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The Ski-binding Protein C184M Negatively Regulates Tumor Growth Factor-β Signaling by Sequestering the Smad Proteins in the Cytoplasm

2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês

10.1074/jbc.m210855200

ISSN

1083-351X

Autores

Kenji Kokura, Hyungtae Kim, Toshie Shinagawa, Md Matiullah Khan, Teruaki Nomura, Shunsuke Ishii,

Tópico(s)

Heterotopic Ossification and Related Conditions

Resumo

Ski is a transcriptional co-repressor and is involved in the negative regulation of tumor growth factor-β (TGF-β) signaling. To understand more fully the role of Ski in TGF-β signaling, we searched for novel Ski-interacting proteins. The identified C184M protein consists of 189 amino acids and contains the leucine-rich region. An association between Ski and C184M involving the leucine-rich region of C184M and the C-terminal coiled-coil motif of Ski was confirmed by glutathione S-transferase pull-down and immunoprecipitation assays. The C184M protein is located in the cytosol, and the C184M and Ski signals are co-localized in the cytoplasm when C184M was co-expressed with Ski in CV-1 cells. The cytoplasmic C184M-Ski complex inhibited the nuclear translocation of Smad2. Consistent with this, the activity of promoter containing the Smad-binding sites was repressed by C184M, and the TGF-β-induced growth inhibition of mink lung Mv1Lu cells was attenuated by the ectopic expression of C184M. Thus, C184M inhibits TGF-β signaling in concert with Ski. In hepatocytes, which express significant levels of C184M, the Ski signals were found only in the cytoplasm, supporting the notion that C184M forms a complex with Ski in the cytosol. Ski is a transcriptional co-repressor and is involved in the negative regulation of tumor growth factor-β (TGF-β) signaling. To understand more fully the role of Ski in TGF-β signaling, we searched for novel Ski-interacting proteins. The identified C184M protein consists of 189 amino acids and contains the leucine-rich region. An association between Ski and C184M involving the leucine-rich region of C184M and the C-terminal coiled-coil motif of Ski was confirmed by glutathione S-transferase pull-down and immunoprecipitation assays. The C184M protein is located in the cytosol, and the C184M and Ski signals are co-localized in the cytoplasm when C184M was co-expressed with Ski in CV-1 cells. The cytoplasmic C184M-Ski complex inhibited the nuclear translocation of Smad2. Consistent with this, the activity of promoter containing the Smad-binding sites was repressed by C184M, and the TGF-β-induced growth inhibition of mink lung Mv1Lu cells was attenuated by the ectopic expression of C184M. Thus, C184M inhibits TGF-β signaling in concert with Ski. In hepatocytes, which express significant levels of C184M, the Ski signals were found only in the cytoplasm, supporting the notion that C184M forms a complex with Ski in the cytosol. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, tumor growth factor-β; GST, glutathione S-transferase; LR, leucine-rich region; MMTV, mouse mammary tumor virus; BrdUrd, bromodeoxyuridine; HA, hemagglutinin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate.1The abbreviations used are: TGF-β, tumor growth factor-β; GST, glutathione S-transferase; LR, leucine-rich region; MMTV, mouse mammary tumor virus; BrdUrd, bromodeoxyuridine; HA, hemagglutinin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate. plays an important role in growth, differentiation, adhesion, and apoptosis (for review, see Ref. 1Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar). TGF-β acts as a potent growth inhibitor for most types of cells. Secreted TGF-β binds to the TGF-β type II receptor, which phosphorylates the type I receptor. The activated type I receptor phosphorylates the receptor-regulated Smads (R-Smads), Smad2 and Smad3. Phospho-Smad2 or phospho-Smad3 then forms a complex with Smad4, known as the co-mediator Smad (co-Smad), and migrates into the nucleus, where it regulates the transcription of target genes. In addition to the Smad-mediated pathway, an alternative pathway involving TGF-β-activated kinase (TAK-1) and TAK1-binding protein 1 (TAB1) also mediates TGF-β signaling (2Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (516) Google Scholar). The TAB1-TAK1 pathway activates the mitogen-activated protein kinase cascade including p38 and c-Jun N-terminal kinase (4Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 5Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). The mitogen-activated protein kinase cascades signals are transmitted to transcriptional regulators, such as ATF-2 and c-Jun (AP-1) that in concert with Smad3/4 induce transcriptional activation of some TGF-β-responsive genes (4Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 5Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 6Zhang Y. Feng X.H. Derynck R. Nature. 1998; 396: 909-913Crossref Scopus (679) Google Scholar).To restrict and terminate the response to the TGF-β signal, the TGF-β signaling is negatively regulated by multiple mechanisms (for review, see Ref. 1Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar). First, the inhibitory Smads (I-Smads), including Smad6 and Smad7, negatively regulate the TGF-β signaling (7Hayashi H. Abdollah S. Qui Y. Cai Y. Cai J. Xu Y.-Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar, 8Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1545) Google Scholar, 9Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar). Because I-Smads do not contain phosphorylation sites for TGF-β receptor I, when recruited to the activated type I receptor they do not dissociate from it, which prevents the association of R-Smads with the type I receptor. The expression of Smad7 is induced by the TGF-β stimulus (7Hayashi H. Abdollah S. Qui Y. Cai Y. Cai J. Xu Y.-Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar, 8Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1545) Google Scholar), indicating that Smad7 is involved in negative feedback regulation of TGF-β signaling. Second, Smad7 recruits the Smurf2 protein to the TGF-β receptor and induces the degradation of the receptor (10Kavasak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar). Smurf2 is a member of the HECT-type ubiquitin-protein isopeptide ligase family, and induces the ubiquitination of the TGF-β receptor, which leads to the degradation of the receptor via the proteasome- and lysosome-dependent pathways. The third mechanism is the negative regulation of the Smad-dependent transcriptional activation by co-repressors in the nucleus. The phosphorylated Smad2 and Smad3 in the nucleus bind to transcriptional corepressors such as TGIF (11Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar) and members of the Ski protein family (12Sun Y. Liu X. Eaton E.N. Lane W.S. Lodish H.F. Weinberg R.A. Mol. Cell. 1999; 4: 499-509Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 13Luo K. Stroschein S.L. Wang W. Chen D. Martens E. Zhou S. Zhou Q. Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (389) Google Scholar, 14Akiyoshi S. Inoue H. Hanai J. Kusanagi K. Nemoto N. Miyazono K. Kawabata M. J. Biol. Chem. 1999; 274: 35269-35277Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 15Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar). The co-repressors compete out p300/CBP, the co-activator of Smad2/4 and Smad3/4, and recruit histone deacetylases to the target genes, leading to the inhibition of the Smad2/4- or Smad3/4-induced transcriptional activation.The ski gene family was originally identified as oncogenes carried by the Sloan-Kettering virus (16Li Y. Turck C.M. Termer J.K. Stavnezer E. J. Virol. 1986; 57: 1065-1072Crossref PubMed Google Scholar). v-ski and c-ski induce transformation of chicken fibroblasts and muscle differentiation of quail embryo fibroblasts (17Colmenares C. Stavnezer E. Cell. 1989; 59: 293-303Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 18Colmenares C. Sutrave P. Hughes S.H. Stavnezer E. J. Virol. 1991; 65: 4929-4935Crossref PubMed Google Scholar). The ski gene family consists of two members, ski and sno (ski-related novel gene) (19Nomura N. Sasamoto S. Ishii S. Date T. Matsui M. Ishizaki R. Nucleic Acids Res. 1989; 17: 5489-5500Crossref PubMed Scopus (149) Google Scholar). We have shown that the ski and sno gene products (Ski and Sno) act as co-repressors and bind with other co-repressors, N-CoR/SMRT and mSin3A (20Nomura T. Kahn M.M. Kaul S.C. Dong H.-D. Wadhwa R. Colmenares C. Kohno I. Ishii S. Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar). Ski and Sno recruit the histone deacetylase complex to the target promoters via multiple repressors, including Mad, retinoblastoma, thyroid hormone receptor-β, and MeCP2 (20Nomura T. Kahn M.M. Kaul S.C. Dong H.-D. Wadhwa R. Colmenares C. Kohno I. Ishii S. Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar, 21Tokitou F. Nomura T. Khan M.M. Kaul S.C. Wadhwa R. Yasukawa T. Kohno I. Ishii S. J. Biol. Chem. 1999; 274: 4485-4488Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 22Kokura K. Kaul S.C. Wadhwa R. Nomura T. Khan M.M. Shinagawa T. Yasukawa T. Colmenars C. Ishii S. J. Biol. Chem. 2001; 276: 34115-34121Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Although Ski and Sno directly bind to Smad2/3/4 and negatively regulate Smad-dependent transcriptional activation (12Sun Y. Liu X. Eaton E.N. Lane W.S. Lodish H.F. Weinberg R.A. Mol. Cell. 1999; 4: 499-509Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 13Luo K. Stroschein S.L. Wang W. Chen D. Martens E. Zhou S. Zhou Q. Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (389) Google Scholar, 14Akiyoshi S. Inoue H. Hanai J. Kusanagi K. Nemoto N. Miyazono K. Kawabata M. J. Biol. Chem. 1999; 274: 35269-35277Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 15Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar), Sno is rapidly degraded upon TGF-β stimulation (23Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Science. 1999; 286: 771-774Crossref PubMed Scopus (436) Google Scholar, 24Sun Y. Liu X. Ng-Eaton E. Lodish H.F. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12442-12447Crossref PubMed Scopus (226) Google Scholar). Smad2 and Smad3 bind and recruit, respectively, two types of ubiquitin-protein isopeptide ligases, Smurf2 and anaphase-promoting complex/cyclosome, to the Sno protein, which leads to the ubiquitination and proteasome-dependent degradation of Sno (25Bonni S. Wang H.-R. Causing C.G. Kavasak P. Stroschein S.L. Luo K. Wrana L. Nat. Cell Biol. 2001; 3: 587-595Crossref PubMed Scopus (271) Google Scholar, 26Stroschein S.L. Bonni S. Wrana J.L. Luo K. Genes Dev. 2001; 15: 2822-2836Crossref PubMed Google Scholar, 27Wan Y. Liu X. Kirschner M.W. Mol. Cell. 2001; 8: 1027-1039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The levels of Sno expression increase markedly 2 h after stimulation with TGF-β (23Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Science. 1999; 286: 771-774Crossref PubMed Scopus (436) Google Scholar), indicating that Sno plays a role in the negative feedback regulation of the TGF-β signaling. In contrast, it is not clear whether the Ski protein is degraded upon TGF-β stimulation (24Sun Y. Liu X. Ng-Eaton E. Lodish H.F. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12442-12447Crossref PubMed Scopus (226) Google Scholar). At least Ski protein degradation does not play any role in the negative feedback regulation of the TGF-β signaling.In the present study, we have identified a Ski-interacting protein, C184M. C184M induces the cytoplasmic accumulation of Ski, and the cytosolic C184M/Ski complex negatively regulates TGF-β signaling by inhibiting the nuclear translocation of Smad2.MATERIALS AND METHODSYeast Two-hybrid Screening and GST Pull-down Assay—Yeast two-hybrid screening using a mouse embryonic cDNA library and GST pull-down assays were performed essentially as described previously (20Nomura T. Kahn M.M. Kaul S.C. Dong H.-D. Wadhwa R. Colmenares C. Kohno I. Ishii S. Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar, 22Kokura K. Kaul S.C. Wadhwa R. Nomura T. Khan M.M. Shinagawa T. Yasukawa T. Colmenars C. Ishii S. J. Biol. Chem. 2001; 276: 34115-34121Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). K buffer (20 mm HEPES (pH 7.9), 75 mm KCl, 0.1 mm EDTA, 2.5 mm MgCl2, 0.02% skim milk, 1 mm dithiothreitol, 50 μm ZnCl2, and 0.01% Nonidet P-40) was used for binding between GST-C184M and in vitro translated Ski. For the binding between GST-Ski and in vitro translated C184M, SK-B buffer (20 mm Tris-HCl (pH 8.5), 2.25 mm dithiothreitol, 100 mm NaCl, and 0.01% Nonidet P-40) was used.Co-immunoprecipitation—By using LipofectAMINE PLUS (Invitrogen), 293T cells were transfected with a mixture of plasmids to express FLAG-C184M (N-FLAG-pact-C184M) (2.75 μg), Ski (pact-Ski) (2.75 μg), or SnoN (pact-SnoN) (2.75 μg) and the internal control plasmid pact-β-gal (0.5 μg). Forty eight hours after transfection, cells were suspended with TMLSLD buffer (50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 0.1% Tween 20, 10% glycerol, and 50 mm NaF) and sonicated, and lysates were prepared. After measuring β-galactosidase activity, immunoprecipitation was performed with anti-C184M antiserum or control serum. In some experiments, we used an anti-FLAG M2 antibody (Sigma) or normal mouse IgG (Santa Cruz Biotechnology) followed by the addition of protein G-Sepharose (Amersham Biosciences). After washing the beads with Harlow-150 buffer (50 mm HEPES (pH 7.5), 0.2 mm EDTA, 10 μm NaF, 0.5% Nonidet P-40, and 150 mm NaCl), the precipitated proteins were eluted with 2× SDS sample buffer, and Western blotting was carried out with an anti-Ski or anti-Sno monoclonal antibody. For the experiments described in Fig. 5, 293T cells were transfected with mixture of plasmids to express FLAG-C184M (N-FLAG-pact-C184M) (1.8 μg), Ski (N-HA-pact-Ski) (1.6 μg), Smad2 (N-FLAG-pact-Smad2) (1.15 μg), Smad4 (N-FLAG-pact-Smad4) (0.65 μg), ALK5* (pCDNA3-ALK5*) (0.5 μg), and the internal control plasmid pact-β-gal (0.3 μg).Subcellular Localization of C184M, Ski, and Smads—For the experiments described in Fig. 4, N-FLAG-pact-C184M (1.5 μg) and pact-Ski (1.5 μg) were transfected into CV-1 cells by the CaPO4 method. Forty eight hours after transfection, cells were fixed on cover glasses with 2% paraformaldehyde/PBS for 45 min at room temperature and permeabilized by 0.1% Triton X-100/PBS for 12 min. After blocking with 3% skim milk/PBS, cells were immunostained with anti-Ski (Santa Cruz Biotechnology, N-20 or H-329), anti-Sno (Santa Cruz Biotechnology, H-317), and anti-FLAG M2 antibodies followed by appropriate secondary antibodies conjugated to Alexa488 or TRITC, and analyzed with laser confocal microscopy (Zeiss LSM510). For the experiments described in Fig. 5, the monoclonal anti-Ski, anti-C184M, and anti-Smad2 (Santa Cruz Biotechnology S-20) antibodies, and appropriate secondary antibodies conjugated to Alexa488, TRITC, or Cy5 were used. Cells were counterstained with Hoechst 33258. Preparations were observed with BX60 microscope (Olympus), and captured images were deblurred by two-dimensional blind deconvolution with AutoDeblur software (Auto-Quant Imaging Inc.).Fig. 4Cytoplasmic co-localization of C184M with Ski.A–E, CV-1 cells were transfected with each of the plasmids to express the protein shown above, immunostained, and analyzed by confocal microscopy. Ski and SnoN were visualized with a rhodamine-conjugated secondary antibody. The FLAG-linked C184M proteins were stained with an anti-FLAG antibody and visualized with an Alexa488-conjugated secondary antibody. WT, wild type. F–I, CV-1 cells were transfected with two plasmids to express the proteins shown above. Ski and SnoN were visualized with a rhodamine-conjugated secondary antibody, and FLAG-C184M was visualized with an Alexa488-conjugated secondary antibody. The signals for both proteins are superimposed in the right-most panels. J, C184M increases the Ski protein levels. CV-1 cells were transfected with the HA-Ski or HA-SnoN expression vectors together with C184M expression vectors. Whole-cell extracts were prepared, and then Western blotting was carried out with an anti-HA (upper panel) or anti-C184M antibody (lower panel). K, C184M increases the cytoplasmic Ski levels. CV-1 cells were transfected as described above, and the cell lysates were fractionated into cytoplasmic and nuclear fractions, which were then used for Western blotting with an anti-Ski antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Growth Properties of Mv1Lu Cells Stably Expressing C184M—The C184M cDNA was inserted into the retrovirus vector pMSCV-IRES-neo (28Cosset F.L. Takeuchi Y. Battini J.L. Weiss R.A. Collins M.K. J. Virol. 1995; 69: 7430-7436Crossref PubMed Google Scholar). Retroviruses were produced in the FLYA13 packaging cell line as described previously (29Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Mv1Lu cells were infected with this virus and maintained in 10% fetal calf serum/Dulbecco's modified Eagle's medium containing 600 μg/ml G418. G418-resistant cells were expanded in 96-well plates. To measure the growth rate of each cell line, cells (3 × 103/well in a 96-well plate) were incubated with various concentrations of TGF-β1 (Sigma) for 21 h, and BrdUrd incorporation was measured using the Cell Proliferation enzyme-linked immunosorbent assay system (Amersham Biosciences) in accordance with the manufacturer's instructions.Luciferase Reporter Assay—For the experiments described in Fig. 6C, a mixture of 3TP-Lux (50 ng) and the internal control phRL-TK(int-) (Promega; 10 ng) was transfected into the stable Mv1Lu cell lines, 1-2 (2 × 104/well in a 24-well plate), 2-8 (0.7 × 104), and 3-18 (2 × 104) using LipofectAMINE PLUS. Twenty four hours after transfection, TGF-β1 (final concentration 40 pm) was added, and the cells were incubated for a further 22–24 h. Firefly luciferase activity was measured together with Renilla luciferase activity (for an internal control) using a dual luciferase assay system (Promega).Fig. 6C184M negatively regulates TGF-β signaling.A, expression levels of C184M protein in stable cell lines. Mv1Lu cells were infected with retrovirus vector encoding wild-type C184M (2-8, 2-11, and 2-15), the ΔLR mutant (3-13 and 3-18) of C184M, or no proteins, and were cloned by G418 selection. Whole-cell lysates were used for Western blotting with anti-C184M proteins. The asterisk indicates endogenous C184M. WT, wild type. B, C184M blocks the TGF-β-induced growth inhibition. By using the Mv1Lu cell clones, BrdUrd incorporation was measured in the presence of various concentrations of TGF-β1. The average of three independent experiments is shown. C, C184M inhibits the TGF-β-induced transcriptional activation. The Mv1Lu cell clones were transfected with the 3TP-Lux reporter, and luciferase assays were performed. Open, black, and gray bars represent the data with clones 1-2, 2-8, and 3-18, respectively. The average of three experiments is indicated with the standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Western Blotting—CV-1 cells (5 × 105 cells/10-cm dish) were transfected with a mixture of N-HA-pact-Ski or SnoN (2 μg), N-FLAG-pact-C184M (8.25 μg), and pact-β-galactosidase (0.75 μg) using LipofectAMINE and incubated for 40 h. Cytoplasmic and nuclear extract were prepared as described previously (30Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3907) Google Scholar). Nuclear contamination of the cytoplasmic extracts was not significant since we could not detect the lamin A protein, a typical nuclear protein, in Western blotting using the cytoplasmic extract (data not shown).Histological Analysis—Deparaffinized sections of C57BL/6 mouse liver were stained with an anti-C184M polyclonal antibody (1:50) or a mixture of the anti-Ski monoclonal antibodies 1-1, 9-1, 11-1, and 16-1 (1:50) as described previously (31Shinagawa T. Nomura T. Colmenares C. Ohira M. Nakagawara A. Ishii S. Oncogene. 2001; 20: 8100-8108Crossref PubMed Scopus (80) Google Scholar).RESULTSIdentification of C184M Protein as a Ski-interacting Protein—To search for novel Ski-binding proteins, we performed a yeast two-hybrid screening as described previously (20Nomura T. Kahn M.M. Kaul S.C. Dong H.-D. Wadhwa R. Colmenares C. Kohno I. Ishii S. Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar) using the full-length Ski as the bait and the mouse embryonic cDNA library. From this screen, we identified clones encoding the C184M protein. The c184m gene was originally identified as a gene whose expression is induced during mouse brain development (32Sakuma-Takagi M. Tohyama Y. Kasama-Yoshida H. Sakagami H. Kondo H. Kurihara T. Biochem. Biophys. Res. Commun. 1999; 263: 737-742Crossref PubMed Scopus (6) Google Scholar). The C184M protein is a small protein consisting of 189 amino acids and contains a hydrophobic amino acid-rich region with a putative leucine zipper motif (leucine-rich region (LR)) (Fig. 1A). A splicing variant of C184M has also been reported to be a putative receptor for the mouse mammary tumor virus (MMTV) (33Golovkina T.V. Dzuris J. van den Hoogen B. Jaffe A.B. Wright P.C. Cofer S.M. Ross S.R. J. Virol. 1998; 72: 3066-3071Crossref PubMed Google Scholar); this variant does not contain the LR (Fig. 1A and see “Discussion”). To examine the direct interaction between C184M and Ski, we carried out a GST pull-down assay. The in vitro translated C184M protein bound to a GST-Ski fusion protein (Fig. 1, B–D). To identify which region of C184M interacts with Ski, we used deletion mutants of C184M. A small fragment of C184M containing only the LR interacted with GST-Ski, but a ΔLR mutant that lacks the LR did not. Some mutants in which the leucines in the LR were mutated to prolines or alanines exhibited the impaired binding to GST-Ski (data not shown).Fig. 1The LR of C184M is required for binding to Ski.A, amino acid sequence of the C184M protein. Asterisks indicate the leucine residues that can form the leucine zipper motif. The dotted line above the sequence shows the hydrophobic (leucine-rich) region. The amino acids deleted in the splicing variant form of C184M (a putative MMTV receptor) are underlined. B, schematic representation of the C184M deletion proteins used. The results of the binding assays shown in D are indicated on the right. WT, wild type. C, analysis of the GST-Ski fusion protein containing full-length Ski. The GST-Ski fusion proteins bound to glutathione-Sepharose resin were analyzed by 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. D, deletion mutants of in vitro translated 35S-C184M were mixed with the GST-Ski resin. The captured proteins were analyzed by SDS-PAGE, followed by autoradiography. The relative binding activities of various forms of C184M are designated + and -, which indicate the relative binding efficiency to GST-Ski and GST is more than 20-fold and less than 5-fold, respectively. The amount of C184M protein in the input lane was 2.5% that used for the binding assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To identify the specific region of the Ski protein that interacts with C184M, we performed similar GST pull-down assays using various forms of in vitro translated Ski and a GST-C184M fusion protein (Fig. 2). The full-length Ski protein binds to GST-C184M, and a C-terminal fragment containing the coiled-coil region (residues 556–728) also bound (Fig. 2C). A mutant lacking the C-terminal coiled-coil region (Δ493–728) did not bind to GST-C184M.Fig. 2The coiled-coil region of Ski interacts with C184M.A, schematic representation of the Ski protein used. The domain structure of Ski is shown in the top panel. The results of the binding assay shown in C are indicated on the right. The binding activities are designated + and -, which indicate the binding of 5–10% and <1% of the input protein, respectively. WT, wild type; aa, amino acids. B, analysis of the GST-C184M fusion protein. The GST-C184M fusion proteins that bound to the glutathione-Sepharose resin were analyzed on 10% SDS-PAGE, followed by Coomassie Brilliant Blue staining. C, various forms of in vitro translated 35S-Ski were mixed with GST-C184M resin. The bound proteins were analyzed with 10% (upper panel) or 15% SDS-PAGE (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In Vivo Interaction between Ski and C184M—To confirm further the interaction between Ski and C184M, we carried out co-immunoprecipitation assays. The expression vectors for both Ski and FLAG-linked C184M were transfected into 293T cells, and whole-cell lysates were prepared for co-immunoprecipitation. The full-length Ski protein was co-precipitated with wild-type C184M but not with the ΔLR mutant (Fig. 3A). The Δ493–728 mutant, which lacks the coiled-coil region of Ski, was not immunoprecipitated with wild-type C184M protein (Fig. 3A). When we used SnoN instead of Ski, SnoN was not efficiently co-precipitated with C184M, suggesting that C184M preferentially binds to Ski (Fig. 3B).Fig. 3In vivo association between C184M and Ski.A, co-immunoprecipitation assays. 293T cells were transfected with the Ski and FLAG-linked C184M expression vectors shown above. Whole-cell lysates were subjected to immunoprecipitation (IP) using anti-C184M antibody or the control IgG. The immunocomplexes were analyzed by Western blotting with the anti-Ski or anti-FLAG antibody. In lanes 1,4, and 7, 20% of the lysates used for immunoprecipitation was directly used for Western blotting. WT, wild type. B, SnoN does not co-immunoprecipitate with C184M. Lysates were prepared from 293T cells that were transfected with the expression vectors shown above. Immunoprecipitation was performed with an anti-FLAG antibody or control IgG followed by Western blotting with an anti-Ski (left panel) or anti-Sno antibody (right panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Co-localization of Ski and C184M—To confirm further the in vivo association between Ski and C184M, we examined the subcellular localization of Ski and C184M in transfected CV-1 cells. When we used full-length Ski, Δ493–728 Ski, or full-length SnoN alone, these proteins localized in the nucleus (Fig. 4, A–C). As reported previously (20Nomura T. Kahn M.M. Kaul S.C. Dong H.-D. Wadhwa R. Colmenares C. Kohno I. Ishii S. Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar), full-length Ski and SnoN tended to be condensed into a nuclear dot-like structure, but Δ493–728 Ski was not. In contrast, wild-type C184M alone localized mainly in the cytoplasm, although in some of the cells (about 10%), C184M localized in both the cytoplasm and nucleus (Fig. 4D and data not shown). The ΔLR mutant of C184M alone localized in both the cytoplasm and nucleus in all cells (Fig. 4E). When wild-type Ski was co-expressed with wild-type C184M, the Ski signals were mainly seen in the cytoplasm (Fig. 4F1). The Ski signals partially co-localized with C184M signals (Fig. 4F1–3). We observed that the intensity of the Ski signals increased with co-expression of C184M, suggesting that C184M not only blocks the nuclear entry of Ski but also increases the levels of cytoplas

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