title: Adjacent carboxyterminal tyrosine phosphorylation events identify functionally distinct ErbB2 receptor subsets: implications for molecular diagnostics.
abstract: Site-directed mutagenesis can define the effects of altering one or more amino acids within a protein, but this technique may lack sensitivity when used to characterize proteins which differ conformationally or posttranslationally at multiple sites. A novel alternative approach involves the direct characterization of wild-type protein isoforms identified by site-specific immunodetection. To this end we have developed antibodies which recognize ErbB2 subsets characterized by adjacent tyrosine phosphorylation events (Y1222 and Y1248) in the C-terminal tail of the oncoprotein. Here we use these phosphoantibodies to demonstrate the existence of tyrosine-phosphorylated ErbB2 subsets which differ in their patterns of heterooligomer formation, in vitro autophosphorylation, and recruitment of SH2-containing substrates. Furthermore, Y1222 and/or Y1248 phosphoantibody immunoreactivity is readily detectable in ErbB2-overexpressing human breast tumors, in which context these phosphorylation events exhibit significant discordance. These data confirm the value of site-specific immunodetection as a strategy for characterizing phosphoprotein function in vitro and in vivo and suggest that multisite phosphotyping of human tumors may contribute novel clinicopathologic insights into the significance of the ErbB2 overexpression phenotype.
authors.affiliation: Department of Metabolic Medicine, Imperial College School of Medicine, London, W12 0NN, United Kingdom.
authors.forename: X
authors.initials: X
authors.lastname: Ouyang
authors.forename: G C
authors.initials: GC
authors.lastname: Huang
authors.forename: A
authors.initials: A
authors.lastname: Chantry
authors.forename: R J
authors.initials: RJ
authors.lastname: Epstein
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism*; D005260:Female; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases