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Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

Oncogene volume 21, pages 8029–8036 (2002)Cite this article

Abstract

We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.

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Abbreviations

EGFR:

epidermal growth factor receptor

MS/MS:

tandem mass spectrometry

PTB:

phosphotyrosine binding domain

SH2:

src homology 2

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Acknowledgements

Work at the Center for Experimental Bioinformatics is supported by a generous grant from the Danish National Research Foundation to the Center for Experimental Bioinformatics (CEBI) at the University of Southern Denmark. A Pandey is supported by a Howard Temin Award from the National Cancer Institute (KO1 CA75447) and by a travel award from the Plasmid Foundation, Roskilde, Denmark. We thank Drs Stuart Decker and Andrius Kazlauskas for providing constructs for EGFR and PDGFR respectively. We would also like to thank Dr Stéphane Richard for the Grb2 expression vector.

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Author notes

  1. Akhilesh Pandey

    Present address: Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark

  2. Akhilesh Pandey and Blagoy Blagoev: These authors contributed equally to this work.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142, Massachusetts, USA

    Akhilesh Pandey & Harvey F Lodish

  2. Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, D-5230, Denmark

    Blagoy Blagoev, Irina Kratchmarova, Minerva Fernandez, Mogens Nielsen, Troels Zakarias Kristiansen, Alexandre V Podtelejnikov & Matthias Mann

  3. Kazusa DNA Research Institute and RIKEN Research Center for Allergy and Immunology, Yana, Kisarazu, 1532-3, 292-0812, Chiba, Japan

    Osamu Ohara

  4. Centre de Recherche de Biochimie Macromoléculaire, 1919 route de Mende, Montpellier, 34293, France

    Serge Roche

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  1. Akhilesh Pandey
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Correspondence to Matthias Mann.

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Pandey, A., Blagoev, B., Kratchmarova, I. et al. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases. Oncogene 21, 8029–8036 (2002). https://doi.org/10.1038/sj.onc.1205988

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