Thrombopoietin-Mediated Ubiquitination and Degradation of C-Mpl

Thrombopoietin (Tpo), acting through its receptor c-Mpl, is critical for the support of hematopoietic stem and progenitor cell survival and proliferation, in addition to being the principal regulator of megakaryopoiesis. Consequently, Tpo signaling must be tightly regulated to prevent uncontrolled proliferation of a number of hematopoietic lineages. Several mechanisms to negatively regulate hematopoietic signaling have previously been identified, including suppressors of cytokine signaling, protein phosphatases and negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation, via either lysosomes or proteasomes. In this study we investigated the mechanisms that regulate Tpo-mediated c-Mpl degradation in both c-Mpl-expressing cell lines and primary mouse cells. Using a factor-dependent hematopoietic cell line, BaF3, that were engineered to express human c-Mpl, we found that Tpo-stimulation caused both lysosomal and proteasomal degradation of c-Mpl. These findings were subsequently confirmed using murine megakaryocytes. We also found that Tpo-stimulation caused the rapid ubiquitination of c-Mpl, which is essential for targeting proteins to the proteasome. As ubiquitination occurs only on lysine residues of target proteins, we next determined which of the two intracellular domain lysine residues of c-Mpl are ubiquitinated by generating BaF3-Mpl cell lines bearing lysine to arginine (K to R) mutations at K 553 and K 573 . Tpo-mediated ubiquitination was unaltered in both single mutations (K 553 R or K 573 R), but ubiquitination was completely ablated in cell lines bearing c-Mpl with both mutations (K 553+573 R), indicating that both sites are ubiquitinated following Tpo-stimulation. Additionally, c-Mpl K 553+573 R displays greatly reduced Tpo-stimulated degradation and significantly greater proliferation in response to Tpo, strongly suggesting a critical role for c-Mpl ubiquitination in the negative regulation of Tpo-mediated proliferation. We also found that following Tpo-stimulation, c-MplK 553+573 R remained phosphorylated significantly longer than wild type (WT), indicating that ubiquitination and degradation, in addition to protein phosphatase activity, is essential for extinguishing the activity of phosphorylated c-Mpl. We observed no significant difference in internalization of c MplK 553+573 R compared to WT following Tpo stimulation, showing that monoubiquitination, which can mediate endocytosis in a number of receptor types, does not regulate c-Mpl internalization. Ubiquitin E3 ligases are required for covalently attaching ubiquitin to lysine residues of target proteins. C-Cbl is one such E3 ligase that has previously been shown to be activated by Tpo. Using siRNA, we significantly attenuated c-Cbl protein expression in BaF3-Mpl cells, which resulted in a marked reduction of c-Mpl degradation (c-Cbl siRNA = 18% degradation; control siRNA = 51% degradation) and ubiquitination after Tpo-stimulation for 60 minutes, compared to cells transfected with non-targeting siRNA. Taken together, these data demonstrate a significant role for Tpo-stimulated c-Mpl proteasomal degradation in the negative regulation of Tpo signaling and proliferation. We have shown that both c-Mpl intracellular lysine residues are ubiquitinated and are vital for the proteasomal degradation of the activated receptor. Further, we have also identified c-Cbl as a potential E3 ligase responsible for the ubiquitination of c-Mpl. Our findings provide novel insights into the negative regulation of Tpo signaling, which greatly enhance our understanding of the biology of c-Mpl signaling and could potentially highlight new targets for the treatment of myeloproliferative diseases.