotopes (15N) to label yeast proteins for obtaining quantitative information about phosphorylation events.7 Stable isotope containing amino acids have been used by our group previously to obtain similar quantitative information about phosphorylation events8 as well as to identify the targets of protein tyrosine kinases.9 Affinity techniques are also used in conjunction with labeling methods to enrich phosphoproteins or phosphopeptides from complex mixtures prior to mass spectrometric analysis. Phosphotyrosine antibodies have been quite successfully used for enrichment of tyrosine phosphorylated proteins.10,11 Immobilized metal affinity chromatography (IMAC) has also been used by investigators for purification of serine/threonine as well as tyrosine phosphorylated peptides.12-15 Over 95 protein tyrosine kinases and 107 protein tyrosine phosphatase genes have been described in Homo sapiens.16,17 Although phosphorylation on tyrosine residues of proteins accounts for a relatively small fraction of total phosphorylation, it is quite significant in a number of biological processes. Our objective was to selectively visualize tyrosine phosphoproteins using stable isotope labeling in cell culture (SILAC), followed Journal of Proteome Research 2005, 4, 1661-1671

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research articles by identification using mass spectrometry. We first induced hyperphosphorylation of proteins on tyrosine residues by an inhibitor of cellular protein tyrosine phosphatases, sodium pervanadate,18 and subsequently enriched tyrosine phosphoproteins followed by SDS-PAGE and LC-MS/MS analysis. Mass spectra from a total of 118 proteins, which exhibited an enrichment among the proteins derived from pervanadate treated (heavy isotope containing) cells, were manually analyzed. Forty-five of these proteins have been described to be tyrosine phosphorylated while the remaining 73 are not known to be tyrosine phosphorylated. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel sites. A bioinformatics analysis of the phosphorylation sites identified in our experiments revealed that most of the tyrosine residues were not predicted by two commonly used phosphorylation site prediction programs, Netphos19 and Scansite.20 Importantly, using this approach, we were able to categorize several proteins of unknown function (e.g., chromosome 3 open reading frame 6 (C3ORF6), KIAA0918, and newly identified cDNA from the epidermal differentiation complex-4 (NICE-4) as potential signaling molecules. We validated the phosphorylation status of a subset of the identified proteins including RNA binding motif protein 4 (RBM4), clathrin assembly lymphoid-myeloid leukemia protein (CALM) and transferrin receptor, using antibodies against endogenous proteins, or, by using epitope-tagged cDNAs in the case of NICE-4, cytoskeleton-associated protein 1 (CKAP1), breast cancer anti-estrogen resistance 3 (BCAR3), WW domain binding protein 2, and C3ORF6. As expected, all of these proteins were highly phosphorylated upon pervanadate treatment. In addition, three of these proteins, BCAR3, CKAP1, and C3ORF6, were found to be tyrosine kinase substrates in the EGF receptor signaling pathway as well. Further experiments on the rest of the identified proteins would help unravel their function in protein signaling cascades.

Experimental Section Chemicals and Reagents. Stable isotope containing amino acids, 13C9-tyrosine, 13C6-arginine and 13C6-lysine, were purchased from Cambridge Isotope Labs (Andover, MA). Complete protease inhibitor cocktail tablets were purchased from Roche (Indianapolis, IN), sodium orthovanadate from Sigma-Aldrich Co (St. Louis, MO), anti-phosphotyrosine antibodies (4G10) agarose-conjugate and streptavidin-agarose beads from Upstate Biotechnology (Lake Placid, NY), antiphosphotyrosine-RC20 biotin conjugate from BD transduction laboratories (Lexington, KY) and colloidal Coomassie staining kit from Invitrogen (Carlsbad, CA). Sequencing grade trypsin was purchased from Promega (Madison, WI). Stable Isotope Labeling with Amino Acids in Cell Culture. HeLa cells were grown in Dulbecco’s modified Eagle’s medium containing ‘light’ tyrosine, arginine and lysine or ‘heavy’ 13C9tyrosine, 13C6-arginine and 13C6-lysine supplemented with 10% dialyzed fetal bovine serum plus antibiotics.21 Detailed instructions about this protocol are available at http://www.silac.org. Heavy arginine and lysine were used together in the media. The HeLa cells were adapted to growth in isotope-containing medium supplemented with dialyzed serum prior to initiating these experiments. Pervanadate was freshly prepared before the experiment by dissolving equimolar (100 mM) solutions of sodium orthovanadate and hydrogen peroxide. For each experiment, 20 dishes (15 cm) of confluent HeLa cells were used per condition (untreated or pervanadate-treated). Five percent 1662

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of the HeLa cell population grown in light media was ‘spiked’ by treating one dish out of 20 dishes with pervanadate. The cells were serum starved for 2 h before treatment with 1 mM pervanadate for 30 min and were subsequently lysed in a modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM sodium orthovanadate in the presence of protease inhibitors) or in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1% SDS and 1 mM sodium orthovanadate in the presence of protease inhibitors). Cloning and Transfection Experiments. NICE-4 (NP•055662), WW domain binding protein 2 (NP•036610), BCAR3 (NP•003558), cytoskeleton-associated protein 1 (NP•001272) and chromosome 3 open reading frame 6 (NP•777568) were subcloned into a Flag epitope-tagged mammalian expression vector, pCMVtag4A. 293T cells were transfected with cDNAs encoding the above-mentioned proteins alone using the calcium phosphate transfection method (Invitrogen, Carlsbad, CA) for the cells to be treated by pervanadate (1 mM) or were cotransfected with EGFR cDNA for the cells to be treated by EGF. The cells were harvested and the immunoprecipitation with anti-Flag antibody was carried out in modified RIPA buffer. Immunoprecipitation and Western Blotting. Light and heavy cell lysates were precleared with protein A-agarose, mixed and incubated with 400 µg of 4G10 monoclonal antibodies coupled to agarose beads, 75 µg of biotin-conjugated RC20 antibody and streptavidin-agarose beads overnight at 4 °C as described earlier.9,21 Precipitated immune complexes were then washed three times with lysis buffer and eluted three times with 100 mM phenyl phosphate in lysis buffer at 37 °C. The eluted phosphoproteins were dialyzed and resolved by 10% SDSPAGE. The gels were stained using colloidal Coomassie stain. Western blotting experiments were performed using antiphosphotyrosine antibody (4G10) and reprobing was carried out to detect individual proteins using specific antibodies. For validation of phosphorylation of individual proteins (CALM, RBM4, and transferrin receptor), HeLa cells were grown in 10 cm dishes. One dish was left untreated as control, one was treated with EGF for 5 min and the other with 1 mM pervanadate for 30 min. Cells were lysed in RIPA buffer. Antibodies against CALM were kindly provided by Dr. Ernst J. Ungewickell, Hanover Medical School, Hanover, Germany, and antibodies against RBM4 were a gift from Dr. Woan-Yuh Tarn, Institute of Biomedical Sciences, Taipei, Taiwan. LC-MS/MS Analysis. The colloidal Coomassie blue stained protein bands were excised and digested with trypsin as described previously.21 Briefly, the gel slices were excised, and incubated with trypsin overnight at 37 °C to allow digestion of proteins after a reduction and alkylation step. After in-gel digestion, the tryptic peptides were extracted. The supernatant from the in-gel digestion containing the peptide mixture was separated and partially dried down in a vacufuge to approximately 10 µL. The extracted peptide mixture was centrifuged for 2 min, 12 000 × g at 4 °C to prevent any small pieces of gel from being loaded onto the liquid chromatography system. The peptide mixture was analyzed by reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS). The LC system (Agilent 1100 Series, Agilent Technologies, Palo Alto, Ca) was equipped with a well plate sampler, a vacuum degasser, and a capillary pump. Reversed-phase chromatography was

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performed by loading peptides using automated sampler onto 2 fused silica capillary columns (OD-360, ID-75 µm) in tandem, a precolumn (5 cm in length) packed with 12 µm C18 ODS-A, (YMC Co, Kyoto, Japan) followed by an analytical column (10 cm in length) packed with 5 µm Vydac C18 resin (Nest Group, Southboro, MA). Columns were washed with 95% mobile phase A (0.4% acetic acid and 0.005% heptafluorobutyric acid, v/v) and 5% mobile phase B (90% acetonitrile, 0.4% acetic acid, and 0.005% heptafluorobutyric acid, v/v). The peptides were loaded onto the precolumn (C18 ODS-A) using a linear gradient of 5% mobile phase B. During sample loading the flow rate was kept at 4 µL/min and for peptide separation the flow rate was decreased to 250 nL/min. Subsequently, separation of peptides was carried out in an analytical column using a linear gradient elution from 87% mobile phase A (0.4% acetic acid and 0.005% heptafluorobutyric acid, v/v) to 40% mobile phase B (90% acetonitrile, 0.4% acetic acid and 0.005% heptafluorobutyric acid, v/v) in 34 min. A potential of 2.5 kV was applied to the emitter in the ion source. The spectra were acquired on a Micromass Q-TOF US-API mass spectrometer (Manchester, UK) equipped with an ion source sample introduction system designed by Proxeon Biosystems (Odense, Denmark). Data Analysis. The acquisition of data was performed using MassLynx (version 4.0). All spectra were obtained in the positive ion mode. The settings used for the automated data collection were as follows: Ion mass window was set to 2.5 Da. The MS/ MS to MS switch criteria was set to intensity below a threshold of 5 counts per second and the MS to MS/MS switch criteria was set to a threshold corresponding to intensity of 6 counts per second. Scan times of 1 s for MS experiments and 3 s for MS/MS experiments were used and number of MS/MS per cycle used was 3 and total cycle time was 10 s with an interscan interval of 0.1 s. Peptide and protein identification from the MS/MS spectra was carried out as follows. MassLynx was employed to generate a peak list (pkl files) from the raw data using the following parameters: smooth window: 4.00; number of smooths: 2 (smooth mode: Savitzky Golay); percentage of peak height to calculate the centroid spectra: 80%; with no baseline subtraction. MS/MS spectra were searched the human RefSeq database22 (build 33) using MASCOT23 (version 1.9) on a Linux cluster. The following settings were used: number of tryptic missed cleavages allowed: 2; peptide window tolerance: (1.0 Da; and fragment mass tolerance: (0.3 Da. Amino acid modifications allowed were oxidation of methionine (+16 Da), carbamidomethylcysteine (+57 Da) and phosphorylation of tyrosine residues (+80 Da). Variable modifications of 80 Da for tyrosine phosphorylation and 6 and 9 Da for stable isotope containing amino acids, 13C6-Arginine, 13C6-Lysine, and 13 C9-Tyrosine, respectively, were used. In general, only peptides with clear mass spectra with a Mascot score >30 and containing a sequence tag of at least four consecutive amino acids were considered in this study. Peptides with lower score presenting a clear tandem mass spectrum were manually interpreted. The intensity ratios were calculated by comparing the extracted ion chromatograms of the light and heavy peptides.21 The tandem mass spectra were manually verified to assign the sequence and phosphorylation sites for all peptides identified in this study. The phosphorylation sites were manually verified and assigned after confirmation of a mass difference of 243 Da corresponding to phosphotyrosine residue. Furthermore, presence of an ion marker for phosphotyrosine immonium ion at m/z 216.043 was also verified. MASCOT results were parsed using an in-house algorithm developed

using C++ to sort peptides with score >30 for further analysis. Sequence coverage for proteins was calculated based on these peptides only. For all purposes, repetition of peptides with similar sequence information was eliminated except in situations where the peptide contained variable modifications (oxidation (M), phosphorylation (Y), 13C9-tyrosine, 13C6-arginine, or 13C6-lysine). If a single peptide was observed for a protein, then its N and C termini were verified for tryptic cleavage ends (arginine or lysine). Ion ratios were calculated for all the peptides with score >30. Further analysis was done on peptides which showed increase in the relative intensity (ion ratio). Sequence alignment of human SLITRK family of proteins was performed and cladogram was made using an online version of ClustalW.24

Results and Discussion SILAC for Differential Labeling of Proteins. SILAC is a simple, in vivo, labeling procedure for investigation of protein dynamics by mass spectrometry and has been successfully used for differential labeling of growing cell populations for tackling the phosphoproteome and quantitation of phosphorylation events.8,9,25 HeLa cells were adapted in media containing stable isotope containing amino acids. Two different labeling experiments were carried out. In one set of experiments, HeLa cells grown in light medium were compared to those grown in 13C6arginine plus 13C6-lysine. In a second set of experiments, cells grown in light medium were compared to those grown in 13C9tyrosine.9 We labeled the cells with 13C6-arginine and 13C6-lysine because it provides a better coverage of labeled peptides. Labeling with 13C9-tyrosine allowed us direct identification of the labeled tyrosine containing peptides and also to facilitate direct identification of sites of phosphorylation on peptides containing labeled tyrosine. In both cases, the stable isotope labeled cells were treated with a tyrosine phosphatase inhibitor, sodium pervanadate which causes hyperphosphorylation of tyrosine residues. The cell populations were harvested and cell lysates were mixed and tyrosine phosphorylated proteins immunoprecipitated using a combination of monoclonal antibodies directed against phosphotyrosine residues as shown in the schematic in Figure 1. Identification of Tyrosine-Phosphorylated Proteins by LCMS/MS. Mixing of light and heavy isotope labeled cell lysates allowed us to compare the profile of proteins in a single experiment. In MS/MS spectra, fragmentation patterns generated by light and heavy peptide pairs are identical except for the expected mass shift of the fragment ions. The peptides containing a single 13C9-tyrosine should be heavier by 9 Da, whereas those containing 13C6-arginine or 13C6-lysine residues should be heavier by 6 Da. The ratio of the intensity of the heavy versus the light peptides provides information about the degree of phosphorylation upon pervanadate treatment. Thus, the greater the extent of phosphorylation of a protein, the higher is its abundance in anti-phosphotyrosine antibody immunoprecipitates. Peptide pairs with a little or no increase in intensity indicate that the protein is not different in abundance in the two states being compared. These proteins were not investigated further as they are likely nonspecifically bound proteins. An increase in heavy/light intensity ratio, indicating an increase in total phosphotyrosine content upon pervanadate treatment, was found in peptides derived from 118 proteins by mass spectrometry (Table 1, 2). As an example, we observed a ratio of 4:1 for a peptide derived from BCAR3 (Figure 2 C, D) and 5:1 for a peptide derived from Emerin (Figure 2A,B). Journal of Proteome Research • Vol. 4, No. 5, 2005 1663

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Figure 1. Schematic of the strategy used for identification of tyrosine-phosphorylated proteins. Cells were either grown in light or heavy stable isotope containing media as indicated. The cell population marked as ‘heavy’ was subjected to pervanadate treatment followed by mixing of cell lysates and immunoprecipitation of phosphotyrosine containing proteins. The proteins were resolved by SDS-PAGE as shown and the protein bands excised, digested with trypsin, and analyzed by LC-MS/MS. The intensity ratios of peptide pairs were calculated from the mass spectra.

Seventy-three of the identified proteins have not previously been shown to be phosphorylated on tyrosine residues. From a total of 13 proteins, we identified 42 phosphorylated tyrosine residues of which 34 were novel (Table 3, 4). The 73 potential phosphoproteins that we identified in this study were not known to be phosphorylated and represent putative tyrosine 1664


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kinase substrates. Though a significant fraction of the proteins identified seem to be highly abundant proteins, a number of them are adapter molecules in signaling pathways, which are generally less abundant. Also, we expect that addition of another fractionation step would allow identification of an even greater number of phosphoproteins. Relative Quantitation of Phosphorylation Events. In addition to identifying phosphorylation sites, mass spectrometry has been used to deduce the stoichiometry of phosphorylation events. Absolute quantitative comparison necessitates the use of internal standards,26 which is often impossible while analyzing complex mixtures of cell lysates. The isotopically labeled peptides identified by MASCOT search engine were manually verified for the quality of the spectra and sequence. Subsequently, the partner of the peptide pair was located. Once the peaks for the peptide pairs were identified, extracted ion chromatograms were generated for each peptide pair and the degree of increase or decrease in signal was calculated. We calculated ion ratios (heavy/light isotope) for multiple peptides corresponding to 92 proteins from tyrosine labeling experiment and peptide pairs corresponding to 184 proteins from arginine and lysine labeling experiments (Supplementary Table 1 in the Supporting Information). Together from two labeling experiments, we have 118 proteins whose intensity was increased after pervanadate treatment. On the basis of a literature search, we noted that 45 of these proteins were previously described to be tyrosine-phosphorylated proteins which fall into various classes of molecules (Table 1). To the best of our knowledge, the remaining 73 have not been described to be tyrosinephosphorylated to date. Comparison of Arginine Plus Lysine Labeling with Tyrosine Labeling. In the case of arginine and lysine labeling, all the peptides occurring from trypsin cleavage resulted in pairs (light and heavy) allowing quantitation of phosphorylation events. In the case of tyrosine labeling, only those peptides that contained tyrosine resulted in peptide pairs. Thus, the coverage for the purposes of quantitation was better in arginine plus lysine labeling. The total number of peptide assignments in this study was 2675, of which 1083 peptides (assigned to 203 proteins) were from arginine plus lysine labeling and 1592 peptides (assigned to 339 proteins) were from tyrosine labeling. The number of proteins identified from arginine plus lysine labeling with peptides showing an increase in intensity was 98 (39 known + 59 novel) proteins whereas that from tyrosine labeling was 80 (35 known + 45 novel) proteins. The overlap of proteins from both experiments was 52 (25 known + 27 novel) proteins (Figure 5A). The number of phosphopeptides identified from arginine plus lysine labeling was 37 that provided 41 phosphorylation sites. In contrast, the number of phosphopeptides identified from tyrosine labeling was 12 that provided 12 phosphorylation sites. The number of overlapping phosphopeptides was 10. Total number of peptide pairs identified from arginine plus lysine labeling was 837 and from tyrosine labeling was 275. The number of peptide pairs showing an increase in the heavy/ light ion ratio was 186 from tyrosine labeling and 655 from arginine plus lysine labeling experiments. Identification of Tyrosine Phosphorylation Sites. The mass spectra of the peptides for which the phosphorylation sites were assigned by Mascot23 were manually verified for the phosphotyrosine residue. An immonium ion at approximately m/z 216.043 that is characteristic of phosphorylated tyrosine residues served as a diagnostic ion for phosphotyrosine con-

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Tyrosine Phosphoproteome of HeLa Cells Table 1. List of Novel Potential Tyrosine Phosphorylated Proteins Identified in This Study protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

RefSeq accession

Cellular Communication and Signal Transduction BAI1-associated protein 2 isoform 3 NP•006331 Brain-enriched guanylate kinase-associated protein NP•065887 Breast cancer antiestrogen resistance 3 NP•003558 Caveolin-3 NP•001225 Cytoplasmic FMR1 interacting protein 1 NP•055423 Notch 2 preproprotein NP•077719 Palmdelphin NP•060204 Partitioning-defective protein 3 homolog NP•062565 Pleckstrin homology-like domain, family B, member 2 NP•665696 Programmed cell death 6 interacting protein NP•037506 Prohibitin NP•002625 Rho guanine nucleotide exchange factor 7 isoform a NP•003890 Sorcin isoform a NP•003121 TRK-fused gene NP•006061 Tumor necrosis factor type 1 receptor associated protein NP•057376 Cellular Organization Actin, gamma 1 propeptide NP•001605 Actin-binding LIM protein 1 isoform s NP•006711 Actin-related protein 2 NP•005713 Actin-related protein 3 NP•005712 Calponin-3 NP•001830 Destrin (actin depolymerizing factor) NP•006861 Echinoderm microtubule associated protein like 4 NP•061936 Emerin NP•000108 Epiplakin 1 NP•112598 Kindlin 1 NP•060141 Laminin M NP•000417 LIM and SH3 protein 1 NP•006139 Myosin IE NP•004989 Plakophilin 4 isoform a NP•003619 Plectin 1 isoform 1 NP•000436 Similar to Myosin heavy chain, nonmuscle type B XP•290747 Spectrin, beta, nonerythrocytic 1 isoform 1 NP•003119 Tight junction protein 2 NP•004808 Tubulin, alpha, 6 NP•116093 WAS protein family, member 2 NP•008921 Protein Synthesis Processing and Protein Fate Actin, beta NP•001092 Cytoskeleton-associated protein 1 NP•001272 Eukaryotic translation elongation factor 2 NP•001952 Eukaryotic translation initiation factor 4B NP•001408 HBS1-like NP•006611 Ubiquitin and ribosomal protein S27a NP•002945 Ubiquitin associated protein 2 isoform 1 NP•060919 WW domain binding protein 2 NP•036610

taining peptides.27 We identified 42 phosphorylation sites (Supplementary Figure 1 in the Supporting Information) in 13 proteins out of which 5 are well-known tyrosine phosphorylated proteins (Table 4). Thirty-four sites out of the 42 tyrosine phosphorylation sites identified were novel (Table 3). Below, we will briefly discuss some of the interesting proteins where we were able to identify novel phosphorylation sites. Cortactin: Cortactin is Src substrate28 and has 3 known phosphorylation sites (Y421, 470 and 486). We identified 3 additional phosphotyrosine sites (Y334, 446, 453) in this protein (Figure 3A). Laminin M: Laminin M/alpha 2 an extracellular protein, is a major component of the basement membrane.29 This represents a rare case of an extracellular protein being phosphorylated on a tyrosine residue (Y1249) (Figure 3B). The significance of this phosphorylation is not clear. MAP1B: Microtubule-associated protein 1B isoform 1 is a cytoskeletal protein involved in microtubule assembly and the phosphorylated form of MAP1B plays a crucial role in the development of the nervous system.30,31 We have mapped 13 new tyrosine phosphorylation sites (Y1062, 1762, 1796, 1830,

protein

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

Nucleic Acid Synthesis and Processing Cleavage and polyadenylation specific factor 5 LIM domain only 7 Poly(rC)-binding protein 2, isoform b Polymerase I and transcript release factor Pre-mRNA cleavage factor I RNA binding motif protein 4 SH2 domain binding protein 1 Energy and Metabolism Acetyl-Coenzyme A carboxylase alpha Aspartate aminotransferase 2 ATP synthase, H+ transporting, beta High-glucose-regulated protein 8 Inosine monophosphate dehydrogenase 2 Phosphogluconate dehydrogenase Propionyl Coenzyme A carboxylase, beta polypeptide Propionyl-Coenzyme A carboxylase, alpha polypeptide Protein-L-isoaspartate O-methyltransferase Pyruvate carboxylase precursor Storage and Transport Clathrin assembly lymphoid-myeloid leukemia protein Phosphate carrier precursor, isoform 1b SEC23A SEC24C Solute carrier family 25 (adenine nucleotide translocator) Transferrin receptor/CD71 Voltage-dependent anion channel 2 Immunity DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3 p53 inducible protein Unclassified Chromosome 3 open reading frame 6 KIAA0918 NICE-4 protein Similar to KIAA0310 protein

RefSeq accession

NP•008937 NP•005349 NP•114366 NP•036364 NP•079087 NP•002887 NP•055448 NP•000655 NP•002071 NP•001677 NP•057342 NP•000875 NP•002622 NP•000523 NP•000273 NP•005380 NP•000911 NP•009097 NP•002626 NP•006355 NP•006314 NP•001142 NP•003225 NP•003366 NP•001347 NP•055191 NP•777568 NP•056382 NP•055662 XP•088459

1870, 1872, 1904, 1905, 1938, 1940, 1957, 1974, 2025) in MAP1B (Figure 3C). N-Cadherin: N-cadherin is a calcium-dependent transmembrane adhesion molecule which is known to play a crucial role in regulating FGF receptor signaling pathway leading to metastasis.32 N-Cadherin is known to be a tyrosine-phosphorylated protein. We have identified one new tyrosine phosphorylation site (Y785) in the cytosolic tail of this protein. Desmoglein 2: Desmoglein 2 (Dsg2) is a calcium-binding transmembrane glycoprotein and is a member of desmosomal cadherins.33 Dsg2 is not a known tyrosine phosphorylated protein. We have identified a novel tyrosine phosphorylation site in this protein (Y967). Catenin, Delta 1: Delta catenin belongs to a group of structurally related proteins involved in cell-cell adhesion. It is a known phosphoprotein but the phosphorylation sites on this protein have not yet been identified.34 We have identified 7 new phosphorylation sites in this protein (Y96, 174, 217, 228, 257, 302, 859), which could play a role in cell adhesion. Aspartate Aminotransferase 2: This is a pyridoxal phosphatedependent enzyme localized to inner-membrane of mitochonJournal of Proteome Research • Vol. 4, No. 5, 2005 1665

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Table 2. List of Known Tyrosine Phosphorylated Proteins Identified in This Study protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

RefSeq accession

Cellular Communication and Signal Transduction Annexin VII isoform 1 NP•001147 Annexin XI NP•001148 Breast cancer anti-estrogen resistance 1 (p130CAS) NP•055382 Catenin, delta 1 NP•001322 Ephrin receptor EphB4 NP•004435 ERBB2 interacting protein isoform 2 NP•061165 G protein-coupled receptor kinase interactor 1 NP•054749 GAP-associated tyrosine phosphoprotein NP•006550 Hematopoietic cell-specific Lyn substrate 1 NP•005326 Hepatocyte growth factor-regulated tyrosine NP•004703 kinase substrate Integrin, beta 4 isoform 1 NP•000204 N-cadherin NP•001783 NCK-associated protein 1 NP•038464 Plakoglobin NP•002221 Proline-serine-threonine phosphatase NP•077748 interacting protein 2 PTK2 protein tyrosine kinase 2 isoform a NP•722560 Signal transducing adaptor molecule 1 NP•003464 Signal transducing adaptor molecule 2 NP•005834 Src homology 3 domain-containing protein NP•054782 HIP-55 Target of Myb1-like 1 NP•005477 Viral oncogene yes-1 homologue 1 NP•005424 Cellular Organization Actinin, alpha 1 NP•001093 Actinin, alpha 4 NP•004915 Clathrin, heavy chain 1 NP•004850 Cofilin 1 NP•005498 Cortactin, isoform a NP•005222 Cortactin, isoform b NP•612632 Desmoglein 2 NP•001934 Filamin 1 NP•001447 Microtubule-associated protein 1B isoform 1 NP•005900 Myosin, heavy polypeptide 9, nonmuscle NP•002464 Paxillin NP•002850 Spectrin, alpha, nonerythrocytic 1 NP•003118 Tight junction protein 1, isoform a NP•003248 Tight junction protein 1, isoform b NP•783297 Tubulin, alpha, ubiquitous NP•006073 Tubulin, beta, 2 NP•006079 Villin 2 NP•003370 Vimentin NP•003371 Protein Synthesis Processing and Protein Fate Valosin-containing protein NP•009057 Nucleic Acid Synthesis and Processing Ewing sarcoma breakpoint region 1 NP•005234 Heterogeneous nuclear ribonucleoprotein K NP•112552 NS1-associated protein 1 NP•006363 Energy and Metabolism Lactate dehydrogenase A NP•005557 Storage and Transport Nephrin related gene NP•060710

dria that plays a role in glutamate metabolism, urea and tricarboxylic acid cycles.35 It is not known to be phosphorylated on tyrosine residues. We have identified a phosphorylation site (Y401) in this enzyme. Emerin: Emerin is a serine-rich nuclear membrane protein associated with nuclear lamina.36 Emerin is considered to be a serine phosphorylated protein and we have added a novel fetature to this protein through the mapping of 5 tyrosine phosphorylation sites (Y59, 74, 85, 161, 167). Valosin-Containing Protein: Valosin-containing protein (VCP) is yeast Cdc48p homologue37 and is a known tyrosine phosphorylated protein. We have identified a phosphotyrosine residue in its C-terminus (Y805). KIAA0918: This protein was originally isolated as a cDNA fragment from a brain library.38 It belongs to the human SLITRK 1666


family of transmembrane signaling molecules. Our analysis identified a phosphorylated tyrosine residue (Y833) in the cytoplasmic domain of this transmembrane protein (Figure 3, Panel D). Like the cytoplasmic residues found in several other transmembrane receptors, it is likely that this tyrosine phosphorylation site is involved in signaling by this novel receptor. Experimental Validation of Tyrosine Phosphorylation and Functional Elucidation of Identified Proteins in EGF Receptor Signaling. As commercial antibodies were not available for validation of all of the potential phosphoproteins identified, it was difficult to confirm their tyrosine phosphorylation using Western blotting. To validate our findings, we selected a subset of proteins (RBM4, CALM, and transferrin receptor) against which immunoprecipitating antibodies were available and which were not previously shown to be phosphorylated. We subjected these proteins to immunoprecipitation using specific antibodies followed by Western blotting with anti-phosphotyrosine antibodies (Figure 4A). We also analyzed a subset of proteins against which no antibodies are available. For this purpose, we first obtained full-length cDNA clones and generated Flag epitope-tagged versions of NICE-4, WW domain binding protein 2, BCAR3, CKAP1, and C3ORF6. We transfected these Flag tagged cDNAs into 293T cells and treated them with pervanadate (Figure 4B). As these proteins were identified from HeLa cells which express EGF receptors, we also chose to investigate the functional relevance of these novel phosphoproteins in EGF receptor signaling by either stimulating HeLa cells with EGF or by coexpressing EGF receptor with these proteins in 293T cells (Figure 4B). As expected, all the proteins that were tested, exhibited increased tyrosine phosphorylation upon pervanadate treatment. Additionally, BCAR3, CKAP1, and C3ORF6 were phosphorylated upon EGF treatment thereby implicating them as molecules that are involved in EGF signaling pathway. RBM4 is a protein with RNA recognition motifs and zinc finger domain characteristic of RNA binding proteins.39,40 We have identified this protein to be tyrosine phosphorylated (Figure 4A). RBM4 has a proline rich segment in its C-terminus which has known ligands containing protein interaction domains such as WW41 and SH3 domains.42 Transferrin receptor is a receptor for transferrin whose main function is to serve as a sensor for the iron in serum bound to transferrin and taking up the iron by endocytosis.43 Transferrin receptor is not known to be a phosphoprotein. We have established transferrin receptor as a tyrosine phosphorylated protein (Figure 4A). We also identified CALM, a phosphatidylinositol and clathrin heavy chain binding protein to be phosphorylated (Figure 4A). A C-terminal segment of CALM probably interacts with proteins in the plasma membrane, possibly contributing to the regulation of the endocytotic activity, mediated by phosphoinositol pathways.44 From our experiments, we were able to implicate 3 molecules, BCAR3, CKAP1, and C3ORF6, as novel tyrosine kinase substrates in the EGF receptor signaling pathway (Figure 4B). BCAR3, contains a src homology 2 (SH2) domain, a hallmark of cellular tyrosine kinase signaling molecules and was initially discovered in a search for genes involved in antiestrogen resistance of human breast cancer cells, which makes it a very interesting candidate because of the involvement of the EGF receptor family in breast cancer progression.45 Cytoskeleton associated protein 1 (CKAP1), contains CAP-GLY domain which is a glycine region highly conserved among several cytoskel-


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Figure 2. Identification of tyrosine phosphorylated proteins by mass spectrometry. (A) A mass spectrum showing a peptide pair (containing 12C9 and 13C9-tyrosine, respectively) differing by 9 Da and exhibiting a ratio of 1:5. (B) Product ion mass spectrum (MS/MS) of the doubly charged ion at m/z 547.78 (from panel A). The spectrum corresponds to the peptide sequence (with 13C9-tyrosine), IFEYETQR, derived from the protein, emerin. (C) A mass spectrum showing a peptide pair (containing 12C6 and 13C6-arginine, respectively) differing by 6 Da and exhibiting a ratio of 1:4. (D) Product ion mass spectrum (MS/MS) of the doubly charged ion at m/z 598.80 (from panel C) which corresponds to the peptide sequence (with 13C6-arginine), GGSGATLEDLDR, derived from the protein, breast cancer antiestrogen resistance 3 (BCAR3).

eton-associated proteins.46 We have also identified CKAP1 to be tyrosine phosphorylated in response to EGF stimulation (Figure 4B). Finally, tyrosine phosphorylation of C3ORF6 upon activation by EGF indicates that C3ORF6 is likely to be an adapter molecule in the EGF signaling pathway (Figure 4B). Progress in finding tyrosine kinase substrates in signaling pathways is limited in part by a need for experimental approaches that can isolate and identify tyrosine-phosphorylated peptides in large numbers. Current proteomic approaches generally reveal only small numbers of tyrosine phosphorylation sites. Direct analysis of EGF receptor signaling pathway25,47 and FGF receptor signaling pathway48 have been carried out. Further experiments need to be carried out to dissect the role of these three molecules in the EGF receptor signaling cascade. While this manuscript was in preparation Hinsby et al. used a similar strategy to investigate phosphotyrosine containing proteins in FGF receptor signaling pathway.48 Two other global approaches that have recently been published include analysis of the yeast pheromone signaling49 pathway using SILAC and immunoaffinity profiling of cancer cells in pervanadate-treated cells using phosphospecific antibodies.50 Nevertheless, our results are distinctive and the tyrosine phosphorylation sites

that we identified have not been reported in these studies again indicating that complementary approaches are necessary for a comprehensive analysis of the phosphoproteome. Bioinformatics Analysis of Tyrosine Phosphoproteins and Phosphorylation Sites. Gene ontology (GO) terms have been widely used to categorize and classify proteins based on biological process and cellular component to assign molecular functions to proteins.51 A functional annotation of the proteins identified in this study was carried out by categorizing the proteins into divisions based on biological processes (Figure 5B). Both known and novel proteins were categorized and classified on the basis of GO biological process terms (Tables 1, 2). Out of the 118 proteins identified, ∼60% of the proteins were cytoskeletal and signaling proteins. On the basis of the published literature, one would expect signaling proteins and proteins associated with the cytoskeleton to be tyrosinephosphorylated. Surprisingly, however, our results also revealed eight key metabolic enzymes to be probable substrates of tyrosine kinases although lactate dehydrogenase was the only one that was previously known to be phosphorylated. We were able to identify a phosphorylation site in the metabolic enzyme, aspartate aminotransferase 2. We found several proteins with Journal of Proteome Research • Vol. 4, No. 5, 2005 1667

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Table 3. List of Novel Tyrosine Phosphorylation Sites Identified in This Study protein

Microtubule-associated protein 1B isoform 1

Identified phosphopeptide Sequence

Refseq

NP•005900

N-cadherin 1 Aspartate aminotransferase 2 Desmoglein 2 Laminin M Catenin, delta 1

NP•001783 NP•002071 NP•001934 NP•000417 NP•001322

Cortactin, isoform a

NP•005222

Emerin

NP•000108

KIAA0918/SLITRK5 Valosin-containing protein Hepatocyte growth factorregulated tyrosine kinase substrate

NP•056382 NP•009057 NP•004703

netphos

scansite

DMSLpYASLTSEK

yes

yes

TPEEGGYSpYDISEK TPEDGDpYSpYEIIEK ESSPLpYSPTFSDSTSAVK ITSFPESEGYSpYETSTK AAEAGGAEEQpYGFLTTPTK TTSPPEVSGYSpYEKTER TSDVGGpYpYYEKIER TATCHSSSSPPIDAASAEPpYGFR TPGDFSpYApYQKPEETTR TPGDFSpYAYQKPEETTR YDEEGGGEEDQDpYDLSQLQQPDTVEPDAIKPVGIR EFSIpYMTK VpYAPASTLVDQPYANEGTVVVTER KLMApYGGK LNGPQDHSHLLpYSTIPR QDVpYGPQPQVR SQSSHSpYDDSTLPLIDR HYEDGYPGGSDNpYGSLSR HpYEDGYPGGSDNpYGSLSR TVQPVAMGPDGLPVDASSVSNNpYIQTLGR SMGYDDLDYGMMSDpYGTAR NASTFEDVTQVSSApYQK GPVSGTEPEPVpYSMEAADYR GPVSGTEPEPVpYSMEAADpYR KEDALLpYQSK DSApYQSITHpYRPVSASR IFEYETQRRLSPPSSSAASSpYSFSDLNSTR GDADMpYDLPKKEDALLYQSK SPApYSVSTIEPR FPSGNQGGAGPSQGSGGGTGGSVYTEDNDDDLpYG AEPMPSASSAPPASSLpYSSPVNSSAPLAEDIDPELAR

yes yes no yes yes yes no yes no no yes yes yes no yes no yes yes yes no yes no yes no yes 1-yes yes yes yes yes yes

yes yes no no yes yes no no no yes no no no no no no no no no no no no no no no no no no no no no

Table 4. List of Known Tyrosine Phosphorylation Sites Identified in This Study protein

Refseq

Breast cancer anti-estrogen resistance 1 (p130CAS)

NP•055382

Paxillin

NP•002850

identified phosphopeptide sequence

netphos

scansite

AQQGLpYQVPGPSPQFQSPPAK

no

no

RPGPGTLpYDVPR HLLAPGPQDIpYDVPPVR VGQGpYVpYEAAQPEQDEYDIPR VGQGYVYEAAQPEQDEpYDIPR FIHQQPQSSSPVpYGSSAK VGEEEHVpYSFPNK

yes yes yes yes yes yes

yes yes no no no yes

a functional role in nucleic acid and protein synthesis and processing to be potential phosphoproteins. We have experimentally proven the tyrosine phosphorylation of an RBM4, a novel conserved zinc finger-containing RNA-binding protein, which is found to be involved in RNA processing39 and of a storage and transport protein, CALM (Figure 4A). We have identified several phosphotyrosine residues in MAP1B. Likewise, our results also revealed 7 new phosphorylated tyrosine residues in delta catenin and 5 in emerin. Investigation of the significance of these modifications will help in further understanding the role of these proteins. For all the proteins where we were able to map tyrosine phosphorylation sites, an analysis was carried out using two popular phosphorylation prediction programs, Netphos19 and Scansite20 (Table 3, 4). In searches using Netphos, an output score of 0.5 was used as cutoff to ensure that the site was a bona fide phosphorylation site. We used a more sensitive approach using Scansite by selecting a low stringency output. Remarkably, as we have reported earlier,47 these programs failed to predict most of the novel tyrosine-phosphorylated sites. 1668


There are a large number of hypothetical proteins in protein database for which functions cannot be easily assigned. However, if they are identified as tyrosine phosphorylated proteins, it is possible to test their specific role more systematically in various tyrosine kinase signaling pathways. In this study, we have identified 4 uncharacterized proteins, which existed only as cDNA sequences in database, as phosphotyrosine containing proteins. The KIAA0918 protein has a signal peptide, several LRR repeats and a transmembrane domain (Figure 6A). An alignment with related protein shows that this protein belongs to the human SLITRK family of signaling proteins (Figure 6B). Aruga et al. have previously pointed out the conservation of several tyrosine residues in the SLITRK family although there is no published evidence for phosphorylation for any of these sites.52 We identified Y833, which is located in the cytosolic tail of SLITRK5 and conserved in some members of this family, as a phosphorylation site.53 The similarity between C-terminal domain of SLITRK family of proteins and trk neurotrophin receptor suggests a possible involvement in PLCγ-mediated signaling53 and could involve


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Figure 3. Mapping of tyrosine phosphorylation sites. MS/MS spectra of four different tyrosine phosphorylated peptides containing either 13C6-arginine or 13C6-lysine. (A) The peptide sequence, GPVSGTEPEPVpYSMEAADpYR, is derived from cortactin and contains two novel tyrosine phosphorylation sites as indicated, Y446 and Y453. (B) The peptide sequence KLMApYGGK, is derived from laminin M. (C) The peptide sequence TPEEGGYSpYDISEK, is derived from microtubule-associated protein 1B isoform 1 (MAP1B). (D) The peptide sequence SPApYSVSTIEPR, is derived from a novel protein (KIAA0918/SLITRK5). pY denotes phosphorylated tyrosine residues.

Figure 4. Experimental validation of tyrosine phosphorylation status by Western blotting. HeLa cells were treated with 1 mM pervanadate for 30 min or 10 ng/mL EGF for 5 min. (A) Antibodies against RNA binding motif protein 4 (RBM4), transferrin receptor and clathrin assembly lymphoid-myeloid leukemia protein (CALM) were used for immunoprecipitation followed by SDS-PAGE and Western blotting. The blots were first probed with anti-phosphotyrosine antibody and then stripped and reprobed with antibodies against the individual proteins as shown. (B) 293T cells were transfected with Flag-tagged cDNAs of BCAR3 (breast cancer anti-estrogen resistance 3), CKAP1 (cytoskeleton-associated protein 1), WW domain binding protein 2 (WWBP2), chromosome 3 open reading frame 6 (C3ORF6) and NICE-4, and were treated with 1 mM pervanadate for 30 min or cotransfected with EGF receptor and stimulated with EGF. The expressed proteins were immunoprecipitated using anti-Flag antibody, followed by SDS-PAGE and Western blotting. The blots were probed with anti-phosphotyrosine antibody followed by reprobing with anti-Flag as shown.

binding of phosphorylated SLITRK5 to SH2 and/or PTB domain containing proteins. C3ORF6 was originally identified by

computational methods followed by experimental validation by PCR amplification.54 It contains a coiled-coil domain and is Journal of Proteome Research • Vol. 4, No. 5, 2005 1669

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Conclusions

Figure 5. Functional annotation of the tyrosine phosphoproteome. (A) A comparison of the two different labeling approaches and the overlap of proteins and phosphorylation sites from the two experiments. (B) The pie chart shows the distribution of biological processes to the known and potential phosphoproteins identified in this study using Gene Ontology terms.

well conserved across species.54 We have validated its tyrosine phosphorylation status both in response to pervanadate treatment and in the EGF signaling pathway. NICE-4, identified as part of human epidermal differentiation complex,55 has a ubiquitin associated domain and we have demonstrated its phosphorylation upon pervanadate treatment.

Our results demonstrate the utility of the SILAC method for in vivo differential labeling of proteins for the identification of tyrosine-phosphorylated proteins and localization of phosphorylation sites. In addition to identification of known tyrosine phosphorylation sites on p130CAS, Hrs and paxillin, we have identified novel sites on MAP1B, N-cadherin, aspartate aminotransferase 2, desmoglein 2, laminin M, catenin delta 1, cortactin, emerin and valosin-containing protein. Nevertheless, we have missed a number of known phosphorylation proteins perhaps owing to their lack of expression in HeLa cells, low abundance levels and/or lack of adequate enrichment using our methods. We have verified 3 novel proteins as downstream intermediates in the EGF receptor signaling pathway. We identified novel hypothetical proteins KIAA0918 and C3ORF6 and confirmed that they are phosphoproteins. A focus on these individual proteins would reveal the specific role of these proteins and novel sites in signaling cascades and the significance of tyrosine phosphorylation in the regulation of their function. Mass spectrometry followed by experimental validation of phosphorylation and verification of the sites of phosphorylation and their significance would provide biological information. Because of its sensitivity and selectivity, this strategy will be useful in proteomic approaches to investigate tyrosine as well as serine/threonine phosphorylation in signaling. Finally, modifications of this strategy could be used to investigate protein-protein interactions and multiprotein complexes that are formed in response to specific stimuli.

Acknowledgment. This work was supported by NHLBI Contract HV-28180 from the National Institutes of Health and by a Career Development Award from the Breast Cancer SPORE (CA 88843) at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins. We thank Pratap Vedula for his assistance with programming.

Figure 6. Functional annotation of an uncharacterized/novel protein, KIAA0918/SLITRK5. (A) The domain architecture of a novel human protein KIAA0918/SLITRK5 shows a signal peptide, numerous leucine-rich repeats (LRR), LRR C and N-terminal domains (LRR CT and LRR NT) and a transmembrane domain. (B) A cladogram of the human SLITRK family of proteins. 1670


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