1. Ubiquitination and degradation of the thrombopoietin receptor c-Mpl
by Sebastian J. Saur, Veena Sangkhae, Amy E. Geddis, Kenneth Kaushansky, and Ian S. Hitchcock
Blood
Volume 115(6):
February 11, 2010
©2010 by American Society of Hematology

2. Tpo-stimulated c-Mpl is degraded by both the proteasome and the lysosome.
Tpo-stimulated c-Mpl is degraded by both the proteasome and the lysosome. (A) BaF-Mpl cells were treated with Chx for 0 to 240 minutes to inhibit synthesis of new protein in conjunction with or without rhTpo, MG-132, and NH4Cl. c-Mpl degradation was determined by the presence or absence of the mature 85-kDa form of c-Mpl. (B) Similar experiments were also performed on bone marrow–derived murine megakaryocytes and human platelets. The data shown are representative of 3 independent experiments.
Sebastian J. Saur et al. Blood 2010;115:
©2010 by American Society of Hematology

3. Tpo stimulates ubiquitination of c-Mpl.
Tpo stimulates ubiquitination of c-Mpl. (A) BaF-Mpl cells were treated with or without MG-132 and rhTpo for 0 to 120 minutes and c-Mpl ubiquitination was analyzed by c-Mpl immunoprecipitation (IP) and immunoblot (IB) analysis using an anti-mono/polyubiquitin antibody. Ubiquitination is seen as a high-molecular-weight smear. The data shown are representative of 3 independent experiments. The equivalence of protein loading was analyzed by reprobing the IB with an anti–c-Mpl antibody. (B) Bone marrow–derived megakaryocytes were pretreated with MG-132 for 0 or 60 minutes, with or without rhTpo. Megakaryocyte c-Mpl ubiquitination was analyzed as described in panel A and the blot shown is representative of 2 independent experiments. (C) BaF-Mpl cells were pretreated with vehicle control (DMSO) or the JAK2 inhibitor JAKI for 30 minutes before Tpo stimulation (50 ng/mL, 60 minutes) and c-Mpl ubiquitination was analyzed. The effectiveness of JAKI to inhibit JAK2 activity was determined by analyzing levels of phosphor (p)–STAT5. Data are representative of 2 independent experiments.
Sebastian J. Saur et al. Blood 2010;115:
©2010 by American Society of Hematology

4. c-Mpl is ubiquitinated at K553 and K573.
c-Mpl is ubiquitinated at K553 and K573. (A) Schematic representation of the human c-Mpl intracellular domain, showing the position of the 2 intracellular lysine (K) residues in relation to tyrosine (Y) and box 1 and box 2. (B) Western blot analysis of total c-Mpl expression in 2 WT c-Mpl clones and 2 c-MplK R clones. (C) Cells expressing c-Mpl with a single K to R mutation (c-Mpl K553R and c-Mpl K573R) display normal levels of c-Mpl ubiquitination in response to Tpo stimulation. (D) c-Mpl ubiquitination after 60-minute Tpo stimulation in BaF cells expressing WT c-Mpl or c-MplK R. The data shown are representative of 3 independent experiments. (E) Biotinylation of mature, membrane-localized c-Mpl in BaF cells expressing WT c-Mpl and c-MplK R to determine normal turnover over a time period of 6 hours. The Western blot shown is representative of 3 independent experiments. The graph displays a quantitation of the turnover of mature, 85-kDa c-Mpl in the 2 cell lines as determined by densitometry, compared with time 0 (100%). Protein loading is standardized by comparison to surface Na+/K+ ATPase α1 expression. The data points represent the mean ± SE of 3 independent experiments (*P < .05, ***P < .001; Student t test).
Sebastian J. Saur et al. Blood 2010;115:
©2010 by American Society of Hematology

5. c-MplK553+573R exhibits altered Tpo-induced proliferation and degradation.
c-MplK R exhibits altered Tpo-induced proliferation and degradation. (A) MTT proliferation assay using BaF cells expressing WT c-Mpl or c-Mpl R. Cells were treated with rhTpo at increasing concentrations for up to 48 hours. The data represent the mean (± SE) of 3 individual experiments using 2 stably expressing clones for each group (*P < .05, ***P < .001). (B) Western blot analysis of Tpo-induced c-Mpl phosphorylation with and without MG-132. The data are representative of 3 individual experiments. (C) Western blot analysis of Tpo-induced c-Mpl degradation of WT c-Mpl and c-MplK R. Cells were pretreated with Chx for 60 minutes before Tpo stimulation for up to 120 minutes. (D) Graphic representation of c-Mpl degradation, comparing mature (85-kDa) c-Mpl at time 0 using densitometry.
Sebastian J. Saur et al. Blood 2010;115:
©2010 by American Society of Hematology

6. c-Cbl siRNA reduces Tpo-induced c-Mpl ubiquitination and degradation.
c-Cbl siRNA reduces Tpo-induced c-Mpl ubiquitination and degradation. (A) c-Cbl siRNA significantly reduced expression of c-Cbl in BaF-Mpl cells 48 hours after transfection. (B) c-Mpl ubiquitination was reduced in cells treated with c-Cbl compared with those treated with nontargeting siRNA. The data shown are representative of 4 individual experiments. (C) Tpo-stimulated c-Mpl degradation was analyzed by Western blot using lysates from BaF-WT-Mpl and BaF-MplK R cells treated with c-Cbl–specific and nontargeting siRNAs and pretreated with Chx for 60 minutes before Tpo stimulation for up to 60 minutes. (D) Graphic representation of degradation, comparing the mature (85-kDa) form of c-Mpl in cells treated with c-Cbl–specific siRNA compared with nontargeting siRNA. The data points represent the mean ± SE of 3 independent experiments (*P < .05, **P < .01; Student t test).
Sebastian J. Saur et al. Blood 2010;115:
©2010 by American Society of Hematology

7. c-Cbl acts as a ubiquitin E3 ligase in the Tpo-stimulated ubiquitination of c-Mpl.
c-Cbl acts as a ubiquitin E3 ligase in the Tpo-stimulated ubiquitination of c-Mpl. (A) BaF-Mpl cells overexpressing WT-Cbl or the E3 ligase dead CblC379A. (B) WT-Cbl– and CblC379A-expressing cells were pretreated with MG-132, then with or without rhTpo for 60 minutes, and c-Mpl ubiquitination was analyzed by IP and Western blot. The data shown are representative of 3 independent experiments. (C) These cells were analyzed for proliferation using an MTT assay at increasing concentrations of rhTpo. The data shown represent mean (± SE) of 3 independent experiments (*P < .05, ***P < .001).
Sebastian J. Saur et al. Blood 2010;115:
©2010 by American Society of Hematology