IDENTIFICATION OF MUTATIONS IN THE CODING SEQUENCE OF THE PROTOONCOGENE C-KIT IN A HUMAN MAST-CELL LEUKEMIA-CELL LINE CAUSING LIGAND-INDEPENDENT ACTIVATION OF C-KIT PRODUCT

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding o f c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is k nown to activate c-kitR tyrosine kinase, thereby leading to autophosph orylation of c-kitR on tyrosine and to association of c-kitR with subs trates such as phosphatidylinositol 3-kinase (PI3K). In a human mast c ell leukemia cell line HMC-1, c-kitR was found to be constitutively ph osphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 ce lls was not detectable by means of PCR after reverse transcription (RT -PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDN A revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resultin g in intracellular amino acid substitutions of Gly-560 for Val and Val -816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In or der to determine the causal role of these mutations in the constitutiv e activation, murine c-kit mutants encoding Gly-559 and/or Val-814, co rresponding to human Gly-560 and/or Val-816, were constructed by site- directed mutagenesis and expressed in a human embryonic kidney cell li ne, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-81 4) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, wh ereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or w ild-type c-kitR was modest or little, respectively. These results sugg est that conversion of Asp-816 to Val in human c-kitR may be an activa ting mutation and responsible for the constitutive activation of c-kit R in HMC-1 cells.