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• W2022210208 abstract "Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor β suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo. Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor β suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo. The phenotype of an individual patient’s cancer is a product of the somatic genetic mutations underlying the tumor. One or more of these genetic changes provides a survival advantage for the cancer cells in the context of the tissue microenvironment. Thus, at a functional level, protein-mediated signaling is directly and indirectly influenced by both the genetic underpinnings and the microecology of the tumor. The “oncogene addiction theory” postulates that genetic mutations cause an oncogenic protein to dominate the signaling control of the cancer cell clone, thereby driving it to survive, grow, invade, and metastasize (1Jain M. Arvanitis C. Chu K. Dewey W. Leonhardt E. Trinh M. Sundberg C.D. Bishop J.M. Felsher D.W. Sustained loss of a neoplastic phenotype by brief inactivation of MYC.Science. 2002; 297: 102-104Crossref PubMed Scopus (536) Google Scholar, 2Weinstein I.B. Disorders in cell circuitry during multistage carcinogenesis: the role of homeostasis.Carcinogenesis. 2000; 21: 857-864Crossref PubMed Scopus (231) Google Scholar). Mutations in the ERBB 1The abbreviations used are: ERBB, family of epidermal growth factor receptors; AKT, protein kinase B; COX2, cyclooxygenase2; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FKHR, Forkhead homolog1; HE, human endothelial; HER2, receptor tyrosine-protein kinase ERBB2; IGF-1R, insulin-like growth factor receptor; IRS-1, insulin receptor substrate; K-RAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; L858R, leucine to arginine mutation at amino acid 858 of EGFR; LCM, laser capture microdissection; Lys-C, lysozyme C serine protease; MTOR, mammalian target of rapamycin; NSCLC, non-small cell lung cancer; RPPA, reverse phase protein microarray; WT, wild type; VEGFR, vascular endothelial growth factor receptor; CREB, cAMP-response element-binding protein; ENOS, endothelial nitric-oxide synthase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FAK, focal adhesion kinase; T-PER, Tissue Protein Extraction Reagent. 1The abbreviations used are: ERBB, family of epidermal growth factor receptors; AKT, protein kinase B; COX2, cyclooxygenase2; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FKHR, Forkhead homolog1; HE, human endothelial; HER2, receptor tyrosine-protein kinase ERBB2; IGF-1R, insulin-like growth factor receptor; IRS-1, insulin receptor substrate; K-RAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; L858R, leucine to arginine mutation at amino acid 858 of EGFR; LCM, laser capture microdissection; Lys-C, lysozyme C serine protease; MTOR, mammalian target of rapamycin; NSCLC, non-small cell lung cancer; RPPA, reverse phase protein microarray; WT, wild type; VEGFR, vascular endothelial growth factor receptor; CREB, cAMP-response element-binding protein; ENOS, endothelial nitric-oxide synthase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FAK, focal adhesion kinase; T-PER, Tissue Protein Extraction Reagent. family of protein receptors are associated with a significant proportion of carcinomas of all types. This class of receptors is particularly poised to play a role in cancer because their normal function involves growth stimulation, prosurvival, motility and migration, and stem cell recruitment (3Boockvar J.A. Kapitonov D. Kapoor G. Schouten J. Counelis G.J. Bogler O. Snyder E.Y. McIntosh T.K. O'Rourke D.M. Constitutive EGFR signaling confers a motile phenotype to neural stem cells.Mol. Cell. Neurosci. 2003; 24: 1116-1130Crossref PubMed Scopus (104) Google Scholar, 4Ayuso-Sacido A. Graham C. Greenfield J.P. Boockvar J.A. The duality of epidermal growth factor receptor (EGFR) signaling and neural stem cell phenotype: cell enhancer or cell transformer?.Curr. Stem Cell Res. Ther. 2006; 1: 387-394Crossref PubMed Scopus (20) Google Scholar, 5Haugh J.M. Huang A.C. Wiley H.S. Wells A. Lauffenburger D.A. Internalized epidermal growth factor receptors participate in the activation of p21(ras) in fibroblasts.J. Biol. Chem. 1999; 274: 34350-34360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 6Jaramillo M.L. Banville M. Collins C. Paul-Roc B. Bourget L. O'Connor McCourt M. Differential sensitivity of A549 non small lung carcinoma cell responses to epidermal growth factor receptor pathway inhibitors.Cancer Biol. Ther. 2008; 7: 557-568Crossref PubMed Scopus (27) Google Scholar, 7Mattoon D.R. Lamothe B. Lax I. Schlessinger J. The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway.BMC Biol. 2004; 2: 24Crossref PubMed Scopus (141) Google Scholar). Current goals of molecular oncology research are to understand how an oncogenic mutation manifests itself at the level of the signal transduction network of the cell, why the altered network provides a survival benefit for the tumor, and how this knowledge can be exploited for individualization of therapy (for a review, see Riedel and Febbo (8Riedel R.F. Febbo P.G. Epidermal growth factor receptor mutations predict sensitivity to gefitinib in patients with non-small-cell lung cancer.Future Oncol. 2005; 1: 461-466Crossref PubMed Scopus (6) Google Scholar)).Epidermal growth factor receptor (EGFR) is a member of the ERBB family of receptor tyrosine kinases that regulates cellular growth, survival, and proliferation. EGFR has been extensively characterized regarding its kinase activity (9Cohen S. Purification of the receptor for epidermal growth factor from A-431 cells: its function as a tyrosyl kinase.Methods Enzymol. 1983; 99: 379-387Crossref PubMed Scopus (26) Google Scholar), amino acid sequence (10Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Downward J. Mayes E.L.V. Whittle N. Waterfield M.D. Seebura P.H. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells.Nature. 1984; 309: 418-425Crossref PubMed Scopus (1979) Google Scholar), receptor abundance (11Dittadi R. Gion M. Brazzale A. Bruscagnin G. Radioligand binding assay of epidermal growth factor receptor: causes of variability and standardization of the assay.Clin. Chem. 1990; 36: 849-854Crossref PubMed Scopus (9) Google Scholar), autophosphorylation properties (12Weber W. Bertics P.J. Gill G.N. Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation.J. Biol. Chem. 1984; 259: 14631-14636Abstract Full Text PDF PubMed Google Scholar), substrates (13Haigler H.T. Schlaepfer D.D. Burgess W.H. Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors.J. Biol. Chem. 1987; 262: 6921-6930Abstract Full Text PDF PubMed Google Scholar, 14Citri A. Yarden Y. EGF-ERBB signalling: towards the systems level.Nat. Rev. Mol. Cell Biol. 2006; 7: 505-516Crossref PubMed Scopus (1578) Google Scholar, 15Burgess A.W. Cho H.S. Eigenbrot C. Ferguson K.M. Garrett T.P. Leahy D.J. Lemmon M.A. Sliwkowski M.X. Ward C.W. Yokoyama S. An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors.Mol. Cell. 2003; 12: 541-552Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar), and mutation sites (16Sharma S.V. Bell D.W. Settleman J. Haber D.A. Epidermal growth factor receptor mutations in lung cancer.Nat. Rev. Cancer. 2007; 7: 169-181Crossref PubMed Scopus (2384) Google Scholar). Overexpression of EGFR in various malignancies (17Hwang D.L. Tay Y.C. Lin S.S. Lev-Ran A. Expression of epidermal growth factor receptors in human lung tumors.Cancer. 1986; 58: 2260-2263Crossref PubMed Scopus (35) Google Scholar) as well as the identification of specific EGFR mutations that enhance therapy response to small molecule inhibitors, notably in lung adenocarcinoma patients, and the observation that patients without detectable EGFR kinase domain mutations respond to tyrosine kinase inhibitor therapy qualifies EGFR as a promising molecular end point for individualized therapy (18Lynch T.J. Bell D.W. Sordella R. Gurubhagavatula S. Okimoto R.A. Brannigan B.W. Harris P.L. Haserlat S.M. Supko J.G. Haluska F.G. Louis D.N. Christiani D.C. Settleman J. Haber D.A. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N. Engl. J. Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9920) Google Scholar, 19Paez J.G. Janne P.A. Lee J.C. Tracy S. Greulich H. Gabriel S. Herman P. Kaye F.J. Lindeman N. Boggon T.J. Naoki K. Sasaki H. Fujii Y. Eck M.J. Sellers W.R. Johnson B.E. Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8403) Google Scholar, 20Sordella R. Bell D.W. Haber D.A. Settleman J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways.Science. 2004; 305: 1163-1167Crossref PubMed Scopus (1473) Google Scholar, 21Takano T. Ohe Y. Sakamoto H. Tsuta K. Matsuno Y. Tateishi U. Yamamoto S. Nokihara H. Yamamoto N. Sekine I. Kunitoh H. Shibata T. Sakiyama T. Yoshida T. Tamura T. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer.J. Clin. Oncol. 2005; 23: 6829-6837Crossref PubMed Scopus (675) Google Scholar). The dichotomy of objective treatment response to EGFR mutation status in a subset of patients suggests possible alternative mechanisms of receptor or downstream protein activation independent of the EGFR kinase domain, further emphasizing the goals of this study in assessing the phosphoprotein profile of mutant and wild type EGFR lung carcinoma cell populations.Very little is known about the state of lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the human lung tissue microenvironment. Within a growing human carcinoma, the tumor cell population is influenced by autocrine and paracrine signaling and cell-cell interactions within the heterogeneous tissue microenvironment. The concentration, source, or temporal fluctuations of cytokines and growth factors influencing the carcinoma cells is unknown. In any individual tumor, ligands that influence the ERBB family of receptors can be generated by the tumor cells themselves or by local host cells. Each individual patient’s carcinoma specimen contains a variable, heterogeneous proportion of stroma, lung parenchyma, bronchial epithelium, inflammatory cells, and endothelial cells. All of these non-carcinoma subpopulations may contain EGF receptors, participate in EGFR signaling, or contribute EGFR-related ligands. Furthermore it is unknown what proportion of the carcinoma population at any point in time is undergoing active signaling for a specific pathway. Consequently the state of the EGFR kinase signaling network within the lung carcinoma cells in a tumor specimen cannot be adequately studied using heterogeneous, ground-up tumor tissue or cultured cell lines (22Espina V. Wulfkuhle J.D. Calvert V.S. VanMeter A. Zhou W. Coukos G. Geho D.H. Petricoin III, E.F. Liotta L.A. Laser-capture microdissection.Nat. Protoc. 2006; 1: 586-603Crossref PubMed Scopus (484) Google Scholar, 23Espina V. Heiby M. Pierobon M. Liotta L.A. Laser capture microdissection technology.Expert Rev. Mol. Diagn. 2007; 7: 647-657Crossref PubMed Scopus (134) Google Scholar, 24Wulfkuhle J.D. Speer R. Pierobon M. Laird J. Espina V. Deng J. Mammano E. Yang S.X. Swain S.M. Nitti D. Esserman L.J. Belluco C. Liotta L.A. Petricoin III, E.F. Multiplexed cell signaling analysis of human breast cancer applications for personalized therapy.J. Proteome Res. 2008; 7: 1508-1517Crossref PubMed Scopus (119) Google Scholar). Laser capture microdissection (LCM) addresses the problem of sample cellular heterogeneity by providing a means to separate tumor cells from the large number of non-tumor cells within the complex microenvironment (22Espina V. Wulfkuhle J.D. Calvert V.S. VanMeter A. Zhou W. Coukos G. Geho D.H. Petricoin III, E.F. Liotta L.A. Laser-capture microdissection.Nat. Protoc. 2006; 1: 586-603Crossref PubMed Scopus (484) Google Scholar, 23Espina V. Heiby M. Pierobon M. Liotta L.A. Laser capture microdissection technology.Expert Rev. Mol. Diagn. 2007; 7: 647-657Crossref PubMed Scopus (134) Google Scholar, 24Wulfkuhle J.D. Speer R. Pierobon M. Laird J. Espina V. Deng J. Mammano E. Yang S.X. Swain S.M. Nitti D. Esserman L.J. Belluco C. Liotta L.A. Petricoin III, E.F. Multiplexed cell signaling analysis of human breast cancer applications for personalized therapy.J. Proteome Res. 2008; 7: 1508-1517Crossref PubMed Scopus (119) Google Scholar, 25Emmert-Buck M.R. Bonner R.F. Smith P.D. Chuaqui R.F. Zhuang Z. Goldstein S.R. Weiss R.A. Liotta L.A. Laser capture microdissection.Science. 1996; 274: 998-1001Crossref PubMed Scopus (2113) Google Scholar, 26Bonner R.F. Emmert-Buck M. Cole K. Pohida T. Chuaqui R. Goldstein S. Liotta L.A. Laser capture microdissection: molecular analysis of tissue.Science. 1997; 278: 1481-1483Crossref PubMed Scopus (781) Google Scholar). Microdissection also lends itself to studying tissue heterogeneity in respect to 1) spatial orientation of the tissue, i.e. invasive front, necrotic center, and distal portions, or 2) composite diseased/uninvolved cell populations. In this study, we microdissected serial sections of lung adenocarcinoma samples, at various depths of the tissue block, to provide a composite portrait, at the protein level, of the entire tumor cell population.In the present study we quantitatively profiled the phosphorylation (abundance) of signal pathway proteins relevant to the EGF receptor signal pathway (see Table II) within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) of known EGFR mutation status. Evaluating the combination of specific receptor protein phosphorylation sites in a tumor sample provides direct functional evidence that the receptor has changed its three-dimensional shape, dimerized, or undergone autophosphorylation on the cytoplasmic region of the receptor. The existence of phosphorylation on the EGFR is transient and may only occur if the receptor is engaged in signaling. Such phosphorylation provides sites of interaction for downstream signaling pathways that drive the growth, survival, differentiation, and motility of cells (3Boockvar J.A. Kapitonov D. Kapoor G. Schouten J. Counelis G.J. Bogler O. Snyder E.Y. McIntosh T.K. O'Rourke D.M. Constitutive EGFR signaling confers a motile phenotype to neural stem cells.Mol. Cell. Neurosci. 2003; 24: 1116-1130Crossref PubMed Scopus (104) Google Scholar, 6Jaramillo M.L. Banville M. Collins C. Paul-Roc B. Bourget L. O'Connor McCourt M. Differential sensitivity of A549 non small lung carcinoma cell responses to epidermal growth factor receptor pathway inhibitors.Cancer Biol. Ther. 2008; 7: 557-568Crossref PubMed Scopus (27) Google Scholar, 27Morgillo F. Woo J.K. Kim E.S. Hong W.K. Lee H.Y. Heterodimerization of insulin-like growth factor receptor/epidermal growth factor receptor and induction of survivin expression counteract the antitumor action of erlotinib.Cancer Res. 2006; 66: 10100-10111Crossref PubMed Scopus (293) Google Scholar, 28Ciardiello F. Troiani T. Bianco R. Orditura M. Morgillo F. Martinelli E. Morelli M.P. Cascone T. Tortora G. Interaction between the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF) pathways: a rational approach for multi-target anticancer therapy.Ann. Oncol. 2006; 17: vii109-vii114Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 29Kobayashi S. Shimamura T. Monti S. Steidl U. Hetherington C.J. Lowell A.M. Golub T. Meyerson M. Tenen D.G. Shapiro G.I. Halmos B. Transcriptional profiling identifies cyclin D1 as a critical downstream effector of mutant epidermal growth factor receptor signaling.Cancer Res. 2006; 66: 11389-11398Crossref PubMed Scopus (97) Google Scholar, 30Liu X. Carlisle D.L. Swick M.C. Gaither-Davis A. Grandis J.R. Siegfried J.M. Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib.Exp. Cell Res. 2007; 313: 1361-1372Crossref PubMed Scopus (47) Google Scholar, 31Pawson T. Gish G.D. Nash P. SH2 domains, interaction modules and cellular wiring.Trends Cell Biol. 2001; 11: 504-511Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 32Schlessinger J. Common and distinct elements in cellular signaling via EGF and FGF receptors.Science. 2004; 306: 1506-1507Crossref PubMed Scopus (362) Google Scholar). Thus, measurement of the phosphorylation sites provides functional information not obtainable by genomics or transcriptomics measurement of the receptor. For analysis of microdissected carcinoma cells we measured six phosphorylation sites on the EGF receptor and 20 selected downstream signal proteins to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation or combination of specific multiple phosphorylation sites on the EGFR.Table IIValidated primary antibodies used for reverse phase protein microarray analysisAntibody/subcellular locationaMany proteins are known to translocate within the cell depending on post-translational modification status. The subcellular location listed is one cellular compartment that the protein may occupy at some point in time in a cell.Sourceb1, Cell Signaling Technology; 2, BD Biosciences; 3, Upstate/Millipore, Billerica, MA; 4, Zymed Laboratories Inc., South San Francisco, CA; 5, Biosource/Invitrogen; 6, Lab Vision/ThermoFisher, Fremont, CA; 7, Promega Biosciences, San Luis Obispo, CA.LCM non-small cell lung cancerA549, H1975, and H1650 cell line time courseCell line screening time coursecCell line screening time course was a single replicate experiment for global evaluation of cell signaling differences among multiple protein end points. The goal was to determine which end points changed ±20% after EGF ligand stimulation and use this subset of proteins for further in-depth analysis.A549 and H1975 cell line time course (triplicate)NucleusATF-2 (Thr-71)1XBUB32XCHK1 (Ser-345)1XCHK2 (Ser-33/35)1XCleaved PARP (Asp-214)1XXCREB (Ser-133)1XXCyclin D12XCyclin E2XELK-1 (Ser-383)1XFOX01 (FKHR) (Ser-256)1XFOX01 (FKHR) (Thr-24)1XXXXFOX03 (FKHRL1) (Ser-253)1XHistone H3 (Ser-10) mitosis marker3XMSK1 (Ser-360)1Xβ-Catenin (Ser-33/37/Thr-41)1Xβ-Catenin (Thr-41/Ser-45)1XSTAT1 (Tyr-701)1XXXSTAT3 (Ser-727)1XXSTAT5 (Tyr-694)1XSTAT6 (Tyr-641)1XMitochondriaBAK1XXBCL-2 (Ser-70)1XXBCL-2 (Thr-56)1XBAD (Ser-112)1XXBAD (Ser-136)1XBAD (Ser-155)1XXMembraneAdducin (Ser-662)3XXβ-Actindβ-Actin was included as a control protein and was analyzed in microdissected cells and cell lines. There was not a significant difference for β-actin in the mutant cells compared with wild type.1XXXc-KIT (Tyr-703)4XXc-KIT (Tyr-719)1Xc-KIT (Tyr-721)4XEGFR1XXXEGFR L858R1XXXEGFR (Tyr-1045)1XXXXEGFR (Tyr-1068)1XXXXEGFR (Tyr-1148)5XXXXEGFR (Tyr-1173)5XXXXEGFR (Tyr-845)1XEGFR (Tyr-992)1XXXXEstrogen receptor α (Ser-118)1XHER2 (Tyr-1248)1XXXXHER3 (Tyr-1289)1XIGF-1 rec. (Tyr-1131)/insulin rec. (Tyr-1146)1XIRS-11XIRS-1 (Ser-612)1XXXXMET (Tyr-1234/1235)1XPDGF receptor β (Tyr-716)3XXPDGF receptor β (Tyr-751)1XPLC-γ-11XPLC-γ-1 (Tyr-783)1XVEGFR 2 (Tyr-1175)1XVEGFR 2 (Tyr-951)1XVEGFR 2 (Tyr-996)1XCytoplasm14-3-3 ζ/γ/η3XX4EBP1 (Ser-65)1XX4EBP1 (Thr-37/46)1XXAKT (Ser-473)1XXXXAKT (Thr-308)1XXXAMPKα1 (Ser-485)1XAMPKβ1 (Ser-108)1XAPC26XXA-RAF (Ser-299)1XASK1 (Ser-83)1XB-RAF (Ser-445)1XXc-ABL (Thr-735)1XXCleaved caspase 3 (Asp-175)1XCleaved caspase 6 (Asp-162)1XCleaved caspase 7 (Asp-198)1XCleaved caspase 9 (Asp-330)1XCOX23XXXC-RAF (Ser-338)1XEIF4E (Ser-209)1XEIF4G (Ser-1108)1XXXENOS (Ser-1177)1XXXENOS/NOS III (Ser-116)3XXERK1/2 (Thr-202/Tyr-204)1XXXXETK (Tyr-40)1XFAK (Tyr-576/577)1XXGAB1 (Tyr-627)1XXGSK3αβ (Ser-21/9)1XXXGSK3α (Ser-21)1XIκBα (Ser-32)1XJAK1 (Tyr-1022/1023)1XLCK (Tyr-505)5XXLKB1 (Ser-334)1XLKB1 (Ser-428)1XXMAPK (pTEpY)7XXMARCKS (Ser-152/156)1XXMEK1 (Ser-298)1XMEK1/2 (Ser-217/221)1XXMTOR (Ser-2448)1XMTOR (Ser-2481)1XXXNF-κβ p65 (Ser-536)1Xp38 MAP kinase (Thr-180/Tyr-182)1Xp70S6 kinase (Ser-371)1XXp70S6 kinase (Thr-389)1XXp70S6 kinase (Thr-412)3XXp90RSK (Ser-380)1XXXPAK1 (Ser-199/204)/PAK2 (Ser-192/197)1XPDK1 (Ser-241)1XPI 3-kinase1XPKA C (Thr-197)1XPKC (pan) (βII Ser-660)1XXPKC α (Ser-657)3XXPKC α/β (Thr-638)1XXPKC ζ/λ (Thr-410/403)1XPKR (Thr-446)1XpRAS 40 (Thr-246)5XPTEN (Ser-380)1XXRAS-GRF1 (Ser-916)1XRSK3 (Thr-356/Ser-360)1XXS6 ribosomal protein (Ser-235/236)1XS6 ribosomal protein (Ser-240/244)1XSAPK/JNK (Thr-183/Tyr-185)1XXSEK1/MKK4 (Ser-80)1XSHC (Tyr-317)3XXXXSHIP1 (Tyr-1020)1XSMAD2 (Ser-465/467)1XXXSRC (Tyr-527)1XXXSRC family (Tyr-416)1XXXTuberin/TSC2 (Tyr-1571)1XTYK2 (Tyr-1054/1055)1XXa Many proteins are known to translocate within the cell depending on post-translational modification status. The subcellular location listed is one cellular compartment that the protein may occupy at some point in time in a cell.b 1, Cell Signaling Technology; 2, BD Biosciences; 3, Upstate/Millipore, Billerica, MA; 4, Zymed Laboratories Inc., South San Francisco, CA; 5, Biosource/Invitrogen; 6, Lab Vision/ThermoFisher, Fremont, CA; 7, Promega Biosciences, San Luis Obispo, CA.c Cell line screening time course was a single replicate experiment for global evaluation of cell signaling differences among multiple protein end points. The goal was to determine which end points changed ±20% after EGF ligand stimulation and use this subset of proteins for further in-depth analysis.d β-Actin was included as a control protein and was analyzed in microdissected cells and cell lines. There was not a significant difference for β-actin in the mutant cells compared with wild type. Open table in a new tab EGFR tyrosine kinase domain gene sequencing was performed for each lung adenocarcinoma case. Thus the EGFR tyrosine kinase domain mutation status of each case in this study set was known and could therefore be compared with the phosphoprotein/signaling protein profile of each case. This is the first comparison of the phosphoprotein profile with the kinase domain mutation status in vivo. 10–20% of non-small cell lung cancer patients in the United States have sensitizing mutations in the EGFR. Specific somatic mutations in the ATP binding pocket of epidermal growth factor receptor have been identified in lung cancer patients who responded favorably to treatment with tyrosine kinase inhibitors directed against EGFR (18Lynch T.J. Bell D.W. Sordella R. Gurubhagavatula S. Okimoto R.A. Brannigan B.W. Harris P.L. Haserlat S.M. Supko J.G. Haluska F.G. Louis D.N. Christiani D.C. Settleman J. Haber D.A. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N. Engl. J. Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9920) Google Scholar, 19Paez J.G. Janne P.A. Lee J.C. Tracy S. Greulich H. Gabriel S. Herman P. Kaye F.J. Lindeman N. Boggon T.J. Naoki K. Sasaki H. Fujii Y. Eck M.J. Sellers W.R. Johnson B.E. Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8403) Google Scholar). Deletion mutation DelE746A750 in exon 19 and point mutation L858R in exon 21 increase enzyme activity and increase the tyrosine kinase Vmax and Km for ATP (33Gilmer T.M. Cable L. Alligood K. Rusnak D. Spehar G. Gallagher K.T. Woldu E. Carter H.L. Truesdale A.T. Shewchuk L. Wood E.R. Impact of common epidermal growth factor receptor and HER2 variants on receptor activity and inhibition by lapatinib.Cancer Res. 2008; 68: 571-579Crossref PubMed Scopus (62) Google Scholar). 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