Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain

Abstract

A peculiar point mutation results in constitutive activation of c-kit receptor tyrosine kinase (KIT) in three different tumor mast cell lines; ie, the HMC-1, P-815, and RBL-2H3. Because constitutive activation of KIT was also observed in the FMA3 mouse mastocytoma cell line, we investigated the molecular mechanism. Sequencing of the whole coding region of the c-kit showed that the point mutation found in HMC- 1, P-815, and RBL-2H3 cells was absent in FMA3 cells and that the c-kit cDNA of FMA3 cells carried an in-frame deletion of 21 base pairs (bp) encoding Thr-Gln-Leu-Pro-Tyr-Asp-His at codons 573 to 579 at the juxtamembrane domain. The FMA3-type c-kit cDNA with 21 bp deletion was introduced into the IC-2 cell line, which was derived from murine cultured mast cells. IC-2 cells were dependent on interleukin (IL)-3 and did not express KIT on the surface. In IC-2 cells introduced with the FMA3-type c-kit cDNA, KIT was constitutively phosphorylated on tyrosines and activated. Moreover, the FMA3-type KIT was dimerized without the stimulation by stem cell factor (SCF), a ligand for KIT. The spontaneously dimerized FMA3-type KIT without SCF binding was not internalized even after the activation. IC-2 cells expressing the FMA3- type KIT grew in suspension culture without IL-3 and SCF and became leukemic in nude athymic mice. The deletion of seven amino acids at the juxtamembrane domain appeared to be a new activating mutation of KIT that might be involved in neoplastic growth of mast cells.

Mutations in the Juxtamembrane Domain of c-KIT Are Associated with Higher Grade Mast Cell Tumors in Dogs

“…In this study and that of London et al, 9 only the juxtamembrane domain was screened for mutations; however, Ma et al 10 also tested the phosphotransferase domain. The phosphotransferase domain of c-KIT has been reported to harbor mutations in some human 7 and rodent [15][16][17] mast cell tumors. In addition to the duplications and deletions, our identification of numerous SNPs in c-KIT, both in the juxtamembrane domain and in other regions of the c-KIT gene, suggests that c-KIT as a whole may be a hotspot for mutations in dogs, with the potential for mutations in mast cell tumors outside of the juxtamembrane domain.…”

“…18 The genes involved in the development of mast cell tumors are currently under study. Analyses of human 7 and rodent [15][16][17] mast cell tumor cell lines have revealed a number of mutations in the proto-oncogene c-KIT. The c-KIT gene encodes for a cell surface receptor, which upon binding of its cognate ligand induces a signal transduction cascade responsible for the development, maturation, and survival of many cell lineages, including hematopoietic stem cells, melanocytes, and mast cells.…”

Early signaling pathways activated by c-Kit in hematopoietic cells

“…The c-Kit juxtamembrane region (amino acids 544±577) is the site of multiple mutations that constitutively activate c-Kit. Substitution of glycine for valine 559 of murine c-Kit results in constitutive dimerization and activation of c-Kit as well as increases in its oncogenic potential [55,57,60,61]. Deletion of seven amino acids in the juxtamembrane region also generates a constitutively active form of c-Kit that spontaneously dimerizes [61].…”

“…Substitution of glycine for valine 559 of murine c-Kit results in constitutive dimerization and activation of c-Kit as well as increases in its oncogenic potential [55,57,60,61]. Deletion of seven amino acids in the juxtamembrane region also generates a constitutively active form of c-Kit that spontaneously dimerizes [61]. Patients with gastrointestinal stromal cell tumors have a series of mutations in the c-Kit juxtamembrane region that also leads to constitutive activation [22].…”

“…Mutations in the juxtamembrane region have also been associated with constitutively active forms of c-Kit. Substitution of valine for glycine 560 activates human c-Kit [55,57,60,61]. Murine c-Kit lacking amino acids 573±579 is also constitutively active [61].…”