Autophosphorylation of the Fes tyrosine kinase. Evidence for an intermolecular mechanism involving two kinase domain tyrosine residues

J Biol Chem. 1996 Jul 19;271(29):17519-25. doi: 10.1074/jbc.271.29.17519.

Abstract

The human c-fes proto-oncogene encodes a cytoplasmic tyrosine kinase (Fes) that is associated with multiple hematopoietic cytokine receptors. Fes tyrosine autophosphorylation sites may regulate kinase activity and recruit downstream signaling proteins with SH2 domains. To localize the Fes autophosphorylation sites, full-length Fes and deletion mutants lacking either the unique N-terminal or SH2 domain were autophosphorylated in vitro and analyzed by CNBr cleavage. Identical phosphopeptides of 10 and 4 kDa were produced with all three proteins, localizing the tyrosine autophosphorylation sites to the C-terminal kinase domain. Substitution of kinase domain tyrosine residues 713 or 811 with phenylalanine resulted in a loss of the 10- and 4-kDa phosphopeptides, respectively, identifying these tyrosines as in vitro autophosphorylation sites. CNBr cleavage analysis of Fes isolated from 32PO4-labeled 293T cells showed that Tyr-713 and Tyr-811 are also autophosphorylated in vivo. Mutagenesis of Tyr-713 reduced both autophosphorylation of Tyr-811 and transphosphorylation of Bcr, a recently identified Fes substrate, supporting a major regulatory role for Tyr-713. Wild-type Fes transphosphorylated a kinase-inactive Fes mutant on Tyr-713 and Tyr-811, suggesting that Fes autophosphorylation occurs via an intermolecular mechanism analogous to receptor tyrosine kinases.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cyanogen Bromide
  • Glutathione Transferase
  • Humans
  • Molecular Sequence Data
  • Mutagenesis
  • Oligopeptides
  • Peptide Fragments / chemistry
  • Peptides
  • Phosphopeptides / chemistry
  • Phosphorylation
  • Polymerase Chain Reaction
  • Protein-Tyrosine Kinases / biosynthesis
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins / biosynthesis
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-fes
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Restriction Mapping
  • Sequence Deletion
  • Spodoptera
  • Transfection
  • Tyrosine

Substances

  • Oligopeptides
  • Peptide Fragments
  • Peptides
  • Phosphopeptides
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Tyrosine
  • FLAG peptide
  • Glutathione Transferase
  • Protein-Tyrosine Kinases
  • FES protein, human
  • Proto-Oncogene Proteins c-fes
  • Cyanogen Bromide