Phosphotyrosine Signaling Networks in Epidermal Growth Factor Receptor Overexpressing Squamous Carcinoma Cells
2005; Elsevier BV; Volume: 4; Issue: 4 Linguagem: Inglês
10.1074/mcp.m400118-mcp200
ISSN1535-9484
AutoresApril Thelemann, Filippo Petti, Graeme Griffin, Ken Iwata, Tony Hunt, Tina Settinari, David Fenyö, Neil W. Gibson, John D. Haley,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoOverexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded μLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cγ (PLCγ), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6. Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded μLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cγ (PLCγ), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6. Overexpression and enhanced activation of the epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; HNSCC, head and neck squamous carcinoma; EGFR, epidermal growth factor receptor; IGF-1, insulin-like growth factor-1; FAK, focal adhesion kinase; HB-EGF, heparin-binding epidermal growth factor; SH2, Src homology 2 domain; PTB, phosphotyrosine binding domain; LPA, lysophosphatidic acid; TGFα, transforming growth factor α; IC50, half maximal inhibitory concentration; pY, phosphotyrosine; SCX, strong cation exchange; PLCγ, phospholipase Cγ; PI3-kinase, phosphatidyl inositol-3 kinase; GO, Gene Ontology database; OSI-774, erlotinib (Tarceva). receptor are frequently observed in human cancers (1.Yarden Y. The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic opportunities.Eur. J. Cancer. 2001; 37: S3-S8Google Scholar, 2.Prenzel N. Zwick E. Leserer M. Ullrich A. Tyrosine kinase signalling in breast cancer. Epidermal growth factor receptor: Convergence point for signal integration and diversification.Breast Cancer Res. 2000; 2: 184-190Google Scholar), and abnormal activation of the receptor's intrinsic tyrosine phosphotransferase activity correlates with poor prognosis (3.Nicholson R.I. Gee J.M. Harper M.E. EGFR and cancer prognosis.Eur. J. Cancer. 2001; 37: S9-S15Google Scholar). Overexpression of EGF receptor is a common event in tumors of the breast, bladder, lung, head and neck, and central nervous system. Inhibitors of EGF receptor function have shown clinical utility, and the definition of key EGF receptor signaling pathways has become increasingly important in understanding the consequences of drug action (2.Prenzel N. Zwick E. Leserer M. Ullrich A. Tyrosine kinase signalling in breast cancer. Epidermal growth factor receptor: Convergence point for signal integration and diversification.Breast Cancer Res. 2000; 2: 184-190Google Scholar). The EGF receptor family of receptor tyrosine kinases, EGFR, ErbB2, ErbB3, and ErbB4, can heterodimerize to allow a diversity of ligand responses with accompanying changes in the rates and routes of internalization and degradation (4.Yarden Y. Sliwkowski M.X. Untangling the ErbB signalling network.Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Google Scholar). The EGF receptor is a membrane glycoprotein having an external cysteine-rich ligand binding domain, linked by a short single transmembrane sequence to intracellular tyrosine kinase and carboxyl-terminal scaffolding domains (5.Downward J. Yarden Y. Mayes E. Scrace G. Totty N. Stockwell P. Ullrich A. Schlessinger J. Waterfield M.D. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences.Nature. 1984; 307: 521-527Google Scholar, 6.Schlessinger J. Ligand-induced, receptor-mediated dimerization and activation of EGF receptor.Cell. 2002; 110: 669-672Google Scholar). The binding of ligands including EGF, TGFα, amphiregulin, and HB-EGF results in an activation of the receptor tyrosine kinase activity and autophosphorylation at multiple tyrosine residues located in the C-terminal domain (1.Yarden Y. The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic opportunities.Eur. J. Cancer. 2001; 37: S3-S8Google Scholar). When phosphorylated, the C-terminal domain serves as a scaffold for the binding of Src homology 2 (SH2)- and phosphotyrosine binding (PTB)-containing adaptor proteins, for example Grb2, Shc, NSP1, and NSP2, which can transduce mitogenic and cell survival signals. Substrate binding in turn can stimulate additional protein-protein interactions to assemble competent signaling complexes required to coordinate the diverse responses elicited by ligand binding (7.Pawson T. Nash P. Assembly of cell regulatory systems through protein interaction domains.Science. 2003; 300: 445-452Google Scholar), and the tyrosine phosphorylation of transiently interacting substrates can establish scaffolds for SH2 and PTB complex formation at distant sites. The compartmentalization of EGF receptors also has marked effects on the repertoire of substrates and interacting factors through which receptor signaling is achieved. For example EGF receptors have been shown to cluster in caveolae (8.Carpenter G. The EGF receptor: A nexus for trafficking and signaling.Bioessays. 2000; 22: 697-707Google Scholar) where autophosphorylation results in interactions with phospholipid and calcium-dependent substrates enriched within this lipid-raft-like microenvironment. Similarly the translocation and internalization of the receptor into early endosomes place the receptor in an important subcellular localization for the transduction of signals through the Ras-Raf-Mek-Erk pathway important in the mitogenic effects of EGF (8.Carpenter G. The EGF receptor: A nexus for trafficking and signaling.Bioessays. 2000; 22: 697-707Google Scholar). Thus both EGF receptor protein interactions as well as the cellular location of receptor complexes determine the downstream signals produced. In vitro and clinical studies have shown considerable variability between cell lines and tumors in their cellular responses to EGF receptor inhibition, which in part has been shown to derive from EGF receptor-independent activation of the phosphatidyl inositol 3-kinase (PI3-kinase) pathway, leading to the continued phosphorylation of the anti-apoptotic serine-threonine kinase Akt (9.Vivanco I. Sawyers C.L. The phosphatidylinositol 3-kinase AKT pathway in human cancer.Nat. Rev. Cancer. 2002; 2: 489-501Google Scholar). The molecular determinants to alternative routes of PI3-kinase activation and consequent EGF receptor inhibitor insensitivity are an active area of investigation (10.Dancey J.E. Predictive factors for epidermal growth factor receptor inhibitors—The bull’s-eye hits the arrow.Cancer Cell. 2004; 5: 411-415Google Scholar). For example the insulin-like growth factor-1 receptor (IGF-1 receptor), which strongly activates the PI3-kinase pathway, has been implicated in cellular resistance to EGF inhibitors. The roles of other tyrosine kinases in mediating insensitivity to selective EGF receptor inhibition are less clear, for example those of the Src family, which participate in the mitogenic and survival signals generated by lysophosphatidic acid (LPA). Similarly cell-cell and cell adhesion networks can also exert survival signals through the PI3-kinase pathway (11.Comoglio P.M. Boccaccio C. Trusolino L. Interactions between growth factor receptors and adhesion molecules: Breaking the rules.Curr. Opin. Cell Biol. 2003; 15: 565-571Google Scholar) and would be postulated to impact cell sensitivity to EGF receptor blockade. The ability of tumor cells to maintain growth and survival signals in the absence of adhesion to extracellular matrix or cell-cell contacts is important not only in the context of cell migration and metastasis but also in maintaining cell proliferation and survival in changing tumor environments where extracellular matrix is being remodeled and cell contact inhibition is abrogated. The EGF receptor and proteins controlling cell adhesion assembly and disassembly have been shown to physically interact and cross-regulate in a complex manner dependent on receptor activity and cell adhesion factors (12.Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. FAK integrates growth-factor and integrin signals to promote cell migration.Nat. Cell Biol. 2000; 2: 249-256Google Scholar, 13.Lu Z. Jiang G. Blume-Jensen P. Hunter T. Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase.Mol. Cell. Biol. 2001; 21: 4016-4031Google Scholar). The principle aim of this study was to 1) better define EGF receptor signaling networks within tumor cells abnormally overexpressing EGF receptors and 2) define those signaling proteins and pathways most sensitive to inhibition of EGF receptor kinase activity. The squamous carcinoma cell line HN5 (14.Knight J. Gusterson B.A. Cowley G. Monaghan P. Differentiation of normal and malignant human squamous epithelium in vivo and in vitro: A morphologic study.Ultrastruct. Pathol. 1984; 7: 133-141Google Scholar) was used as a model system to investigate phosphotyrosine-dependent cellular signaling. HN5 cells show a high basal level of EGF receptor activity, derived in part from autocrine production of TGFα, and are sensitive to EGF receptor inhibition. Receptor overexpression is prevalent in squamous cell carcinomas of the head and neck (HNSCC), occurring in over 40% of cases, and inhibition of EGF receptor signaling has been shown to reduce tumor xenograft growth in vivo (15.Pollack V.A. Savage D.M. Baker D.A. Tsaparikos K.E. Sloan D.E. Moyer J.D. Barbacci E.G. Pustilnik L.R. Smolarek T.A. Davis J.A. Vaidya M.P. Arnold L.D. Doty J.L. Iwata K.K. Morin M.J. Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: Dynamics of receptor inhibition in situ and antitumor effects in athymic mice.J. Pharmacol. Exp. Ther. 1999; 291: 739-748Google Scholar). Erlotinib, a selective EGF receptor kinase inhibitor, has an IC50 for cellular EGF receptor kinase inhibition of ∼50 nm. The inhibition of HN5 cell proliferation is closely correlated to EGF receptor inhibition, with an IC50 of 90 nm. Here anti-phosphotyrosine (anti-pY) and anti-EGF receptor affinity chromatography were coupled with multiple MS approaches (Fig. 1) to define proteins and protein complexes associated with EGF receptor signaling and with kinase inhibition. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Models of EGF receptor signaling in HNSCC were constructed. The relative abundance of anti-pY-selected proteins after EGF receptor kinase inhibition by erlotinib (OSI-774 Tarceva; Ref. 16.Moyer J.D. Barbacci E.G. Iwata K.K. Arnold L. Boman B. Cunningham A. DiOrio C. Doty J. Morin M.J. Moyer M.P. Neveu M. Pollack V.A. Pustilnik L.R. Reynolds M.M. Sloan D. Theleman A. Miller P. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase.Cancer Res. 1997; 57: 4838-4848Google Scholar) and after hyperstimulation of EGF receptor kinase activity by addition of exogenous EGF was measured by protein immunoblot and peptide ICAT methods (17.Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar). Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cγ (PLCγ), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation, suggesting a complex balance between growth factor-induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, LPA signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6. Immunoaffinity resins (anti-pY and anti-EGF receptor) were prepared by covalent coupling to resin followed by low pH washing to remove noncovalently bound antibody, a potential source of significant immunoglobulin heavy and light chain contamination in later steps. Freshly prepared immunoaffinity resins were used for each biological experiment to maximize binding and reduce carryover. Anti-pY antibodies PY20 (Exalpha Biologicals, Inc., Watertown, MA) and PY100 (Cell Signaling Technology, Beverly, MA) were mixed in an 8:1 ratio and bound to Protein G-resin (Pierce, Rockford, IL) for 30 min at room temperature. In separate experiments, anti-EGF receptor antibody, recognizing an extracellular epitope (EGFR.1, Ref. 18.Waterfield M.D. Mayes E.L. Stroobant P. Bennet P.L. Young S. Goodfellow P.N. Banting G.S. Ozanne B. A monoclonal antibody to the human epidermal growth factor receptor.J. Cell. Biochem. 1982; 20: 149-161Google Scholar; BD Bioscience, San Diego, CA), was similarly immobilized. Antibody resins were washed extensively, and 0.02 m dimethylpimelidate dihydrochloride (DMP, in 0.2 m triethanolamine, pH 8.2; Pierce) was added and mixed for 1 h on a rotating platform. Crosslinking was terminated by washing with three resin volumes of 0.2 m triethanolamine, pH 8.2, once with 0.2 m sodium citrate, pH 2.8 (to remove noncovalently bound IgG), and finally with three volumes of 10 mm TrisHCl, pH 8.2. Antibody crosslinked resins were stored at 4 °C until use. In later experiments disuccinimidyl suberate (DSS) crosslinker was substituted for DMP (Pierce) with similar results. Approximately 2 × 108 HN5 cells (14.Knight J. Gusterson B.A. Cowley G. Monaghan P. Differentiation of normal and malignant human squamous epithelium in vivo and in vitro: A morphologic study.Ultrastruct. Pathol. 1984; 7: 133-141Google Scholar) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. HN5 cell extracts were prepared by mild detergent lysis (1% Triton X-100) containing protease and phosphatase inhibitors (see below) to enhance the preservation of protein interactions, lost when deoxycholate-containing lysis buffers (e.g. RIPA) were used. The selective EGF receptor kinase inhibitor erlotinib (1 μm OSI-774) was added to HN5 cells for 60 or 120 min prior to lysis. EGF-treated cells were incubated with ligand (10 ng/ml) for 10 min prior to lysis unless otherwise stated. Cells were washed once with PBS prior to lysis in 50 mm HEPES, pH 7.5, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.8 μm aprotinin, 20 μm leupeptin, 40 μm beestatin, 15 μm pepstatin A, 14 μm E-64 [1:100 dilution of protease inhibitor mixture P8340; Sigma, St. Louis, MO], sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole [1:100 dilution of phosphatase inhibitor mixture P5726; Sigma] for ∼3 min. Insoluable material was removed by centrifugation (13,000 × g, 10 min, 4 °C). Protein concentration was determined by microBCA assay (Pierce). Lysates were precleared by incubation with blank Protein-G resin for 30 min at 4 °C prior to immunoprecipitation to reduce nonspecific binding. Antibody resins were equilibrated with lysis buffer and incubated with HN5 cell lysates for 2–4 h at 4 °C with rotation. Antibody-antigen complexes were washed with >200 volumes of 10 mm TrisHCl, pH 8.0, 150 mm NaCl at 4 °C, proteins eluted with 0.1% TFA, 5% methanol, and dried in vacuo. The initial anti-pY affinity step yielded ∼50–100 μg of protein from 2 × 108 cells, representing an approximate 1,000-fold enrichment, greatly reducing sample complexity for subsequent LC-MS/MS protein identification. Visual inspection of SDS-PAGE-fractioned anti-pY affinity-isolated proteins (Fig. 2A) or nonspecific proteins trapped on resin control (Fig. 2, B and C) or goat anti-rabbit antibody control (data not shown) qualitatively indicated minimal nonspecific binding and generally low IgG release from the resin. Equivalent amounts of protein extract, as determined by BCA assay, were subject to SDS-PAGE. Protein immunodetection was performed by electrophoretic transfer of SDS-PAGE-separated proteins to nitrocellulose, incubation with antibody, and chemiluminescent second step detection (ECL; Amersham, Piscataway, NJ). Antibodies included: p130 Cas (#sc-860; Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Shc (#2434; Cell Signaling, Beverly, MA), phospho-Paxillin (#2541; Cell Signaling), phospho-Akt (#9271; Cell Signaling), phospho-HER2/ErbB2 (#2245; Cell Signaling), phospho-p44/42 MAP kinase (#9101; Cell Signaling), phospho-EGFR (Tyr845) (#2231; Cell Signaling), phospho-EGFR (Tyr992) (#2235; Cell Signaling), phospho-EGFR (Tyr1045) (#2237; Cell Signaling), EGFR (#2232; Cell Signaling), Pyk2 (#06–559-MN; Upstate Biotechnology, Lake Placid, NY), phospho-Caveolin-1 (#3251; Cell Signaling), phospho-p70 S6 kinase (#9205; Cell Signaling), phospho-GSK-3α/β (#9331; Cell Signaling), phospho-EGFR (Tyr1068) (#2236; Cell Signaling), phospho-Src family (Tyr416) (#2101; Cell Signaling), phospho-Src (Tyr527) (#2105; Cell Signaling), FAK (#06–543-MN; Upstate Biotechnology), Karyopherin B1 (#sc-11367; Santa Cruz Biotechnology), 14-3-3ζ (#sc-1019; Santa Cruz Biotechnology), 14-3-3ε (#610542; BD Transduction Laboratories, San Jose, CA), ACK (#sc-323; Santa Cruz Biotechnology), α-catenin (#sc-9988; Santa Cruz Biotechnology), Plakophilin 2 (#610788; BD Transduction Laboratories), GRB2 (#3972; Cell Signaling), δ-catenin (#07-259; Upstate Biotechnology), EF-1α (#05-235; Upstate Biotechnology), Tyk 2 (#sc-169; Santa Cruz Biotechnology), γ-catenin (#sc-8415; Santa Cruz Biotechnology), EphA2 receptor (#34-7400; Zymed Laboratories, South San Francisco, CA), clathrin heavy chain (#CP45; Oncogene Research Products, Boston, MA), c-Yes (#06-514; Upstate Biotechnology), and IGF-1R-β chain (#sc-713; Santa Cruz Biotechnology). Proteins isolated by anti-pY affinity chromatography were denatured, reduced, carboxamidomethylated, and proteolytically cleaved with trypsin. Peptides were introduced into the Q-TOF mass spectrometer either using reverse phase (C18) HPLC or, to further reduce sample complexity and ion suppression, using coupled strong cation exchange-reverse phase (SCX-C18) HPLC. The use of multiple overlapping methods (Fig. 1) greatly improved the breadth of protein identification and peptide coverage, with greatest coverage with cleavable-ICAT and SCX-C18 strategies (data not shown). Two-dimensional SCX-C18 chromatography (19.Wolters D.A. Washburn M.P. Yates 3rd, J.R. An automated multidimensional protein identification technology for shotgun proteomics.Anal. Chem. 2001; 73: 5683-5690Google Scholar) was performed using a 1 × 5-mm cation exchange column packed with polysulfoethyl A resin (SCX; PolyLC, Columbia, MD) and 0.32 × 150-mm column packed with Pepmap C18 resin (LC Packings, San Francisco, CA) loaded and developed at 30 μl/min. Peptides were detected by UV absorbance at 214 nm using a 250-nl internal volume flow cell with a 2-mm path length. Peptides were eluted from the C18 resin then coupled directly to the mass spectrometer, at a flow rate of ∼2 μl/min. One-dimensional C18 chromatography was performed using a 0.1 × 150-mm column packed with C18 resins (MagicC18, Michrom Bioresources, Auburn, CA or Vydac MS218, Nest Group, Southborough, MA) and developed using a 2–70% ACN, 0.1% formic acid gradient with a flow rate of ∼500 nl/min. The electrospray source was fitted with an uncoated tapered fused silica tip (10–15-μm inner diameter; New Objective, Cambridge, MA) to which a voltage of 3.0 kV was applied with nebulizing nitrogen gas. Information-dependent MS and MS/MS acquisitions were made on an orthogonal Q-TOF (qQ-TOF) instrument (Sciex, Toronto, Canada) using a 1-s survey scan (m/z 400–1,200) followed by three consecutive 3-s product ion scans of 2+, 3+, and 4+ parent ions with a 4-min exclusion period. The product ion mass range was typically limited to 60–1,000 Da scanned in two cycles or 60–1,600 Da scanned in three cycles, and where collision energy was dynamically ramped according to mass and charge state, were used to maximize the duty cycle of the instrument. In later experiments, ions were stored in the second quadrapole and released in synchrony with the pulsing of ions in TOF detector, resulting in an ∼ 5–8-fold increase in sensitivity. Data were acquired using Sciex Analyst QS software. Proteins were identified from survey and product ion spectral data, with an MS and MS/MS mass tolerance of 0.15 Da, using both Swiss-Prot and Genbank NR databases and ProID (Version 1.0 EP2; Applied Biosystems, Foster City, CA), Mascot (Matrix Science, London, United Kingdom), and SONAR (Proteometrics, New York, NY) search programs. Mascot and SONAR searches accessed merged dta format files. Sciex wiff files were converted to dta format (“Export IDA Spectra” script) and merged using the Merge function from Matrix Science. Protein sequences for porcine trypsin, mouse immunoglobulin constant regions, and variable regions from the anti-pY antibody PY20 were appended to the human Swiss-Prot database. One missed tryptic cleavage was allowed and post-translational modifications considered included only cysteine derivitization and tyrosine phosphorylation. ProID confidence scores of >90% were considered, after which spectra were manually inspected as no criteria were found for any of the search programs that would allow correct unattended protein assignments without an unacceptably high false-negative rate. Mascot scores of >20 and SONAR expectation values of <1 were considered. Only proteins assigned with two or more peptides were included in Table I. Peptide redundancy was prevented by manually sorting the peptide lists in Excel.Table IProteins and protein classes isolated by anti-pY affinity chromatography from HN5 HNSCC cellsAccession IDProtein class and nameUnique peptidesMean % confidenceMean % scoreSelect domainsCalcium regulationgi‖7656952Calcyclin binding protein49918SGS, p23sp‖Q14257Reticulocalbin 2 precursor (Calcium-binding protein ERC-55)39930EF-hand, ER_TARGET 1sp‖P31949S100 calcium-binding protein A11 (Calgizzarin)29928CaBP_S100, EF-handgi‖4506773S100 calcium-binding protein A9 (Calgranulin B)29926CaBP_S100, EF-handsp‖P16615Sarcoplasmic/endoplasmic reticulum calcium ATPase 229925Calcium_ATPase, HydrolaseCell cyclegi‖4758046Cell cycle progression 2 protein29914FAST_Lau_richsp‖P06493Cell division control protein 2 homolog (p34 protein)49929Ser_thr_pkin_AS, Ser_thr_pkinaseCell proliferation, survivalsp‖P4265514-3-3 protein ε (Mitochondrial import stimulation)3992614-3-3sp‖P3194714-3-3 protein ς (Stratifin) (Epithelial cell marker protein 1)4992214-3-3sp‖P2734814-3-3 protein τ4992614-3-3sp‖P2931214-3-3 protein ζ (PKC inhibitor protein-1)4992814-3-3sp‖P35222Catenin β (88kDa)29219Armadillosp‖P00533Epidermal growth factor receptor precursor859935Furin-like, Tyr_pkinasesp‖P09382Galectin-1 (Beta-galactoside-binding lectin L-14-I)29922Galectin, Gal-bind_lectin 1sp‖P29354Growth factor receptor-bound protein 2 (GRB2 adapter protein)49827Neu_cyt_fact_2, SH2, SH3sp‖P04792Heat shock 27 kDa protein (Stress-responsive protein 27)109939Crystallin_alpha, Hsp20sp‖P08069Insulin-like growth factor I receptor precursor29923FN_III, Furin-like 1, Tyr_pkinasesp‖Q14192LIM-protein 3 (SLIM 3) (LIM-domain protein DRAL)39820LIM domainsp‖Q16539Mitogen-activated protein kinase 1429929p38_MAPK, Ser_thr_pkinasesp‖P27361Mitogen-activated protein kinase 3 (and/or MAPK1; Erk 1,2)29923MAPK, Ser_thr_pkinasesp‖P29597Non-receptor tyrosine-protein kinase TYK229924FERM, Tyr_pkinase, SH2gi‖4885525NSP1; SH2 domain containing 3A; novel SH2-containing protein 149926SH2, GEF for Ras-like small GTPasesgi‖4502371NSP2, breast cancer antiestrogen resistance 3119930SH2, GEF for Ras-like small GTPasessp‖P19174PLCγ1; 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase γ39923C2, EF-hand, PH, PI_PLC, SH2, SH3sp‖O75340Programmed cell death protein 629938EF-handsp‖P07947Proto-oncogene tyrosine-protein kinase YES (p61-YES)79933Prot_kinase, SH2, SH3, Tyr_pkinasesp‖P04626Receptor protein-tyrosine kinase erbB-2 precursor79932Furin-like, Tyr_pkinase, YLP_motifsp‖P29353SHC transforming protein69929PID_domain, PTB_PID, SH2sp‖P40763Signal transducer and activator of transcription 3 (STAT3)89825P53-like, SH2, STATsp‖P12931Src proto-oncogene tyrosine-protein kinase69928SH2, SH3, Tyr_pkinase, Tyr_pkinase_ASCell adhesion, cytoskeletongi‖8922075ACK1, activated p21cdc42Hs kinase49723SH3, Tyr_pkinase, UBA_domainsp‖P02570Actin, cytoplasmic 1 (and/or α, γ)69937Actin, Actin_likesp‖O43707α-actinin 4 (F-actin cross linking; also α-actinin-1)49918Actbind_actnin, Calponin-like, Spectrin, EF-handsp‖P04083Annexin I (lipocortin I; chromobindin 9) (P35)29925Annexinsp‖P07355Annexin II (lipocortin II; chromobindin 8)29936Annexinsp‖P08758Annexin V (lipocortin V; endonexin II; calphobindin I)39924Annexinsp‖Q14161ARF GTPase-activating protein GIT129918ANK, GIT, hRIP_liketrm‖Q99018BPAG2, 180-kDa bullous pemphigoid antigen-229815Collagensp‖P35221Catenin α-1 (Cadherin-associated protein) (α E-catenin)109928Alpha_catenin, Vinculinsp‖O60716Catenin Δ-1 (p120 catenin; cadherin-associated src)99932Armadillosp‖Q03135Caveolin-129944Caveolinsp‖P56945CRK-associated substrate (p130Cas)169937SH3gi‖30410805CUB domain-containing protein 1 isoform 1 (SIMA 135/CDCP1)79721CUB domaingi‖17975768Ephrin receptor EphB3 precursor; EPH-like tyrosine kinase-239729Tyrosine kinase, TNFR domain, FN_IIIsp‖P29317Ephrin type-A receptor 2 precursor79927FN_III, SAM, Tyr_pkinasesp‖P54760Ephrin type-B receptor 4 precursor29929FN_III-like, Gal_bind_like, SAM, Tyr_pkinasesp‖P98172Ephrin-B1 precursor (EPH-related receptor tyrosine kinase ligand 2)29925Cupredoxin, Ephrinsp‖Q05397Focal adhesion kinase 1 (FADK 1) (pp125FAK, PTK2)199931Band_4, Focal_AT, Tyr_pkinasesp‖Q12931Heat shock protein 75 kDa (HSP75; TRAP-1)29928ATPbind_A
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