Characterization of Chicken Kit Tyrosine Kinase Receptor in Cos Cell Transfectants and in Chicken Brain

Abstract

The Kit tyrosine kinase (Kit) encoded by the c-kit proto-oncogene is a receptor for stem cell factor (SCF). Kit proteins of mice and humans are expressed in various kinds of hematopoietic progenitor cells and are essential for the growth of these cells. Wild-type chKit (chKit+) and a mutant chKit (chKit42) that contained an amino acid change from Asp⁷⁷⁷ to Asn corresponding to that in Kit of the W⁴² mutant mice were produced in Cos-1 cells transfected with expression plasmids containing the chicken c-kit cDNA, and characterized using two kinds of anti-chKit antisera. The W⁴² mutant Kit has previously been shown to be defective for kinase activity. The chKit+ of 145 kilodalton (kDa) and 130 kDa with varying degrees of N-linked glycosylation were detected. Western blot analysis using an anti-phosphotyrosine monoclonal antibody showed that autophosphorylation of chKit+ was greatly enhanced upon chicken SCF induction. The chKit+ did not respond to mouse SCF. The kinase activity of chKit42 was abolished by the amino acid substitution, indicating the Asp⁷⁷⁷ residue was essential for the activity. In addition, 145 kDa chKit conjugated with sialic acid residue(s) was detected in chicken brain by immunoprecipitation using the antisera. An in vitro kinase assay showed the kinase activity of this protein. These structural and functional similarities of chKit to mammalian Kit proteins shown in this study implicate a possible role of chKit in chicken hematopoietic system.