Abstract
The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.
Highlights
From the Department of Medicine, Division of Endocrinology and Metabolism, and The Center for Molecular Genetics, University of California, San Diego, La Jollu, California, 92093
A role for theEGF receptor in ulated by epidermal growth factor (EGF)-stimulated autophosphorylation and by tumorigenesis has been suggested by elevated expression phorbol ester-stimulated, protein kinaseC (Ca2+/phos- of the receptor in human tumors (6b),y correlation of receptor pholipid-dependent enzyme)mediated phosphorylation overexpression with tumor growth in athymic mice (7), and at identified sites
We have identified three of these sites in EGF receptor purified from 92P-labeled A431cells
Summary
Tryptic peptides were separated by reverse-phase HPLC on a C18 Determination of Amino Acid Sequence of Phosphopeptides column (Vydac) equilibrated in 10 mM potassium phosphate, pH 6.0, and 3% acetonitrile. Same tryptic peptide phosphorylated at serine and threonine or threoninealone, respectively These phosphopeptides were separated inthe initial HPLC purification in phosphate buffer at pH 6.0 (Fig. l), but they eluted a t a similar acetonitrile concentration inthe second HPLC stepin trifluoroacetic acid at pH 2.3. This tryptic peptide is located between the inner boundary of the transmembranedomain and theATP-binding site of the EGF receptor (35). EGF treatment did not significantly increase the level of P2 phosphorylation in this mutant EGrFeceptor
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