ubiquitinationanddegradationofthethrombopoietinreceptorc-mpl · via its receptor c-mpl, supports...

PLATELETS AND THROMBOPOIESIS

Ubiquitination and degradation of the thrombopoietin receptor c-MplSebastian J. Saur,1 Veena Sangkhae,1 Amy E. Geddis,1 Kenneth Kaushansky,1 and Ian S. Hitchcock1

1Department of Medicine, University of California San Diego, La Jolla

Regulation of growth factor and cytokinesignaling is essential for maintainingphysiologic numbers of circulating hema-topoietic cells. Thrombopoietin (Tpo), act-ing through its receptor c-Mpl, is requiredfor hematopoietic stem cell maintenanceand megakaryopoiesis. Therefore, thenegative regulation of Tpo signaling iscritical in many aspects of hematopoi-esis. In this study, we determine themechanisms of c-Mpl degradation in thenegative regulation of Tpo signaling. We

found that, after Tpo stimulation, c-Mpl isdegraded by both the lysosomal and pro-teasomal pathways and c-Mpl is rapidlyubiquitinated. Using site-directed mu-tagenesis, we were able to determine thatc-Mpl is ubiquitinated on both of its intra-cellular lysine (K) residues (K553 and K573).By mutating these residues to arginine,ubiquitination and degradation were sig-nificantly reduced and caused hyperpro-liferation in cell lines expressing thesemutated receptors. Using short interfer-

ing RNA and dominant negative overex-pression, we also found that c-Cbl, whichis activated by Tpo, acts as an E3 ubiq-uitin ligase in the ubiquitination of c-Mpl.Our findings identify a previously un-known negative regulatory pathway forTpo signaling that may significantly im-pact our understanding of the mecha-nisms affecting the growth and differentia-tion of hematopoietic stem cells andmegakaryocytes. (Blood. 2010;115:1254-1263)

Introduction

Hematopoiesis is tightly regulated by several cytokines and growthfactors to ensure that numbers of circulating blood cells remainconstant under normal conditions. In many hematologic disorders,cytokine and growth factor signaling is dysfunctional, resulting inthe overproduction or underproduction of 1 or more blood celllineages. Thrombopoietin (Tpo) is a hematopoietic cytokine that,via its receptor c-Mpl, supports hematopoietic stem cell mainte-nance and proliferation and is the primary regulator of megakaryo-poiesis.1,2 Absence of Tpo signaling results in thrombocytopenia,reduced numbers of transplantable stem cells, and eventuallyaplastic anemia in humans.3-5 Conversely, excessive Tpo signaling,usually due to mutations in c-Mpl or its secondary signalingproteins, results in hyperproliferation of numerous cell lineages,causing myeloproliferative syndromes.6-8 Therefore, the control ofTpo-mediated signaling is critical in maintaining physiologicnumbers of circulating blood cells.

Protein phosphatases, suppressors of cytokine signaling (SOCS)proteins, and inhibitory intracellular mediators are all mechanismsthat contribute to the negative regulation of cytokine signaling.9-12

However, the process of receptor internalization and degradation isone of the quickest and most effective ways in which activatedreceptors are negatively regulated. We recently demonstrated amechanism for Tpo-stimulated c-Mpl internalization, through theinteraction of adaptor protein 2 with YRRL motifs located at Y521

and Y591 in the c-Mpl intracellular domain; elimination of thesesites significantly reduced degradation of the receptor.13

Ubiquitination is a posttranslational modification involving thecovalent attachment of the small (� 8 kDa) protein ubiquitin tolysine residues of target proteins. Ubiquitination relies on theactivities of 3 groups of enzymes; ubiquitin-activating enzymes(E1), ubiquitin carrier proteins (E2), and ubiquitin protein ligases

(E3).14 The attachment of single ubiquitin molecules to targetproteins (monoubiquitination) has previously been shown to medi-ate protein trafficking and intracellular signaling.15 However,activated ubiquitin is also able to form direct interactions with otherubiquitin molecules via 1 of the 7 lysine residues (usually ly-sine 48). Once 4 or more ubiquitins are conjugated in a polyubiq-uitin chain, the protein is targeted to the proteasome and degraded.The process of ubiquitination and proteasomal degradation oftransmembrane growth factor receptors is a common regulatorymechanism and has been identified in several different systems(reviewed in Marmor and Yarden16). Studies of the ubiquitinationand degradation of the epidermal growth factor receptor andplatelet derived growth factor receptor, colony-stimulatingfactor-1, ErbB-2, and Met have identified c-Cbl as one of the E3ligases responsible for receptor ubiquitination in response togrowth factor stimulation.17-21 In these examples, stimulation of thereceptor leads to recruitment of Cbl, either directly throughphosphorylated tyrosine residues in the receptor or indirectly viaadaptor proteins.22,23 Cbl is then phosphorylated, stimulating its E3ligase activity.

In the current studies, we further explored the molecularmechanisms of c-Mpl degradation, specifically testing the role ofubiquitination as a negative regulator of Tpo signaling in c-Mpl–expressing cell lines, murine megakaryocytes, and human platelets.We then identified which lysines in the intracellular domain ofc-Mpl are ubiquitinated and determined their effects on c-Mpldegradation, Tpo-dependent cell growth, and levels of receptorphosphorylation in response to Tpo. Finally, using short interferingRNA (siRNA) and dominant negative overexpression, we deter-mined that c-Cbl significantly contributes to c-Mpl ubiquitinationand degradation through its E3 ubiquitin ligase activity. Our

Submitted June 10, 2009; accepted October 6, 2009. Prepublished online asBlood First Edition paper, October 30, 2009; DOI 10.1182/blood-2009-06-227033.

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findings identify a previously unknown negative regulatory mecha-nism for Tpo signaling that may significantly impact our understand-ing of the mechanisms affecting the growth and differentiation ofhematopoietic stem cells and megakaryocytes.

Methods

Chemicals and reagents

The proteasome inhibitor MG-132 and Janus kinase 2 (JAK2)–specificinhibitor JAKI were purchased from Calbiochem. The lysosome inhibitorNH4Cl, cycloheximide, and mouse monoclonal anti-Na�/K�-ATPase �1antibody were purchased from Sigma-Aldrich. N-terminal–specific poly-clonal rabbit anti–human c-Mpl antibody was provided by Amgen Pharma-ceuticals, and C-terminal–specific polyclonal rabbit anti–human c-Mplantibody was purchased from Millipore. The mouse monoclonal antipolyu-biquitin antibody was purchased from Biomol. Rabbit anti–c-Cbl, rabbitanti-phospho–signal transducer and activator of transcription 5 (STAT5)and rabbit anti-STAT5 were purchased from Cell Signaling Technologies.Monoclonal mouse antiactin was purchased from Sigma-Aldrich. Second-ary antibodies, goat anti–rabbit horseradish peroxidase and rabbit anti–mouse horseradish peroxidase were purchased from Santa Cruz Biotechnol-ogy and fluorescently labeled goat anti–rabbit 488 was purchased fromInvitrogen. Membrane-impermeable sulfo-N-hydroxysuccinimido-biotin andneutravidin-coupled agarose beads were purchased from Pierce. Recombi-nant human (rh) Tpo was a gift from Don Foster (Zymogenetics,Seattle, WA).

Cell lines, cell culture, and platelet isolation

The interleukin-3 (IL-3)–dependent prolymphoid cell line BaF3 expressinghuman c-Mpl (BaF-Mpl) was maintained in RPMI1640 medium (Invitro-gen) containing 10% fetal bovine serum (FBS) supplemented with IL-3(2 �L/mL of conditioned medium from IL-3–producing baby hamsterkidney cells). The retroviral packaging cell line Platinum-E was maintainedin Dulbecco modified Eagle medium supplemented with 10% FBS. Togenerate BaF-Mpl clones, subconfluent Platinum-E cells were transfectedwith pMX-puro-c-Mpl constructs using Lipofectamine 2000 transfectionreagent (Invitrogen) for 48 hours, prior to viral supernatant collection. BaF3cells lines were then incubated in viral supernatant supplemented with IL-3for 48 hours before selection with puromycin (2 �g/mL; Invitrogen).Clonal populations were generated by limiting dilution. Total c-Mpl proteinexpression was determined by Western blot and cell surface expression wasconfirmed using flow cytometry for membrane-localized c-Mpl as previ-ously described.13 Platelets were isolated after collection of venous bloodfrom healthy volunteers and resuspended in Walsh buffer (137mM NaCl,20mM N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid, 5.6mM dex-trose, 1 mg/mL bovine serum albumin, 1mM MgCl2, 2.7mM KCl, 3.3mMNaH2PO4, pH 7.4) prior to treatment.

Receptor constructs

Wild-type human c-Mpl (NM_005373.1) was cloned into the retroviralexpression vector pMX-puro using EcoRI and XhoI cloning sites. The c-Mplintracellular domain point mutations (c-Mpl K553R, c-Mpl K573R, andc-Mpl K553 � 573R) were introduced using the QuikChange mutagenesis kit(Stratagene) with the following oligonucleotides: K553R forward primer:5�-CCTGAGCCCGCCCAGGGCCACAGTC-3�, K553R reverse primer:5�-GACTGTGGCCCTGGGCGGGCTCAGG-3�, K573R forward primer:5�-CTTGAAATCCTCCCCAGGTCCTCAGAGAGGACTCC-3�; andK573R reverse primer: 5�-GGAGTCCTCTCTGAGGACCTGGGGAG-GATTTCAAG-3�. Mutations were confirmed by sequencing of genomicDNA isolated from individual clones. Wild-type mouse c-Cbl(NM_007619.2) cDNA was amplified from the cDNA of BaF3-Mpl andwas cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen)using NotI and XhoI cloning sites. The c-Cbl point mutation in theRING-finger domain (c-Cbl C379A) was introduced using the QuikChangemutagenesis kit (Stratagene) with the following oligonucleotides: C379A

forward primer: 5�-GCTATCAACAAGGCGGAGGTGCCACTGCTAA-CCCTGTGGCC-3�, C379A reverse primer: 5�-GGCCACAGGGTTA-GCAGTGGCACCTCCGCCTTGTTGATAGC-3�.

Immunoblotting and immunoprecipitation

After chemical treatment or Tpo stimulation, BaF-Mpl cell lines were lysedin Nonidet P40 (NP-40) lysis buffer (50mM tris(hydroxymethyl)aminometh-ane-HCl, pH 7.4, 1% NP-40, 150mM NaCl, 1mM ethylenediaminetetraace-tic acid, 10mM �-glycerolphosphate, 1mM Na3VO4, 10mM NaF) contain-ing 1% protease inhibitors (Sigma-Aldrich). Denatured proteins werefractionated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electro-phoresis and transferred to polyvinylidene fluoride membranes. Proteinexpression was detected by incubating with specific antibodies andvisualized by chemiluminescent detection reagent (ECL-plus; GE Health-care). Western blots were quantified by densitometry using ImageJ analysissoftware (National Institutes of Health, http://rsbweb.nih.gov/ij). For immu-noprecipitations, cells were lysed in NP-40 lysis buffer. Supernatants wereprecleared by preincubating for 1 hour at 4°C with protein A beads(Millipore) before antibody incubation overnight. Immunoprecipitates werewashed before resuspension in Laemmli buffer (125mM tris(hydroxymethyl-)aminomethane-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol,0.004% bromophenol blue), and boiled for 5 minutes at 100°C. Sampleswere all subjected to SDS–polyacrylamide gel electrophoresis and trans-ferred to polyvinylidene fluoride membranes.

c-Mpl turnover assay

BaF-Mpl and BaF-MplK553�573R cells growing in log phase in IL-3 werewashed 3 times in phosphate-buffered saline before membrane proteinswere labeled with sulfo-N-hydroxysuccinimido-biotin for 1 hour at 4°C aspreviously described.13 The biotinylation reaction was quenched by theaddition of 100mM glycine and unbound biotinylation reagent wasremoved by repeated washing. Cells were then returned to media containing10% FBS without cytokines for 0 to 6 hours before cell lysis. Biotinylatedprotein was pulled down using neutravidin-labeled agarose beads andprecipitates were analyzed by Western blot for c-Mpl. Loading of mem-brane proteins was controlled by reprobing for the ubiquitous membranelocalized Na�/K� ATPase �1.

MTT proliferation assays

BaF-Mpl clones in log-phase growth were washed 3 times with phosphate-buffered saline to remove IL-3, resuspended in RPMI1640 supplementedwith 2% FBS, and plated into 96-well plates at a concentration of1000 cells/well. rhTpo was then added at concentrations ranging from1 pg/mL to 100 ng/mL. Cells were incubated for 48 hours before treatmentwith 2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) reagent (Sigma) and incubated at 37°C for 5 hours. Cells werelysed and formazan crystals dissolved in 100 �L of MTT lysis solution(dH2O, 20% SDS, 40% N,N-dimethyl formamide, 2% acetic acid, 0.2%HCl) for 6 hours at 37°C. Absorbance was then read on a colorimetric platereader at 570 nm. Each data point is expressed as a percentage ofproliferation stimulated by a maximal dose of murine IL-3 (4 �L/mLmurine IL-3 supernatant). Each experiment was performed in triplicate with2 different clones for each wild-type or mutant c-Mpl construct.

RNA interference

siRNA to murine c-Cbl was purchased from Thermo Scientific. A combina-tion of 4 specific siRNAs was used (siGenome SMARTpool M-040165-00-0005): (1) GAUCUGACCUGCAAUGAUU, (2) GGAGACACUUUCCG-GAUUA, (3) GGCGAAACCUGACCAAAUU, and (4) GAAGAGG-ACACAGAAUAUA. siRNA was transfected into cells using an Amaxanucleofector (Lonza) as described previously.13 Protein knockdown wasanalyzed by Western blot 48 hours after transfection.

c-Mpl UBIQUITINATION AND PROTEASOMAL DEGRADATION 1255BLOOD, 11 FEBRUARY 2010 � VOLUME 115, NUMBER 6




Results

Tpo stimulation mediates proteasomal and lysosomaldegradation of c-Mpl

First, we determined whether c-Mpl was degraded via the proteaso-mal and/or lysosomal pathways after Tpo stimulation. Afterincubation for 12 hours in starvation medium (RPMI, 2% FBSwithout cytokines), BaF-Mpl cells were treated with the protea-some inhibitor MG-132, or the lysosome inhibitor NH4Cl, incombination with cycloheximide (Chx) to prevent de novo proteinsynthesis, and rhTpo for the times indicated in Figure 1.

The mature cell surface c-Mpl receptor displays a molecularweight of 85 kDa due to extensive glycosylation, compared withthat localized to the secretory pathway, which is an 80-kDaform.13,24 In the absence of rhTpo, BaF-Mpl cells slowly degradedthe surface receptor form of c-Mpl protein (Figure 1A). In contrast,cells treated with rhTpo exhibited extensive degradation of maturec-Mpl protein within 60 minutes, which was greatly inhibited bypretreatment with MG-132. Although the expression of c-Mpl didnot vary between control BaF-Mpl cells and those pretreated withthe lysosomal inhibitor NH4Cl for the first 60 minutes after theaddition of Tpo to the cultures, significantly less receptor degrada-tion was observed between 60 and 120 minutes. To confirm thesefindings in a more physiologically relevant setting, we purified andstudied murine megakaryocytes from C57/Bl6 mice (Figure 1B left

panel). In contrast to BaF-Mpl cells, murine megakaryocytesdisplay proportionately far more of the mature 85-kDa protein thanthe immature intracellular 80-kDa form, which was almost absent.Megakaryocytic c-Mpl protein was significantly reduced after60 minutes of Tpo stimulation, which was blocked by adding bothMG-132 and NH4Cl. Interestingly, c-Mpl was not degraded afterTpo stimulation of isolated human platelets (Figure 1B right panel).

Tpo stimulation causes ubiquitination of c-Mpl

As proteasomal degradation of proteins requires polyubiquitina-tion, we next examined the effects of Tpo stimulation on c-Mplubiquitination. BaF-Mpl cells were treated with MG-132 for135 minutes and Tpo was added 10 to 120 minutes before the endof incubation with MG-132. Control cells were incubated withMG-132 alone for 135 minutes. Whole-cell lysates were immuno-precipitated with an anti-Mpl antibody and analyzed by Westernblotting using an antiubiquitin antibody. Ubiquitinated c-Mpl wasdetected 10 to 30 minutes after Tpo stimulation as a high-molecular-weight smear (Figure 2A). Using murine megakaryocytes we alsofound increased c-Mpl ubiquitination 60 minutes after Tpo stimula-tion (Figure 2B). Next we determined the role of Janus kinase 2(JAK2) in Tpo-stimulated c-Mpl ubiquitination by pretreatingBaF-Mpl cells with the JAK2 inhibitor JAKI for 30 minutes beforeTpo stimulation. We found no significant difference in Tpo-stimulated c-Mpl ubiquitination in the presence of JAKI (Figure2C). The effectiveness of JAKI was determined by performing

Figure 1. 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 ofnew 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 independentexperiments.

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Western blots for phospho-STAT5, which is directly phosphory-lated by active JAK2. We found that the inhibitor was effective infully blocking STAT5 phosphorylation.

To further investigate the effects of monoubiquitination and polyubiq-uitination on the function of c-Mpl, we mutated the 2 intracellular lysine(K) residues K553 and K573 in the wild-type receptor to arginine (R;Figure 3A). We next generated 2 wild-type c-Mpl and 2 mutantc-MplK553�573R clones and confirmed equal total expression of c-Mplprotein by Western blotting (Figure 3B) as well as equal cell surfaceexpression by flow cytometry (data not shown). Receptors bearing only1 mutation (c-MplK553R and K573R) exhibited normal ubiquitination,indicating that both lysines are sites of ubiquitination (Figure 3C). Bymutating both intracellular lysine residues, we prevented Tpo-stimulatedubiquitination of c-Mpl (Figure 3D). All of the 3 mutated cell lines(K553R, K573R, and K553�573R) exhibited normal Tpo-stimulated c-Mplinternalization compared with control cells (data not shown). Next, wedetermined whether ubiquitination of c-Mpl is involved in the turnoverof mature, cell surface–localized receptor in the absence of Tpo, usingcells expressing wild-type (WT)–Mpl and MplK553�573R. To detect the

level of cell surface c-Mpl, we developed a sensitive membrane proteinbiotinylation assay, in which all surface proteins are first biotinylated,and after various time intervals, the cells were lysed, the remainingsurface proteins were specifically captured with avidin beads, and c-Mplwas recognized by Western blotting. We found a significant decrease inthe normal cell surface turnover of c-MplK553�573R compared with WTc-Mpl (Figure 3E). For example, after 4 hours, 58% (� 12%) ofc-MplK553�573R remained on the cell surface, compared with just 22%(� 2%) of WT c-Mpl. A similar pattern was observed at 6 hours(c-MplK553�573R 34% (� 7%) versus WT c-Mpl 6% (� 1%).

BaF-MplK553�573R cells are hyperproliferative in response toTpo

Next, we used the BaF-Mpl and BaF-MplK553�573R cells todetermine the biologic significance of c-Mpl ubiquitination onTpo-stimulated proliferation and signaling. Treatment with rhTpocaused the cells to proliferate in a concentration-dependent mannerin all clones. Overall, BaF-MplK553�573R cells displayed increased

Figure 2. 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 wasanalyzed 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) Bonemarrow–derived megakaryocytes were pretreated with MG-132 for 0 or 60 minutes, with or without rhTpo. Megakaryocyte c-Mpl ubiquitination was analyzed as described inpanel 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 for30 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 byanalyzing levels of phosphor (p)–STAT5. Data are representative of 2 independent experiments.





Tpo-stimulated proliferation compared with BaF-Mpl cells. Atsubmaximal concentrations of rhTpo (0.1 ng/mL), BaF-MplK553�573R reached 53% (� 1.3%) of IL-3–induced maximalcell proliferation compared with 28% (� 1.5%) in BaF-Mpl. Atmaximal concentrations of rhTpo (1 ng/mL), BaF-MplK553�573Rcells proliferated to 98% (� 3.5%) of IL-3–induced maximalconcentration, compared with 80% (� 2.8%) in BaF-Mpl cells

(Figure 4A). To determine whether Tpo-induced signaling ofc-MplK553�573R cells differed from BaF-Mpl cells, we examinedseveral major signaling pathways normally stimulated by Tpo,including JAK2, STAT5, extracellular signal-related kinase 1/2,and phosphoinositide-3 kinase/AKT. Levels of activation at increas-ing concentrations of Tpo and signaling kinetics over a period of180 minutes were determined, and no significant difference in

Figure 3. 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-MplK553�573R clones. (C) Cellsexpressing 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-Mplubiquitination after 60-minute Tpo stimulation in BaF cells expressing WT c-Mpl or c-MplK553�573R. 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-MplK553�573R to determine normal turnover over a time period of 6 hours. TheWestern 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 determinedby densitometry, compared with time 0 (100%). Protein loading is standardized by comparison to surface Na�/K� ATPase �1 expression. The data points represent themean � SE of 3 independent experiments (*P � .05, ***P � .001; Student t test).





these signaling pathways was observed (data not shown). However,c-Mpl phosphorylation was prolonged in BaF-Mpl cells pretreatedwith MG-132 60 minutes after Tpo stimulation (Figure 4B). Inaddition, compared with BaF-Mpl cells, BaF-MplK553�573R cellsexhibited greatly increased (5 minutes) and prolonged (60 minutes)c-Mpl phosphorylation after Tpo stimulation. In contrast, MG-132had no effect on prolonging phosphorylation in BaF-MplK553�573Rcells. We next investigated the effects of c-MplK553�573R on c-Mpldegradation. BaF-Mpl and BaF-MplK553�573R cells were pretreatedwith Chx for 60 minutes, prior to stimulation with rhTpo for 0 to120 minutes before c-Mpl protein was analyzed by Westernblotting (Figure 4C-D). After 60 minutes, 49% of the wild-typec-Mpl protein remained, whereas 89% of the c-MplK553�573R wasstill present in Chx- and Tpo-treated cells. Degradation ofc-MplK553�573R continued after 60 minutes, with only 32% ofc-Mpl still detectable after 120 minutes.

C-Cbl acts as an E3 ligase in the ubiquitination of c-Mpl

E3 ubiquitin ligases are required to covalently attach ubiquitin tolysine residues of target proteins. C-Cbl has previously been shownto act as an E3 ligase and is expressed in megakaryocytes and otherhematopoietic cells. C-Cbl can also be activated by Tpo.25,26 Toexamine whether c-Cbl is involved in the degradation of c-Mpl, wefirst used specific siRNA to attenuate c-Cbl protein expression inBaF-Mpl cells (Figure 5A). Reduced c-Cbl protein expressionresulted in a marked reduction of c-Mpl ubiquitination after Tpostimulation compared with nontargeted siRNA-treated controlBaF-Mpl cells (Figure 5B). We then determined the effects of c-CblsiRNA on Tpo-stimulated c-Mpl degradation by preincubatingnontargeted and c-Cbl siRNA-treated cells with Chx for 30 minutes

before stimulating with Tpo for up to 60 minutes. After 60 minutes,82% of mature (85-kDa) c-Mpl remained in the c-Cbl siRNA-treated cells, compared with 49% in the sample treated with anontarget siRNA (Figure 5C-D top panels). c-Cbl siRNA experi-ments were also performed on BaF-MplK553�573R cells, whichcannot be ubiquitinated (Figure 3D). In these cells, c-Cbl siRNAhad no significant effect on Tpo-stimulated degradation (Figure5C-D bottom panels). To confirm that c-Cbl is an E3 ligase forc-Mpl and not part of an indirect ubiquitination pathway, wedetermined the effects of c-CblC379A on c-Mpl turnover, a RING-finger domain E3 function inactivating mutant of c-Cbl. StableWT-c-Cbl or c-CblC379A–overexpressing BaF-Mpl cells (Figure6A) were first analyzed for Tpo-stimulated c-Mpl ubiquitinationafter 60 minutes after MG-132 pretreatment for 30 minutes. Wefound that ubiquitination was markedly reduced in cells thatoverexpressed c-CblC379A (Figure 6B) compared with wild-typec-Cbl. To examine whether overexpression of c-CblC379A wouldmimic the hyperproliferative phenotype found in BaF-MplK553�573Rcells (Figure 4A), we performed an MTT proliferation assay withBaF-Mpl cells overexpressing the 2 forms of c-Cbl. We found asignificant increase in cell growth at 1 and 10 ng/mL of rhTpo inBaF-Mpl/c-CblC379A compared with cells overexpressing wild-type c-Cbl (Figure 6C), consistent with our findings in theBaF-MplK553�573R cells.

Discussion

After ligand-induced activation of growth factor receptors,several negative feedback mechanisms modulate the strength

Figure 4. c-MplK553�573R exhibits altered Tpo-induced proliferation and degradation. (A) MTT proliferation assay using BaF cells expressing WT c-Mpl or c-Mpl553�573R.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 clonesfor 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 individualexperiments. (C) Western blot analysis of Tpo-induced c-Mpl degradation of WT c-Mpl and c-MplK553�573R. Cells were pretreated with Chx for 60 minutes before Tpostimulation for up to 120 minutes. (D) Graphic representation of c-Mpl degradation, comparing mature (85-kDa) c-Mpl at time 0 using densitometry.





and duration of intracellular signaling events to attain theappropriate cellular response.27-29 Lack or gain of function canlead to uncontrolled cell growth and malignancies.30 Althoughnegative regulatory mechanisms have been extensively studiedfor several different membrane receptors, the mechanisms ofregulation of activated c-Mpl after Tpo stimulation, in particular

those of receptor degradation, are largely unknown. In thiswork, we demonstrate that c-Mpl is degraded by both proteaso-mal and lysosomal pathways after Tpo stimulation and thatinhibition of receptor ubiquitination (and consequently proteaso-mal degradation) results in a hyperproliferative phenotype inhematopoietic cell lines.

Figure 5. 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 hoursafter 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 of4 individual experiments. (C) Tpo-stimulated c-Mpl degradation was analyzed by Western blot using lysates from BaF-WT-Mpl and BaF-MplK553�573R cells treated withc-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 of3 independent experiments (*P � .05, **P � .01; Student t test).





Previous work on the erythropoietin receptor (EpoR), withwhich c-Mpl shares significant homology, has similarly shownroles for both proteasomal and lysosomal degradation of thereceptor after stimulation with Epo.31 Indeed, kinetics of bothdegradation and ubiquitination of the receptors after activation alsoseem similar. However, Walrafen et al found that c-Cbl–specificsiRNA and overexpression of mutated c-Cbl had no effect on EpoRubiquitination.31 In this study, we demonstrated that c-Cbl, whichcan act as an E3 ligase, is important for Tpo-mediated ubiquitina-tion of c-Mpl. Taken together, these 2 findings suggest thatdifferent molecular pathways mediate the ubiquitination of the2 receptors. Although we were unable to demonstrate directinteractions between c-Mpl and c-Cbl by coimmunoprecipitation(data not shown), this may be due to the transient nature of theinteraction between c-Cbl and its targets, or may indicate that anadaptor protein is required to associate c-Cbl and the c-Mplreceptor. The phosphatase SHP2 has been identified as an adaptorprotein for the signaling subunit of the IL-6 receptor, gp130, andc-Cbl, enabling the ubiquitination of the receptor by c-Cbl.32 Wehave found that SHP2 can be recruited to activated c-Mpl (I.S.H.,unpublished data), making a similar mechanism for the recruitmentof c-Cbl to c-Mpl plausible. Interestingly, we also found thatTpo-stimulated c-Mpl ubiquitination appears to be a JAK2-independent pathway. JAK2-independent c-Mpl ubiquitinationalso supports our finding that ubiquitination and degradation ofc-Mpl occur in the absence of cytokines. The results presented inFigure 3E suggest that surface-localized c-Mpl has a level ofbackground turnover in which the majority of mature receptor isreplaced roughly every 6 hours. Presumably, stimulation with Tpogreatly increases the kinetics of this turnover, by stimulating thesame or different internalization and degradation pathways. It

should be noted that reduced c-Cbl function, either by siRNA oroverexpression of c-CblC379A, reduced rather than entirely pre-vented c-Mpl ubiquitination. Therefore it is highly likely that otherubiquitin E3 ligases, of which there are hundreds, also contribute toc-Mpl ubiquitination. We are currently investigating the signalingpathways responsible for unstimulated and Tpo-stimulated c-Mplturnover, c-Mpl and c-Cbl interactions, and identification of otherE3 ligases potentially involved in c-Mpl ubiquitination.

Because monoubiquitination of some membrane receptors haspreviously been described as critical for their internalization(reviewed in Hitchcock et al13), we addressed this question using aBaF-MplK553�573R cell model, which cannot be ubiquitinated inthe intracellular domain. Using this cell line, we found thatinternalization of c-Mpl in response to Tpo was normal, demonstrat-ing that alternative mechanisms are required for c-Mpl internaliza-tion. We recently described the importance of the intracellularmotif Y591RRL for the recruitment of adaptor protein, which allowsthe rapid internalization of c-Mpl from the cell surface in aclathrin-dependent manner.33-37 Other receptors, such as those forepidermal growth factor, growth hormone, leptin, and transferrin,require ubiquitination for clathrin-dependent internalization.38,39

These receptors contain conserved intracellular tyrosine, serine,and lysine residues that are necessary for their ubiquitin-dependentinternalization.6 In the case of c-Mpl, the replacement of bothintracellular lysine residues with arginine did not alter the internal-ization kinetics. Consequently, we conclude that the ubiquitinationof c-Mpl is not required for its internalization.

We also identified a role for c-Mpl ubiquitination as a negativefeedback regulator of Tpo signaling. BaF-MplK553�573R cells,which cannot undergo ubiquitination in the intracellular domain,are hyperproliferative in response to Tpo. Although we found no

Figure 6. 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 Westernblot. The data shown are representative of 3 independent experiments. (C) These cells were analyzed for proliferation using an MTT assay at increasing concentrations ofrhTpo. The data shown represent mean (� SE) of 3 independent experiments (*P � .05, ***P � .001).





significant difference in the major signaling pathways betweenc-MplK553�573R and WT-c-Mpl (data not shown), we did findincreased and prolonged receptor phosphorylation. The prolongedpresence of phosphorylated docking sites for other signalingproteins, particularly at intracellular Y625 and Y631, known bindingsites for STATs and adaptor proteins growth factor receptor-boundprotein 2, Son of sevenless, and SHC (reviewed in Rathinam etal40), may explain the observed hyperproliferative phenotype.Confirming our observations with the mutated c-Mpl, we found asimilar, but not as potent, hyperproliferative phenotype afteroverexpression of c-Cbl with a disabled RING-finger domain.These findings are concurrent with the study by Rathinam et al, inwhich they described a hyperproliferative phenotype in hematopoi-etic stem cells that lack endogenous c-Cbl and instead expressc-Cbl with an inactive RING-finger domain. The researchersdescribed increased levels of STAT5 phosphorylation after Tpostimulation and subsequently augmented c-Myc expression.41,42

Not only do our data support these findings, we hypothesize thatone of the roles of c-Cbl is to act directly on c-Mpl as an ubiquitinE3 ligase, therefore mediating signaling after stimulation with Tpo.We are currently investigating the role of c-Cbl in the ubiquitina-tion of c-Mpl in hematopoietic stem cells and primary maturemegakaryocytes.

Interestingly, novel mutations affecting the E3 ligase functionof c-Cbl have recently been described in patients with acutemyeloid leukemia, suggesting a significant role for c-Cbl as ahematopoietic oncogene. In this regard, it would be extremelyinteresting to determine whether identical or different mutations ofc-Cbl are present in the myeloproliferative disorders that are relianton Tpo signaling, such as essential thrombocythemia and primarymyelofibrosis.

Our studies provide novel insights into the regulatory mecha-nisms of Tpo signaling, determine the intracellular lysine residuesof c-Mpl that are ubiquitinated after Tpo stimulation, and identify arole for c-Cbl as an E3 ubiquitin ligase for c-Mpl. Our findingsprovide novel insights into the regulation of Tpo signaling anddegradation of c-Mpl. Similar mechanisms may also be of impor-tance in regulating the signaling of numerous hematopoieticgrowth factors, critically affecting the proliferation and differentia-tion of hematopoietic lineages.

Acknowledgment

This research was supported by National Institutes of Health grantsR01DK049855 and P01 HL078784-04.

Authorship

Contribution: All authors have substantially contributed to thecontent of the paper and have agreed to the submission in its currentformat; V.S. performed research and analyzed data; A.E.G. andK.K. designed experiments, interpreted data, and edited the manu-script; and S.J.S. and I.S.H. designed and performed research,analyzed data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Ian S. Hitchcock, Department of Medicine,University of California San Diego, 9500 Gilman Dr, MD 0726, LaJolla, CA 92093; e-mail: [email protected].

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online October 30, 2009 originally publisheddoi:10.1182/blood-2009-06-227033

2010 115: 1254-1263

Sebastian J. Saur, Veena Sangkhae, Amy E. Geddis, Kenneth Kaushansky and Ian S. Hitchcock Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

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