Identification of signaling molecules involved in Epidermal Growth Factor Receptor (EGFR) signaling

Abstract

Lung cancer is the leading cause of mortality in India with cancers of the lung constituting 8.73% of relative proportion of all cancers in Bangalore (NCRP, 2008). The development of specific tyrosine kinase inhibitors (TKIs) such as erlotinib against the Epidermal growth factor receptor (EGFR) has been promising in targeted therapy of lung cancers. Specific driver mutations in EGFR kinase domain are responsible for determining sensitivity or resistance to TKIs. More than 50% of the mutations have a leucine to arginine substitution at position 858 (L858R) that confers sensitivity to erlotinib. EGFR activation at the membrane triggers a series of tyrosine and serine/threonine phosphorylation cascades. The goal of this study was to identify the sites of phosphorylation of proteins and quantify the degree of phosphorylation upon ligand stimulation or TKI inhibition of a lung adenocarcinoma cell line harboring a TKI-sensitizing mutant EGFR. H3255, a lung adenocarcinoma cell line harboring L858R EGFR was SILAC (Stable isotope labeling with amino acids in cell culture) labeled and phosphorylated serine/threonine/tyrosine peptides were enriched using a titanium dioxide based chemical method and also an antibody enrichment method (Phosphoscan). A 3-plex SILAC labeling method where control cells were labeled ?light?, cells treated with EGF labeled ?medium? and cells treated with EGF and erlotinib were labeled ?heavy? was used. From the Phosphoscan enrichment protocol we could identify a total of 460 tyrosine (pY), 64 serine (pS) and 49 threonine sites (pT) that represents 207 proteins while in tandem, in the TiO2 enrichment protocol we could identify of a total of 3586 phosphosites (3167 phosphoserine sites, 395 phosphothreonine sites and 24 phosphotyrosine sites) that represents 1434 proteins. The higher counts for the serine and threonine residues are representative of the biological stoichiometry where serine/threonine residues are more abundant than tyrosine phosphorylated residues.