A transforming mutation induces dimerization and enhances activity of the c-kit soluble tyrosine kinase domain

grantor: University of TorontoAn activating mutation (DY814) located in the catalytic domain of the c-kit receptor has been found in mastocytomas. I characterized purified WT and DY814 kinase domains expressed in Pichia pastoris. A Flag epitope was fused to c-kit cDNAs enabling purification with anti-Flag antibodies. Yeast expressing DY814, but not WT, contained multiple tyrosine phosphorylated proteins. The mutation was found to increase the apparent k$\rm\sb{cat}$ by 10-fold and decrease the apparent K$\rm\sb{m}$ for ATP by 9-fold. WT activity displayed a non-linear dependence on enzyme concentration that could be enhanced by anti-Flag antibodies. In contrast, DY814 activity dependence on enzyme concentration was linear and marginally enhanced by anti-Flag antibodies. The WT kinase migrated as a monomer in gel filtration whereas the DY814 mutant migrated as a dimer. These results indicate that DY814 promotes dimerization of the c-kit kinase, potentially contributing to its transforming potential.M.Sc