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Identification and biochemical characterization of novel putative substrates for the epidermal growth factor receptor
F Fazioli1, D P Bottaro, L Minichiello
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
To gain insight into the mechanisms which control the mitogenic response to epidermal growth factor (EGF), we have partially purified and characterized several intracellular proteins which are phosphorylated on tyrosine residues following activation of the epidermal growth factor receptor (EGFR). Partial purification was achieved by immunoaffinity chromatography using immobilized anti-phosphotyrosine antibodies. Antisera generated against the partially purified proteins were used to identify at least five novel EGFR putative substrates, designated, on the basis of their apparent molecular weight, p97, p68, p61, p56, and p23. All of these proteins became specifically phosphorylated on tyrosine after EGF treatment of intact cells, as assessed by phosphoamino acid analysis, and none of them represented an EGFR degradation product. The phosphorylation of these proteins appeared to be relatively specific for the EGFR. In particular, an EGFR-related kinase, erbB-2 was much less efficient than EGFR at phosphorylating p97, p56, and p23 and incapable of phosphorylating p68. The identification of these novel EGFR putative substrates should lead to a better understanding of the mechanisms controlling the specificity of EGFR-mediated mitogenic signaling.
Insights
Researchers identified five novel intracellular proteins phosphorylated by epidermal growth factor (EGF) signaling. These findings enhance understanding of EGF receptor (EGFR) mediated cell growth and specificity.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Biochemistry
Background:
- Epidermal Growth Factor Receptor (EGFR) signaling is crucial for cell proliferation.
- Understanding the specific intracellular proteins involved in EGFR-mediated mitogenic responses is essential.
Purpose of the Study:
- To identify and characterize novel intracellular proteins phosphorylated by EGFR upon EGF stimulation.
- To investigate the specificity of EGFR in phosphorylating these identified substrates.
Main Methods:
- Immunoaffinity chromatography using anti-phosphotyrosine antibodies for partial purification of proteins.
- Generation of antisera against purified proteins for identification.
- Phosphoamino acid analysis to confirm tyrosine phosphorylation after EGF treatment.
Main Results:
- Identification of five novel putative EGFR substrates: p97, p68, p61, p56, and p23.
- All identified proteins were specifically phosphorylated on tyrosine residues following EGF stimulation.
- Phosphorylation of p97, p56, and p23 was less efficient with erbB-2 compared to EGFR; p68 was not phosphorylated by erbB-2.
Conclusions:
- The study identified novel substrates of EGFR, contributing to the understanding of EGFR-mediated mitogenic signaling.
- The findings highlight the specificity of EGFR in phosphorylating these substrates, differentiating it from related kinases like erbB-2.