Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation

Abstract

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is known to play a crucial role in hematopoiesis, especially in mast cell growth and differentiation. Although a number of dominant loss-of- function mutations of c-kit gene have been well characterized in mice, rats, and humans, little is known about the c-kit mutations contributing to ligand-independent activation of the c-kit receptor tyrosine kinase (KIT). In a murine mastocytoma cell line, P-815, KIT has been found to be constitutively phosphorylated on tyrosine and activated in a ligand-independent manner. Sequencing of the whole coding region of c-kit cDNA showed that c-kit cDNA of P-815 cells carries a point mutation in codon 814, resulting in amino acid substitution of Tyr for Asp. Murine wild-type c-kit cDNA and mutant- type c-kit cDNA encoding Tyr in codon 814 were expressed in cells of a human embryonic kidney cell line, 293T. In the transfected cells, mutant-form KITTyr814 was strikingly phosphorylated on tyrosine and activated in immune complex kinase reaction regardless of stimulation with a ligand for KIT (stem cell factor), whereas tyrosine phosphorylation and activation was barely detectable in wild-form KIT. The data presented here provide evidence for a novel activating mutation of c-kit gene that might be involved in neoplastic growth or oncogenesis of some cell types, including mast cells.

Identification of novel FLT‐3 Asp835 mutations in adult acute myeloid leukaemia

“…For example, Val and Tyr substitutions of human c-kit Asp816 have been reported in mast cell lines (Kitayama et al, 1995), as well as in systemic mastocytosis and its associated leukaemia (Longley et al, 1999;Sotlar et al, 2000). Furthermore, Tsujimura et al (1994) identified a mutation of the equivalent residue in a mouse mastocytoma cell line P-815 that changed Asp814 to Tyr, while Glover et al (1995) demonstrated that cassette mutagenesis of the c-fms homologue, Asp802, created receptors that are potently transforming in the factor-dependent, mouse haematopoietic progenitor cell line FDC-P1. Interestingly, Morley et al (1999) have recently shown that the oncogenic effects of cfms Asp802 mutations are restricted to hydrophobic amino acid substitutions.…”

“…The FLT3 Asp838Val mutation was selected because of its homology with known activating mutations involving codons 814 and 816 in mouse and human c-kit respectively. Such c-kit mutations have been described in human (Furitsu et al, 1993) and mouse mast cell lines (Tsujimura et al, 1994), as well in human systemic mastocytosis and its associated leukaemia (Longley et al, 1999;Sotlar et al, 2000).…”

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c‐kit proto‐oncogene

“…These alterations, that convert the receptor into a constitutive active state, have been reported for the RBL-2H3, P-815, C2, and HMC-1 mast cell lines. [23][24][25][26] Activating mutations within the ckit gene appear to be causative in most gastrointestinal stromal tumours (GISTs) 27 and some types of mastocytosis, [28][29][30] the latter of which is a heterogeneous group of disorders characterized by accumulation of mast cells in tissues. Several studies of cells obtained from patients with mastocytosis have described a point mutation in the catalytic domain of Kit (for reviews see Vliagoftis et al 31 and Nilsson et al 32 ).…”

Mastocytosis cells bearing a c‐kit activating point mutation are characterized by hypersensitivity to stem cell factor and increased apoptosis

“…The Asp816!Val substitution is caused by an A!T transversion at nucleotide 2468 of the c-kit coding region (Nagata et al, 1995;Longley et al, 1996). Constitutive activation of the c-Kit receptor containing this mutation has been described in several mast cell leukaemic lines (Furitsu et al, 1993;Tsujimura et al, 1994Tsujimura et al, , 1995. Using the IC2 mast cell line, we have previously demonstrated that the mutation leads to the following.…”