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

T. Furitsu, T. Tsujimura, T. Tono, H. Ikeda, H. Kitayama, U. Koshimizu, H. Sugahara, J. H. Butterfield, L. K. Ashman, Y. Kanayama, Y. Matsuzawa, Y. Kitamura, Y. Kanakura

Research output: Contribution to journalArticle

695 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1736-1744
Number of pages9
JournalJournal of Clinical Investigation
Volume92
Issue number4
DOIs
StatePublished - 1993

Keywords

  • activation
  • leukemia
  • point mutation
  • protooncogene c-kit
  • tyrosine kinase

ASJC Scopus subject areas

  • Medicine(all)

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    Furitsu, T., Tsujimura, T., Tono, T., Ikeda, H., Kitayama, H., Koshimizu, U., Sugahara, H., Butterfield, J. H., Ashman, L. K., Kanayama, Y., Matsuzawa, Y., Kitamura, Y., & Kanakura, Y. (1993). 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. Journal of Clinical Investigation, 92(4), 1736-1744. https://doi.org/10.1172/JCI116761