Premium
Large‐scale phosphoproteomic comparison between epidermal growth factor and insulin‐like growth factor‐1 signaling
Author(s) -
Stevens Tracy Alison,
Brill Laurence M,
Williams Scott,
Peters Eric C,
Mustelin Tomas
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a527-c
Subject(s) - tyrosine phosphorylation , phosphorylation , epidermal growth factor , tyrosine , phosphoproteomics , signal transduction , protein phosphorylation , growth factor , chemistry , tyrosine kinase , immunoprecipitation , protein tyrosine phosphatase , microbiology and biotechnology , biochemistry , biology , receptor tyrosine kinase , sh2 domain , protein kinase a , receptor , gene
Reversible tyrosine phosphorylation of proteins is crucial for signal transduction pathways regulating many cell processes. A large‐scale tyrosine phosphoproteomics approach was used to compare the set of cellular proteins phosphorylated on specific tyrosine residues in HeLa cells treated with epidermal growth factor (EGF) and insulin‐like growth factor‐1 (IGF‐1). The technology platform combines immunoprecipitation using phosphotyrosine‐specific antibodies with methyl‐esterification and immobilized metal affinity chromatography (IMAC) to enrich for phosphotyrosine‐containing peptides, followed by nano‐flow liquid chromatography and electrospray ionization tandem mass spectrometry (nano‐LC/ESI‐MS/MS). Database searching identified 87 phosphopeptides with assigned tyrosine phosphorylated sites representing 40 proteins in response to EGF, with 56% uniquely phosphorylated in EGF‐treated cells. In response to IGF‐1, 119 tyrosine phosphopeptides were identified from 53 proteins, 67% of which were unique to IGF‐1 treatment. Many novel proteins and their associated tyrosine phosphorylation sites were identified. EGF induced phosphorylation of transcription factors and proteins involved in cell proliferation, while IGF‐1 induced phosphorylation of proteins involved in cell adhesion and survival. Immunoprecipitation of selected proteins confirmed the differential tyrosine phosphorylation between growth factor responses. The sensitivity and broad applicability of this strategy makes it useful for profiling the dynamics of tyrosine phosphorylation in signal transduction networks. Supported by NIH grant U54 RR020843.