Identification of sites on epidermal growth factor receptors which are phosphorylated by pp60src in vitro

Biochim Biophys Acta. 1996 Jun 13;1312(2):85-93. doi: 10.1016/0167-4889(96)00027-4.

Abstract

The Epidermal Growth Factor Receptor (EGF-R) becomes constitutively tyrosine phosphorylated on two novel sites in v-Src transformed cells, and these phosphorylations are associated with enhanced signaling activity [1]. To determine whether Src could directly phosphorylate these sites, we have examined the ability of the Src kinase to phosphorylate both wild-type and kinase-defective EGF-Rs in vitro. Although purified Src could phosphorylate EGF-Rs, the pattern of phosphorylation sites was not identical to what was previously found in vivo [1]: Src in vitro directly phosphorylated EGF-Rs on one autophosphorylation site (Tyr 1173) which was not a site of re-induced in vivo phosphorylation, suggesting the in vivo inaccessibility of this site. One Src-specific in vitro phosphorylation site (Tyr 03) appeared to correspond to one of the in vivo Src-induced sites (sPY2), but the other Src-specific in vivo site (sPY1) was not significantly phosphorylated in vitro, raising the possibility of a Src-induced tyrosine kinase cascade. The ability of Src to phosphorylate the EGF-R is consistent with the suggestion that the receptor can function as a kinase substrate independent of its intrinsic enzymatic activity, as implied by recent studies on signaling by kinase-defective EGF-Rs.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Cell Line
  • Cell Line, Transformed
  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • Electrophoresis, Polyacrylamide Gel
  • ErbB Receptors / chemistry
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Humans
  • Molecular Sequence Data
  • Mutation / genetics
  • Oncogene Protein pp60(v-src) / metabolism
  • Oncogene Protein pp60(v-src) / pharmacology
  • Peptides / chemistry
  • Phosphorylation
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • Receptor Protein-Tyrosine Kinases / chemistry
  • Receptor Protein-Tyrosine Kinases / genetics
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Trypsin / metabolism
  • Tyrosine / metabolism

Substances

  • Peptides
  • Tyrosine
  • ErbB Receptors
  • Receptor Protein-Tyrosine Kinases
  • Oncogene Protein pp60(v-src)
  • Proto-Oncogene Proteins pp60(c-src)
  • Trypsin