A Truncated Isoform of c-Mpl with an Essential C-terminal Peptide Targets the Full-length Receptor for Degradation
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m401386200
ISSN1083-351X
AutoresJörn Coers, Christina Ranft, Radek C. Skoda,
Tópico(s)HER2/EGFR in Cancer Research
ResumoThrombopoietin and its cognate receptor c-Mpl are the primary regulators of megakaryopoiesis and platelet production. They also play an important role in the maintenance of hematopoietic stem cells. Here, we have analyzed the function of a truncated Mpl receptor isoform (Mpl-tr), which results from alternative splicing. The mpl-tr variant is the only alternate mpl isoform conserved between mouse and humans, suggesting a relevant function in regulating Mpl signaling. Despite the presence of a signal peptide and the lack of a transmembrane domain, Mpl-tr is retained intracellularly. Our results provide evidence that Mpl-tr exerts a dominant-negative effect on thrombopoietin-dependent cell proliferation and survival. We demonstrate that this inhibitory effect is due to down-regulation of the full-length Mpl protein. The C terminus of Mpl-tr, consisting of 30 amino acids of unique sequence, is essential for the suppression of thrombopoietin-dependent proliferation and Mpl protein down-regulation. Cathepsin inhibitor-1 (CATI-1), an inhibitor of cathepsin-like cysteine proteases, counteracts the effect of Mpl-tr on Mpl protein expression, suggesting that Mpl-tr targets Mpl for lysosomal degradation. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function through a proteolytic process directed by a truncated isoform of the same receptor. Thrombopoietin and its cognate receptor c-Mpl are the primary regulators of megakaryopoiesis and platelet production. They also play an important role in the maintenance of hematopoietic stem cells. Here, we have analyzed the function of a truncated Mpl receptor isoform (Mpl-tr), which results from alternative splicing. The mpl-tr variant is the only alternate mpl isoform conserved between mouse and humans, suggesting a relevant function in regulating Mpl signaling. Despite the presence of a signal peptide and the lack of a transmembrane domain, Mpl-tr is retained intracellularly. Our results provide evidence that Mpl-tr exerts a dominant-negative effect on thrombopoietin-dependent cell proliferation and survival. We demonstrate that this inhibitory effect is due to down-regulation of the full-length Mpl protein. The C terminus of Mpl-tr, consisting of 30 amino acids of unique sequence, is essential for the suppression of thrombopoietin-dependent proliferation and Mpl protein down-regulation. Cathepsin inhibitor-1 (CATI-1), an inhibitor of cathepsin-like cysteine proteases, counteracts the effect of Mpl-tr on Mpl protein expression, suggesting that Mpl-tr targets Mpl for lysosomal degradation. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function through a proteolytic process directed by a truncated isoform of the same receptor. Cytokine receptor signaling has profound effects on cell survival, proliferation, and differentiation of the receiving cell (1Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1136) Google Scholar). It is therefore not surprising that components of the signaling cascade are tightly regulated at several levels. An important mechanism for controlling gene expression is alternative splicing, allowing the synthesis of structurally and functionally distinct protein isoforms (2Roberts G.C. Smith C.W. Curr. Opin. Cell Biol. 2002; 6: 375-383Crossref Scopus (110) Google Scholar). Many alternative splice variants of different cytokine receptors have been described, but the function of most of the resulting protein isoforms remains unknown. Cytokine receptor isoforms may be classified according to the presence or absence of a transmembrane domain. Isoforms lacking a transmembrane domain are often termed "soluble cytokine receptors" and can fulfill different physiological functions (3Heaney M.L. Golde D.W. Blood. 1996; 87: 847-857Crossref PubMed Google Scholar, 4Fernandez-Botran R. FASEB J. 1991; 5: 2567-2574Crossref PubMed Scopus (256) Google Scholar). In general, soluble receptors may function as agonists by stabilizing their ligands, e.g. growth hormone and tumor necrosis factor (5Veldhuis J.D. Johnson M.L. Faunt L.M. Mercado M. Baumann G. J. Clin. Investig. 1993; 91: 629-641Crossref PubMed Scopus (103) Google Scholar, 6Aderka D. Engelmann H. Maor Y. Brakebusch C. Wallach D. J. Exp. Med. 1992; 175: 323-329Crossref PubMed Scopus (761) Google Scholar), or contrarily act as antagonists by competing with the membrane-bound receptor for ligand binding, e.g. epidermal growth factor and interleukin (IL)-1 1The abbreviations used are: IL, interleukin; ALLN, N-acetyl-Leu-Leu-Nle-CHO; CHO, Chinese hamster ovary; CATI-1, cathepsin inhibitor-1; CT, threshold cycles; EpoR, erythropoietin receptor; EST, (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte-macrophage colony-stimulating factor; c-mpl, cellular homolog of the myeloproliferative leukemia oncogene; TPO, thrombopoietin; Q-PCR, quantitative-PCR; XTT, 2,3-bis[2-methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5-carboxanilide; Z, benzyloxycarbonyl; Bz, benzophenone.1The abbreviations used are: IL, interleukin; ALLN, N-acetyl-Leu-Leu-Nle-CHO; CHO, Chinese hamster ovary; CATI-1, cathepsin inhibitor-1; CT, threshold cycles; EpoR, erythropoietin receptor; EST, (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte-macrophage colony-stimulating factor; c-mpl, cellular homolog of the myeloproliferative leukemia oncogene; TPO, thrombopoietin; Q-PCR, quantitative-PCR; XTT, 2,3-bis[2-methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5-carboxanilide; Z, benzyloxycarbonyl; Bz, benzophenone. (7Basu A. Raghunath M. Bishayee S. Das M. Mol. Cell. Biol. 1989; 9: 671-677Crossref PubMed Scopus (113) Google Scholar, 8Arend W.P. Malyak M. Smith M.F. Whisenand T.D. Slack J.L. Sims J.E. Giri J.G. Dower S.K. J. Immunol. 1994; 153: 4766-4774PubMed Google Scholar). Soluble receptors can arise from alternative splicing or from proteolytic receptor shedding on the cell surface. Isoforms generated by alternative splicing often contain additional protein sequence due to unspliced intron sequence and/or a shift of the reading frame. Generally, no biological function has been attributed to these additional stretches of amino acids. Thrombopoietin (TPO) and its receptor "cellular homolog of myeloproliferative leukemia" (c-Mpl) are the primary regulators of megakaryopoiesis (9Kaushansky K. Drachman J.G. Oncogene. 2002; 21: 3359-3367Crossref PubMed Scopus (142) Google Scholar). The c-mpl gene is composed of 12 exons (see Fig. 1A) (10Mignotte V. Vigon I. Boucher de Crevecoeur E. Romeo P.H. Lemarchandel V. Chretien S. Genomics. 1994; 20: 5-12Crossref PubMed Scopus (86) Google Scholar). In the mouse, two distinct alternate mRNA isoforms are known. The transmembrane variant mpl-II is due to usage of a cryptic splice acceptor in exon 4 resulting in an in-frame deletion of 60 amino acids (11Sabath D.F. Lofton-Day C. Lin N. Lok S. Kaushansky K. Broudy V.C. Biochim. Biophys. Acta. 2002; 1574: 383-386Crossref PubMed Scopus (2) Google Scholar). No function has yet been assigned to this isoform. The second mRNA variant encodes a truncated soluble receptor, Mpl-tr, and is the only one found both in human and mouse. This variant results from splicing of exon 8 directly to exon 11, eliminating the juxtamembrane extracellular part and the transmembrane domain (12Skoda R.C. Seldin D.C. Chiang M.K. Peichel C.L. Vogt T.F. Leder P. EMBO J. 1993; 12: 2645-2653Crossref PubMed Scopus (166) Google Scholar, 13Vigon I. Florindo C. Fichelson S. Guenet J.L. Mattei M.G. Souyri M. Cosman D. Gisselbrecht S. Oncogene. 1993; 8: 2607-2615PubMed Google Scholar). Due to an altered reading frame at the splice acceptor site of exon 11, Mpl-tr protein terminates in a short stretch of novel amino acid sequences (see Fig. 1). mpl-tr mRNA accounts for ∼30% of mpl mRNA in mouse spleen (12Skoda R.C. Seldin D.C. Chiang M.K. Peichel C.L. Vogt T.F. Leder P. EMBO J. 1993; 12: 2645-2653Crossref PubMed Scopus (166) Google Scholar). Despite the presence of a signal sequence and the lack of a transmembrane domain, Mpl-tr is not secreted into the cell supernatant when ectopically expressed in cell line (12Skoda R.C. Seldin D.C. Chiang M.K. Peichel C.L. Vogt T.F. Leder P. EMBO J. 1993; 12: 2645-2653Crossref PubMed Scopus (166) Google Scholar). In humans, two alternate mRNA mpl species are known in addition to mpl-tr. The mpl-K variant is due to a readthrough beyond the exon 10 splice donor site (14Vigon I. Mornon J.P. Cocault L. Mitjavila M.T. Tambourin P. Gisselbrecht S. Souyri M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5640-5644Crossref PubMed Scopus (463) Google Scholar). The resulting K-form of the receptor diverges from the native sequence after the ninth cytoplasmic amino acid and terminates within intron 10. mpl-del, a second isoform, arises as a consequence of alternative splicing between exons 8 and 9 and encodes a protein with an in-frame deletion of 24 amino acids and unknown function (15Li J. Sabath D.F. Kuter D.J. Cytokine. 2000; 12: 835-844Crossref PubMed Scopus (20) Google Scholar). Because of the lack of secretion of Mpl-tr, we analyzed whether Mpl-tr plays a physiological role intracellularly. Here, we demonstrate that Mpl-tr specifically inhibits TPO-dependent proliferation and survival. We show that Mpl-tr is responsible for initiating protein down-regulation of the full-length Mpl receptor by a cathepsin-like cysteine protease activity. As a consequence, the amount of total Mpl protein in the cell is drastically reduced. Further, our data show that for this effect, a short peptide sequence at the C terminus of Mpl-tr is essential. The ability of Mpl-tr to antagonize Mpl function represents a novel mechanism by which cytokine signaling is regulated. DNA Constructs—The plasmid pCD4 (pMICD4) is a gift from Dr. Harvey A. Lodish. It contains an internal ribosomal entry site followed by a truncated cDNA of the human CD4 gene and is derived from the retroviral expression vector pMX (16Liu X. Constantinescu S.N. Sun Y. Bogan J.S. Hirsch D. Weinberg R.A. Lodish H.F. Anal. Biochem. 2000; 280: 20-28Crossref PubMed Scopus (122) Google Scholar). To generate pCD4-mpl-tr, mpl-tr was cloned into the restriction sites XhoI and blunted BamHI of the multiple cloning site of pCD4. For the generation of the mpl-tr mutants, site-directed mutagenesis was performed using the QuikChange XL mutagenesis kit (Stratagene, Cedar Creek, TX) according to the manufacturer's protocol. The following primers were used: 5′-GAAGGCCGTGAGGACTGGAAGTAGACTGAGGCAAGCTTTGTGG-3′ (sense) and 5′-CCACAAAGCTTGCCTCAGTCTACTTCCAGTCCTCACGGCCTTC-3′ (antisense) for the stop codon in Δpep30; 5′-GAAGGCCGTGAGGACTGGAAGAGACTGAGGCAAGCTTTGTGG-3′ (sense) and 5′-CCACAAAGCTTGCCTCAGTCTCTTCCAGTCCTCACGGCCTTC-3′ (antisense) for the frameshift in tr-pepmpl; 5′-GCCCTAAGTCCTTCTTAAGGCCACGGTTACCGATAGCTGTG-3′ (sense) and 5′-CACAGCTATCGGTAACCGTGGCCTTAAGAAGGACTTAGGGC-3′ (antisense) for the stop codon in tr-pepmpl. mpl, mpl-tr, and pep30 cDNAs were cloned into the 5Myc-pcDNA1 vector (gift from Dr. Eva Reinhard, Biozentrum, University of Basel), which contains at its 5′ end a sequence encoding a hemagglutinin signal sequence followed by five Myc epitopes (17Bormann P. Roth L.W. Andel D. Ackermann M. Reinhard E. Mol. Cell Neurosci. 1999; 13: 167-179Crossref PubMed Scopus (41) Google Scholar). For stable transfections, Myc-tagged mpl cDNA was cloned into the pGD expression vector (18Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1908) Google Scholar) as a XhoI-NotI fragment. For transient transfections into human kidney 293T cells, mouse mpl, Myc-mpl, and mpl-tr cDNAs were subcloned into the pcDNA3 expression vector (Invitrogen) as XhoI-NotI fragments. Cell Transfection and Culture—BaF3 cells (19Palacios R. Steinmetz M. Cell. 1985; 41: 727-734Abstract Full Text PDF PubMed Scopus (583) Google Scholar) were cultured as described (20Stoffel R. Wiestner A. Skoda R.C. Blood. 1996; 87: 567-573Crossref PubMed Google Scholar). UT-7 cells (21Komatsu N. Nakauchi H. Miwa A. Ishihara T. Eguchi M. Moroi M. Okada M. Sato Y. Wada H. Yawata Y. Suda T. Miura Y. Cancer Res. 1991; 51: 341-348PubMed Google Scholar) were grown in RPMI 1640 supplemented with 10% fetal calf serum and 2 ng/ml recombinant human granulocyte-macrophage colony stimulatory factor (GM-CSF) (PromoCell, Heidelberg, Germany). For transfections of BaF3 and UT-7 cells, 0.5–1 × 107 cells were electroporated at 270 V and 975 microfarads at ambient temperature in the presence of 20 μg of plasmid. UT-7/Myc-mpl cells were cultured in the presence of 450 μg/ml G418. A pool of stably transfected UT-7/Myc-mpl cells was used for the transfection with different pCD4 constructs. The BaF3/mpl cell clone TM17, which has been described (20Stoffel R. Wiestner A. Skoda R.C. Blood. 1996; 87: 567-573Crossref PubMed Google Scholar), was used for transfections with different pCD4 constructs. Cells expressing human CD4 were selected by the usage of anti-CD4 microbeads according to the manufacturer's protocol (Miltenyi, Auburn, CA). mpl-tr conditioned medium was obtained from UT-7 cells expressing pCD4-mpl-tr, which were cultured at exponential growth phase in human GM-CSF for 2 days. Control medium was harvested from untransfected UT-7 cells. For transient transfections of 293T cells, the transfection reagent FuGENE was used according to the manufacturer's protocol (Roche Applied Science). Where indicated, protease inhibitors (Calbiochem) were added at a final concentration of 25 μm each, and cells were cultured for 6–8 h before harvesting total cell lysate for immunoblot analysis. The inhibitors used were the cathepsin inhibitor cysteine cathepsin inhibitor I (CATI-1) (Z-Phe-Gly-NHO-Bz), the proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO), and the calpain inhibitors ALLN (N-Acetyl-Leu-Leu-Nle-CHO) and EST ((2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester). Proliferation Assay—An XTT proliferation kit (Roche Applied Science) was used according to the manufacturer's protocol to determine cytokine-dependent cell proliferation. In brief, cells were plated in 96-well plates at 104 cells/well in 100 μl of medium containing the indicated concentrations of cytokine. After 3.5 days of stimulation for BaF3 cells and 5 days of stimulation of UT7 cells, 50 μl of a 1 mg/ml stock solution of XTT with 5 mmol/liter phenazine methosulfate, an electron coupling agent, was added to each well. The product of XTT reduction by viable cells, reflecting the number of cells per well, was measured at 4 h at 450 nm. Protein Expression Analyses—Surface expression of Myc-Mpl was determined by flow cytometry. For this analysis, 5 × 105 cells were incubated with the mouse monoclonal antibody 9E10 (22Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2151) Google Scholar) directed against the N-terminal Myc tag of Myc-Mpl for 60 min on ice followed by incubation with the fluorescein isothiocyanate (FITC)-labeled goat anti-mouse antibody (BD Biosciences). Untransfected UT-7 cells served as a control. Total amounts of c-Mpl and Mpl-tr were determined by immunoblot analysis using a purified rabbit polyclonal antibody directed against Mpl, as described (12Skoda R.C. Seldin D.C. Chiang M.K. Peichel C.L. Vogt T.F. Leder P. EMBO J. 1993; 12: 2645-2653Crossref PubMed Scopus (166) Google Scholar). The same polyclonal antibody and 9E10 were used for the detection of Myc-Mpl protein by immunoblot analysis. To control for protein loading, the membranes were reprobed using the mouse monoclonal antibody AC-40 directed against actin (Sigma). Pulse-chase Analysis—293T cells cultured in 60-mm dishes were transiently transfected with 3 μg of Myc-mpl and 3 μg of mpl-tr expressed off the pcDNA3 expression vector. Pulse-chase analysis was begun 40 h after transfection. To perform the pulse-chase, cell monolayers were washed twice with warm phosphate-buffered saline and starved of methionine and cysteine by incubation for 40 min at 37 °Cin 1 ml of methionine/cysteine-free Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% dialyzed fetal calf serum (Invitrogen). Following amino acid starvation, cellular proteins were pulse-labeled by incubating each plate of cells with 400 μCi of methionine/cysteine (Tran35S-label; PerkinElmer Life Sciences) for 30 min at 37 °C. The radioactive medium was then removed, and the cells were washed twice in warm phosphate-buffered saline, re-fed with Dulbecco's modified Eagle's medium supplemented with 2 mm methionine and 2 mm cysteine, and incubated for the indicated times. Cells were collected, and labeled proteins were recovered by denaturing immunoprecipitation using the 9E10 antibody and the method of Hofmann et al. (23Hofmann F. Martelli F. Livingston D.M. Wang Z. Genes Dev. 1996; 10: 2949-2959Crossref PubMed Scopus (212) Google Scholar). Immune complexes were analyzed on 10% SDS-PAGE gels. The dried gels were quantitated on a Molecular Imager FX (Bio-Rad) using Quantity One software (Bio-Rad). Apoptosis—For quantification of apoptosis, UT-7/Myc-Mpl cells were grown at 2 × 105 cells/ml in the presence of either 2 ng/ml human GM-CSF or 2 ng/ml or 20 ng/ml human TPO (gift from Dr. Frederic J. de Sauvage). After 48 h, cells were washed twice and stained with annexin V with the use of the annexin V-FITC kit (Roche Applied Science). FITC-positive cells were quantitated by flow cytometry. Generation of cDNA and Quantitative-PCR (Q-PCR)—RNA was extracted from cell lines by means of TRIzol reagent (Invitrogen). RNA was treated with RNase-free DNase (Promega, Madison, WI) for 90 min at 37 °C, heat-inactivated, and then purified with RNAeasy (Qiagen) according to the manufacturer's protocol. For reverse-transcriptase-PCR, 1 μg of RNA was reverse-transcribed after random hexamer priming in a 30-μl reaction mix containing 100 units of omniscript reverse transcriptase (Qiagen). The differential quantification of c-mpl and c-mpl-tr was performed on an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA), as described (24Kralovics R. Buser A.S. Teo S.S. Coers J. Tichelli A. van der Maas A.P. Skoda R.C. Blood. 2003; 102: 1869-1871Crossref PubMed Scopus (127) Google Scholar). The sequences for the isoform specific primers and for MGB probes were as follows: for the exon 10/11 boundary of murine c-mpl, 5′-GCAATTTCCTGCGCACTACA-3′ (sense), 5′-GGAAGCGAGGGCCACAA-3′ (antisense), 5′-GAGACTGAGGCATGC-3′ (probe); for mpl-tr, 5′-AGCGAGGGCCACAAAGC-3′ (sense), 5′-CAGCTCAAGAGACCTGCTACCA-3′ (antisense), 5′-CCTCAGTCTCCTTCCAGT-3′ (probe). The ΔCT values were derived by subtracting the threshold cycle (CT) values for c-mpl and c-mpl-tr from the CT value for ribosomal protein L19 (RPL19), which serves as an internal control (24Kralovics R. Buser A.S. Teo S.S. Coers J. Tichelli A. van der Maas A.P. Skoda R.C. Blood. 2003; 102: 1869-1871Crossref PubMed Scopus (127) Google Scholar, 25Ghilardi N. Li J. Hongo J.A. Yi S. Gurney A. de Sauvage F.J. J. Biol. Chem. 2002; 277: 16831-16836Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). All reactions were run in duplicates. Co-expression of mpl-tr with c-mpl Inhibits TPO-dependent Mitogenic and Survival Signaling—To study the effect of mpl-tr on TPO-dependent proliferation, we used two cytokine-dependent cell lines for cell growth assays: the murine cell line BaF3 and the human megakaryoblastic cell line UT-7. Neither cell line expresses Mpl protein endogenously. To assay the potential effects of mpl-tr on the function of the full-length Mpl protein, we used BaF3 cells stably transfected with murine mpl (BaF3/mpl) (20Stoffel R. Wiestner A. Skoda R.C. Blood. 1996; 87: 567-573Crossref PubMed Google Scholar) (Fig. 1). These cells were subjected to a second round of transfection with an expression vector containing the cDNA for murine mpl-tr. The presence of an intraribosomal entry site followed by a truncated form of human CD4 in the vector allowed selection of mpl-tr-expressing cells using magnetic anti-CD4 microbeads. This procedure was repeated 3–4 times to enrich for CD4-positive cells, and expression of CD4 was confirmed by flow cytometry (data not shown). The sorted cells were cultured with different concentrations of either IL-3 or TPO. As expected, parental BaF3 cells failed to respond to TPO (Fig. 2A). On the other hand, BaF3/mpl cells and BaF3/mpl cells transfected with the parental pCD4 plasmid proliferated in dependence on TPO concentration. In contrast, BaF3/mpl cells expressing mpl-tr failed to show a proliferative response to TPO, behaving similarly to parental BaF3 cells. Importantly, mpl-tr did not have a general inhibitory effect on proliferation because in the presence of IL-3, BaF3/mpl cells expressing mpl-tr grew as efficiently as control cells. The same result was obtained when mpl-tr was expressed in human UT-7 cells that were stably transfected with mpl. In this experiment, we used an mpl construct with five Myc tags at the N terminus (UT-7/Myc-mpl) (Fig. 1). The Myc-Mpl protein conveyed TPO-responsiveness to UT-7 cells, demonstrating that the Myc tag did not interfere with Mpl function (Fig. 2B). When mpl-tr was expressed in UT-7/Myc-mpl cells, TPO-mediated proliferation was abolished, but GM-CSF-dependent proliferation remained unchanged. To test whether the observed inhibitory effect could be mediated by secreted Mpl-tr protein, medium conditioned by mpl-tr-expressing cells was transferred onto BaF3/mpl cells. TPO-dependent proliferation of BaF3/mpl cells was not inhibited by the presence of this conditioned medium (Fig. 2C), demonstrating that no secreted inhibitory activity exists. Since Mpl signaling exerts an anti-apoptotic effect, we analyzed the effect of mpl-tr on TPO-dependent cell survival. UT-7/Myc-mpl cells transfected with either mpl-tr or control vector were cultured with GM-CSF or with TPO. After 48 h, cells were incubated with annexin V and analyzed by flow cytometry. In the presence of GM-CSF, mpl-tr had no effect on the number of annexin V-positive cells. However, with TPO, most cells coexpressing mpl and mpl-tr stained positive for annexin V (Fig. 3). This indicates that mpl-tr expression inhibits the anti-apoptotic signal delivered by TPO.Fig. 3Effect of mpl-tr on cell survival. UT-7/Myc-mpl cells were transfected with pCD4 (open bars) or pCD4-mpl-tr (black bars) and sorted with anti-CD4 microbeads. Cells were cultured in the presence of GM-CSF or TPO at the indicated concentrations for 48 h, stained with FITC-labeled annexin V, and analyzed by flow cytometry. The results of three independent experiments are shown. Error bars indicate the standard deviation.View Large Image Figure ViewerDownload (PPT) mpl-tr Mediates Down-modulation of Mpl Protein Expression in a Post-transcriptional Manner—To investigate the inhibitory mechanism exerted by mpl-tr, we analyzed the effects of mpl-tr on Mpl protein expression. First, we asked whether Mpl protein surface expression was affected by the presence of mpl-tr. Cells were stained with anti-Myc antibodies (Fig. 4A). As expected, UT-7/Myc-Mpl cells showed marked Myc-mpl surface expression. In contrast, the staining of UT-7/Myc-mpl cells expressing mpl-tr did not significantly differ from the control staining of the parental UT-7 cells, indicating that mpl-tr interferes with Myc-mpl cell surface expression. We then asked whether the lack of detectable Myc-Mpl surface expression correlated with a decrease in total Myc-Mpl protein. Immunoblot analysis of total cell lysates demonstrated a massive reduction of Myc-Mpl protein in cells that expressed mpl-tr (Fig. 4B). The decrease in Myc-Mpl protein was observed irrespective of whether the cells were grown with TPO or GM-CSF. Importantly, mpl-tr did not alter mpl mRNA levels, as indicated by the unchanged ΔCT values for mpl (Fig. 4B). Expression of mpl-tr also led to a dramatic decrease in expression of the untagged Mpl protein in BaF3/mpl cells without affecting mRNA levels, showing that mpl-tr targets mpl protein regardless of the presence of an N-terminal Myc tag (see Fig. 6C). To determine whether this phenomenon was limited to hematopoietic cells, we performed transient co-transfections of mpl and mpl-tr cDNAs into human 293T cells. As shown in Fig. 4C, expression of mpl-tr in 293T cells lowered the amount of Mpl protein without altering mpl mRNA expression.Fig. 6Functional analysis of the C-terminal peptide of Mpl-tr. A, a schematic drawing of Mpl-tr protein and two mpl-tr mutants used in this study. The amino acid sequence of the junction and the C-terminal 30 amino acids are shown. In the tr-Δpep30 mutant, all C-terminal 30 amino acids of Mpl-tr are removed. In the tr-pepmpl mutant, the pep30 peptide is replaced by 30 amino acids derived from the mpl reading frame of exon 11. B, proliferation of BaF3/mpl cells stably transfected with pCD4 (open squares), pCD4-mpl-tr (filled triangles), pCD4-Δpep30 (open triangles), and pCD4-tr-pepmpl (filled diamonds) in the presence of increasing concentrations of IL-3 or TPO (numbers indicate concentration in ng/ml). Error bars indicate the standard deviation. C, immunoblot analysis of total lysates from the same cells as in B. Expression of mpl mRNA is indicated by the ΔCT values below the corresponding lanes.View Large Image Figure ViewerDownload (PPT) mpl-tr Mediates Dose-dependent Reduction of Mpl Protein Expression and a Decrease of Mpl Protein Half-life, whereas Steady-state Levels of mpl-tr Remain Unchanged—To quantify the effect of mpl-tr on Mpl expression, we transiently co-transfected 293T cells with a constant amount of plasmid encoding mpl and varying amounts of plasmid for the expression of mpl-tr. In this experiment, Mpl protein amount was affected by mpl-tr in a dose-dependent manner (Fig. 5A). Measuring mRNA levels by Q-PCR confirmed that mRNA expression correlated with the amount of plasmid DNA transfected. For example, the mpl-tr (ΔCT) value for cells transfected with 0.1 μg of mpl-tr was –4. Cells transfected with 30 times the amount of mpl-tr (3 μg) had an mpl-tr (ΔCT) value of –9. This corresponds to a decrease of 5 CT, which equals 25 = 32 times higher expression of mpl-tr (Fig. 5A). In the converse experiment, we transfected 293T cells with a constant amount of mpl-tr and varied the concentration of mpl. We found that steady-state expression of Mpl-tr was not altered by the presence of increasing amounts of mpl (Fig. 5B). Since mpl-tr affects the steady-state levels of Mpl protein, we determined the half-life of Mpl by a pulse-chase experiment (Fig. 5C). In the presence of mpl-tr, the half-life of Mpl was decreased from 5 to 6 h to 2 to 3 h (Fig. 5C). CATI-1 Restores Mpl Protein Expression in the Presence of mpl-tr—To identify the mechanism that underlies diminished Mpl protein expression in the presence of mpl-tr, we treated mpl-tr-transfected UT-7/Myc-mpl cells with the cathepsin inhibitor CATI-1, the proteasome inhibitor MG132, or the calpain inhibitors ALLN or EST. Inhibitors were added at a final concentration of 25 μm each, and cells were cultured for 6–8 h in the presence of GM-CSF. Only CAPI-1 restored Mpl protein expression (Fig. 5D), and a weak Mpl band was detectable with MG132 treatment, whereas the other inhibitors had no effect. None of the inhibitors changed the steady-state Mpl protein levels in the UT-7/Myc-mpl cells lacking mpl-tr (Fig. 5E). To confirm these results in a different cell system, we also treated 293T cells with CATI-1 and determined Mpl protein expression. Similar to UT-7/Myc-mpl, 293T cells co-transfected with mpl and mpl-tr showed a rescue of Mpl protein expression in the presence of CATI-1 (Fig. 5F). These results argue that mpl-tr mediates Mpl protein degradation by a cathepsin-like protease activity. The C-terminal Peptide Sequence of Mpl-tr Is Necessary but Not Sufficient for the Inhibition of Cell Proliferation and for Mpl Protein Degradation—The amino acid sequence of Mpl-tr is identical to the N terminus of Mpl except for a stretch at the C terminus of Mpl-tr, 30 amino acids in length (Fig. 1). We therefore speculated that this unique C-terminal peptide in Mpl-tr could be of functional importance. To test this hypothesis, we made two mutants of mpl-tr. In the first mutant, Δpep30, we introduced a stop codon at position 427, removing the entire C-terminal peptide (Fig. 6A). In the second mutant, tr-pepmpl, the sequence of the C-terminal peptide was changed by adding 2 bp, which restored the reading frame of full-length mpl, and by introducing a stop codon terminating the reading frame after 30 amino acids (Fig. 6A). With these mpl-tr mutants, we stably transfected BaF3/mpl cells and assayed TPO-dependent proliferation. As shown in Fig. 6B, only mpl-tr abrogated mpl-mediated cell growth, whereas tr-Δpep30 and tr-pepmpl did not interfere with TPO-dependent proliferation. As expected, the mpl-tr mutants did not interfere with the proliferative responses to IL-3 (Fig. 6B). Immunoblot analysis showed that Δpep30 and tr-pepmpl were expressed at levels similar to Mpl-tr (Fig. 6C). This indicates that the degree of expression does not explain the failure of the mutants to inhibit TPO-dependent growth. Importantly, the levels of Mpl protein in cells expressing the mpl-tr mutants were similar to BaF3/mpl control cells, but Mpl was undetectable in cells expressing mpl-tr. These results demonstrate that the C-terminal peptide sequence from Mpl-tr is required for the ability of Mpl-tr to promote a decrease in Mpl protein and to inhibit TPO-dependent cell proliferation. Fu
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