Differentiation of megakaryocyte precursors requires sustained ERK activation. (A) Thrombopoietin (TPO) activation of ERKs has 2 components, a Ras-dependent transient ERK activation and a Rap1-dependent sustained ERK activation. TPO binds its receptor (Mpl) to activate the JAK family kinases that promote tyrosine phosphorylation of the cytoplasmic tail. These phosphorylations serve as binding sites for signaling molecules, including Shc, which recruits Grb2 (growth factor receptor-bound protein-2)/SOS to activate Ras (Ras-GTP) resulting in a transient activation of ERKs (pERK). Sustained pERK requires the activation Rap1 (Rap1-GTP) and B-Raf. The recruitment of a Cbl/CrkL/C3G complex to Mpl has been proposed to trigger the activation of Rap1. (B) Stimulation of megakaryocytes with TPO induces sustained activation/phosphorylation of ERKs (pERK), which is required for megakaryocyte precursor differentiation (+). The sustained ERK activation by TPO has 2 components mediated by Ras and Rap1, as described in panel A. In contrast, stimulation of megakaryocytes with GM-CSF or EPO induces a Ras-dependent transient pERK, which does not lead to cellular differentiation (–). Direct contact between stromal cells and megakaryocytes inhibits the sustained pERK to prevent differentiation (–) by specifically disrupting Rap1/B-Raf signals. Shc indicates SH2-containing protein; STAT, signal transducer and activator of transcription.