Arkadia Induces Degradation of SnoN and c-Ski to Enhance Transforming Growth Factor-β Signaling
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m701294200
ISSN1083-351X
AutoresYoshiko Nagano, Konstantinos J. Mavrakis, Kian Leong Lee, Tomoko Fujii, Daizo Koinuma, Hitoshi Sase, Keiko Yuki, Kazunobu Isogaya, Masao Saitoh, Takeshi Imamura, Vasso Episkopou, Kohei Miyazono, Keiji Miyazawa,
Tópico(s)NF-κB Signaling Pathways
ResumoTransforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators that target either signaling receptors or activated Smad complexes. Among the negative regulators, Smad7 antagonizes TGF-β signaling mainly through targeting the signaling receptors, whereas SnoN and c-Ski repress signaling at the transcriptional level through inactivation of Smad complexes. We previously found that Arkadia is a positive regulator of TGF-β signaling that induces ubiquitin-dependent degradation of Smad7 through its C-terminal RING domain. We report here that Arkadia induces degradation of SnoN and c-Ski in addition to Smad7. Arkadia interacts with SnoN and c-Ski in their free forms as well as in the forms bound to Smad proteins, and constitutively down-regulates levels of their expression. Arkadia thus appears to effectively enhance TGF-β signaling through simultaneous down-regulation of two distinct types of negative regulators, Smad7 and SnoN/c-Ski, and may play an important role in determining the intensity of TGF-β family signaling in target cells. Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators that target either signaling receptors or activated Smad complexes. Among the negative regulators, Smad7 antagonizes TGF-β signaling mainly through targeting the signaling receptors, whereas SnoN and c-Ski repress signaling at the transcriptional level through inactivation of Smad complexes. We previously found that Arkadia is a positive regulator of TGF-β signaling that induces ubiquitin-dependent degradation of Smad7 through its C-terminal RING domain. We report here that Arkadia induces degradation of SnoN and c-Ski in addition to Smad7. Arkadia interacts with SnoN and c-Ski in their free forms as well as in the forms bound to Smad proteins, and constitutively down-regulates levels of their expression. Arkadia thus appears to effectively enhance TGF-β signaling through simultaneous down-regulation of two distinct types of negative regulators, Smad7 and SnoN/c-Ski, and may play an important role in determining the intensity of TGF-β family signaling in target cells. Members of the transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF-β, transforming growth factor-β; Smurf, Smad ubiquitin regulatory factor; HDACs, histone deacetylases; CBP, CREB-binding protein; ES cell, embryonic stem cell; IP, immunoprecipitation; APC, anaphase promoting complex; WT, wild-type; RT, reverse transcription; E3, ubiquitin-protein isopeptide ligase. family have a diverse array of activities, including regulation of growth, motility, extracellular matrix production, differentiation, and apoptosis, in various target cells. TGF-β signal is transduced through two different types of serine/threonine kinase receptors, termed type I and type II, on the cell surface (1Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (481) Google Scholar, 2Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4442) Google Scholar, 3Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4940) Google Scholar, 4ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar). Upon binding of TGF-β to the type II receptor, the type I receptor is recruited to the ligand-receptor complex and is phosphorylated by the constitutively active type II receptor kinase. Type I receptor is then activated and phosphorylates the receptor-regulated Smads, Smad2 and Smad3. Phosphorylated Smad2 and Smad3 form oligomeric complexes with Smad4, a common Smad, and translocate into the nucleus. The activated Smad complexes then bind to promoter regions of target genes either directly or together with other transcription factors and regulate their transcription in cooperation with transcriptional coactivators and corepressors (5Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Crossref PubMed Scopus (595) Google Scholar, 6Miyazono K. Maeda S. Imamura T. ten Dijke P. Heldin C.-H. Smad Signal Transduction. Vol. 5. Springer, Dordrecht, The Netherlands2006: 277-293Google Scholar). TGF-β signaling must be tightly controlled, because its aberration has been reported to cause progression of various diseases, including cancer and fibrosis (7Massagué J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2112) Google Scholar, 8Blobe G.C. Schiemann W.P. Lodish H.F. N. Engl. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2218) Google Scholar). TGF-β signaling is regulated by various proteins that target either signaling receptors or activated Smad complexes. Smad7, an inhibitory Smad, appears to play a central role in down-regulation of the activity of signaling receptors. Smad7 is located in the nucleus and is translocated to the plasma membrane in a Smurf (Smad ubiquitin regulatory factor) 1/2-dependent fashion (9Kavsak 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 (1130) Google Scholar, 10Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar, 11Suzuki C. Murakami G. Fukuchi M. Shimanuki T. Shikauchi Y. Imamura T. Miyazono K. J. Biol. Chem. 2002; 277: 39919-39925Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12Tajima Y. Goto K. Yoshida M. Shinomiya K. Sekimoto T. Yoneda Y. Miyazono K. Imamura T. J. Biol. Chem. 2003; 278: 10716-10721Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Smad7 then competitively inhibits phosphorylation of Smad2 and Smad3 through binding to activated type I receptor kinase (13Hayashi H. Abdollah S. Qiu 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 (1180) Google Scholar, 14Nakao A. Afrakhte M. Morén 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 (1588) Google Scholar). In addition, Smad7 recruits Smurf1/2 to the type I receptor. Because Smurf1 and Smurf2 are HECT-type E3 ubiquitin ligases, they down-regulate the levels of type I receptor proteins through ubiquitylation and proteasomal degradation (9Kavsak 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 (1130) Google Scholar, 10Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). In contrast, SnoN and c-Ski, which are nuclear corepressor proteins of the Ski family, are potent regulators that target the activated Smad complexes (15Akiyoshi 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 (339) Google Scholar, 16Luo K. Stroschein S.L. Wang W. Chen D. Martens E. Zhou S. Zhou Q. Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (392) Google Scholar, 17Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Science. 1999; 286: 771-774Crossref PubMed Scopus (442) Google Scholar, 18Sun 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 (227) Google Scholar, 19Xu 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). They have been shown to recruit histone deacetylases (HDACs) to the Smad complexes and to compete with transcriptional coactivator p300/CBP for binding to the Smad complexes (15Akiyoshi 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 (339) Google Scholar, 16Luo K. Stroschein S.L. Wang W. Chen D. Martens E. Zhou S. Zhou Q. Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (392) Google Scholar, 19Xu 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). Misexpression of these negative molecules has been implicated in various pathological conditions. Reduction of Smad7 protein has been reported in human fibroblasts of patients with scleroderma (20Dong C. Zhu S. Wang T. Yoon W. Li Z. Alvarez R.J. ten Dijke P. White B. Wigley F.M. Goldschmidt-Clermont P.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3908-3913Crossref PubMed Scopus (228) Google Scholar) and in tissues with renal fibrosis in mice (21Fukasawa H. Yamamoto T. Togawa A. Ohashi N. Fujigaki Y. Oda T. Uchida C. Kitagawa K. Hattori T. Suzuki S. Kitagawa M. Hishida A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8687-8692Crossref PubMed Scopus (183) Google Scholar). Systemic deletion of one copy of the Sno or Ski gene causes increased susceptibility to chemical carcinogens (22Shinagawa T. Dong H-D. Xu M. Maekawa T. Ishii S. EMBO J. 2000; 19: 2280-2291Crossref PubMed Scopus (94) Google Scholar, 23Shinagawa T. Nomura T. Colmenares C. Ohira M. Nakagawara A. Ishii S. Oncogene. 2001; 20: 8100-8108Crossref PubMed Scopus (80) Google Scholar). Increased expression of Smad7 has been found in inflammatory bowel disease (24Monteleone G. Kumberova A. Croft N.M. McKenzie C. Steer H.W. MacDonald T.T. J. Clin. Investig. 2001; 108: 601-609Crossref PubMed Scopus (534) Google Scholar) and pancreatic cancer (25Kleeff J. Ishiwata T. Maruyama H. Friess H. Truong P. Büchler M.W. Falb D. Korc M. Oncogene. 1999; 18: 5363-5372Crossref PubMed Scopus (246) Google Scholar). Increased expression of SnoN or c-Ski has been implicated in the progression of esophageal squamous cell carcinomas (26Imoto I. Pimkhaokham A. Fukuda Y. Yang Z-Q. Shimada Y. Nomura N. Hirai H. Imamura M. Inazawa J. Biochem. Biophys. Res. Commun. 2001; 286: 559-565Crossref PubMed Scopus (72) Google Scholar, 27Fukuchi M. Nakajima M. Fukai Y. Miyazaki T. Masuda N. Sohda M. Manda R. Tsukada K. Kato H. Kuwano H. Int. J. Cancer. 2004; 108: 818-824Crossref PubMed Scopus (82) Google Scholar), melanomas (28Reed J.A. Bales E. Xu W. Okan N.A. Bandyopadhyay D. Medrano E.E. Cancer Res. 2001; 61: 8074-8078PubMed Google Scholar), estrogen receptor-positive breast carcinomas (29Zhang F. Lundin M. Ristimäki A. Heikkilä P. Lundin J. Isola J. Joensuu H. Laiho M. Cancer Res. 2003; 63: 5005-5010PubMed Google Scholar), and colorectal carcinomas (30Buess M. Terracciano L. Reuter J. Ballabeni P. Boulay J-L. Laffer U. Metzger U. Herrmann R. Rochlitz C. Neoplasia (Bratisl.). 2004; 6: 207-212Crossref PubMed Scopus (59) Google Scholar). Maintenance of expression levels of these negative regulators within the appropriate ranges thus appears to be important. Arkadia was originally isolated by gene-trap mutagenesis in mice as a factor required for induction of the mammalian node in the extraembryonic lineages (31Episkopou V. Arkell R. Timmons P.M. Walsh J.J. Andrew R.L. Swan D. Nature. 2001; 410: 825-830Crossref PubMed Scopus (85) Google Scholar), and it was found to induce mesendoderm by enhancing nodal-related signaling (32Niederländer C. Walsh J.J. Episkopou V. Jones C.M. Nature. 2001; 410: 830-834Crossref PubMed Scopus (60) Google Scholar). Arkadia is a nuclear protein with 989 amino acid residues, with a characteristic RING domain in its C terminus. We previously found that Arkadia is an E3 ubiquitin ligase that enhances TGF-β signaling by targeting a negative regulator, Smad7 (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar). Another mechanism of enhancement of TGF-β signaling involving degradation of phospho-Smad2 and Smad3 has also recently been proposed (34Mavrakis K.J. Andrew R.L. Lee K.L. Petropoulou C. Dixon J.E. Navaratnum N. Norris D.P. Episkopou V. Plos Biol. 2007; 5: 586-603Crossref Scopus (80) Google Scholar). Arkadia is the first example of an E3 ubiquitin ligase that positively regulates TGF-β family signaling. We report here that Arkadia targets SnoN and c-Ski in addition to Smad7. Arkadia thus effectively enhances TGF-β signaling through simultaneous down-regulation of two distinct types of negative regulators. Cell Culture—HepG2, 293T, HeLa, COS7, and NMuMG cells were obtained from the American Type Culture Collection. HepG2, 293T, HeLa, and COS7 cells were maintained in Dulbecco's modified Eagle's medium (Sigma or Invitrogen) containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. NMuMG cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 10 μg/ml insulin. Wild-type and Arkadia-/- embryonic stem (ES) cells (31Episkopou V. Arkell R. Timmons P.M. Walsh J.J. Andrew R.L. Swan D. Nature. 2001; 410: 825-830Crossref PubMed Scopus (85) Google Scholar) were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mm l-glutamine (Invitrogen), 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate (Invitrogen), 9 mg/liter β-mercaptoethanol (Sigma), 20% fetal bovine serum, and leukemia inhibitory factor. cDNA Construction—Expression constructs encoding mouse Arkadia, human SnoN, and human c-Ski were described previously (15Akiyoshi 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 (339) Google Scholar, 33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar, 35Mizuide M. Hara T. Furuya T. Takeda M. Kusanagi K. Inada Y. Mori M. Imamura T. Miyazawa K. Miyazono K. J. Biol. Chem. 2003; 278: 531-536Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Mutants of Arkadia and c-Ski were described previously (15Akiyoshi 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 (339) Google Scholar, 33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar) or generated using a PCR-based method. SnoN mutants were also generated using a PCR-based approach. Expression constructs encoding a constitutively active mutant of the TGF-β type I receptor, ALK-5-TD, ubiquitin, Smad7, Smad4, and Smad2, were described previously (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar). RNA Interference—RNA interference was principally performed as described by Brummelkamp et al. (36Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3979) Google Scholar). To generate pSUPER constructs, oligonucleotides corresponding to Smad7-pSUPER (forward, 5′-gatccccGAGGCTGTGTTGCTGTGAAttcaagagaTTCACAGCAACACAGCCTCtttttggaaa-3′; reverse, 5′-agcttttccaaaaaGAGGCTGTGTTGCTGTGAAtctcttgaaTTCACAGCAACACAGCCTCggg-3′), c-Ski-pSUPER (forward, 5′-gatccccGCTTCTACTCCTACAAGAGttcaagagaCTCTTGTAGGAGTAGAAGCtttttggaaa-3′; reverse, 5′-agcttttccaaaaaGCTTCTACTCCTACAAGAGtctcttgaaCTCTTGTAGGAGTAGAAGCggg-3′), and SnoN-pSUPER (forward, 5′-gatccccGTTGGAGGAGAAAAGAGACttcaagagaGTCTCTTTTCTCCTCCAACtttttggaaa-3′; reverse, 5′-agcttttccaaaaaGTTGGAGGAGAAAAGAGACtctcttgaaGTCTCTTTTCTCCTCCAACggg-3′) were annealed, followed by ligation into the pSUPER vector, which was digested with BglII/HindIII. Target sequences for NC1 were described previously (37Maeda S. Hayashi M. Komiya S. Imamura T. Miyazono K. EMBO J. 2004; 23: 552-563Crossref PubMed Scopus (286) Google Scholar). To confirm knockdown of these proteins, 293T cells were transfected with FLAG-tagged Smad7, c-Ski, or SnoN and corresponding pSUPER constructs. Levels of expression of proteins were determined by immunoblotting using anti-FLAG (M2) antibody (supplemental Fig. 1). Luciferase Assay—A TGF-β-responsive reporter, (CAGA)9-MLP-Luc (38Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar), was used. Cells were transiently transfected with an appropriate combination of promoter-reporter constructs, pSUPER constructs, and expression plasmids, including pcDEF3-ALK-5-TD-HA, using FuGENE 6 transfection reagent (Roche Diagnostics). Total amounts of transfected DNAs were the same in each experiment. Cells were cultured for 24 h after transfection. Cell lysates were then prepared, and luciferase activities in the lysates were measured by the dual-luciferase reporter system (Promega) using a luminometer (MicroLumat Plus LB96V, Berthold). Values were normalized using Renilla luciferase activity under the control of thymidine kinase promoter. Antibodies—The antibodies used were as follows: anti-SnoN H-317 (rabbit polyclonal, Santa Cruz Biotechnology) for immunoblotting of endogenous SnoN; anti-Arkadia 65 (see below) for immunoprecipitation of endogenous Arkadia; normal rabbit IgG (Santa Cruz Biotechnology) as a negative control for immunoprecipitation using anti-Arkadia 65; anti-Arkadia 62 (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar) for immunoblotting of transfected Arkadia in the ubiquitylation assay; anti-Arkadia 3AP4 (see below) for detection of Arkadia in mouse ES cell lines; anti-tubulin DM 1A (Sigma); anti-FLAG M2 (Sigma); anti-Myc 9E10 (Pharmingen); and anti-hemagglutinin 3F10 (Roche Diagnostics). Anti-Arkadia 65 was prepared by immunizing a rabbit with mouse Arkadia (amino acid residues 854-936) expressed as a fusion protein with glutathione S-transferase. Anti-Arkadia 3AP4 was prepared by immunizing a rabbit with a peptide derived from mouse Arkadia (amino acid residues 742-756) conjugated to bovine thyroglobulin followed by affinity purification. Immunoprecipitation and Immunoblotting—293T and COS7 cells were transiently transfected using FuGENE 6 (Roche Diagnostics) and incubated for 24 h before analysis. Cells were lysed with a buffer containing 1% Nonidet P-40, 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, and 1% Trasylol (Bayer). In some experiments, 50 μm MG132 (Peptide Institute) and 5 mm EDTA were added to the lysis buffer. Cleared lysates were incubated with anti-FLAG antibody for 1 h or overnight at 4 °C. NMuMG cells were lysed with a buffer containing 0.5% Nonidet P-40, 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1% protease inhibitor mixture (EDTA-free) (Nacalai Tesque), 1% phosphatase inhibitor mixture 1 (Sigma), 1% phosphatase inhibitor mixture 2 (Sigma), and 50 μm MG132. Lysates containing 300 μg of total protein were used for immunoprecipitation. Mouse ES cells were lysed with a buffer containing 1% Nonidet P-40, 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1% Trasylol, 50 μm MG-132 (Calbiochem), and 5 mm EDTA. Immunoprecipitates or cleared cell lysates were separated by SDS-PAGE and transferred to Fluoro Trans W membrane (Pall). Immunoblotting was performed using the indicated antibodies. Immunofluorescence Labeling—Immunofluorescence labeling was performed using HeLa cells as described previously (39Nagata M. Goto K. Ehata S. Kobayashi N. Saitoh M. Miyoshi H. Imamura T. Miyazawa K. Miyazono K. Genes Cells. 2006; 11: 1267-1280Crossref PubMed Scopus (36) Google Scholar). The primary antibodies used were anti-FLAG M2 (×250) and anti-Arkadia 62 (×1000). Pulse-Chase Analysis—Pulse-chase analysis was performed as described previously (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar). RT-PCR—Total RNAs from wild-type and Arkadia-/- mouse ES cells were extracted using the RNeasy mini kit (Qiagen). RNAs were reverse-transcribed by random hexamer priming using SuperScript™ II RT (Invitrogen). Semiquantitative RT-PCR was performed as follows: 35 cycles of 94 °C (15 s), 52 °C (30 s), and 72 °C (1 min) for SnoN, 40 cycles of 94 °C (15 s), 58 °C (30 s), and 72 °C (1 min) for Arkadia, and 25 cycles of 94 °C (15 s), 60 °C (30 s), and 72 °C (1 min) for glyceraldehyde-3-phosphate dehydrogenase. The primer sequences used were as follows: mouse SnoN, forward 5′-TCATTTTTACACCCCAGCTACTACCT and reverse 5′-GCGACACATTCGGTGCAA; and mouse Arkadia, forward 5′-TCATATTCATGTGCCTCAAACCA and reverse 5′-CCCAGTTCCCAGGCAGTTC. The primers for mouse glyceraldehyde-3-phosphate dehydrogenase were described previously (37Maeda S. Hayashi M. Komiya S. Imamura T. Miyazono K. EMBO J. 2004; 23: 552-563Crossref PubMed Scopus (286) Google Scholar). DNA Affinity Precipitation—Cell lysates were prepared in 1% Nonidet P-40, 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1% Trasylol, 50 μm MG132, and 5 mm EDTA. Precipitation of proteins from the lysates using 3× CAGA probe was performed as described previously (40Nishihara A. Hanai J. Imamura T. Miyazono K. Kawabata M. J. Biol. Chem. 1999; 274: 28716-28723Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Arkadia May Have Substrates Other Than Smad7—We previously reported that Arkadia induces ubiquitylation and degradation of Smad7 to augment TGF-β signaling (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar). To determine whether Smad7 is the major substrate of Arkadia in enhancement of TGF-β signaling, we first examined the effect of Arkadia on TGF-β signaling when expression of Smad7 was silenced by RNA interference. HepG2 cells were transfected with Smad7-pSUPER and FLAG-Arkadia and stimulated by cotransfection of ALK-5-TD. TGF-β signaling was determined by the TGF-β-responsive reporter (CAGA)9-MLP-Luc. As shown in Fig. 1, silencing of Smad7 resulted in enhancement of TGF-β signaling, but Arkadia further enhanced TGF-β signaling even after silencing of Smad7, suggesting that Arkadia may have substrates other than Smad7 for enhancement of TGF-β signaling. Arkadia Binds to SnoN and c-Ski—Because SnoN and c-Ski have been shown to be important negative regulators of TGF-β signaling, these proteins could be candidates for novel substrates of Arkadia. To test this possibility, we first examined the physical interaction of Arkadia with SnoN and c-Ski. Arkadia-C937A (CA), a mutant lacking ubiquitin ligase activity (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar), was used to avoid degradation of proteins bound to Arkadia. When 293T cells were transfected with SnoN and Arkadia, Arkadia was coprecipitated with SnoN (Fig. 2A). As shown in Fig. 2B, Arkadia also bound to c-Ski. These interactions were observed in both the presence and absence of ALK-5-TD, indicating that they are minimally affected by TGF-β signaling (Fig. 2, A and B). Interaction of endogenous Arkadia and SnoN proteins was also examined in NMuMG mouse mammary epithelial cells, because endogenous Arkadia and SnoN proteins are detectable in this cell line, and expression of SnoN was induced by treatment with BMP-4 (data not shown). Cells were treated with MG132 to prevent proteasomal degradation of proteins bound to Arkadia. When Arkadia was immunoprecipitated by anti-Arkadia antibody, coprecipitation of SnoN was detected by immunoblotting (Fig. 2C). We then examined the subcellular localization of Arkadia and SnoN or c-Ski in HeLa cells transfected with Arkadia-CA and SnoN or c-Ski. As shown in Fig. 2D, Arkadia was colocalized with SnoN (upper panels) and c-Ski (lower panels) in the nucleus of transfected HeLa cells. Arkadia Promotes Ubiquitylation and Degradation of SnoN and c-Ski—Because Arkadia interacted with SnoN and c-Ski, and was colocalized with them in the nucleus, we next examined whether SnoN and c-Ski serve as substrates of Arkadia. Ubiquitylation of SnoN was observed in the presence of wild-type Arkadia but not in the presence of inactive mutant Arkadia-CA or Arkadia ΔC (amino acid residues 1-936), which lacks C-terminal RING domain (Fig. 3A and data not shown). Ubiquitylated SnoN was considerably decreased when cells were not treated with MG132, suggesting that the SnoN ubiquitylated by Arkadia was degraded by proteasomes. Arkadia induced ubiquitylation of Smad7 as reported previously (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar) but failed to induce that of Smad4, which does not interact with Arkadia (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar). Similar results were obtained for c-Ski (Fig. 3B). Because Arkadia induced ubiquitylation of SnoN and c-Ski, we next used pulse-chase analysis to examine whether Arkadia promotes degradation of SnoN and c-Ski. Turnover of SnoN protein was accelerated in the presence of Arkadia but not in the presence of Arkadia-CA (Fig. 3C). Similar results were obtained in the case of c-Ski (Fig. 3D). These findings indicate that Arkadia promotes degradation of SnoN and c-Ski through ubiquitylation. Arkadia-/- ES Cells Express Higher Levels of SnoN Protein Than Do Wild-type ES Cells—Because Arkadia enhanced the degradation of SnoN, we compared the levels of expression of SnoN protein between wild-type and Arkadia-/- ES cell lines (31Episkopou V. Arkell R. Timmons P.M. Walsh J.J. Andrew R.L. Swan D. Nature. 2001; 410: 825-830Crossref PubMed Scopus (85) Google Scholar) (Fig. 4). Lack of Arkadia protein as well as mRNA was confirmed by immunoblotting and semi-quantitative RT-PCR (Fig. 4, 2nd and 5th panels). We found that levels of expression of the SnoN protein were higher in Arkadia-/- ES cell lines than in the wild-type ES cell lines (Fig. 4, top panel). However, levels of expression of SnoN mRNA in the Arkadia-/- cell lines were similar to those in wild-type cells (Fig. 4, 4th panel). These findings suggest that absence of Arkadia results in accumulation of SnoN protein through a post-translational mechanism. We thus conclude that endogenous Arkadia contributes to degradation of SnoN. We also examined the expression levels of c-Ski, but it was not detectable in these cell lines. We have previously reported that knockdown of Arkadia increases Smad7 protein in HaCaT cells (33Koinuma D. Shinozaki M. Komuro A. Goto K. Saitoh M. Hanyu A. Ebina M. Nukiwa T. Miyazawa K. Imamura T. Miyazono K. EMBO J. 2003; 22: 6458-6470Crossref PubMed Scopus (190) Google Scholar); however, it was not detectable in the ES cell lines. Interaction of Arkadia with Its Substrates Is Mediated through Its C-terminal Region—We next examined which regions in Arkadia are responsible for interaction with its substrates. Arkadia protein was divided into five fragments, and constructs expressing Arkadia fragments were designed (Fig. 5D). The interaction of these fragments with substrates of Arkadia was examined in transfected 293T cells (Fig. 5, A-C). As shown in Fig. 5A, SnoN interacted with the C-terminal region of Arkadia (amino acid residues 772-936), whereas c-Ski and Smad7 mainly interacted with both the N- and C-terminal regions of Arkadia (amino acid residues 1-291 and 772-936, respectively; Fig. 5, B and C). These findings suggest that the C-terminal region of Arkadia is important for the interaction with its substrates, although the N-terminal region of Arkadia also participates in the interaction with c-Ski and Smad7. SnoN and c-Ski Interact with Arkadia through Regions Containing the SAND Domain—We next determined Arkadia-interacting regions in SnoN and c-Ski. SnoN (684 amino acid residues) was divided into three fragments (residues 1-262, 263-479, and 480-684), and interaction of them with Arkadia was determined in transfected 293T cells. As shown in Fig. 6A (upper panels), Arkadia interacted with the central region of SnoN (residues 263-479). Further analysis was performed using two fragments derived from the central region of SnoN (residues 263-355 and 356-479). Arkadia interacted with the SnoN fragment containing amino acid residues 263-355 (Fig. 6A, lower panels), which includes the SAND domain in SnoN. c-Ski (728 amino acid residues) was also divided into three fragments (residues 1-210, 211-490, and 491-728), each of which corresponds to that of SnoN in Fig. 6A. Similar to SnoN, Arkadia interacted with the central region of c-Ski (amino acids 211-490), which contains the SAND domain in c-Ski (Fig. 6B). Arkadia Interacts with SnoN and c-Ski in Complexes with Smads—Because the Arkadia-interacting regions in SnoN and c-Ski overlap with the Smad4-interacting regions in them (the SAND domain) (41Wu J.-W. Krawitz A.R. Chai J. Li W. Zhang F. Luo K. Shi Y. Cell. 2002; 111: 357-367Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), we examined whether SnoN or c-Ski interacts with the Smad complex a
Referência(s)