Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway

Hanno Steen, Bernhard Kuster, Minerva Fernandez, Akhilesh Pandey, Matthias Mann

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1031-1039
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number2
DOIs
StatePublished - Jan 11 2002
Externally publishedYes

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Phosphorylation
Epidermal Growth Factor Receptor
Tyrosine
Phosphotyrosine
Threonine
Proteins
Proteomics
Serine
Signal transduction
Electrophoresis
Eukaryota
Immunoprecipitation
Epidermal Growth Factor
Mass spectrometry
Anti-Idiotypic Antibodies
Signal Transduction
Mass Spectrometry
Gels
Ions
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. / Steen, Hanno; Kuster, Bernhard; Fernandez, Minerva; Pandey, Akhilesh; Mann, Matthias.

In: Journal of Biological Chemistry, Vol. 277, No. 2, 11.01.2002, p. 1031-1039.

Research output: Contribution to journalArticle

Steen, Hanno ; Kuster, Bernhard ; Fernandez, Minerva ; Pandey, Akhilesh ; Mann, Matthias. / Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 2. pp. 1031-1039.
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