Phosphorylation at threonine-654 is not required for negative regulation of the epidermal growth factor receptor by non-phorbol tumor promoters.
Phosphorylation of the epidermal growth factor (EGF) receptor following activation of protein kinase C appears to negatively regulate EGF binding and the receptor-associated tyrosine kinase activity. We have identified two agents, the calcium ionophore A23187 and the non-phorbol tumor promoter thapsigargin, that similarly inhibit the EGF receptor binding and kinase activities through protein kinase C-independent pathways. Both agents activate protein kinases that phosphorylate the EGF receptor in A431 cells. To test the hypothesis that negative regulation of the EGF receptor always occurs through phosphorylation of threonine-654, a site uniquely phosphorylated by protein kinase C, we analyzed the tryptic phosphopeptides of EGF receptors isolated from cells treated with these agents. While limited phosphorylation of threonine-654 results from the A23187 treatment, no significant phosphorylation of this residue is detected after thapsigargin treatment. These results suggest that EGF receptor phosphorylation is a general mechanism for altering receptor properties and that site(s) of phosphorylation other than threonine-654 may negatively regulate the kinase activity as well as the binding of the EGF receptor.
Friedman
B A
BA
Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge 02139.
van Amsterdam
J
J
Fujiki
H
H
Rosner
M R
MR
D000001:Calcimycin / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010766:Phosphorylation; D010936:Plant Extracts / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D019284:Thapsigargin; D013912:Threonine; D014357:Trypsin
D002273:Carcinogens; D010446:Peptide Fragments; D010936:Plant Extracts; D013912:Threonine; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D004815:Epidermal Growth Factor; D019284:Thapsigargin; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D014357:Trypsin