c-Ab1 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

Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unpho sphorylated murine type IV c-Abl and measured the kinetic parameters of c-A bl tyrosine kinase activity in a solution with a peptide-based assay. Unpho sphorylated c-Abl exhibited substantial peptide kinase activity with K-m of 204 muM and V-max of 33 pmol min(-1). Contrary to previous observations us ing immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (abo ut 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K-m = 91 muM, 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 autophosphoryla tion rate was dependent on Abl protein concentration consistent with an int ermolecular reaction, A tyrosine to phenylalanine mutation (Y412F) at the c -Abl residue homologous to the c-Src catalytic domain autophosphorylation s ite impaired the activation of wildtype 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 o f wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was m inimally activated by autophosphorylation, These results support a model wh ere c-Abl is inhibited in part through an intramolecular Src homology 3-lin ker interaction and stimulated to full catalytic activity by sequential pho sphorylation at Tyr-412 and Tyr-245.