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The process of megakaryopoiesis and platelet production is complex, with the potential for regulation at multiple stages. Megakaryocytes are derived from the hematopoietic stem cell through successive lineage commitment steps, and they undergo a unique maturation process that includes polyploidization, development of an extensive internal demarcation membrane system and finally formation of proplatelet processes. Platelets are shed from these processes into vascular sinusoids within the bone marrow. Megakaryocyte differentiation is regulated both positively and negatively by transcription factors and cytokine signaling. Thrombopoietin is the most important hematopoietic cytokine for platelet production. Clinically, acquired and inherited mutations affecting megakaryocytic transcription factors and thrombopoietin signaling have been identified in disorders of thrombocytopenia and thrombocytosis.

Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K 553 and K 573 ). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes. (Blood. 2010;115: 1254-1263) IntroductionHematopoiesis is tightly regulated by several cytokines and growth factors to ensure that numbers of circulating blood cells remain constant under normal conditions. In many hematologic disorders, cytokine and growth factor signaling is dysfunctional, resulting in the overproduction or underproduction of 1 or more blood cell lineages. Thrombopoietin (Tpo) is a hematopoietic cytokine that, via its receptor c-Mpl, supports hematopoietic stem cell maintenance and proliferation and is the primary regulator of megakaryopoiesis. 1,2 Absence of Tpo signaling results in thrombocytopenia, reduced numbers of transplantable stem cells, and eventually aplastic anemia in humans. [3][4][5] Conversely, excessive Tpo signaling, usually due to mutations in c-Mpl or its secondary signaling proteins, results in hyperproliferation of numerous cell lineages, causing myeloproliferative syndromes. 6-8 Therefore, the control of Tpo-mediated signaling is critical in maintaining physiologic numbers of circulating blood cells.Protein phosphatases, suppressors of cytokine signaling (SOCS) proteins, and inhibitory intracellular mediators are all mechanisms that contribute to the negative regulation of cytokine signaling. [9][10][11][12] However, the process of receptor internalization and degradation is one of the quickest and most effective ways in which activated receptors are negatively regulated. We recently demonstrated a mechanism for Tpo-stimulated c-Mpl internalization, through the interaction of adaptor protein 2 with YRRL motifs located at Y 521 and Y 591 in the c-Mpl intracellular domain; elimination of these sites significantly reduced degrad...

Mature megakaryocytes are large, polyploid bone marrow cells that give rise to circulating platelets (1). Thrombopoietin (TPO) 1 has been characterized as the primary hematopoietic cytokine regulating normal megakaryocyte (MK) development. The receptor for TPO is encoded by the c-mpl proto-oncogene, a member of the hematopoietic growth factor receptor family, which in altered form causes a myeloproliferative syndrome in mice (2). In addition to its effects on MK development, TPO has been found to play a significant role in promoting stem cell survival, and in conjunction with other cytokines, it can support stem cell expansion (3-5). Characterization of the pathways by which TPO signals to promote survival and proliferation in stem cells and developing MKs is critical to a better understanding of the physiologic, pathologic, and potentially therapeutic roles of the cytokine in stem cell expansion, myeloproliferative disorders, and states of bone marrow failure.Many of the effects of TPO on cell survival and proliferation have been ascribed to activation of the Jak/STAT and Ras/Raf/ MAPK pathways (reviewed in Ref. 6). Activation of Jak2 leads to tyrosine phosphorylation of Mpl as well as STAT3 and STAT5 (7,8), and the phosphorylated STATs dimerize and translocate to the nucleus, where they stimulate transcription. In addition, phosphorylation of distal tyrosine residues on Mpl creates docking sites for SHC, which undergoes phosphorylation and then recruits Grb2/SOS, thus activating the Ras/Raf/ MAPK pathway (8 -10). Activation of the Ras/Raf/MAPK pathway can also occur by an alternative, as yet undetermined mechanism, independent of the distal receptor phosphotyrosine residues (11).Although these studies and others have shown that activation of the Jak/STAT and Ras/Raf/MAPK pathways is important for signaling by Mpl, other pathways likely contribute to the cellular response to TPO. One such pathway that may contribute to Mpl signaling is phosphatidylinositol 3-kinase (PI3K). PI3K has been shown to be activated by many growth factors involved in hematopoiesis, including stem cell factor, platelet-derived growth factor, erythropoietin, interleukin 3 (IL-3), IL-2, granulocyte-macrophage colony-stimulating factor, granulocyte colony stimulating factor, insulin-like growth factor 1, and TPO (12-16). It has been well established that activation of PI3K plays an important role in promoting cell survival (17-19). However, although there are ample data supporting a requirement for PI3K in proliferation of nonhematopoietic cells (20 -22), data showing specific requirements for PI3K in the cytokine-dependent proliferation of hematopoietic cells are inconclusive. Studies in cell lines with IL-2, IL-3, and erythropoietin have supported a role for PI3K in the proliferative response (23-26); however, studies with Flt-3L have not (27). Importantly, the conclusions from studies performed in primary cells often differ from those performed in cell lines. For example, data from cell lines support the role of PI3K in erythropoietin-...

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