Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor.
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGF alpha-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.
Guo
Lin
L
Amgen Corporation, Seattle, Washington 98101, USA.
Kozlosky
Carl J
CJ
Ericsson
Lowell H
LH
Daniel
Thomas O
TO
Cerretti
Douglas P
DP
Johnson
Richard S
RS
D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D008024:Ligands; D011994:Recombinant Proteins; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors