Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Abstract

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...

Thrombopoietin and hematopoietic stem cells

“…55 Mpl is then ubiquinated and degraded by Cbl through its E3 ubiquitin ligase activity. 56 Cbl's important role as a negative regulator of TPO signaling is shown, because it is one of the few negative regulators that has a significant effect on steady state megakaryopoiesis; Cbl -/-mice have a thrombocytosis and excessive numbers of splenic megakaryocytes. 57 Not all Mpl significantly contributes to clearance of TPO.…”

Dynamin 2–dependent endocytosis is required for normal megakaryocyte development in mice

“…Because Mpl surface expression is regulated by clathrin-dependent endocytosis, [37][38][39][40] we investigated the role of DNM2 in platelet Mplmediated endocytosis. Dnm2-null platelets expressed Mpl normally, as indicated by immunoblot analysis ( Figure 4A).…”

Regulation of Tyrosine Kinase Signaling by Cbl in Hematopoietic Stem Cells

“…Thrombopoietin (TPO) is indispensable for the maintenance of HSC quiescence (Yoshihara et al, 2007;Qian et al, 2007). Although its receptor (TPO-R, also known as Mpl or c-Mpl) is not an RTK, stimulation with TPO induce phosphorylation of Cbl , Mpl have been shown to be ubiquitinylated (Saur et al, 2010) and Cbl loss alters the signal transduction downstream of TPO (Rathinam et al, 2008;Naramura et al, 2010). Therefore, Mpl may interact with Cbl indirectly.…”