Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf-1-and Rap1-B-Raf-dependent pathways

Thrombopoietin (TPO) regulates growth and differentiation of megakaryocytes . We previously showed that extracellular signal-regulated kinases (ERKs) a re required for TPO-mediated full megakaryocytic maturation in both normal progenitors and a megakaryoblastic cell line (UT7) expressing the TPO recep tor (Mpl), In these cells, intensity and duration of TPO-induced ERK signal are controlled by several regions of the cytoplasmic domain of Mpl, In thi s study, we explored the signaling pathways involved in this control. We sh ow that the small GTPases Ras and Rap1 contribute together to TPO-induced E RK activation in UT7-Mpl cells and that they do so by activating different Raf kinases as downstream effecters: a Ras-Raf-P pathway is required to ini tiate ERK activation while Rap1 sustains this signal through B-Raf, Indeed, (i) in cells expressing wild-type or mutant Mpl, TPO-induced Ras and Rap1 activation correlates with early and sustained phases of ERK signal, respec tively; (ii) interfering mutants of Ras and Rap1 both inhibit ERK kinase ac tivity and ERK-dependent Elk1 transcriptional activation in response to TPO ; (iii) the kinetics of activation of Raf-1 and B-Raf by TPO follow those o f Ras and Rap1, respectively (iv) RasV12-mediated Elk1 activation was modul ated by the wild type or interfering mutants of Raf-1 but not those of B-Ra f; (v) Elk1 activation mediated by a constitutively active mutant of Rap1 ( Rap1V12) is potentiated by B-Raf and inhibited by an interfering mutant of this kinase, UT7-Mpl cells represent the second cellular model in which Ras and Rap1 act in concert to modulate the duration of ERK signal in response to a growth factor and thereby the differentiation program. This is also, to our knowledge, the first evidence suggesting that Rap1 may play an activ e role in megakaryocytic maturation.