MOLECULAR-BASES OF DOMINANT NEGATIVE AND LOSS OF FUNCTION MUTATIONS AT THE MURINE C-KIT WHITE SPOTTING LOCUS - W37, WV, W41 AND W

The proto-oncogene c-kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white-spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. W(v) and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co-culture assay. We show that W37, W(v) and W41 are the result of missense mutations in the kinase domain of the c-kit coding sequence (W37 E→K at position 582; W(v) T→M position 660 and W41 V→M position 831), which affect the c-kit associated tyrosine kinase to varying degrees. The c-kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c-kit(W37) protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain in the c-kit protein. A 125 kd c-kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface. These results provide an explanation for the dominant nature of the W37, W(v) and W41 mutations and for the null phenotype of the original W mutation and they also provide insight into the mechanism of c-kit-mediated signal transduction.