Activating mutation in the catalytic domain of c-kit elicits hematopoietic transformation by receptor self-association not at the ligand-induced dimerization site.

The c-kit receptor tyrosine kinase (KIT) is constitutively activated by naturally occurring mutations in either the juxtamembrane domain or the kinase domain. Although the juxtamembrane domain mutations led to ligand-independent KIT dimerization, the kinase domain mutations (Asp814 --> Val or Tyr) did not. In an effort to determine if the kinase domain mutant could transfer oncogenic signaling without receptor dimerization, we have constructed the truncated types of c-kitWild and c-kitTyr814 cDNAs (c-kitDel-Wild and c-kitDel-Tyr814 cDNAs, respectively), in which ligand-binding and ligand-induced dimerization domains were deleted. When c-kitDel-Wild and c-kitDel-Tyr814 genes were introduced into a murine interleukin-3 (IL-3)-dependent cell line Ba/F3, KITDel-Tyr814 was constitutively phosphorylated on tyrosine and activated, whereas KITDel-Wild was not. In addition, Ba/F3 cells expressing KITDel-Tyr814 (Ba/F3(Del-Tyr814)) grew in suspension culture without the addition of exogenous growth factor, whereas Ba/F3 cells expressing KITDel-Wild (Ba/F3(Del-Wild)) required IL-3 for growth. The factor-independent growth of Ba/F3(Del-Tyr814) cells was virtually abrogated by coexpression of KITW42 that is a dominant-negative form of KIT, but not by that of KITWild, suggesting that KITDel-Tyr814 may not function as a monomer but may require receptor dimerization for inducing factor-independent growth. Furthermore, KITDel-Tyr814 was found to be coimmunoprecipitated with KITWild or KITW42 by an ACK2 monoclonal antibody directed against the extracellular domain of KIT. Moreover, KITW42 was constitutively associated with a chimeric FMS/KITTyr814 receptor containing the ligand-binding and receptor dimerization domain of c-fms receptor (FMS) fused to the transmembrane and cytoplasmic domain of KITTyr814, but not with a chimeric FMS/KITWild receptor even after stimulation with FMS-ligand. These results suggest that constitutively activating mutation of c-kit at the Asp814 codon may cause a conformation change that leads to receptor self-association not in the extracellular domain and that the receptor self-association of the Asp814 mutant may be important for activation of downstream effectors that are required for factor-independent growth and tumorigenicity.