Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells.
We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells.
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Sawyer
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Cohen
D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D011506:Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D002118:Calcium