c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines

J Biol Chem. 2000 Nov 10;275(45):35631-7. doi: 10.1074/jbc.M005401200.

Abstract

Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unphosphorylated murine type IV c-Abl and measured the kinetic parameters of c-Abl tyrosine kinase activity in a solution with a peptide-based assay. Unphosphorylated c-Abl exhibited substantial peptide kinase activity with K(m) of 204 microm and V(max) of 33 pmol min(-1). Contrary to previous observations using immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K(m) = 91 microm, V(max) = 112 pmol min(-1)). Autophosphorylation stimulated the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates. The autophosphorylation rate was dependent on Abl protein concentration consistent with an intermolecular reaction. A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impaired the activation of wild-type c-Abl by 90% but reduced activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibited the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was minimally activated by autophosphorylation. These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245.

Publication types

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

MeSH terms

  • Animals
  • Binding Sites
  • Catalytic Domain
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Kinetics
  • Mice
  • Models, Biological
  • Mutation*
  • Peptides / metabolism
  • Phenylalanine / chemistry
  • Phosphorylation
  • Point Mutation
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins c-abl / chemistry*
  • Proto-Oncogene Proteins c-abl / metabolism*
  • Time Factors
  • Tyrosine / metabolism*
  • src Homology Domains / genetics*

Substances

  • DNA, Complementary
  • Peptides
  • Tyrosine
  • Phenylalanine
  • Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins c-abl