ACTIVATING MUTATIONS OF THE C-KIT PROTOONCOGENE IN A HUMAN MAST-CELL LEUKEMIA-CELL LINE

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is kn own to play a crucial role in mast cell growth and differentiation. In a human mast cell leukemia cell line (HMC-1), KitR was found to be co nstitutively phosphorylated on tyrosine, activated and associated with phosphatidylinositol 3-kinase (PI3K) in the absence of autocrine prod uction of SCF. Sequencing of c-kit cDNA revealed that c-kit genes of H MC-1 cells were composed of a normal, wild-type allele and a mutant al lele with two point mutations in codon 560 and codon 816, resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp, respectively. Murine c-kit mutants encoding Gly-559 and/or V al-814, corresponding to human Gly-560 and/or Val-816, were constructe d by site-directed mutagenesis and expressed in cells of a human embry onic kidney cell line (293T). In the transfected cells, KitR (Gly-559 + Vat-814) and KitR (Val-814) were strikingly phosphorylated on tyrosi ne and activated in the absence of SCF, whereas tyrosine phosphorylati on and activation of KitR (Gly-559) or wild-type KitR was modest or li ttle, respectively. These results suggest that constitutive activation of KitR in HMC-1 results from the activating mutations of c-kit gene, and raise the possibility that the activating mutations, particularly at codon 814 of murine c-kit or at codon 816 of human c-kit, may part icipate in oncogenesis of mast cells.