Differentiation induced by the c-Mpl cytokine receptor is blocked by mutant Shc adaptor protein.

c-Mpl, a member of the cytokine receptor superfamily, induces both proliferative and differentiation responses when stimulated with its ligand thrombopoietin (TPO). To examine signal transduction pathways associated with differentiation versus proliferation, 32D clone 3 cells, a murine interleukin 3-(IL-3)-dependent cell line capable of granulocytic differentiation, were engineered to express human c-Mpl (designated 32DM.2). Human TPO-containing medium was produced by transient transfection of 293 cells. Treatment of 32DM.2 cells with human TPO induced cellular aggregates within 12 h of exposure to ligand. 32DM.2 cells maintained 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 differentiation effect of TPO was observed in the absence and presence of the mitogen IL-3. Evaluation of protein tyrosine phosphorylation following exposure to ligand revealed that TPO stimulation induced an elevated level of tyrosine phosphorylation of the adaptor protein Shc 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 Shc in c-Mpl differentiation, we transfected 32DM.2 cells with a mutant Shc gene that lacked the region coding for the phosphotyrosine interaction domain (delta PI-Shc). Expression of the delta PI-Shc protein in 32DM.2 cells blocked the TPO differentiation response with no effect on IL-3-stimulated proliferation. These studies demonstrate that c-Mpl-induced differentiation results from the activation of signal transduction pathways that are dominant to the IL-3 proliferative response and independent of the Ras/MAPK signal transduction pathway. The ability of the delta PI-Shc protein to block TPO-induced differentiation implicates Shc as a mediator of signal transduction pathways leading to differentiation, which is distinct from its role as a mediator in activating the Ras/MAPK pathway.