Substitution of an Aspartic Acid Results in Constitutive Activation of c-<i>kit</i>Receptor Tyrosine Kinase in a Rat Tumor Mast Cell Line RBL-2H3

Abstract

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 KIT^Tyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KIT^Tyr817 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 anti-sense 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.

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation

“…Similarly, Val and Tyr substitutions of the equivalent Asp802 residue of the closely related c-Kit receptor have been reported in mast cell leukaemia (Kitayama et al, 1995;Tsujimura et al, 1995). Here, we found that all hydrophobic amino acid substitutions of Asp802 of the M-CSFr were highly transforming.…”

Retinoids Induce Growth Inhibition and Apoptosis in Mast Cell Tumor Cell Lines

“…Uncontrolled proliferation and the accumulation of mast cells occurs in MCTs, whereas the normal process of proliferation and differentiation of mast cells are under the control of cytokines, particularly stem cell factor (SCF), also termed as a Kit ligand [8]. Several years ago, point mutations in c-kit that lead to constitutive activation of Kit in the absence of ligand binding were identified in three malignant mast cell lines (human HMC-1, RBL and mouse P815) [3,25,26]. This provides an indication that dysregulation of Kit may promote uncontrolled growth of survival of mast cells.…”

STAT3 activation is required for Asp816 mutant c-Kit induced tumorigenicity

“…The 293 cell line was established by transduction of human embryonic kidney cells with the adenovirus 5 DNA (Graham et al, 1977) and has been used as a model for studying signal transduction and oncogenesis, including some observations of the biological properties of Asp 816(human)/814(mouse) mutant c-Kit (Furitsu et al, 1993;Kanakura et al, 1994;Moriyama et al, 1996;Tsujimura et al, 1994Tsujimura et al, , 1995Yajima et al, 1998). Furthermore, this cell line has been shown in nude mice to rarely form tumors when injected in low cell numbers (6610 5 ) and to give rise to small-sized tumors when injected in higher numbers of cells (4610 6 ) (Arrick et al, 1992).…”