Introduction of a loss-of-function point mutation from the SH3 region of the Caenorhabditis elegans sem-5 gene activates the transforming ability of c-abl in vivo and abolishes binding of proline-rich ligands in vitro

Oncogene. 1995 May 18;10(10):1977-88.

Abstract

We have introduced two loss-of-function point mutations from highly conserved regions of the src homology 3 (SH3) domains of the Caenorhabditis elegans sem-5 gene into the SH3 domain of the murine type IV c-abl tyrosine kinase proto-oncogene. One of the mutations, P131L, activated abl to transform fibroblasts while the other, G128R, did not. When combined with independent activating mutations in the c-abl kinase domain or NH2-terminus, the G128R mutation blocked transformation by the double mutant, suggesting that the G128R mutant was unable to transform cells for trivial reasons. The c-Abl G128R mutant, like wild type c-Abl protein, was localized to the nucleus and actin cytoskeleton and had normal tyrosine kinase activity in vitro, while the transforming c-Abl P131L protein was localized exclusively to the cytoplasm and exhibited decreased in vitro kinase activity. By real-time biospecific interaction analysis, the wild type Abl SH3 domain bound to two proteins containing proline-rich motifs with dissociation constants of 0.2 and 17 microM; the G128R mutant bound with 50-fold lower affinity, and no binding was detected by the P131L mutant. Both mutations completely abolished binding of the Abl SH3 domain to proline-rich target proteins in a filter-binding assay. These results suggest that the transforming activity of Abl is regulated in vivo by an inhibitor protein which associates with the SH3 domain via a proline-rich sequence.

Publication types

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

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Caenorhabditis elegans
  • Cell Nucleus / metabolism
  • Cytoplasm / metabolism
  • Genes, Helminth / genetics
  • Genes, Helminth / physiology*
  • Genes, abl / genetics
  • Genes, abl / physiology*
  • Mice
  • Molecular Sequence Data
  • Phosphorylation
  • Phosphotyrosine
  • Point Mutation / physiology*
  • Protein-Serine-Threonine Kinases / genetics*
  • Proto-Oncogene Proteins c-abl / metabolism*
  • Transfection
  • Tyrosine / analogs & derivatives*
  • Tyrosine / metabolism

Substances

  • Phosphotyrosine
  • Tyrosine
  • Proto-Oncogene Proteins c-abl
  • Protein-Serine-Threonine Kinases