Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

TY - JOUR

T1 - Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

AU - Saur, Sebastian J.

AU - Sangkhae, Veena

AU - Geddis, Amy E.

AU - Kaushansky, Kenneth

AU - Hitchcock, Ian S.

PY - 2010/2/11

Y1 - 2010/2/11

N2 - 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.

AB - 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.

KW - Animals

KW - Blood Platelets

KW - Blotting, Western

KW - Cell Differentiation

KW - Cell Proliferation

KW - Cells, Cultured

KW - Hematopoietic Stem Cells

KW - Humans

KW - Immunoblotting

KW - Immunoprecipitation

KW - Lysine

KW - Megakaryocytes

KW - Mice

KW - Mutagenesis, Site-Directed

KW - Proto-Oncogene Proteins c-cbl

KW - RNA, Messenger

KW - RNA, Small Interfering

KW - Receptors, Thrombopoietin

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Thrombopoietin

KW - Ubiquitin

KW - Ubiquitin-Protein Ligases

KW - Ubiquitination

U2 - 10.1182/blood-2009-06-227033

DO - 10.1182/blood-2009-06-227033

M3 - Article

C2 - 19880496

SN - 1528-0020

VL - 115

SP - 1254

EP - 1263

JO - Blood

JF - Blood

IS - 6

ER -