Autophosphorylation of gp185erbB-2 in vivo is confined to its carboxy terminus and is required for optimal erbB-2 transforming activity under conditions of receptor overexpression. It remains unresolved, however, to what extent autophosphorylation regulates erbB-2 mitogenic signaling in normal cells, nor is the biochemical basis for such a regulatory function known. To address these issues, we utilized a chimeric molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and intracellular domains of the erbB-2 product. In this EGFR/erbB-2 chimera, erbB-2 kinase activity is regulated by EGF binding. An EGFR/erbB-2 mutant bearing multiple Tyr----Phe substitutions at erbB-2 autophosphorylation sites (EGFR/erbB-2 5P) displayed markedly reduced phosphotyrosine content following EGF stimulation in comparison with the non-mutated chimera. When expressed in NR6 cells, the EGFR/erbB-2 5P mutant was unable to deliver a sizeable mitogenic signal when activated by EGF at physiological levels. In intact cells, the 5P mutant was still able to stimulate phosphorylation of the gamma isozyme of phospholipase C (PLC-gamma), a prototype erbB-2 substrate, although with a delayed time course, indicating that the 5P mutation decreased the affinity of the erbB-2 kinase for this substrate. This conclusion was further supported by the inability of the 5P mutant to associate with PLC-gamma in co-immunoprecipitation experiments. We infer that a major role of autophosphorylation is to increase the affinity of the erbB-2 kinase for its cellular substrates, so that, under physiological conditions, autophosphorylation is absolutely required for erbB-2 mitogenic signaling.
|Titolo:||erbB-2 autophosphorylation is required for mitogenic action and high-affinity substrate coupling|
DI FIORE, PIER PAOLO (Ultimo)
|Parole Chiave:||Proto-Oncogene Proteins; Phosphorylation; Receptor, erbB-2; Receptor, Epidermal Growth Factor; Growth Substances; Molecular Sequence Data; Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Amino Acid Sequence; Mutation; Signal Transduction; Cell Line|
|Settore Scientifico Disciplinare:||Settore MED/04 - Patologia Generale|
|Data di pubblicazione:||lug-1992|
|Appare nelle tipologie:||01 - Articolo su periodico|