Role of threonine residues in regulation of the epidermal growth factor receptor by protein kinase C and mitogen-activated protein kinase

J Biol Chem. 1993 Jul 25;268(21):15536-43.

Abstract

Epidermal growth factor (EGF) receptor tyrosine kinase activity is down-regulated by a number of growth-modulating agents that activate protein kinase C and/or mitogen-activated protein (MAP) kinases. Although the mechanism is unclear, it has been hypothesized that phosphorylation of specific threonine residues leads to inhibition of the EGF receptor tyrosine kinase. Two sites phosphorylated on the EGF receptor in response to phorbol esters are possible mediators of this effect: threonine 654, the target of protein kinase C, and threonine 669, the target of MAP kinase and the major site of phosphorylation on the EGF receptor. In order to investigate the role of these residues in receptor regulation, we substituted glutamic acid to mimic the negative charge introduced by phosphorylation at these sites. The wild-type and mutant receptor cDNAs were then transfected into CHO cells that lack endogenous EGF receptor. The EGF binding properties of the mutant receptors were similar to those of the wild-type EGF receptors. EGF stimulated tyrosine kinase activity and DNA synthesis in cells expressing both mutant receptors, indicating that the mutant EGF receptors are biologically active. Treatment of cells with phorbol esters inhibited the high affinity EGF binding and tyrosine kinase activities of both mutant and wild-type EGF receptors. These results indicate that acidic residues at either the Thr-654 or Thr-669 site modulate but do not block EGF receptor signalling. Furthermore, this data demonstrates that the mutant EGF receptors are still a target for inhibition by phorbol esters. Thus, events other than phosphorylation of Thr-654 or Thr-669 appear to be required for receptor down-regulation by protein kinase C or MAP kinase.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • CHO Cells
  • Cloning, Molecular
  • Cricetinae
  • Epidermal Growth Factor / metabolism
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / chemistry
  • ErbB Receptors / drug effects
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Mitogen-Activated Protein Kinase 1
  • Mutation
  • Peptide Mapping
  • Phorbol 12,13-Dibutyrate / pharmacology
  • Phosphorylation
  • Protein Kinase C / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / metabolism*
  • Threonine / metabolism*
  • Transfection

Substances

  • Threonine
  • Phorbol 12,13-Dibutyrate
  • Epidermal Growth Factor
  • ErbB Receptors
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Protein Kinase C
  • Mitogen-Activated Protein Kinase 1