ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Figure 1Loading Img
ADDITION / CORRECTIONThis article has been corrected. View the notice.

Phosphoproteomic Profiling of NSCLC Cells Reveals that Ephrin B3 Regulates Pro-survival Signaling through Akt1-Mediated Phosphorylation of the EphA2 Receptor

View Author Information
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
Cite this: J. Proteome Res. 2011, 10, 5, 2566–2578
Publication Date (Web):March 17, 2011
Copyright © 2011 American Chemical Society

    Article Views





    Read OnlinePDF (5 MB)
    Supporting Info (4)»


    Abstract Image

    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.

    Supporting Information

    Jump To

    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

    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system:

    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
    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.
    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.
    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.
    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.
    6. Dimitar B. Nikolov, Juha P. Himanen. From genetic data and structures to drug development: New approaches to target Eph receptors. 2021, 227-249.
    7. Chung-Ting Jimmy Kou, Raj P. Kandpal. Differential Expression Patterns of Eph Receptors and Ephrin Ligands in Human Cancers. BioMed Research International 2018, 2018 , 1-23.
    8. Catherine Cheng, Velia M. Fowler, Xiaohua Gong. EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens. Experimental Eye Research 2017, 162 , 9-17.
    9. Alireza Azimi, Rainer Tuominen, Fernanda Costa Svedman, Stefano Caramuta, Maria Pernemalm, Marianne Frostvik Stolt, Lena Kanter, Pedram Kharaziha, Janne Lehtiö, Carolina Hertzman Johansson, Veronica Höiom, Johan Hansson, Suzanne Egyhazi Brage. Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells. Cell Death & Disease 2017, 8 (8) , e3029-e3029.
    10. Maria Ruzzene, Jessika Bertacchini, Alex Toker, Sandra Marmiroli. Cross-talk between the CK2 and AKT signaling pathways in cancer. Advances in Biological Regulation 2017, 64 , 1-8.
    11. Yue Zhou, Hiroaki Sakurai. Emerging and Diverse Functions of the EphA2 Noncanonical Pathway in Cancer Progression. Biological & Pharmaceutical Bulletin 2017, 40 (10) , 1616-1624.
    12. Hamidreza Pazoki-Toroudi, Hamed Amani, Marjan Ajami, Seyed Fazel Nabavi, Nady Braidy, Pandima Devi Kasi, Seyed Mohammad Nabavi. Targeting mTOR signaling by polyphenols: A new therapeutic target for ageing. Ageing Research Reviews 2016, 31 , 55-66.
    13. Ghazal Efazat, Metka Novak, Vitaliy O. Kaminskyy, Luigi De Petris, Lena Kanter, Therese Juntti, Per Bergman, Boris Zhivotovsky, Rolf Lewensohn, Petra Hååg, Kristina Viktorsson. Ephrin B3 interacts with multiple EphA receptors and drives migration and invasion in non-small cell lung cancer. Oncotarget 2016, 7 (37) , 60332-60347.
    14. Cristina Girardi, Maria Ruzzene. CK2 Function in the Regulation of Akt Pathway. 2015, 125-140.
    15. Mohamed Amessou, Mustapha Kandouz. Role of the Family of Ephs and Ephrins in Cell-Cell Communication in Cancer. 2015, 255-286.
    16. K Viktorsson, R Lewensohn, B Zhivotovsky. Systems biology approaches to develop innovative strategies for lung cancer therapy. Cell Death & Disease 2014, 5 (5) , e1260-e1260.
    17. Ed Dudley, A. Elizabeth Bond. Phosphoproteomic Techniques and Applications. 2014, 25-69.
    18. Satu Mäki‐Nevala, Virinder Kaur Sarhadi, Katja Tuononen, Sonja Lagström, Pekka Ellonen, Mikko Rönty, Aino Wirtanen, Aija Knuuttila, Sakari Knuutila. Mutated Ephrin Receptor Genes in Non‐Small Cell Lung Carcinoma and Their Occurrence with Driver Mutations—Targeted Resequencing Study on Formalin‐Fixed, Paraffin‐Embedded Tumor Material of 81 Patients. Genes, Chromosomes and Cancer 2013, 52 (12) , 1141-1149.
    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.
    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.
    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.
    22. S Ståhl, V O Kaminskyy, G Efazat, A Hyrslova Vaculova, S Rodriguez-Nieto, A Moshfegh, R Lewensohn, K Viktorsson, B Zhivotovsky. Inhibition of Ephrin B3-mediated survival signaling contributes to increased cell death response of non-small cell lung carcinoma cells after combined treatment with ionizing radiation and PKC 412. Cell Death & Disease 2013, 4 (1) , e454-e454.
    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.
    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.
    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.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect