DIFFERENTIATION-INDUCED BY THE C-MPL CYTOKINE RECEPTOR IS BLOCKED BY MUTANT SHC ADAPTER PROTEIN

c-Mpl, a member of the cytokine receptor superfamily, induces both pro liferative and differentiation responses when stimulated with its liga nd thrombopoietin (TPO). To examine signal transduction pathways assoc iated with differentiation versus proliferation, 32D clone 3 cells, a murine interleukin 3-(IL-3)-dependent cell line capable of granulocyti c differentiation, were engineered to express human c-Mpl (designated 32DM.2). Human TPO-containing medium was produced by transient transfe ction of 293 cells, Treatment of 32DM.2 cells with human TPO induced c ellular aggregates within 12 h of exposure to ligand. 32DM.2 cells mai ntained in the presence of TPO did not change in cell number over a 72 -h period and acquired characteristics of granulocytic differentiation as evidenced by metamyelocytic cellular morphology, The differentiati on effect of TPO was observed in the absence and presence of the mitog en IL-3, Evaluation of protein tyrosine phosphorylation following expo sure to ligand revealed that TPO stimulation induced an elevated level of tyrosine phosphorylation of the adaptor protein She when compared with IL-3. However, treatment of 32DM.2 cells with TPO did not result in the phosphorylation of mitogen-activated protein kinase (MAPK), To evaluate the potential role of She in c-Mpl differentiation, we transf ected 32DM.2 cells with a mutant She gene that lacked the region codin g for the phosphotyrosine interaction domain (Delta PI-Shc). Expressio n of the Delta PI-Shc protein in 32DM.2 cells blocked the TPO differen tiation response with no effect on IL-3-stimulated proliferation, Thes e studies demonstrate that c-Mpl-induced differentiation results from the activation of signal transduction pathways that are dominant to th e IL-3 proliferative response and independent of the Ras/MAPK signal t ransduction pathway, The ability of the Delta PI-Shc protein to block TPO-induced differentiation implicates She as a mediator of signal tra nsduction pathways leading to differentiation, which is distinct from its role as a mediator in activating the Ras/MAPK pathway.