Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells

摘要: Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF mutants in which autophosphorylation sites, either alone or combination, were replaced by phenylalanine. Binding Shc to lacking single residues at 1148 1173 decreased approximately 60 15%, respectively, whereas other point bound wild-type level Shc. markedly both and 1173. In peptide inhibition assay, phosphorylated nonameric representing 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited glutathione S-transferase-Shc SH2 domain fusion protein vitro autophosphorylated suggesting that N-terminal sequences adjacent phosphotyrosine are necessary association Based on results assays peptides tyrosines 992, receptor, we constructed another mutant one these was retained. The amount receptors retaining 1173, 992 80, 40, 10% level, respectively. These indicate activated human is a major site secondary intact cells.