Tohru Tsujimura

Identification of mutations in the coding sequence of the proto-oncogene 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 of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells 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 cDNA 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 resulting 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 order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.

Role of c-kit receptor tyrosine kinase in the development, survival and neoplastic transformation of mast cells

The c‐kit gene is allelic with the dominant spotting (W) locus on mouse chromosome 5 and encodes a receptor tyrosine kinase. The llgand for c‐klt receptor is stem cell factor (SCF), which is the principal growth factor for mast cells. The loss‐of‐function mutations of c‐kit receptor affect the development of mast cells, thereby resulting in a depletion of mast cells. The abundant expression of c‐ktt receptor is indispensable for the survival of mast cells. In addition, the galn‐of‐function mutations of c‐kit receptor were found in several tumor mast cell lines. When these galn‐of‐function mutations were introduced to cells of murine interleukin (IL)‐3‐dependent cell lines, the expression of c‐kit receptor with constitutive tyrosine kinase activity not only abrogated the IL‐3 requirement of the cells, but also caused them to become tumorlgenic in nude athymic mice. The gain‐of‐function mutations of c‐kit receptor appear to result in the malignant transformation of mast cells. Taken together, the signals from the c‐ktt receptor are essential for the development, survival, and malignant transformation of mast cells.

Molecular cloning of cDNA encoding the c-kit receptor of Shiba goats and a novel alanine insertion specific to goats and sheep in the kinase insert region

The complete open reading frame (ORF) of the c-kit cDNA was cloned from a cerebellar cDNA library of the Shiba goat (Capra hircus var Shiba) with the dominant black-eyed white phenotype. The analysis of the deduced amino acid sequence revealed the presence of a single amino acid insertion (alanine) in the kinase insert (KI) region. While the newly found alanine insertion is not correlated with the coat color phenotype of goats, it appears to be characteristic of the c-kit genes in goats and sheep. Although the biological significance of the insert remains to be investigated, its phylogenetically limited distribution will provide us with a useful and interesting tool to analyze the problems of evolution of sheep and goats in bovidae.