Ubiquitination and degradation of the thrombopoietin receptor c-Mpl.
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.
Saur
Sebastian J
SJ
Department of Medicine, University of California San Diego, La Jolla, CA, USA.
Sangkhae
Veena
V
Geddis
Amy E
AE
Kaushansky
Kenneth
K
Hitchcock
Ian S
IS
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D053628:Receptors, Thrombopoietin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013926:Thrombopoietin / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053628:Receptors, Thrombopoietin; D025801:Ubiquitin; C082218:MPL protein, human; D013926:Thrombopoietin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C067419:CBL protein, human; D008239:Lysine