Title

Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation

UMMS Affiliation

Program in Molecular Medicine

Publication Date

2-25-1990

Document Type

Article

Subjects

Amino Acid Sequence; Amino Acids; Animals; Cell Line; DNA Replication; Epidermal Growth Factor; Humans; Molecular Sequence Data; *Mutation; Oligonucleotide Probes; Peptide Fragments; Phosphopeptides; Phosphorylation; Plasmids; Platelet-Derived Growth Factor; Receptor, Epidermal Growth Factor; *Serine; *Threonine; Thymidine

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

The major sites of serine and threonine phosphorylation of the human epidermal growth factor (EGF) receptor observed in intact cells are Thr654, Thr669, Ser1046, and Ser1047. Phosphorylation of the EGF receptor is increased at these sites in cells treated with platelet-derived growth factor or phorbol ester. This increase in EGF receptor phosphorylation is associated with an inhibition of the high affinity binding of EGF to cell surface receptors and an inhibition of the receptor tyrosine protein kinase activity. In order to test the hypothesis that the phosphorylation of the EGF receptor is mechanistically related to the modulation of EGF receptor function, we replaced the major sites of serine and threonine phosphorylation with alanine residues. EGF receptors containing single point mutations or multiple mutations were expressed in Chinese hamster ovary cells. Analysis of the regulation of the EGF receptor tyrosine protein kinase activity demonstrated that phorbol ester caused an inhibition of the tyrosine phosphorylation of wild-type receptors and receptors lacking Thr669, Ser1046, or Ser1047. In contrast, the inhibition of EGF receptor tyrosine phosphorylation caused by phorbol ester was not observed for any of the mutated EGF receptors that lacked Thr654. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at Thr654 is required for the inhibition of the receptor tyrosine protein kinase activity caused by phorbol ester. Investigation of the apparent affinity of the EGF receptor demonstrated that treatment with phorbol ester caused an inhibition of the high affinity binding of 125I-EGF to cells expressing wild-type EGF receptors and each of the mutated EGF receptors examined. We conclude that the regulation of the apparent affinity of the EGF receptor is independent of the major sites of serine and threonine phosphorylation of the EGF receptor.

Rights and Permissions

Citation: J Biol Chem. 1990 Feb 25;265(6):3407-16.

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of biological chemistry

PubMed ID

2105948