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.
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.
Brasher
B B
BB
Center for Blood Research, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115-5717, USA.
Van Etten
R A
RA
D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics*
D018076:DNA, Complementary; D010455:Peptides; D014443:Tyrosine; D010649:Phenylalanine; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl