Systemic Analysis of Tyrosine Phosphorylated Proteins in

Systemic Analysis of Tyrosine Phosphorylated Proteins in Angiopoietin-1 Induced Signaling Pathway of Endothelial Cells Young Mee Kim,† Jawon Seo,† Yung Hee Kim,† Jaeho Jeong,† Hye Joon Joo,† Dong-Hee Lee,† Gou Young Koh,‡ and Kong-Joo Lee*,† Center for Cell Signaling & Drug Discovery Research, College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea, and Biomedical Center and Department of Biological Sciences, KAIST, Daejon, 305-701, Korea Received March 23, 2007

Angiogenesis is an essential process in physiological and pathological processes and is well-regulated to maintain the cellular homeostasis by balancing the endothelial cells in proliferation and apoptosis. Angiopoietin-1 (Ang1) regulates angiogenesis as a ligand of Tie 2 receptor tyrosine kinase. However, the regulation pathways are not well-understood. To date, only a few of the signaling molecules involved in the Tie 2 receptor tyrosine kinase-mediated angiogenesis have been identified. In this study, we systematically identified tyrosine-phosphorylated proteins in Ang1-induced signaling cascade in human umbilical vein endothelial cells (HUVECs), employing proteomic analyses combining two-dimensional gel electrophoresis, Western analysis using phosphotyrosine antibody and mass spectrometry (MALDITOF MS and nanoLC-ESI-q-TOF tandem MS). We report here the identification, semiquantitative analysis, and kinetic changes of tyrosine-phosphorylated proteins in response to Ang1 in HUVECs and identified 66 proteins among 69 protein spots showing significant changes. Of these, p54nrb was validated as a molecule involved in cell migration. These results suggest that Ang1 induces stabilization of neo-vessel network by regulating the phosphorylations of metabolic and structural proteins. Keywords: COMP-Ang1 • tyrosine phosphorylation • 2D gel electrophoresis • proteomics • p54nrb • HUVECs • cell migration • angiogenesis

Introduction Angiogenesis, the generation of new capillaries from the preexisting, is essential to various physiological and pathological processes, that is, embryonic development, the menstrual cycle, wound healing, tumor growth and metastasis, rheumatoid arthritis, diabetic retinopathy, atherosclerosis, and vascularization of the myocardium. The process of angiogenesis is regulated to maintain cellular homeostasis by balancing proliferation and apoptosis of endothelial cells. Endothelial survival is controlled by several angiogenic growth factors: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiopoietin-1 (Ang1).1-3 VEGF induces the formation of new capillary vessels in early stage of angiogenesis, while Ang1 induces vessel maturation and stabilization of neovascular networks,4,5 which suggests that VEGF and Ang1 act to complement each other in angiogenesis. Ang1 plays an important regulatory role in angiogenesis, as a ligand of Tie 2 receptor tyrosine kinase.6,7 Recent reports8,9 suggest that stimulation of Ang1, in contrast to VEGF, has no * To whom correspondence should be addressed. College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea. Tel, 82-2-3277-3038; Fax, 82-2-3277-3760; e-mail, [email protected]. † Ewha Womans University. ‡ KAIST.

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mitogenic effect on endothelial cells; Ang1 and Tie2 receptor act in the later steps of the angiogenic process such as the remodeling and the maturation of newly formed vascular system and stabilization of the capillaries. A series of in vitro studies showed that Ang1-promoted endothelial cell survival is mediated mainly by tyrosine phosphorylation of Tie 2 receptor and activation of PI3 kinase/Akt.7,9-11 Ang1 stimulates recruitment of Dok-R (downstream of kinase) in a Tie 2-dependent manner; and phosphorylation of Dok-R in turn recruits Ras GTPase-activating protein and adaptor molecule Nck, to regulate cell motility and rearrange cytoskeletal proteins.7,12 However, how these pathways are regulated is not wellunderstood.10 To date, only a few of signaling molecules involved in the Tie 2 receptor tyrosine kinase-mediated angiogenesis have been identified. Recently, proteomics has been used to understand the signaling pathways in various systems.13-16 We examined the profiles of tyrosine-phosphorylated proteins in primary human umbilical vein endothelial cells (HUVECs) by stimulation with VEGF.16 Post-translational modification (PTM), especially tyrosine phosphorylation by tyrosine kinase receptor, has been shown to be an important process for regulating protein activities in signaling process involved in cell survival, apop10.1021/pr070168k CCC: $37.00

 2007 American Chemical Society

Tyr-Phosphorylated Proteins in Ang1-Induced Signaling Pathway

tosis, differentiation, proliferation, gene expression, morphological change, and cell motility.14,17-22 However, the studies of tyrosine-phosphorylated proteins are difficult because the fraction of tyrosine-phosphorylated proteins is extremely low.23 We employed proteomic analyses, and systematically identified tyrosine-phosphorylated proteins involved in Ang1-signaling cascades with the goal of developing insights into angiogenesis. To validate correlation of tyrosine phosphorylation and angiogenesis, we selected p54nrb among tyrosine-phosphorylated proteins by Ang1. p54nrb (non-POU in mouse) has been known as multifunctional nuclear protein implicated in processes such as transcriptional regulation, splicing, DNA unwinding, viral RNA processing, and control of cell proliferation, among others. It is not known whether p54nrb was involved in angiogenesis. This is the first report that tyrosine-phosphorylated p54nrb is involved in the regulation of angiogenesisrelated responses.

Materials and Methods Cell Culture. Primary endothelial cells (HUVECs) from AngioLab, Daejon, Korea, were prepared 80% confluent in 100 mm dishes at passage 4-6 in Medium 199 (GIBCO BRL) containing 10% fetal bovine serum (FBS), heparin (100 µg/mL, Sigma), endothelial cell growth supplement (ECGS, 50 µg/mL, Sigma), 100 µg/mL streptomycin, and 100 units/mL penicillin G at 37 °C in an atmosphere of 5% CO2 and 95% air. The primary cultured cells used in the present study were always obtained at passage 6. Treatment of HUVECs with COMP-Ang1. HUVECs were incubated in Medium 199 containing 2% FBS overnight (>16 h), then treated with 200 ng/mL COMP-Ang1 in M-199 containing 2% FBS for 30 min at 37 °C,7,9 and recovered at various times. Because native Ang1 recombinant protein is insoluble and aggregates, we used COMP-Ang1 (cartilage oligomeric matrix protein-Ang1), a soluble and stable variant of Ang1.24,25 Treated cells were harvested after washing twice with ice-cold PBS. Two-Dimensional Gel Electrophoresis. 1. IPG Ready DriStrips Gel. The cells treated with COMP-Ang1 were harvested and washed with cold PBS three times and subsequently lysed for 1 h at room temperature in a buffer containing 9.5 M urea, 2% Triton X-100, 5% β-mercaptoethanol, 0.4% ampholyte 3-10, 1.6% ampholyte 5-7, 1 mM phenylmethylsulfonyl fluoride, 5 µg /mL aprotinin, 10 µg/mL pepstatin A, 10 µg/mL leupeptin, 1 mM EDTA, 10 mM Na3VO4, and 10 mM NaF. Protein concentrations were measured by the Bradford assay. Equal amounts of proteins (50 µg) were then electrofocused in 7-cm Immobiline DryStrips (pH 4-7) with the GE Healthcare Biosciences IPGphor using the following protocol with 50 µA per strip at 20 °C: (1) rehydration for 12 h; (2) 500 V for 1 h (step and hold); (3) 1000 V for 1 h (step and hold); and (4) 8000 V for 3 h (step and hold). After electrofocusing, the strips were shaken for 15 min with an equilibration buffer (1.5 M Tris-Cl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 10 mg/mL dithiothreitol) and loaded onto 11% second-dimensional SDS-PAGE. 2. Micro-Tube Gel. The cells treated with COMP-Ang1 were harvested and washed with cold PBS three times and subsequently lysed for 1 h at room temperature in a buffer containing 9.5 M urea, 2% Triton X-100, 5% β-mercaptoethanol, 1% ampholyte 3-10, 1% ampholyte 6-8, 1 mM phenylmethylsulfonyl fluoride, 5 µg /mL aprotinin, 10 µg /mL pepstatin A, 10 µg/mL leupeptin, 1 mM EDTA, 10 mM Na3VO4, and 10 mM NaF, and equal amounts of proteins (50 µg) were focused to

research articles separate basic proteins in 10 cm microtube gel (9.2 M urea, 2% Triton X-100, 4% acrylamide, 1% 3.5-10 ampholyte, and 1% 6-8 ampholyte). The following focusing protocol was used: (1) 500 V for 10 min, (2) 600 V in upper chamber buffer (10 mM H3PO4) and low chamber buffer (20 mM NaOH) for 2.5 h. An electrode was used to controvert anode and cathode. After focusing, the gels were soaked in 1× gel sample buffer on ice and loaded onto 11% second-dimensional SDS-PAGE. Proteins from SDS-PAGE were stained with silver or electroblotted onto a PVDF membrane. Immunoblot Analysis. Proteins from control and COMPAng1-treated cells were separated by SDS-PAGE under reducing conditions, transferred to PVDF membrane, and probed with monoclonal antibody to phospho-tyrosine (4G10, Upstate Biotechnology, Inc., pY100, Cell Signaling Technology), polyclonal antibody to thioredoxin Reductase 1 (Labfrontier, Korea), monoclonal antibody to PDI (Stressgene, Canada), monoclonal antibody to p54nrb (non-POU, BD Transduction Lab.), monoclonal antibody to ZAP 70 (Upstate Biotechnology, Inc.), and monoclonal antibody to hnRNP K (provided by Dr. Y. M. Kim). The protein-antibody complexes were visualized with HRP (horseradish peroxidase)-conjugated secondary antibody at a 1:2000 dilution. The blots were incubated for 30 s in the ECL plus kit (GE Healthcare Biosciences, Sweden) and exposed to LAS1000 (Fuji photofilm Co., Japan) or X-ray film (Hyper-film, GE Healthcare Biosciences, Sweden). Quantitative Analysis of Tyrosine Phosphorylation. Images were scanned using an image scanner (PowerLook 2100XL, UMAX) and semiquantitatively analyzed using the software package ImageMaster two-dimensional software (GE Healthcare Biosciences, Sweden). Each sample of anti-phosphotyrosine antibody was run together with internal standards (phosphotyrosine molecular weight standard, Upstate Biotechnology, Inc., #12-2566) and the ratio of intensity between sample and internal standard was used for quantitative analysis. MS Peptide Fingerprinting Analysis and Sequencing by ESI-q-TOF Tandem MS. The cellular proteins were separated on two-dimensional gel electrophoresis and silver stained. Each spot was digested in-gel. The gel spots were excised with a scalpel, crushed, and destained by washing with 25 mM ammonium bicarbonate in 50% acetonitrile (ACN). The silverstained gels were destained by washing with 15 mM K4Fe(CN)6, and 50 mM sodium thiosulfate prior to crushing. The gels were then dehydrated by addition of ACN rehydrated by adding 1020 µL of 25 mM ammonium bicarbonate with 10 ng/µL of sequencing grade trypsin (Promega), and incubated at 37 °C for 12-15 h. Peptides were extracted by adding 30 µL of a solution containing 60% ACN and 0.1% trifluoroacetic acid (TFA). The extraction was repeated three times and completed by adding 20 µL of ACN. The extracted solutions were pooled and evaporated to dryness in a SpeedVac vacuum centrifuge. Samples were reconstituted in 10 µL of 0.1% TFA and treated with ZipTips containing C18 resin (Millipore) according to the manufacturer’s instructions. The washed peptides were eluted with saturated matrix solution (R-cyano-4-hydroxycinnamic acid in 60% ACN and 0.1% TFA). Peptide mixtures were analyzed with MALDI-TOF MS using a delayed ion extraction and ion mirror reflector mass spectrometer (Voyager-DE STR; Applied Biosystems, Inc.). External calibration was carried out using Sequazyme Peptide Mass Standard Kit (Perspective Biosystems), and internal calibration was performed using the autolytic peaks of trypsin. This procedure typically results in Journal of Proteome Research • Vol. 6, No. 8, 2007 3279

research articles mass accuracies of 50 ppm. For interpretation of the mass spectra, we used the MS-Fit program available on the Web site of the University of California, San Francisco (prospector.ucsf.edu/). For protein identifications by sequencing using nanoLC-ESI-q-TOF tandem MS, the rest of the digested peptides were reconstituted in 10 µL of 0.1% TFA and desalted with ZipTip C18 (Millipore Co.) according to the manufacturer’s instructions. The peptides were fractionated with 10 µL of 5, 30, 50, and 80% ACN/0.1% TFA. ESI-MS/MS coupled with reverse-phase liquid chromatography (15 cm × 75 µm, C18 column) performed with a Q-TOF equipped with a nano flow Z-spray ion source (Waters Inc., U.K.). Mass data acquisitions were piloted using MassLynx (Version 4.0, Waters Inc., U.K.) software “survey scan” modes. In this mode, MS spectra were acquired, followed by MS/MS spectra of the four most intense ions within a 10 s window, in cycle manner. When the intensity of a peak rose above a threshold of counts, tandem mass spectra were acquired. The internal parameters of Q-TOF were set as follows. The electrospray capillary voltage was set to 3.0 kV, the cone voltage was set to 100 V, and the source temperature was set to 80 °C. The MS survey scan was m/z 400-1500 with a scan time of 1 s and an inter-scan delay time of 0.1 s. The scan range for MS/MS acquisition was from m/z 50 to 1500 with a scan time of 2 s and an inter-scan delay time of 0.1 s. Normalized collision energies for peptide fragmentation were set using the charge-state recognition files for +2, +3, and +4 peptide ions. Fragmentation was performed using the collision gas and with a collision energy profile optimized for various mass ranges of precursor ions. The acquired spectra were automatically processed and searched against the nonredundant Swiss-Prot protein sequence database using ProteinLynx Global Server 2.1 (Waters Inc., U.K.), with the following parameters: smoothing type, Savitzk-Golay; centroid top, 80%; precursor-ion mass accuracy, 0.2 Da; fragment-ion mass accuracy, 0.2 Da; variable modification, Phosphoryl STY; 1 missed cleavage; trypsin was used as the proteolytic enzyme. Cross-Linking of Antibody-Protein A Sepharose. Protein-A sepharose (40 µL, Sigma) was incubated with 20 µL of antiphosphotyrosine antibody (4G10, Upstate Biotechnology; pY100, Cell Signaling Technology) for 2 h at room temperature and washed with 0.2 M sodium borate (pH 9.0). Antiphosphotyrosine antibody-protein-A sepharose complex was cross-linked with 20 mM DMP (dimetylpimelimidate, Sigma) in 0.2 M sodium borate (pH 9.0) for 1 h at room temperature, and the reaction was stopped using 0.2 M ethanolamine (pH 8.0). Immunoprecipitation. The cells treated with COMP-Ang1 were harvested and washed with cold PBS three times and subsequently lysed in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM PMSF, 5 µg/ mL aprotinin, 1 µg/mL pepstatin A, 20 µM/mL leupeptin, 5 mM Na3VO4, and 5 mM NaF) with 3 times the original packed cell volume for 30 min in ice. After centrifugation for 15 min at 4000 rpm, the supernatant (cytosol fraction) was separated, and the pellet was resuspended in half-packed cell volume of low salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 20 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 5 µg/mL aprotinin, 1 µg/mL pepstatin A, 20 µM leupeptin, 5 mM Na3VO4, and 5 mM NaF). High salt buffer (same as above except that 20 M KCl was replaced with 1.2 M KCl) was added to half of the packed cell volume dropwise, incubated for 30 min in ice, and centrifuged for 30 min at 14 500 rpm. The supernatant (nuclear fraction) was added to the first supernatant. 3280

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Protein concentrations were measured by the Bradford assay. Equal amounts of proteins (1 mg) were added to an immunoprecipitation buffer containing 20 mM HEPES, pH 7.4, 15% glycerol, 150 mM KCl, 0.5% NP-40, 1 mM EDTA, 1 mM PMSF, 5 µg/mL aprotinin, 1 µg/mL pepstatin A, 20 µM leupeptin, 5 mM Na3VO4, abd 5 mM NaF. A total of 40 µL of cross-linked anti-phosphotyrosine antibody-protein A sepharose was added and incubated at 4 °C overnight. The precipitated immune complexes were washed three times with the immunoprecipitation buffer. In Vitro Cell Migration Assay. The migration assays were performed using a 24 well chemotaxis chamber (transwell, Costar). The upper chamber of transwell was coated with 50 µg of matrigel (Matrigel basement membrane matrix, BD Biosciences) for 1 h at 37 °C and then placed into 24 well chambers. The lower chamber was filled with media containing 10% FBS. Cells transiently transfected with flag-p54nrb (105 cells/150 µL) were seeded in each well in the upper chamber, and then incubated for 24 h at 37 °C. The non-migrating cells after incubation were removed from the upper side of the filters with a cotton ball. The filters were stained with Diff-Quik staining solution (Fisher Scientific). After the transwell was set on the measuring-rod, the migrating cells were counted at 50fold magnification using a microscope. All measurements were at least triplicated.

Results Ang1 Stimulates Protein Tyrosine Phosphorylation. We first examined the kinetic analysis of global tyrosine phosphorylation changes in Ang1-treated primary HUVECs. Cells were stimulated with 200 ng/mL COMP-Ang1 for 30 min at 37 °C and recovered at various times (0 ∼ 8 h). VEGF- and Ang1induced tubule formation in terms of both area and length was compared to that in control cells (Supplementary Figure 1A in Supporting Information). The phosphorylated proteins were detected with anti-phosphotyrosine antibody. Tyrosine phosphorylation by Ang1 was initiated after recovery for 30 min, reached maximum level after recovery for 1 h, and continued thereafter until recovery for 8 h (Supplementary Figure 1B in Supporting Information). On the basis of this finding, Ang1treated cells were subsequently recovered for 45 min and 3 h. We then extensively examined tyrosine-phosphorylated proteins in response to Ang1 by proteomic analysis. Cellular proteins were separated on 2D gels and detected both with silver staining and immunostaining using anti-phosphotyrosine monoclonal antibody (Figure 1). We detected 69 protein spots that showed significant changes in phosphorylation in response to Ang1 at two pI ranges (4-7 and 6-10, p-value < 0.05). In total, 48 protein spots were detected in the pI range of 4-7 (Figure 1A) and 21 protein spots in the basic pI range of 6-10 (Figure 1B). The gels of samples obtained after 45 min and 3 h recovery after treatment were visualized by silver staining (Figure 1A,B, left upper panel) and immunostaining using antiphosphotyrosine monoclonal antibody (control, Ang1 45 minR, 3 hR). Among various post-translational modifications such as phosphorylation, acetylation, methylation, and glutathionylation, tyrosine phosphorylation is an important modification because it mainly regulates signal transduction in many cellular processes. To assess the degree of protein phosphorylation in response to Ang1, we performed semiquantitative analyses using immunostaining (Figure 2A). The degree of phosphorylation of each spot was normalized over phosphotyrosine standard markers as described previously.13 The relative in-


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Figure 1. 2D gel profiles of tyrosine-phosphorylated proteins induced by COMP-Ang1. Serum-starved cells were stimulated with 200 ng/mL COMP-Ang1 for 30 min at 37 °C and then recovered at 45 min and 3 h. The proteins were as loaded in 2D gel, and gel images were visualized by silver staining and immunostaining with anti-phosphotyrosine antibody. Panels A and B images on upper panel are silver-stained and immunostained gel with anti-phosphotyrosine antibody. Panels A and B, bottom panels, show detailed images. pI range of (A) images is 4-7, and pI range of (B) images is 6-10. Numbered spots were excised and analyzed by in-gel digestion and MALDI-TOF MS or nanoLC-ESI-q-TOF tandem MS.

tensity of each of the 69 spots after treatment was compared to the intensity of untreated controls. We performed five independent experiments, and the quantitative data of reproducible spots were represented from at least three sets. The results are presented as mean ( SD. Protein spots detected on the immunoblots were cut out from the silver-stained gels after matching the X-ray films with the silver-stained gels and subjected to in-gel digestion with trypsin followed by mass spectrometric analyses. We identified 66 proteins from 69 protein spots using MALDI-TOF MS (Table 1) and confirmed the results by sequencing using nanoLCESI-q-TOF tandem MS (supplementary Table 1 in Supporting Information). The results were analyzed using gene ontology (www.godatabase.org) to predict the various functions of proteins. We identified 26 proteins (28 spots) acting in cell growth or maintenance; 9 proteins (9 spots) acting in nucleic acid binding; 2 proteins (2 spots) acting as chaperones/ mediators of protein folding; 3 proteins (6 spots) with cell motility and structural functions; 8 proteins (8 spots) acting as signal transducers; 4 proteins (4 spots) related to proteasome degradation; 2 proteins (3 spots) acting as ligand binders or carriers; and 12 with uncertain functions (12 spots). Figure 2B shows the identified proteins that were classified by their functions. Forty percent of the identified proteins function in cell growth or maintenance, and proteins with uncertain function (uncertain proteins) made up 17% of identified proteins. To identify the proteins with uncertain functions, we examined their alignments using NCBI BLAST homology search

(supplementary Table 2 in Supporting Information). Spots 33, 38, 39, and 63 were found to have homology with cytoskeletal proteins (actin, tubulin, and keratin). Spot 36 (similar to neurotrimin) has homology with neuronal cell adhesion molecules, and spot 59 (unnamed protein product) has a potential to function as a transcription factor. Spot 37 (small glutaminerich tetratricopeptide) is a cochaperone that binds directly to Hsc70/Hsp70 and regulates their ATPase activity. Spot 1 (cell death 6 interacting protein) plays a role in regulating cell adhesion and morphology.26 Spot 2a (integrin beta 2) mediates cell adhesion or cytoskeletal organization, and regulates cell growth, differentiation, migration, and apoptosis through transducing signals. Spot 4 (cytoplasmic dynein) interacts with microtubules to generate force and transport particles and organelles along the microtubules. These findings suggest that Ang1 regulates the stabilization of endothelial cells network by modulating tyrosine phosphorylation of angiogenesis-related downstream proteins. The phosphorylated proteins could be sorted into three groups depending on the kinetics of tyrosine phosphorylation (Figure 3A). These groups clustered the raw data using GeneSight (version 4.0) program. Group I includes proteins with increased extents of tyrosine phosphorylation according to recovery time in response to Ang1. These include 34 spots, which correspond to 49.2% of the total protein spots. Group II includes proteins with increased extents of tyrosine phosphorylation only after 45 min of recovery time and decrease thereafter. This group includes 31 spots, corresponding to 45% of the total protein spots. Group III includes proteins with Journal of Proteome Research • Vol. 6, No. 8, 2007 3281

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Figure 2. Quantitation and classification of protein tyrosine phosphorylation after COMP-Ang1 treatment. (A) Images were quantitated as described under Materials and Methods. The ratio of intensities of sample and internal standard was used for the quantitation of 69 tyrosine-phosphorylated proteins. (B) Classification of differentially phosphorylated proteins in response to COMP-Ang1 treatment.

increased tyrosine phosphorylation only after 3 h of recovery time. Groups of proteins classified by function are listed in Figure 3B. Tyrosine phosphorylation of most proteins in response to Ang1 was increased at 45 min recovery time, and 3282


thereafter, phosphorylation of half of them was decreased. We also found that only spot 17, TR 3 (thioredoxin reductase 3) had slightly decreased tyrosine phosphorylation. Because TR is known as ROS target, its dephosphorylation by Ang1 implies

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Tyr-Phosphorylated Proteins in Ang1-Induced Signaling Pathway Table 1. List of Tyrosine-Phosphorylated Proteins by COMP-Ang1a

spot no.

2 2ab 3 4 5 9 11 12 17 18 20, 21 22 30, 31 32 40 44 47

52 53 56 57 58 60

61 62 65 13 16 23 26 29 34 50 51 55 14 15 10, 24, 25, 28 42 54

1 6 19b

identified protein

accession no.

MW (Da)

Proteins That Act in Cell Growth or Maintenance Histone deacetylase 7A variant 3 32482808 99093 Integrin beta 2 4557886 84792 PYK2 N-terminal domain-interacting receptor 1 13654296 106810 Cytoplasmic dynein intermediate chain 2C 13649465 68427 Transglutaminase A chain 119720 83268 Lon protease-like protein 414046 95195 CTP synthase 20981706 66691 GYS1 protein 13112017 76484 Serum albumin precursor 6013427 69226 Thioredoxin reductase (TR1) 2500118 54420 Thioredoxin reductase 3 5107031 63430 Serine/threonine protein phosphatase 2A 4506019 51693 PDI ER60 precursor 2507461 56797 Adenylosuccinate synthetase 415849 49916 LIM domains containing 1 13548632 52338 Guanine nucleotide binding protein alpha 11 subunit 2286217 42090 Mannan-binding lectin serine protease 1 isoform 2 21264359 81861 Thioredoxin peroxidase 5453549 30540 Glyoxalase II(hydroxyacyl glutathione hydrolase) 4885389 28860

pl

MS coverage (%)

6.9 6.7 6.7 5.2 5.7 5.9 6.0 6.1 5.9 6.1 6.4 5.8 6.0 6.0 6.4 5.5 5.0 5.9 6.9

11 26 19 27 21 26 22 20 15 22 16 19 52 19 17 38 18 21 26

5.4

75

8.4 8.0 8.5 7.8

25 23 18 10

6.0 8.9

18 43

8.3 9.5 9.8 8.7 8.3 8.3 9.4

31 18 31 22 45 44 38

5.2 8.2 5.9 5.5 5.4 5.2 5.6 5.4 8.9 8.9 9.0

MS/MS coverage (%)

number of the matched peptides (MS/MS)

24.6/37.6

11/19

32 25 54 30 29 31 42 27 25 45 40

21.4

8

53.5

13

30 31 56

24.4 16.4

9 7

5.1

81

2/22/21/7

31351 65866 55874 66080

5.4 8.1 8.5 7.1

58 32 22 20

5.4/47.2/ 47.4/16.7 32.5

Proteins That Act as Signal Transducers Programmed cell death 6 interacting protein(HP95) 13375569 95997 Zinc finger protein 397 29840864 61165 Aberrant epidermal growth factor receptor 181985 72396

6.1 6.9 7.0

29 28 17

A Chain A, Crystal Structure Of Human Glutathione S-Transferase P1--1[v104] Complexed 2554831 23342 With (9r,10r)-9-(S-Glutathionyl)-10- Hydroxy-9,10Dihydrophenanthrene 3′-phosphoadenosine 5′-phosphosulfate synthase 2 alpha 12642584 69429 NF protein isoform 219940 62301 ZAP 70 24657846 55874 ADAM 30 precursor (A disintegrin and 14423636 88925 metalloproteinase domain 30) Mitochondrial GTP-binding protein 1 13650157 52059 Similar to ATP synthase, H+ transporting, 13111901 40286 mitochondrial F1 complex, alpha subunit, Glutaryl-Coenzyme A dehydrogenase isoform a 4503943 48128 KIAA1493 protein 7959247 46824 Regulator of Fas-induced apoptosis 4885641 43147 Serine (or cysteine) proteinase inhibitor 32454741 46441 Phosphoglycerate kinase 1 129902 44728 Aldolase A 4557305 39420 GAPDH 19718751 34646 Proteins That Act as Nucleic Acid Binding Molecules hnRNP K 48429104 51029 Elongation factor for selenoproein translation 24308275 62579 HnRNP H1 5031753 49230 RuvB-like 2 5730023 51157 HnRNPF 4826760 45672 Eukaryotic translation initiation factor 3, subunit 5 epsilon 4503519 37564 Actin, beta 14250401 41005 Eukaryotic translation initiation factor 3 2494895 36502 HnRNP M 1710636 77470 HnRNP M isoform b 14141154 73562 p54nrb 13124797 54101

Proteins That Act as Chaperones/Mediators of Protein Folding Heat shock 60 kDa protein 1 (chaperonin) 129379 61055 5.7 HnRNP K isoform a 48429104 51029 5.2 TCP1, subunit 5 (epsilon) 24307939 59672 5.5 Vimentin

Proteins That Function in Structural Molecules 55977767 53652

F-actin capping protein beta subunit (CapZ beta) Keratin 2a ZAP70 protein Unnamed protein product

13124696 4557703 24657846 34532022

7

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Table 1 (Continued)

pl

MS coverage (%)

69

Proteins That Act as Signal Transducers Rho GDI beta 10835002 22988 Angiopoietin-like 4 protein 21536396 45214 Annexin A2 4757756 38604 lkBL protein 5031199 43257 LOC342892 protein 30851224 40578 Similar to Tyrosine-protein kinase ZAP-70 18600045 69873

5.1 9.1 7.6 7.2 8.3 7.8

46 35 43 12 37 32

35 43 45 48

Proteins That Relate with Proteasome 60S acidic ribosomal protein P0 4506667 34274 Proteasome endopeptidase complex zeta chain 88168 26425 Prohibitin 464371 29804 Proteasome beta 4 subunit 22538467 29205

5.7 4.7 5.6 5.7

52 44 84 56

spot no.

49 64 66 67

7, 8 68 7a 19a 27 33 36 37 38 39 41 46 59 63

identified protein

accession no.

MW (Da)

Proteins That Act as Ligand Binding or Carriers 24308035 74670 6.2 7981297 71982 6.0 6063691 30723 8.6 Uncertainty Hypothetical protein 12053005 65254 6.8 KIAA0776 protein 24308039 89596 6.3 Unnamed protein product 7020772 58140 5.7 Unknown protein 13279176 52488 5.9 Similar to neurotrimin (HNT protein) 30047135 34985 6.5 Small glutamine-rich tetratricopeptide 4506921 34063 4.8 Gastric cancer antigen Ga55 21724162 41240 5.0 Unnamed protein product 34081 30838 4.8 Immunoglobulin kappa light chain VLJ region 21669485 28459 6.7 Hypothetical protein 12224973 28201 4.6 Daxx 2253709 26342 4.9 Unnamed protein product 34533986 49205 9.2 ABLIM 1 protein 12803267 46087 8.3 Serine hydroxymethyltransferase 1 22547189 49028 7.6 Exocyst complex component 7 dJ534K7.2 (novel protein) Porin isoform 1

MS/MS coverage (%)

number of the matched peptides (MS/MS)

11.6 19.5

3 5

30 22 37 29 16 39 22 27 22 14 44 35 23 26 19 24 24

a Proteins phosphorylated following COMP-Ang1 were identified with MALDI-TOF MS (MS) and nanoLC-ESI-q-TOF tandem MS (MS/MS) and grouped by their functions inferred using gene ontology (www.godatabase.org). b After very closely associated protein spots are divided in two, they were identified as two proteins with MALDI-TOF MS, and present as no. and no.a.

that its activity is regulated by ROS induced by Ang1. This is consistent with our previous report that ROS is generated by Ang1 treatment.27 Confirmation of Proteins Identified by MALDI-TOF MS. We confirmed that the proteins having the same molecular weight and pI comigrate in a single spot on 2D gel,28 identified by peptide fingerprinting using MALDI-TOF MS and by sequencing selected spots with nanoLC-ESI-q-TOF tandem MS (supplementary Table 1 in Supporting Information). To validate the tyrosine-phosphorylated proteins in response to Ang1, we performed two sets of experiments to confirm that identified proteins are real tyrosine-phosphorylated proteins and not proteins that comigrated to the same position on 2D gel. First, tyrosine-phosphorylated spots detected by Western analysis using anti-phosphotyrosine antibody were stripped and then reprobed with various specific antibodies against identified proteins by MS (Figure 4A). As shown in Figure 4A, we found that Ser/Thr phosphatase (spot 18), PDI (spots 20 and 21), vimentin (spot 10), hnRNP K (spot 13), and prohibitin (spot 45) were tyrosine-phosphorylated by Ang1. Second, we performed immunoprecipitation assay with anti-phosphotyrosine antibody, and the immunoprecipitates were identified by Western analysis using several specific antibodies on 1D gel in Figure 4B and with silver staining (supplementary Figure 2 in Supporting Information). As shown in Figure 4B, we found that total tyrosine phosphorylation was increased ∼3.5-fold in response to Ang1 compared to control, and the results acquired 3284


from 2D gels were similar. We also showed that hnRNP K (spot 13), TR 1 (spot 12), PDI (spots 20 and 21), p54nrb (non-POU, spot 55), and ZAP 70 (spots 53 and 54) are specifically tyrosine phosphorylated by Ang1. Bands of immunoprecipitated proteins by phosphotyrosine antibody were cut out from the silverstained 1D gel, and followed by nanoLC-ESI-q-TOF tandem MS analysis (supplementary Figure 2 in Supporting Information). Several proteins (keratin, hsp70, actin, vimentin, p54nrb (non-POU), and 60S ribosomal protein) among identified proteins from 2D gel were also identified on 1D gel. Western analysis of several proteins in 1D gel presented in Figure 4B shows the same tendency of increase in 2D gel in response to Ang1 as in Figure 3A. However, we did not make direct comparisons between 1D- and 2D-gels as quantitative analysis of Western analysis after immunoprecipitation using different antibodies is not comparable because of differences in their specificities. These results show that the identified proteins are specifically tyrosine-phosphorylated and wellmatched with the quantitative results in Figure 2A, in response to Ang1 treatment. Tyrosine-Phosphorylated p54nrb Increased Invasiveness of Cells. To validate the effect of the phosphorylated protein identified by proteomics on angiogenesis or cell invasion, we selected p54nrb among Ang1-induced, tyrosine-phosphorylated proteins and examined the effect of overexpression of p54nrb on cell invasion. HUVECs or HeLa cells were transiently transfected with flag-p54nrb, and then an equal number of cells


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Figure 3 (continued) Journal of Proteome Research • Vol. 6, No. 8, 2007 3285

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Figure 3. Clustering and comparison of tyrosine-phosphorylated proteins by COMP-Ang1 (A) Tyrosine phosphorylation levels of COMP-Ang1-treated cells were grouped by their levels of phosphorylation relative to that of control cells. Group I contains proteins with increased tyrosine phosphorylation at recovery; Group II are proteins with increased phosphorylation at 45 min recovery time followed by decreased phosphorylation; Group III are proteins with increased phosphorylation only at 3 h recovery time. (B) The ratio of proteins numbers of each group relative to that of total proteins is shown. Proteins of each group were classified by their functions.

was seeded onto the matrigel. Both p54nrb-overexpressed HUVECs and HeLa cells were specifically increased migrationdependent on the amounts of p54nrb (Figure 5). We also examined the status of tyrosine phosphorylation of p54nrb in HeLa cells and found that p54nrb was specifically phosphorylated in tyrosine residues (Figure 5B-c). These results suggest that p54nrb is involved in the regulation of migration in the angiogenesis process by tyrosine phosphorylation. Prediction of Possible Tyrosine Phosphorylation Sites, Tyrosine Kinases, and Binding Proteins of Phosphorylated Motifs. Analyses of 66 proteins phosphorylated by Ang1 were performed by a computer-assisted program as described previously.13 This allowed us to predict the possible phosphorylation sites, possible protein kinases involved, and the possible proteins that bind to phosphorylated proteins. Prediction of phosphorylation sites was performed by NetPhos (www.cbs.dtu.dk/services/NetPhos, Supplementary Table 3 in Supporting Information). Supplementary Table 3 (Supporting Information) lists the predicted phosphotyrosine residues with possible tyrosine kinases and phosphotyrosine recognition motifs, using Scansite (scansite.mit.edu/). On the basis of these results, we can predict the Ang1-mediated signaling pathways. It appears that the larger the number of proteins, the higher the possibility that kinases and phosphotyrosine recognition motif-containing proteins are activated. As shown in Supplementary Table 4 (Supporting Information), proteins that potentially bind to tyrosine-phosphorylated proteins were predicted from 2 to 32 sites, with ERK D and PDK1 being the most frequent binding 3286


sites. The functions of phosphotyrosine recognition motifcontaining proteins can also be identified; ERK D can be activated in response to extracellular stimulus and induced cell proliferation or cell migration. Nck interacts with focal adhesion kinase and modulates cell motility. Crk affects cell morphology upon translocation to the cell membrane or mediates membrane ruffling and cell motility in a Rac-dependent manner. Cortactin regulates Erk/Src phosphorylation to activate NWASP acting in actin polymerization. Amphiphysin is one of the cytoskeleton components.29-31 These facts suggest that most of the proteins that are tyrosine-phosphorylated by Ang1 were involved in cell morphology or motility to rearrange or stabilize cytoskeletal proteins, and that computer-aided predictions can assist in interpretation of experimental results and help to narrow down the experimental trials.

Discussion Recent reports suggest that endothelial receptor tyrosine kinase is important for signaling in vascular development.32,33 However, the exact signaling mechanism by receptor tyrosine kinase in angiogenesis is not well-understood. Our study employing proteome analysis has identified a number of phosphorylated proteins involved in Ang1-induced angiogenesis. We tried to examine the Ang1’s signaling pathway in primary cultures of HUVECs having same passage, not in model cell culture systems. This limited the total amount of cellular


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Figure 4. Characterization of tyrosine-phosphorylated proteins after COMP-Ang1 treatment. (A) The membranes detected by antiphosphotyrosine antibody were stripped, and then reprobed with antibodies of several specific proteins; Ser/Thr phosphatase, PDI, vimentin, hnRNP K, and prohibitin, followed by HRP-conjugated secondary antibody. (B) The serum-starved cells were incubated with 200 ng/mL COMP-Ang1 for 30 min at 37 °C and then recovered for 45 min and 3 h. Cells were lysed, and the equal amount of protein was immunoprecipitated with anti-phosphotyrosine antibody. Immunoprecipitated proteins were separated by 1D gel electrophoresis, and electroblotted onto PVDF membrane. The blots were incubated with hnRNP K, TR 1, PDI, p54nrb, and ZAP 70 antibodies at a 1:1000 dilution in PBST at 4 °C overnight. The antigen-antibody complexes were visualized with HRP-conjugated secondary antibody. Journal of Proteome Research • Vol. 6, No. 8, 2007 3287

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Figure 5. p54nrb increased migration of HUVECs and HeLa cells. HUVECs (A) and HeLa cells (B) were transiently transfected with flag-p54nrb. Cells (10K or 20K) were seeded onto matrigel-coated transwells, and incubated for 24h. (B-a) Microscopy image of migrated cells, (B-b) number of migrated cells. (C) HeLa cells were transiently transfected with flag-p54nrb and cell lysate was immunoprecipitated by flag antibody. Immunoprecipitated proteins were detected with phosphotyrosine antibody and flag antibody. 3288



samples available. Since we need exact and thorough comparison of protein phosphorylations in control and Ang1 treatments by Western analysis, we chose mini 2D gel electrophoresis for clear separation of each protein and MALDI-TOF MS having 10 times more sensitivity than ESI-q-TOF tandem MS, for the identification of low-abundance proteins. We identified 66 tyrosine-phosphorylated proteins by Ang1/Tie 2 activation using large-scale proteomic analysis combining the separation of proteins on two-dimensional gel and protein identifications with MALDI-TOF MS and nanoLC-ESI-q-TOF tandem MS, and confirmed the identity of some tyrosinephosphorylated proteins through immunoprecipitation assay using anti-phosphotyrosine antibody. This is the first study to report that 66 proteins having various functions are responsive to phosphorylation by Ang1 treatment. However, tyrosine phosphorylation sites of identified proteins could not be identified with MS because of the low abundance of tyrosine phosphorylation (

Systemic Analysis of Tyrosine Phosphorylated Proteins in

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