Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation.
Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATp binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, we propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization.
Honegger
A M
AM
Rorer Biotechnology, Inc., King of Prussia, PA 19406.
Kris
R M
RM
Ullrich
A
A
Schlessinger
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases