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Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Proteome Res. 2011, 10, 5, 2566-2578
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Phosphoproteomic Profiling of NSCLC Cells Reveals that Ephrin B3 Regulates Pro-survival Signaling through Akt1-Mediated Phosphorylation of the EphA2 Receptor

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† Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, SE-171 76 Stockholm, Sweden
§ Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden
‡ Department of Biological Chemistry, Venetian Institute of Molecular Medicine (VIMM), University of Padova, Padova, Italy
∥ Science for Life Laboratory, Stockholm, Box 1031, 17121 Solna, Sweden
*Janne Lehtiö, Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, SE-171 76 Stockholm, Sweden. Phone: +46 8 5177 639. Fax: +46 8 5177 1000. E-mail: Janne.Lehtiö@ki.se
Cite this: J. Proteome Res. 2011, 10, 5, 2566–2578
Publication Date (Web):March 17, 2011
https://doi.org/10.1021/pr200037u
Copyright © 2011 American Chemical Society
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    Abstract

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    The ephrin and Eph signaling circuit has been reported as deregulated in a number of tumor types including nonsmall cell lung cancer (NSCLC). Here we show that suppression of the ephrin-familly member ephrin B3 decreases NSCLC cell proliferation and has profound effects on cell morphology. To reveal which signaling networks ephrin B3 utilize to regulate such effects on growth and morphology, differential regulation of phosphorylated proteins was analyzed in the NSCLC cell line U-1810. Using strong cat ion exchange (SCX) and TiO2-based fractionation followed by nano-LC and mass spectrometry analysis, we identified 1083 unique phosphorylated proteins. Out of these, 150 proteins were found only when ephrin B3 is expressed, whereas 66 proteins were found exclusively in U-1810 cells with silenced ephrin B3. Network analysis of changes in the phosphoproteome with regard to the presence or absence of ephrin B3 expression generated a hypothesis that the site specific phosphorylation on Ser-897 detected on the erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2) is critical for the survival of NSCLC cells. Upstream of the EphA2 phosphorylation, activation of Akt1 on Ser 129 was also revealed as part of the ephrin B3-mediated signaling pathway. Phosphorylation of these sites was further confirmed by immune-based strategies in combination with mass spectrometry. Moreover, by further stepwise pathway walking, annotating the phosphorylated sites and their corresponding kinases upstream, our data support the process in which a Heat shock protein 90 isoform (HSP90AA1) acts as a protector of EphA2, thereby saving it from degradation. In addition, protein kinase CK2 (CK2) is suggested as a dominant kinase, activating downstream substrates to generate the effects on NSCLC proliferation and morphology.

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    Supplementary Figure 1. Top scoring network (score 58) generated from IPA analysis. Highlighting ephrin B3-mediated activation of the EphA2 receptor and PTK2/FAK1 as potential important factors for survival of NSCLC cells. Supplementary Figure 2. Phospho-MS/MS spectra. All possible annotations of spectra from phosphorylated peptides corresponding to AKT1 double and single phosphorylation, FAK1 and HSP90AA1. This material is available free of charge via the Internet at http://pubs.acs.org.

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    Cited By

    This article is cited by 25 publications.

    1. Elena B. Pasquale. Eph receptors and ephrins in cancer progression. Nature Reviews Cancer 2023, 6 https://doi.org/10.1038/s41568-023-00634-x
    2. Shubhashish Chakraborty, Reshita Baruah, Neha Mishra, Ashok K Varma. In-silico and structure-based assessment to evaluate pathogenicity of missense mutations associated with non-small cell lung cancer identified in the Eph-ephrin class of proteins. Genomics & Informatics 2023, 21 (3) , e30. https://doi.org/10.5808/gi.22069
    3. Andreas Lau, Nghia Le, Claudia Nguyen, Raj P. Kandpal. Signals transduced by Eph receptors and ephrin ligands converge on MAP kinase and AKT pathways in human cancers. Cellular Signalling 2023, 104 , 110579. https://doi.org/10.1016/j.cellsig.2022.110579
    4. Iason Psilopatis, Ioannis Karniadakis, Konstantinos Stylianos Danos, Kleio Vrettou, Kleita Michaelidou, Konstantinos Mavridis, Sofia Agelaki, Stamatios Theocharis. May EPH/Ephrin Targeting Revolutionize Lung Cancer Treatment?. International Journal of Molecular Sciences 2023, 24 (1) , 93. https://doi.org/10.3390/ijms24010093
    5. Vitaliy O. Kaminskyy, Petra Hååg, Metka Novak, Ákos Végvári, Vasiliki Arapi, Rolf Lewensohn, Kristina Viktorsson. EPHA2 Interacts with DNA-PKcs in Cell Nucleus and Controls Ionizing Radiation Responses in Non-Small Cell Lung Cancer Cells. Cancers 2021, 13 (5) , 1010. https://doi.org/10.3390/cancers13051010
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    14. Cristina Girardi, Maria Ruzzene. CK2 Function in the Regulation of Akt Pathway. 2015, 125-140. https://doi.org/10.1007/978-3-319-14544-0_8
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    17. Ed Dudley, A. Elizabeth Bond. Phosphoproteomic Techniques and Applications. 2014, 25-69. https://doi.org/10.1016/B978-0-12-800453-1.00002-6
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    19. Giulia Falivelli, Erika Mathes Lisabeth, Elena Rubio de la Torre, Gizeh Perez-Tenorio, Giovanna Tosato, Ombretta Salvucci, Elena B. Pasquale, . Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands. PLoS ONE 2013, 8 (11) , e81445. https://doi.org/10.1371/journal.pone.0081445
    20. Zhi Tang, Erika Bereczki, Haiyan Zhang, Shan Wang, Chunxia Li, Xinying Ji, Rui M. Branca, Janne Lehtiö, Zhizhong Guan, Peter Filipcik, Shaohua Xu, Bengt Winblad, Jin-Jing Pei. Mammalian Target of Rapamycin (mTor) Mediates Tau Protein Dyshomeostasis. Journal of Biological Chemistry 2013, 288 (22) , 15556-15570. https://doi.org/10.1074/jbc.M112.435123
    21. Jing-Jing Li, Dong Xie. The roles and therapeutic potentials of Ephs and ephrins in lung cancer. Experimental Cell Research 2013, 319 (2) , 152-159. https://doi.org/10.1016/j.yexcr.2012.08.005
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    23. Pierre Saintigny, Shaohua Peng, Li Zhang, Banibrata Sen, Ignacio I. Wistuba, Scott M. Lippman, Luc Girard, John D. Minna, John V. Heymach, Faye M. Johnson. Global Evaluation of Eph Receptors and Ephrins in Lung Adenocarcinomas Identifies EphA4 as an Inhibitor of Cell Migration and Invasion. Molecular Cancer Therapeutics 2012, 11 (9) , 2021-2032. https://doi.org/10.1158/1535-7163.MCT-12-0030
    24. Mustapha Kandouz. The Eph/Ephrin family in cancer metastasis: communication at the service of invasion. Cancer and Metastasis Reviews 2012, 31 (1-2) , 353-373. https://doi.org/10.1007/s10555-012-9352-1
    25. Dana M. Brantley-Sieders. Clinical relevance of Ephs and ephrins in cancer: Lessons from breast, colorectal, and lung cancer profiling. Seminars in Cell & Developmental Biology 2012, 23 (1) , 102-108. https://doi.org/10.1016/j.semcdb.2011.10.014
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