Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel Factor correlates with leukemic potential

The authors investigated the roles of PI3-kinase and PLC-gamma in stimulati on by Steel Factor (SLF) through c-Kit, c-Kit mutants YF719, YF728, and a Y F719/F728 double mutant were expressed in 32D myelomonocytic cells. KitYF71 9 fails to recruit PI3-kinase after stimulation with SLF, whereas KitYF728 fails to stimulate PLC-gamma phosphorylation or mobilize Ca++. Both single mutants responded mitogenically to soluble SLF (sSLF) in a manner indisting uishable from wild type (WT), although sSLF failed to stimulate or promote the survival of cells expressing the double mutant. In contrast, although c ells expressing WT or YF719 were mitogenically stimulated by membrane-bound SLF (mSLF), stimulation of cells expressing KitYF728 was impaired, Similar ly, cells expressing WT or YF719 receptors were stimulated by plate-bound a nti-Kit antibodies, Whereas cells expressing the YF728 receptor were not st imulated. Neomycin sulfate, a PLC antagonist, inhibited cells expressing YF 719 receptors stimulated by sSLF, Neomycin also inhibited cells expressing the WT receptor that were stimulated by mSLF or immobilized anti-Kit antibo dies but did not inhibit stimulation of cells expressing WT or YF719 recept ors by sSLF, 32D cells expressing KitWT, KitF719, or KitYF728 were injected into mice and the presence of cells was evaluated by colony assays 6 to 7 weeks later. Although both KitWT and KitYF719 expressing cells could be rec overed from the spleen and bone marrow, recovery of KitYF728 cells from the se organs was severely reduced. These results indicate that Kit tyrosine 72 8 is of particular importance for mitogenic stimulation by mSLF or immobili zed ligand and is required for full maintenance of cells in vivo, likely th rough activation of PLC-gamma. (C) 2000 by The American Society of Hematolo gy.