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.
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.
Van Etten
R A
RA
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115-5717, USA.
Debnath
J
J
Zhou
H
H
Casasnovas
J M
JM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017238:Genes, Helminth / Q000235:genetics / Q000502:physiology*; D016313:Genes, abl / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine; D017354:Point Mutation / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D019000:Phosphotyrosine; D014443:Tyrosine; D016315:Proto-Oncogene Proteins c-abl; D017346:Protein Serine-Threonine Kinases