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

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

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

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

Correspondence to: A Pandey and M Mann, E-mail: Pandey@cebi.sdu.dk and M Mann; E-mail: mann@bmb.sdu.dk

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

^bThese authors contributed equally to this work.

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.

Oncogene (2002) 21, 8029-8036. doi:10.1038/sj.onc.1205988

proteomics; mass spectrometry; bioinformatics

EGFR, epidermal growth factor receptor; MS/MS, tandem mass spectrometry; PTB, phosphotyrosine binding domain; SH2, src homology 2