Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells.
Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the binding sites of Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF receptor mutants in which autophosphorylation sites, either alone or in combination, were replaced by phenylalanine. Binding of Shc to EGF receptor mutants lacking single tyrosine residues at 1148 or 1173 decreased by approximately 60 or approximately 15%, respectively, whereas other single point mutants bound the wild-type level of Shc. Binding of Shc markedly decreased in mutants lacking both tyrosine residues at 1148 and 1173. In peptide inhibition assay, phosphorylated nonameric peptide representing tyrosine 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited the binding of glutathione S-transferase-Shc SH2 domain fusion protein to in vitro autophosphorylated EGF receptors, suggesting that N-terminal sequences adjacent to phosphotyrosine are necessary for the association of Shc. Based on results of peptide inhibition assays in which phosphorylated peptides representing tyrosines 992, 1148, and 1173 inhibited Shc binding to the receptor, we constructed another EGF receptor mutant in which one of these tyrosine residues was retained. The amount of Shc bound to mutant receptors retaining tyrosines 1148, 1173, or 992 was approximately 80, approximately 40, or approximately 10% of wild-type level, respectively. These results indicate that tyrosine 1148 of activated human EGF receptors is a major binding site of Shc and tyrosine 1173 is a secondary binding site in intact cells.
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Y
Y
Okabayashi
Y
Y
Kido
T
T
Okutani
Y
Y
Sugimoto
K
K
Sakaguchi
M
M
Kasuga
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D066246:ErbB Receptors / Q000096:biosynthesis / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010649:Phenylalanine; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine
D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D066246:ErbB Receptors