A transforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain.

An activating mutation (DY814) located in the catalytic domain of the c-Kit receptor has been found in mastocytomas from human, mouse and rat. We evaluated the enzymic properties of purified wild-type (WT) and DY814 tyrosine kinase domains expressed in Pichia pastoris. A linker encoding the Flag epitope was fused to c-Kit cDNA species, enabling affinity purification of the proteins with anti-Flag antibodies. Yeast lysates expressing DY814 contained multiple tyrosine-phosphorylated proteins, whereas WT lysates had no detectable tyrosine phosphorylation. Purification of the WT and mutant kinases in the presence of vanadate demonstrated that both enzymes undergo autophosphorylation. Kinetic analyses of WT and DY814 kinases indicated that at 20 nM enzyme concentration the mutation increases the specific activity 10-fold and decreases the apparent Km for ATP 9-fold. WT activity displayed a hyperbolic dependence on enzyme concentration, consistent with a requirement for dimerization or aggregation for activity. This activity was also enhanced by anti-Flag antibodies. In contrast, the dependence of DY814 activity on enzyme concentration was primarily linear and only marginally enhanced by anti-Flag antibodies. Gel-filtration analysis showed that the WT kinase migrated as a monomer, whereas the DY814 mutant migrated as a dimer. These results indicate that this point mutation promotes dimerization of the c-Kit kinase, potentially contributing to its transforming potential in mast cells.