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J. Biol. Chem. 277 (2): 1031-1039

© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Tyrosine Phosphorylation Mapping of the Epidermal Growth Factor Receptor Signaling Pathway*

Hanno SteenDagger , Bernhard Kuster§, Minerva FernandezDagger , Akhilesh PandeyDagger ||, and Matthias MannDagger §**

From the Dagger  Protein Interaction Laboratory, Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M and § MDS Proteomics A/S, Staermosegaardsvej 6, 5230 Odense M, Denmark

Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.

* This work was supported in part by a grant from the Danish National Research Foundation to the Center for Experimental Bioinformatics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Cellzome GmbH, 69117 Heidelberg, Germany.

|| Supported by a Howard Temin award (Grant CA 75447) from the NCI, National Institutes of Health and by a travel award from the Plasmid Foundation, Denmark. To whom correspondence may be addressed. Tel.: 45-65502366; Fax: 45-65933018; E-mail: pandey@cebi.

** To whom correspondence may be addressed. Tel.: 45-65502364; Fax: 45-65933929; E-mail:

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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