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 (Asp 814 → 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- kit Wild and c- kit Tyr814 cDNAs (c- kit Del-Wild and c- kit Del-Tyr814 cDNAs, respectively), in which ligand-binding and ligand-induced dimerization domains were deleted. When c- kit Del-Wild and c- kit Del-Tyr814 genes were introduced into a murine interleukin-3 (IL-3)–dependent cell line Ba/F3, KIT Del-Tyr814 was constitutively phosphorylated on tyrosine and activated, whereas KIT Del-Wild was not. In addition, Ba/F3 cells expressing KIT Del-Tyr814 (Ba/F3 Del-Tyr814 ) grew in suspension culture without the addition of exogenous growth factor, whereas Ba/F3 cells expressing KIT Del-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 KIT W42 that is a dominant-negative form of KIT, but not by that of KIT Wild , suggesting that KIT Del-Tyr814 may not function as a monomer but may require receptor dimerization for inducing factor-independent growth. Furthermore, KIT Del-Tyr814 was found to be coimmunoprecipitated with KIT Wild or KIT W42 by an ACK2 monoclonal antibody directed against the extracellular domain of KIT. Moreover, KIT W42 was constitutively associated with a chimeric FMS/KIT Tyr814 receptor containing the ligand-binding and receptor dimerization domain of c- fms receptor (FMS) fused to the transmembrane and cytoplasmic domain of KIT Tyr814 , but not with a chimeric FMS/KIT Wild receptor even after stimulation with FMS-ligand. These results suggest that constitutively activating mutation of c- kit at the Asp 814 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 Asp 814 mutant may be important for activation of downstream effectors that are required for factor-independent growth and tumorigenicity.