Flt3 signaling involves tyrosyl-phosphorylation of SHP-2 and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells

Flt3 ligand (FL) is an early-acting potent co-stimulatory cytokine that reg ulates proliferation and differentiation of a number of blood cell lineages . Its receptor Flt3/Flk2 belongs to class III receptor tyrosine kinases tha t also include the receptors for colony-stimulating factor 1. Steel factor, and platelet-derived growth factor. Using CSF-1 receptor/Flt3 chimeras, tw o groups have characterized some of the pout-receptor signaling events and substrate specificity of murine Flt3 receptor. However, there are few studi es on the signaling pathway through human Flt3. We examined human Flt3 sign aling pathways in a murine IL-3-dependent hematopoietic cell line Baf3, whi ch stable expresses full-length human Flt3 receptor. This subline prolifera tes in response to human FL. Like the chimeric murine Flt3, human Flt3 unde rgoes autophosphorylation, associates with Grb2, and leads to tyrosine phos phorylation of Shc on ligand binding. We found that SHP-2, but not SHP-1, i s tyrosine-phosphorylated by FL stimulation, SHP-2 does not associate with Flt3, but binds directly to Grb2. SHIP is also tyrosine-phosphorylated and associates with Shc after FL simulation. We further examined the downstream signaling pathway. FL transiently activates MAP kinase. This activation co uld he blocked by PD98059, a specific MEK inhibitor, PD98059 also blocked c ell proliferation in response to FL. These results demonstrate that SHP-2 a nd SHIP are important components in the human Flt3 signaling pathway and su ggest that SHP-2 and SHIP, by forming complexes with adapter proteins Grb2 and Shc, may modulate MAP kinase activation, which may be necessary for the mitogenic signaling of Flt3.