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• W2101133609 abstract "The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of β-catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling. The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of β-catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling. The Wnt family of secreted signaling molecules is highly conserved among animal species and has been implicated in three major pathways, the canonical pathway and the two noncanonical pathways including planar cell polarity and calcium pathway (1Widelitz R. Wnt signaling through canonical and non-canonical pathways: recent progress.Growth Factors (Chur, Switzerland). 2005; 23: 111-116Crossref PubMed Scopus (173) Google Scholar). The canonical Wnt signaling pathway plays an important role in developmental processes including cell adhesion (2Shariatmadari M. Peyronnet J. Papachristou P. Horn Z. Sousa K.M. Arenas E. Ringstedt T. Increased Wnt levels in the neural tube impair the function of adherens junctions during neurulation.Mol. Cell. Neurosci. 2005; 30: 437-451Crossref PubMed Scopus (17) Google Scholar), morphology (3Dean C.H. Miller L.A. Smith A.N. Dufort D. Lang R.A. Niswander L.A. Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland.Dev. Biol. 2005; 286: 270-286Crossref PubMed Scopus (87) Google Scholar), proliferation (4Rao A.S. Kremenevskaja N. Resch J. Brabant G. Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/β-catenin signalling.Eur. J. Endocrinol. 2005; 153: 929-938Crossref PubMed Scopus (86) Google Scholar), and migration (5Neth P. Ciccarella M. Egea V. Hoelters J. Jochum M. Ries C. Wnt signaling regulates the invasion capacity of human mesenchymal stem cells.Stem. Cells. 2006; 4: 1892-1903Crossref Scopus (143) Google Scholar). Mutational deregulation of the Wnt cascade is closely associated with various tumors and other diseases (6Benhaj K. Akcali K.C. Ozturk M. Redundant expression of canonical Wnt ligands in human breast cancer cell lines.Oncol. Rep. 2006; 15: 701-707PubMed Google Scholar, 7Caricasole A. Bakker A. Copani A. Nicoletti F. Gaviraghi G. Terstappen G.C. Two sides of the same coin: Wnt signaling in neurodegeneration and neuro-oncology.Biosci. Rep. 2005; 25: 309-327Crossref PubMed Scopus (67) Google Scholar). Protein phosphorylation is one of the most important mechanisms for signaling propagation, and there is no exception for canonical Wnt signal transduction (8Swiatek W. Kang H. Garcia B.A. Shabanowitz J. Coombs G.S. Hunt D.F. Virshup D.M. Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation.J. Biol. Chem. 2006; 281: 12233-12241Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 9Yanfeng W.A. Tan C. Fagan R.J. Klein P.S. Phosphorylation of frizzled-3.J. Biol. Chem. 2006; 281: 11603-11609Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 10Patel S. Doble B. Woodgett J.R. Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword?.Biochem. Soc. Trans. 2004; 32: 803-808Crossref PubMed Scopus (125) Google Scholar, 11Rubinfeld B. Tice D.A. Polakis P. Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase 1ε.J. Biol. Chem. 2001; 276: 39037-39045Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 12Sun T.Q. Lu B. Feng J.J. Reinhard C. Jan Y.N. Fantl W.J. Williams L.T. PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling.Nat. Cell Biol. 2001; 3: 628-636Crossref PubMed Scopus (210) Google Scholar, 13Novak A. Dedhar S. Signaling through β-catenin and Lef/Tcf.Cell. Mol. Life Sci. 1999; 56: 523-537Crossref PubMed Scopus (267) Google Scholar). Many regulators or downstream components in the canonical Wnt signaling pathway are known to be phosphorylated or dephosphorylated in the transduction of signals from extracellular Wnt stimulation to intracellular effectors. These include Wnt receptors low density lipoprotein receptor-related protein-6 (8Swiatek W. Kang H. Garcia B.A. Shabanowitz J. Coombs G.S. Hunt D.F. Virshup D.M. Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation.J. Biol. Chem. 2006; 281: 12233-12241Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and Frizzled 3 (9Yanfeng W.A. Tan C. Fagan R.J. Klein P.S. Phosphorylation of frizzled-3.J. Biol. Chem. 2006; 281: 11603-11609Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and signaling components glycogen synthase kinase-3β (GSK3β) 1The abbreviations used are: GSK3β, glycogen synthase kinase-3β; SILAC, stable isotope labeling by amino acids in cell culture; PPI, protein-protein interaction; RRM2, ribonucleoside-diphosphate reductase M2 subunit; HPRD, Human Protein Reference Database; LEF, lymphoid enhancer factor; APC, adenomatous polyposis coli protein; TCF, T cell factor; Dvl, Dishevelled; MEK1, mitogen-activated protein kinase kinase 1; PP2A, protein phosphatase 2A; PP2Ac, α isoform of serine/threonine protein phosphatase 2A catalytic subunit; PKCα, protein kinase Cα; HSP90, heat shock protein 90; hnRNP, heterogeneous nuclear ribonucleoprotein; RanBP3, Ran-binding protein 3; EBP50, ezrin-radixin-moesin-binding phosphoprotein 50; SGN7a, COP9 signalosome complex subunit 7a; Cul1, Cullin 1; Rbx1, Ring box 1; siRNA, small interfering RNA; HEK, human embryonic kidney; HA, hemagglutinin; IPI, International Protein Index; KEGG, Kyoto Encyclopedia of Genes and Genomes; WT, wild type; GFP, green fluorescent protein; TNF, tumor necrosis factor; E1B-AP5, E1B-55 kDa-associated protein; IκBα-SR, I kappa B alpha-super repressor. 1The abbreviations used are: GSK3β, glycogen synthase kinase-3β; SILAC, stable isotope labeling by amino acids in cell culture; PPI, protein-protein interaction; RRM2, ribonucleoside-diphosphate reductase M2 subunit; HPRD, Human Protein Reference Database; LEF, lymphoid enhancer factor; APC, adenomatous polyposis coli protein; TCF, T cell factor; Dvl, Dishevelled; MEK1, mitogen-activated protein kinase kinase 1; PP2A, protein phosphatase 2A; PP2Ac, α isoform of serine/threonine protein phosphatase 2A catalytic subunit; PKCα, protein kinase Cα; HSP90, heat shock protein 90; hnRNP, heterogeneous nuclear ribonucleoprotein; RanBP3, Ran-binding protein 3; EBP50, ezrin-radixin-moesin-binding phosphoprotein 50; SGN7a, COP9 signalosome complex subunit 7a; Cul1, Cullin 1; Rbx1, Ring box 1; siRNA, small interfering RNA; HEK, human embryonic kidney; HA, hemagglutinin; IPI, International Protein Index; KEGG, Kyoto Encyclopedia of Genes and Genomes; WT, wild type; GFP, green fluorescent protein; TNF, tumor necrosis factor; E1B-AP5, E1B-55 kDa-associated protein; IκBα-SR, I kappa B alpha-super repressor. (10Patel S. Doble B. Woodgett J.R. Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword?.Biochem. Soc. Trans. 2004; 32: 803-808Crossref PubMed Scopus (125) Google Scholar), adenomatous polyposis coli protein (APC) (11Rubinfeld B. Tice D.A. Polakis P. Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase 1ε.J. Biol. Chem. 2001; 276: 39037-39045Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), Dishevelled (Dvl) (12Sun T.Q. Lu B. Feng J.J. Reinhard C. Jan Y.N. Fantl W.J. Williams L.T. PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling.Nat. Cell Biol. 2001; 3: 628-636Crossref PubMed Scopus (210) Google Scholar), and β-catenin (13Novak A. Dedhar S. Signaling through β-catenin and Lef/Tcf.Cell. Mol. Life Sci. 1999; 56: 523-537Crossref PubMed Scopus (267) Google Scholar). For example, as a key component in canonical Wnt signaling, β-catenin is phosphorylated by serine/threonine kinases in the absence of Wnt stimulation, and the hyperphosphorylation leads to its ubiquitination and degradation by the proteasome. When Wnt is present, β-catenin is free from phosphorylation, which results in its stabilization and translocation into the nucleus to regulate gene transcription (13Novak A. Dedhar S. Signaling through β-catenin and Lef/Tcf.Cell. Mol. Life Sci. 1999; 56: 523-537Crossref PubMed Scopus (267) Google Scholar). Previous work has demonstrated the indispensable role of reversible phosphorylation in regulation of the canonical Wnt signaling cascade by studying one or several specific proteins (10Patel S. Doble B. Woodgett J.R. Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword?.Biochem. Soc. Trans. 2004; 32: 803-808Crossref PubMed Scopus (125) Google Scholar, 12Sun T.Q. Lu B. Feng J.J. Reinhard C. Jan Y.N. Fantl W.J. Williams L.T. PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling.Nat. Cell Biol. 2001; 3: 628-636Crossref PubMed Scopus (210) Google Scholar). The newly developed proteomics strategies make possible the global characterization of a signaling pathway. Recently phosphoproteomes of many signaling pathways, including those activated by epidermal growth factor, fibroblast growth factor, interferon-α, insulin, and transforming growth factor β, have been analyzed by using immunoaffinity purification and/or IMAC enrichment or [32P]orthophosphate radioactive isotope labeling followed by mass spectrometric identification (14Lim Y.P. Diong L.S. Qi R. Druker B.J. Epstein R.J. Phosphoproteomic fingerprinting of epidermal growth factor signaling and anticancer drug action in human tumor cells.Mol. Cancer Ther. 2003; 2: 1369-1377PubMed Google Scholar, 15Hinsby A.M. Olsen J.V. Bennett K.L. Mann M. Signaling initiated by overexpression of the fibroblast growth factor receptor-1 investigated by mass spectrometry.Mol. Cell. Proteomics. 2003; 2: 29-36Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Zheng H. Hu P. Quinn D.F. Wang Y.K. Phosphotyrosine proteomic study of interferon α signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography.Mol. Cell. Proteomics. 2005; 4: 721-730Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Stasyk T. Dubrovska A. Lomnytska M. Yakymovych I. Wernstedt C. Heldin C.H. Hellman U. Souchelnytskyi S. Phosphoproteome profiling of transforming growth factor (TGF)-β signaling: abrogation of TGFβ1-dependent phosphorylation of transcription factor-II-I (TFII-I) enhances cooperation of TFII-I and Smad3 in transcription.Mol. Biol. Cell. 2005; 16: 4765-4780Crossref PubMed Scopus (41) Google Scholar, 18Wang Y. Li R. Du D. Zhang C. Yuan H. Zeng R. Chen Z. Proteomic analysis reveals novel molecules involved in insulin signaling pathway.J. Proteome Res. 2006; 5: 846-855Crossref PubMed Scopus (23) Google Scholar). These global studies have unraveled the relationships between many novel phosphoproteins or phosphorylation sites and specific signaling pathways, therefore enriching our view of comprehensive phosphorylation regulation in signal transduction. However, many of the earlier approaches would not allow quantitative characterization of the dynamic changes in phosphorylation events and distinguishing induced phosphorylation events from constitutive phosphorylation events (15Hinsby A.M. Olsen J.V. Bennett K.L. Mann M. Signaling initiated by overexpression of the fibroblast growth factor receptor-1 investigated by mass spectrometry.Mol. Cell. Proteomics. 2003; 2: 29-36Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Wang Y. Li R. Du D. Zhang C. Yuan H. Zeng R. Chen Z. Proteomic analysis reveals novel molecules involved in insulin signaling pathway.J. Proteome Res. 2006; 5: 846-855Crossref PubMed Scopus (23) Google Scholar). Although some of the approaches are able to quantitate phosphorylation changes, the quantitation is based on two-dimensional gel electrophoresis or a chemical derivation method (14Lim Y.P. Diong L.S. Qi R. Druker B.J. Epstein R.J. Phosphoproteomic fingerprinting of epidermal growth factor signaling and anticancer drug action in human tumor cells.Mol. Cancer Ther. 2003; 2: 1369-1377PubMed Google Scholar, 16Zheng H. Hu P. Quinn D.F. Wang Y.K. Phosphotyrosine proteomic study of interferon α signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography.Mol. Cell. Proteomics. 2005; 4: 721-730Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Stasyk T. Dubrovska A. Lomnytska M. Yakymovych I. Wernstedt C. Heldin C.H. Hellman U. Souchelnytskyi S. Phosphoproteome profiling of transforming growth factor (TGF)-β signaling: abrogation of TGFβ1-dependent phosphorylation of transcription factor-II-I (TFII-I) enhances cooperation of TFII-I and Smad3 in transcription.Mol. Biol. Cell. 2005; 16: 4765-4780Crossref PubMed Scopus (41) Google Scholar). Recently a mass spectrometry-based quantitation approach termed stable isotope labeling by amino acids in cell culture (SILAC) has been introduced (19Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar). Combined with a phosphoprotein or phosphopeptide enrichment method, SILAC has been widely applied to profile dynamic phosphorylation changes in signal transduction (20Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar, 21Zhang G. Spellman D.S. Skolnik E.Y. Neubert T.A. Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC).J. Proteome Res. 2006; 5: 581-588Crossref PubMed Scopus (71) Google Scholar). In the present study, we investigated the differential phosphoproteome upon stimulation of a canonical Wnt protein, Wnt3a, by using SILAC in combination with phosphoprotein enrichment and LC-MS/MS analysis. Stable isotope containing amino acid [13C6]lysine was purchased from Cambridge Isotope Laboratories (Andover, MA). The RPMI 1640 medium deficient in l-lysine was a custom medium preparation from Chemicon (Temecula, CA). Complete protease inhibitor mixture tablets were purchased from Roche Applied Science, sodium orthovanadate was from Sigma-Aldrich, and the phosphoprotein purification kit was from Qiagen (Valencia, CA). Sequencing grade trypsin was purchased from Promega (Madison, WI). Recombinant mouse Wnt3a was purchased from R&D Systems (Minneapolis, MN). Wnt3a-containing conditioned medium was prepared as previously described (22Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Antibodies against the following proteins were used: heat shock protein 90 (HSP90), mitogen-activated protein kinase kinase 1 (MEK1), and protein kinase Cα (PKCα) from Santa Cruz Biotechnology (Santa Cruz, CA); nucleophosmin and β-tubulin from Sigma-Aldrich; heterogeneous nuclear ribonucleoprotein C (hnRNP C) from ImmuQuest Ltd. (Ingleby Barwick, Cleveland, UK); α isoform of serine/threonine protein phosphatase 2A catalytic subunit (PP2Ac) and β-catenin from BD Biosciences; and ribonucleoside-diphosphate reductase M2 chain (RRM2) from Abnova Corp. (Taipei City, Taiwan). Mouse anti-HA monoclonal antibody was purchased from Covance (Princeton, NJ). HEK293 cells were grown in RPMI 1640 medium containing [12C6]lysine (“light”) or [13C6]lysine (“heavy”) supplemented with 10% dialyzed fetal bovine serum plus antibiotics. Detailed instructions for this protocol are available upon request. Briefly the HEK293 cells were adapted to grow in isotope-containing medium supplemented with dialyzed serum and passaged for six generations to ensure a complete replacement prior to initiating these experiments. The heavy cells were stimulated with Wnt3a (100 ng/ml) for 1 or 30 min. The light, unstimulated cells were divided to serve as a common zero point. A phosphoprotein purification kit (23Metodiev M.V. Timanova A. Stone D.E. Differential phosphoproteome profiling by affinity capture and tandem matrix-assisted laser desorption/ionization mass spectrometry.Proteomics. 2004; 4: 1433-1438Crossref PubMed Scopus (46) Google Scholar, 24Fu J. Naren A.P. Gao X. Ahmmed G.U. Malik A.B. Protease-activated receptor-1 activation of endothelial cells induces protein kinase Cα-dependent phosphorylation of syntaxin 4 and Munc18c: role in signaling p-selectin expression.J. Biol. Chem. 2005; 280: 3178-3184Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 25Nanamori M. Chen J. Du X. Ye R.D. Regulation of leukocyte degranulation by cGMP-dependent protein kinase and phosphoinositide 3-kinase: potential roles in phosphorylation of target membrane SNARE complex proteins in rat mast cells.J. Immunol. 2007; 178: 416-427Crossref PubMed Scopus (37) Google Scholar) from Qiagen (Valencia, CA) was applied to enrich phosphoproteins according to the manufacturer's instructions. Briefly light and heavy cells were lysed in the Lysis Buffer, and the cell lysate was centrifuged at 10,000 × g at 4 °C for 30 min to remove insoluble material. After centrifugation, the protein concentration of the cell lysate was quantitated by Bradford assay, and different cell lysates (light or heavy) were combined at an equal sample amount. The extracted proteins of combined cell lysates were diluted to a concentration of 0.1 mg/ml with Lysis Buffer, and a total of 25 ml of the extracted proteins was applied to a Lysis Buffer-equilibrated phosphoprotein purification column at room temperature. After washing the column with 6 ml of Lysis Buffer, the phosphoproteins were eluted with 2 ml of Phosphoprotein Elution Buffer. All the buffers and the phosphoprotein purification column were provided in the kit by the manufacturer (Qiagen). The eluted phosphoproteins were dialyzed, lyophilized, and resolved by 7.5–17.5% gradient SDS-PAGE. The gel was stained using Coomassie Brilliant Blue stain to visualize the gel lanes. The two gel lanes (“0 min-1 min,” the combined cell samples stimulated with Wnt3a for 0 or 1 min; “0 min-30 min,” the combined cell samples stimulated with Wnt3a for 0 or 30 min) were cut into 26 slices, respectively. The excised slices were subjected to in-gel trypsin digestion as described before (26Jiang X.S. Tang L.Y. Cao X.J. Zhou H. Xia Q.C. Wu J.R. Zeng R. Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells.Electrophoresis. 2005; 26: 4540-4562Crossref PubMed Scopus (25) Google Scholar). For verification of phosphoprotein enrichment and SILAC results, HEK293 cells were grown and treated the same way as the sample for SILAC analysis except for stable isotope [13C6]lysine (heavy) labeling. Briefly the cells were grown in 10-cm2 dishes. One dish of cells was left untreated as control, and two dishes of cells were treated with Wnt3a ligand for 1 and 30 min, respectively. Cell lysis and phosphoprotein enrichment of the three samples were separately performed according to the manufacturer's instruction (Qiagen). Eluted phosphoproteins were dialyzed, lyophilized, and reconstituted by an equal volume (250 μl) of 4× sample buffer (40% glycerol, 8% SDS, 250 mm Tris-HCl, 4% β-mercaptoethanol). To validate the efficacy of phosphoprotein enrichment in the elution fraction, 10 μg of proteins of the total cell lysate, elution fraction, and flow-through fraction from the phosphoprotein purification kit were separated by 12.5% SDS-PAGE followed by silver staining (26Jiang X.S. Tang L.Y. Cao X.J. Zhou H. Xia Q.C. Wu J.R. Zeng R. Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells.Electrophoresis. 2005; 26: 4540-4562Crossref PubMed Scopus (25) Google Scholar) or Western blotting analysis using mixed phosphoserine, phosphothreonine, and phosphotyrosine monoclonal antibodies. For Western blotting verification of SILAC results, the eluted phosphoprotein fractions were loaded at the same volume of 10 μl. After transfer, the nitrocellulose membranes were blocked by 1× Net-gelatin (150 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl, pH 7.5, 0.05% Triton X-100, 0.25% gelatin) and then incubated with the corresponding primary antibodies followed by horseradish peroxidase-conjugated or IRDye 800CW-conjugated affinity-purified secondary antibodies. The membranes were subjected to fluorescent chemiluminescence detection and exposed to films or scanned by the Odyssey Infrared Imaging System 9120 (LI-COR, Lincoln, NE) according to the manufacturer's instructions. The peptide mixtures from each gel slice were separated by reverse phase HPLC followed by tandem mass spectrometry analysis. Reverse phase HPLC was performed using an Agilent 1100 Capillary system (Agilent Technologies) on a C18 column (150-μm inner diameter, 100-mm length; Column Technology Inc., Fremont, CA). The pump flow rate was 1.6 μl/min. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The tryptic peptide mixtures were eluted using a gradient of 2–55% B over 135 min. The mass spectral data shown in this study were acquired on a linear quadrupole ion trap (LTQ) mass spectrometer (Thermo, San Jose, CA) equipped with an electrospray interface operated in positive ion mode. The temperature of the heated capillary was set at 170 °C. A voltage of 3.0 kV applied to the ESI needle resulted in a distinct signal. The normalized collision energy was 35.0. The number of ions stored in the ion trap was regulated by the automatic gain control. Voltages across the capillary and the quadrupole lenses were tuned by an automated procedure to maximize the signal for the ion of interest. The mass spectrometer was set such that one full MS scan was followed by 10 MS/MS scans on the 10 most intense ions from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 2; repeat duration, 30 s; exclusion duration, 90 s. The BioWorks™ 3.1 software suite was used to generate the peak lists of all acquired MS/MS spectra, and then they were automatically searched against the Internation Protein Index (IPI) human database (version 3.04) (27Kersey P.J. Duarte J. Williams A. Karavidopoulou Y. Birney E. Apweiler R. The International Protein Index: an integrated database for proteomics experiments.Proteomics. 2004; 4: 1985-1988Crossref PubMed Scopus (640) Google Scholar) containing 49,078 protein entries using the SEQUEST (University of Washington, licensed to Thermo Finnigan) searching program. Trypsin was designated as the protease, and up to two missed cleavages were allowed. Carbamidomethylation was searched as a fixed modification, and phosphorylation of serine/threonine/tyrosine residues (+79.98 Da) and isotope-labeled lysine (+6.00 Da) were allowed as variable modifications. The mass tolerance of the CID spectra was set as ±1.0 Da. For non-phosphopeptide identification, a rigorously accepted SEQUEST result must have a ΔCn score of at least 0.1 (regardless of charge state). The cross-correlation score must be ≥1.9 for a +1 tryptic peptide, ≥2.2 for a +2 tryptic peptide, and ≥3.75 for a +3 tryptic peptide (28Eng J.K. Mccormack A.L. Yates J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5420) Google Scholar). For phosphorylation site identification, first, defined phosphopeptides should be above the increased Xcorr threshold setting of ≥2.0 for +1 tryptic peptide, ≥2.5 for +2 tryptic peptide, and ≥4.0 for +3 tryptic peptide (previously reported Xcorr thresholds for phosphopeptide filtering were set as 1.9, 2.2, and 3.75 for singly, doubly, and triply charged ions, respectively (29Feng S. Ye M. Zhou H. Jiang X. Jiang X. Zou H. Gong B. Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis.Mol. Cell. Proteomics. 2007; 6: 1656-1665Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), or 2.5 and 3.3 for doubly and triply charged ions, respectively (30Ballif B.A. Villen J. Beausoleil S.A. Schwartz D. Gygi S.P. Phosphoproteomic analysis of the developing mouse brain.Mol. Cell. Proteomics. 2004; 3: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar)). Second, a phosphorylation site(s) was (were) considered to be unique only when a certain phosphopeptide had a ΔCn score of at least 0.1, as ΔCn ≥ 0.1 is significant for discriminating the first (top) candidate peptide from the second candidate peptide (28Eng J.K. Mccormack A.L. Yates J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5420) Google Scholar). Third, all the spectra of the quantitated peptides and phosphopeptides were manually inspected according to the criteria (31Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2071) Google Scholar) that an MS/MS spectrum of good quality must have its fragment ion peaks clearly above baseline noise, show sequential members of the b- or y-ion series with phosphorylation site included, and show intense proline-directed fragment ions. In addition, the phosphoric acid neutral loss peaks were checked for phosphorylation site identification (30Ballif B.A. Villen J. Beausoleil S.A. Schwartz D. Gygi S.P. Phosphoproteomic analysis of the developing mouse brain.Mol. Cell. Proteomics. 2004; 3: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). To eliminate redundancy, we first used the IPI database, which offers complete nonredundant data sets built from the Swiss-Prot, TrEMBL, Ensembl," @default.
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• W2101133609 date "2007-11-01" @default.
• W2101133609 modified "2023-10-15" @default.
• W2101133609 title "Quantitative Phosphoproteome Profiling of Wnt3a-mediated Signaling Network" @default.
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