Preferential detection of catalytically inactive c-erbB-2 by antibodies to unphosphorylated peptides mimicking receptor tyrosine autophosphorylation sites.
The c-erbB-2 tyrosine kinase is often overexpressed in human breast cancer, but correlations of receptor expression with tumour behaviour have proven elusive in patients without metastases at diagnosis. To address the possibility that receptor function may be more informative than expression, we previously developed function-specific c-erbB-2 antibodies using synthetic tyrosine-phosphorylated peptide immunogens (Epstein et al., Proc. Natl. Acad. Sci. USA 1992; 89: 10435-10439). Here the converse approach has been taken to determine the functional status of c-erbB-2 receptors detected by antibodies to dephosphorylated (dep) autophosphorylation sequences. In contrast to antiphosphopeptide (apt) antibodies, dep antibodies to the Tyr1248 autophosphorylation site exhibited preferential, but not exclusive, binding to tyrosine-dephosphorylated c-erbB-2. Consistent with this, catalytically active and inactive receptors could not be clearly distinguished by in vitro autophosphorylation experiments in which c-erbB-2 was immunoprecipitated using a monoclonal Tyr1248 dep antibody. A dep antiserum recognizing autophosphorylation sites N-terminal to Tyr1248 exclusively recognized tyrosine-dephosphorylated c-erbB-2 following antibody preabsorption with homologous phosphopeptides. Although indirect, these data are consistent with a model of sequential c-erbB-2 autophosphorylation in which Tyr1248 is the final residue modified. Moreover, since many studies of c-erbB-2 expression have used antibodies to dephosphorylated autophosphorylation sites, these results caution against automatically equating such receptor immunoreactivity with in vivo function or clinical significance.
Epstein
R J
RJ
Division of Cell, Molecular and Oncology Research, Charing Cross & Westminster Medical School, University of London, UK.
D000042:Absorption; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis; D000918:Antibody Specificity; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D007106:Immune Sera / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013997:Time Factors
D000906:Antibodies; D016895:Culture Media, Serum-Free; D007106:Immune Sera; D010455:Peptides; D020794:Receptor Protein-Tyrosine Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; D013762:Tetrahydrofolate Dehydrogenase