Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.
We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.
Sturani
E
E
Dipartimento di Fisiologia e Biochimica Generali, Università, Milan, Italy.
Zippel
R
R
Toschi
L
L
Morello
L
L
Comoglio
P M
PM
Alberghina
L
L
D002294:Carcinoma, Squamous Cell; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism*; D005347:Fibroblasts; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D007700:Kinetics; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D066246:ErbB Receptors; D011493:Protein Kinase C; D014357:Trypsin