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

The c-kit receptor tyrosine kinase (KIT) is constitutively activated by nat urally occurring mutations in either the juxtamembrane domain or the kinase domain. Although the juxtamembrane domain mutations led to ligand-independ ent KIT dimerization, the kinase domain mutations (Asp(814)-->Val or Tyr) d id not. In an effort to determine if the kinase domain mutant could transfe r oncogenic signaling without receptor dimerization. we have constructed th e truncated types of c-kit(Wild) and c-kitTyr(814) cDNAs (c-kit(Del-Wild) a nd c-kit(Del-Tyr814) cDNAs, respectively), in which ligand-binding and liga nd-induced dimerization domains were deleted. When c.kit(Del-Wild) and c-ki t(Del-Tyr814) genes were introduced into a murine interleukin-3 (IL-3)-depe ndent cell line Ba/F3, KITDel-Tyr814 was constitutively phosphorylated on t yrosine and activated, whereas KITDel-Wild was not. In addition, Ba/F3 cell s expressing KITDel-Tyr814 (Ba/F3(Del-Tyr814)) grew in suspension culture w ithout the addition of exogenous growth factor, whereas Ba/F3 cells express ing KITDel-Wild (Ba/F3(Del-Wild)) required IL-3 for growth. The factor-inde pendent growth of Ba/F3(Del-Tyr814) cells was virtually abrogated by coexpr ession of KITW42 that is a dominant-negative form of KIT, but not by that o f KITWild, suggesting that KITDel-Tyr814 may not function as a monomer but may require receptor dimerization for inducing factor-independent growth. F urthermore, KITDel-Tyr814 was found to be coimmunoprecipitated with KITWild Or KITW42 by an ACK2 monoclonal antibody directed against the extracellula r domain of KIT. Moreover, KITW42 was constitutively associated with a chim eric FMS/KITTyr814 receptor containing the ligand-binding and receptor dime rization domain of c-fms receptor (FMS) fused to the transmembrane and cyto plasmic domain of KITTyr814. but not with a chimeric FMS/KITWild receptor e ven after stimulation with FMS-ligand. These results suggest that constitut ively activating mutation of c-kit at the Asp(814) codon may cause a confor mation change that leads to receptor self-association not in the extracellu lar domain and that the receptor self-association of the Asp(814) mutant ma y be important for activation of downstream effecters that are required for factor-independent growth and tumorigenicity. (C) 1999 by The American Soc iety of Hematology.