and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells

Abstract: Flt3 ligand (FL) is an early-acting po-tent co-stimulatory cytokine that regulates prolifera-tion and differentiation of a number of blood cell lineages. Its receptor Flt3/Flk2 belongs to class III receptor tyrosine kinases that also include the receptors for colony-stimulating factor 1, Steel factor, and platelet-derived growth factor. Using CSF-1 receptor/Flt3 chimeras, two groups have characterized some of the post-receptor signaling events and substrate specificity of murine Flt3 receptor. However, there are few studies on the signaling pathway through human Flt3. We exam-ined human Flt3 signaling pathways in a murine IL-3-dependent hematopoietic cell line Baf3, which stably expresses full-length human Flt3 receptor. This subline proliferates in response to human FL. Like the chimeric murine Flt3, human Flt3 under-goes autophosphorylation, associates with Grb2, and leads to tyrosine phosphorylation of Shc on ligand binding. We found that SHP-2, but not SHP-1, is tyrosine-phosphorylated by FL stimula-tion. SHP-2 does not associate with Flt3, but binds directly to Grb2. SHIP is also tyrosine-phosphory-lated and associates with Shc after FL simulation. We further examined the downstream signaling pathway. FL transiently activates MAP kinase. This activation could be blocked by PD98059, a spe-cific MEK inhibitor. PD98059 also blocked cell proliferation in response to FL. These results dem-onstrate that SHP-2 and SHIP are important com-ponents in the human Flt3 signaling pathway and suggest that SHP-2 and SHIP, by forming com-plexes with adapter proteins Grb2 and Shc, may modulate MAP kinase activation, which may be necessary for the mitogenic signaling of Flt3. J