INTRODUCTION OF A LOSS-OF-FUNCTION POINT MUTATION FROM THE SH3 REGIONOF 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 co nserved regions of the src homology 3 (SH3) domains of the Caenorhabdi tis elegans sem-5 gene into the SH3 domain of the murine type IV c-abl tyrosine kinase proto-oncogene. One of the mutations, P131L, activate d abl to transform fibroblasts while tbe other, G128R, did not. When c ombined with independent activating mutations in the c-abl kinase doma in or NH,terminus, the G128R mutation blocked transformation by the do uble mutant, suggesting that the G128R mutant was unable to transform cells for trivial reasons. The c-Abl G128R mutant, like wild type c-Ab i protein, was localized to the nucleus and actin cytoskeleton and had normal tyrosine kinase activity in vitro, while the transforming c-Ab l P131L protein was localized exclusively to the cytoplasm and exhibit ed decreased in vitro kinase activity. By real-time biospecific intera ction analysis, the wild type Abl SH3 domain bound to two proteins con taining proline-rich motifs with dissociation constants of 0.2 and 17 mu M; the G128R mutant bound with 50-fold lower affinity, and no bindi ng was detected by the P131L mutant. Both mutations completely abolish ed binding of the Abl SH3 domain to proline-rich target proteins in a filter-binding assay, These results suggest that the transforming acti vity of Abl is regulated in vivo by an inhibitor protein which associa tes with the SH3 domain via a proline-rich sequence.