TI - 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 . AB - 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 .