Regulation of Wnt Signaling by the Nuclear Pore Complex
2008; Elsevier BV; Volume: 134; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2008.03.010
ISSN1528-0012
AutoresMiki Shitashige, Reiko Satow, Kazufumi Honda, Masaya Ono, Setsuo Hirohashi, Tesshi Yamada,
Tópico(s)Cancer-related gene regulation
ResumoBackground & Aims: The function of β-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, β-catenin has no nuclear localization signal, and the mechanisms by which β-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. Methods: Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. Results: TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and β-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and β-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. Conclusions: Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells. Background & Aims: The function of β-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, β-catenin has no nuclear localization signal, and the mechanisms by which β-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. Methods: Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. Results: TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and β-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and β-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. Conclusions: Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells. The Wnt-signaling pathway plays important roles in embryogenesis and carcinogenesis.1Peifer M. Polakis P. Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus.Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1154) Google Scholar Secreted Wnt molecules bind to cell surface frizzled and lipoprotein receptor-related protein (LRP) 5/6 receptors and evoke downstream intracellular signaling.2Kikuchi A. Tumor formation by genetic mutations in the components of the Wnt signaling pathway.Cancer Sci. 2003; 94: 225-229Crossref PubMed Scopus (204) Google Scholar, 3Vogelstein B. Kinzler K.W. Cancer genes and the pathways they control.Nat Med. 2004; 10: 789-799Crossref PubMed Scopus (3409) Google Scholar The signal is transmitted into the cytoplasmic multiprotein complex containing the adenomatous polyposis coli (APC) gene product, axin/axin2, β-catenin, casein kinase I (CKI), and glycogen synthase kinase 3β (GSK3β), where β-catenin is phosphorylated by GSK3β and CKI. Phosphorylated β-catenin is subject to rapid degradation via the ubiquitin-proteasome pathway.2Kikuchi A. Tumor formation by genetic mutations in the components of the Wnt signaling pathway.Cancer Sci. 2003; 94: 225-229Crossref PubMed Scopus (204) Google Scholar, 4Aberle H. Bauer A. Stappert J. et al.β-Catenin is a target for the ubiquitin-proteasome pathway.EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2204) Google Scholar, 5Polakis P. Wnt signaling and cancer.Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar The Wnt signal inhibits the GSK3β enzyme activity and results in the accumulation of β-catenin. Excess β-catenin proteins form complexes with T-cell factor (TCF)/lymphoid enhancer factor (LEF) family DNA-binding proteins6Yamada T. Takaoka A.S. Naishiro Y. et al.Transactivation of the multidrug resistance 1 gene by T-cell factor 4/β-catenin complex in early colorectal carcinogenesis.Cancer Res. 2000; 60: 4761-4766PubMed Google Scholar, 7Wong N.A. Pignatelli M. β-Catenin—a linchpin in colorectal carcinogenesis?.Am J Pathol. 2002; 160: 389-401Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar and transactivate their target genes involved in the regulation of cell fate determination, differentiation, proliferation, and death.6Yamada T. Takaoka A.S. Naishiro Y. et al.Transactivation of the multidrug resistance 1 gene by T-cell factor 4/β-catenin complex in early colorectal carcinogenesis.Cancer Res. 2000; 60: 4761-4766PubMed Google Scholar, 7Wong N.A. Pignatelli M. β-Catenin—a linchpin in colorectal carcinogenesis?.Am J Pathol. 2002; 160: 389-401Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar However, β-catenin has no identifiable nuclear localization signal,8Fagotto F. Gluck U. Gumbiner B.M. Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of β-catenin.Curr Biol. 1998; 8: 181-190Abstract Full Text Full Text PDF PubMed Google Scholar and the mechanisms by which cytoplasmic β-catenin is imported into the nucleus and forms complexes with TCF/LEF nuclear proteins are poorly understood. More than 80% of colorectal cancers contain mutations in the APC gene,9Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4322) Google Scholar and half of the remainder has mutations in the GSK3β/CKI-phosphorylation sites of the β-catenin (CTNNB1) gene.10Ilyas M. Tomlinson I.P. Rowan A. et al.β-Catenin mutations in cell lines established from human colorectal cancers.Proc Natl Acad Sci U S A. 1997; 94: 10330-10334Crossref PubMed Scopus (445) Google Scholar, 11Sparks A.B. Morin P.J. Vogelstein B. et al.Mutational analysis of the APC/β-catenin/Tcf pathway in colorectal cancer.Cancer Res. 1998; 58: 1130-1134PubMed Google Scholar As a result, colorectal cancer cells are unable to degrade β-catenin, and β-catenin protein accumulates in the cytoplasm.12Morin P.J. Sparks A.B. Korinek V. et al.Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC.Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3553) Google Scholar TCF-4 is a member of the TCF/LEF family commonly expressed in colorectal cancer cells and implicated in maintenance of the undifferentiated status of intestinal crypt epithelial cells.13Korinek V. Barker N. Moerer P. et al.Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet. 1998; 19: 379-383Crossref PubMed Scopus (1342) Google Scholar, 14van de Wetering M. Sancho E. Verweij C. et al.The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1761) Google Scholar Constitutive transactivation of the target genes of TCF-4 by β-catenin imposes a crypt progenitor phenotype on intestinal epithelial cells and is considered to be crucial for colorectal carcinogenesis.15Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2975) Google Scholar Our recent series of proteomic studies have revealed that a large variety of nuclear proteins participate in the β-catenin and TCF-4 complex and modulate its transcriptional activity.16Idogawa M. Yamada T. Honda K. et al.Poly(ADP-ribose) polymerase-1 is a component of the oncogenic T-cell factor-4/β-catenin complex.Gastroenterology. 2005; 128: 1919-1936Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 17Shitashige M. Naishiro Y. Idogawa M. et al.Involvement of splicing factor-1 in β-catenin/T-cell factor-4-mediated gene transactivation and pre-mRNA splicing.Gastroenterology. 2007; 132: 1039-1054Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 18Idogawa M. Masutani M. Shitashige M. et al.Ku70 and poly(ADP-ribose) polymerase-1 competitively regulate β-catenin and T-cell factor-4-mediated gene transactivation: possible linkage of DNA damage recognition and Wnt signaling.Cancer Res. 2007; 67: 911-918Crossref PubMed Scopus (62) Google Scholar, 19Huang L. Shitashige M. Satow R. et al.Functional interaction of DNA topoisomerase IIα with the β-catenin and T-cell factor-4 complex.Gastroenterology. 2007; 133: 1569-1578Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Furthermore, the protein composition of the β-catenin and TCF-4 nuclear complex is not fixed but dynamically regulated by endogenous programs associated with intestinal epithelial cell differentiation. In the present study, we first searched for other protein components of native complexes containing TCF-4 to obtain further insight into the functional properties of TCF-4. Human embryonic kidney (HEK) cell line 293 and colorectal cancer cell line DLD-1 were obtained from the Health Science Research Resources Bank (Osaka, Japan). Human colorectal cancer cell line HCT-116 was purchased from the American Type Culture Collection (Rockville, MD). Antibodies used in this study and their suppliers are listed in Supplementary Table S1 (see Supplementary Table S1 online at www.gastrojournal.org). Five × 108 cells were lysed with lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 1 mmol/L EDTA, 0.5% Triton X-100, 0.05% SDS, 2 mmol/L TCEP) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 mmol/L NaF, a protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN), and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St. Louis, MO). The cell lysate was spun for 2 hours at 100,000g, and the supernatant was precipitated with anti-TCF-4 mouse monoclonal antibody-coupled Dynabeads Protein G (Dynal Biotech, Oslo, Norway) at 4°C for 5 hours. The precipitates were digested with modified trypsin (Promega, Madison, WI). The resulting tryptic peptides were concentrated and desalted with a 500-μm inner diameter × 1 mm HiQ sil C18-3 trapping column (KYA Technologies, Tokyo, Japan). The peptides were then fractionated with a 0%–80% acetonitrile gradient (200 nL/minute for 3 hours) using a 150-μm inner diameter × 5 cm C18W-3 separation column (KYA Technologies) and analyzed with a Q-Star Pulsar-i mass spectrometer equipped with a nanospray ionization source (Applied Biosystems, Foster City, CA).20Hara T. Honda K. Shitashige M. et al.Mass spectrometry analysis of the native protein complex containing actinin-4 in prostate cancer cells.Mol Cell Proteomics. 2007; 6: 479-491Crossref PubMed Scopus (47) Google Scholar Cell lysates were incubated at 4°C overnight with anti-TCF-4 monoclonal antibody, anti-β-catenin, anti-Nupp62 goat polyclonal antibodies, anti-Ran binding protein-2 (anti-RanBP2), anti-Ran GTPase-activating protein-1 (anti-RanGAP1), anti-Ubc9, anti-histone H4, anti-small ubiquitin-related modifier 1 (anti-SUMO1), anti-Nup98 rabbit polyclonal antibodies, anti-Nup153 sheep polyclonal antibody, normal mouse IgG, normal goat IgG, normal rabbit IgG, or normal sheep IgG and precipitated with Dynabeads Protein G (Dynal Biotech).16Idogawa M. Yamada T. Honda K. et al.Poly(ADP-ribose) polymerase-1 is a component of the oncogenic T-cell factor-4/β-catenin complex.Gastroenterology. 2005; 128: 1919-1936Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Human β-catenin complementary DNA (cDNA) lacking the 134-amino acid sequence in its NH2-terminus was subcloned into pFLAG-CMV4 (Sigma-Aldrich) to prepare pFLAG-β-cateninΔN134.17Shitashige M. Naishiro Y. Idogawa M. et al.Involvement of splicing factor-1 in β-catenin/T-cell factor-4-mediated gene transactivation and pre-mRNA splicing.Gastroenterology. 2007; 132: 1039-1054Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Human SUMO1 and Ubc9 expression constructs (pcDNA3.1-SUMO1[GG] and pcDNA3.1-Ubc9)21Rangasamy D. Wilson V.G. Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein.J Biol Chem. 2000; 275: 30487-30495Crossref PubMed Scopus (55) Google Scholar were kindly provided by Dr Van G. Wilson (Texas A&M University System Health Science Center, College Station, TX). Human RanBP2 cDNA22Yokoyama N. Hayashi N. Seki T. et al.A giant nucleopore protein that binds Ran/TC4.Nature. 1995; 376: 184-188Crossref PubMed Scopus (422) Google Scholar was kindly provided by Drs Takeharu Nishimoto and Tomoyuki Ohba (Kyushu University, Fukuoka, Japan) and subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). pDsRed1-N1-human RanGAP123Joseph J. Tan S.H. Karpova T.S. et al.SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles.J Cell Biol. 2002; 156: 595-602Crossref PubMed Scopus (231) Google Scholar was kindly provided by Dr Mary Dasso (National Institutes of Health, Bethesda, MA). Human sentrin-specific protease (SENP) 1, SENP2, and SENP3 expression constructs (pCMV-Tag2B-SENP1, pCMV-Tag2B-SENP2, and pcDNA-RGS-SENP3)24Cheng J. Wang D. Wang Z. et al.SENP1 enhances androgen receptor-dependent transcription through desumoylation of histone deacetylase 1.Mol Cell Biol. 2004; 24: 6021-6028Crossref PubMed Scopus (158) Google Scholar were kindly provided by Dr Edward T. H. Yeh (University of Texas, MD Anderson Cancer Center, Houston, TX). Human SENP3 cDNA was further subcloned into pCMV-Tag 2B (Stratagene, La Jolla, CA). Small interfering RNA (siRNA) were synthesized and annealed by Dharmacon (Chicago, IL). The sequences of siRNAs used in this study are available in Supplementary Table S2 (see Supplementary Table S2 online at www.gastrojournal.org). Protein samples were fractionated by SDS-PAGE and blotted onto Immobilon-P membrane (Millipore, Billerica, MA). After incubation with the primary antibodies at 4°C overnight, the blots were detected with the relevant horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-goat, or anti-sheep IgG antibody and ECL Western blotting detection reagents (Amersham Biosciences, Amersham, United Kingdom).25Honda K. Yamada T. Hayashida Y. et al.Actinin-4 increases cell motility and promotes lymph node metastasis of colorectal cancer.Gastroenterology. 2005; 128: 51-62Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar In vitro sumoylation was performed in 50-μL reaction mixtures at 30°C for 30 minutes or the indicated time using a SUMOylation kit (BIOMOL, Plymouth Meeting, PA). Purified glutathione S-transferase (GST)-tagged recombinant TCF-4 protein was purchased from Abnova (Taipei City, Taiwan), and GST-β-catenin was purchased from Upstate (Charlottesville, VA). Aos1/Uba2, Ubc9, His-tagged SUMO1[GG], GST-RanBP2ΔFG (amino acids 2553–2838), GST-RanGAP1 (amino acids 418–587), GST-SENP1 (amino acids 415–643), and GST-SENP2 (amino acids 368–549) were obtained from BIOMOL. The reaction was analyzed directly by immunoblotting with anti-TCF-4, anti-SUMO1, and anti-GST antibodies or immunoprecipitated with anti-TCF-4, anti-β-catenin, and anti-SUMO1 antibodies and analyzed by immunoblotting with anti-TCF-4, anti-β-catenin, and anti-SUMO antibodies. Forty-eight hours after transfection with the indicated plasmids and siRNA, total cellular proteins were immunoprecipitated with anti-TCF-4 monoclonal antibody. The precipitates were then analyzed by immunoblotting with anti-TCF-4 rabbit polyclonal antibody. Cells cultured on glass coverslips (Asahi Technoglass, Tokyo, Japan) were fixed with 4% paraformaldehyde at room temperature for 10 minutes and permeabilized with 0.2% Triton X-100. After blocking with 10% normal swine serum (Vector Laboratory, Burlingame, CA), the cells were incubated with primary antibodies at 4°C overnight and subsequently with Alexa fluor-488 anti-mouse antibody (Invitrogen). The specimens were examined with a laser scanning microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany).25Honda K. Yamada T. Hayashida Y. et al.Actinin-4 increases cell motility and promotes lymph node metastasis of colorectal cancer.Gastroenterology. 2005; 128: 51-62Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar Cells were transfected using the Lipofectamine 2000 reagent (Invitrogen). Twenty-four hours later, 750, 300, and 1000 μg/mL G418 (Geneticin; Invitrogen) was added to the culture medium of HEK293, DLD1, and HCT-116 cells, respectively. Cells were stained with Giemsa solution (Wako, Osaka, Japan) after selection for 8 days.17Shitashige M. Naishiro Y. Idogawa M. et al.Involvement of splicing factor-1 in β-catenin/T-cell factor-4-mediated gene transactivation and pre-mRNA splicing.Gastroenterology. 2007; 132: 1039-1054Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar A pair of luciferase reporter constructs, TOP-FLASH and FOP-FLASH (Upstate), was used to evaluate TCF/LEF transcriptional activity. Cells were transiently transfected in triplicate with one of the luciferase reporters and phRL-TK (Promega) by using the Lipofectamine 2000 reagent (Invitrogen). Luciferase activity was measured with the Dual-luciferase Reporter Assay system (Promega) using Renilla reniformis luciferase activity as an internal control.16Idogawa M. Yamada T. Honda K. et al.Poly(ADP-ribose) polymerase-1 is a component of the oncogenic T-cell factor-4/β-catenin complex.Gastroenterology. 2005; 128: 1919-1936Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 18Idogawa M. Masutani M. Shitashige M. et al.Ku70 and poly(ADP-ribose) polymerase-1 competitively regulate β-catenin and T-cell factor-4-mediated gene transactivation: possible linkage of DNA damage recognition and Wnt signaling.Cancer Res. 2007; 67: 911-918Crossref PubMed Scopus (62) Google Scholar Forty-eight hours after transfection, cells were packed in phosphate-buffered saline (PBS) supplemented with 1 mmol/L DTT, 10 mmol/L PMSF, 1 mmol/L NaF, 0.4 mmol/L sodium orthovanadate, 10 mmol/L N-ethylmaleimide, and a protease inhibitor cocktail. Nuclear and total cell lysates were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) and analyzed by immunoblotting with anti-β-catenin, anti-TCF-4, and anti-β-actin antibodies. Blot intensity was quantified using a LAS-3000 scanner and Science Lab 2003 software (Fuji Film, Tokyo, Japan).26Sato S. Idogawa M. Honda K. et al.β-Catenin interacts with the FUS proto-oncogene product and regulates pre-mRNA splicing.Gastroenterology. 2005; 129: 1225-1236Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Total RNA was prepared with RNeasy Mini Kit (Qiagen, Valencia, CA). DNase-I-treated RNA was random primed and reverse transcribed using SuperScript II reverse transcriptase (Invitrogen). The TaqMan universal polymerase chain reaction (PCR) master mix and predesigned TaqMan Gene Expression probe and primer sets were purchased from Applied Biosystems. Amplification data measured as an increase in reporter fluorescence were collected using the PRISM 7000 Sequence Detection system (Applied Biosystems). The messenger RNA (mRNA) expression level relative to the internal control (β-actin) was calculated by the comparative threshold cycle (CT) method.19Huang L. Shitashige M. Satow R. et al.Functional interaction of DNA topoisomerase IIα with the β-catenin and T-cell factor-4 complex.Gastroenterology. 2007; 133: 1569-1578Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, immunoprecipitated with anti-TCF-4 antibody were analyzed directly by nanoflow liquid chromatography (LC) and tandem mass spectrometry (MS/MS).17Shitashige M. Naishiro Y. Idogawa M. et al.Involvement of splicing factor-1 in β-catenin/T-cell factor-4-mediated gene transactivation and pre-mRNA splicing.Gastroenterology. 2007; 132: 1039-1054Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar HCT-116 carries a mutation in the CTNNB1 gene,10Ilyas M. Tomlinson I.P. Rowan A. et al.β-Catenin mutations in cell lines established from human colorectal cancers.Proc Natl Acad Sci U S A. 1997; 94: 10330-10334Crossref PubMed Scopus (445) Google Scholar whereas DLD1 carries the truncational mutation and loss of heterozygosity of the APC gene.6Yamada T. Takaoka A.S. Naishiro Y. et al.Transactivation of the multidrug resistance 1 gene by T-cell factor 4/β-catenin complex in early colorectal carcinogenesis.Cancer Res. 2000; 60: 4761-4766PubMed Google Scholar As a result, both cell lines accumulate nuclear β-catenin protein. The redundant LC-MS/MS data were compiled using in-house software, and 70 proteins were found to be constantly immunoprecipitated with anti-TCF-4 antibody in 4 independent experiments (2 experiments using HCT-116 and 2 experiments using DLD1) (see Supplementary Table S3 online at www.gastrojournal.org). These 70 proteins included TCF-4 (transcription factor 7-like 2) itself, β-catenin (catenin, β 1), and known TCF-4- or β-catenin-associating proteins such as poly(ADP-ribose) synthetase/polymerase (PARP-1), thyroid-lupus autoantigen p70 (Ku70), DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (DDX5), desmoplakin, and heterogeneous nuclear ribonucleoproteins (hnRNPs) A2/B1 and M,16Idogawa M. Yamada T. Honda K. et al.Poly(ADP-ribose) polymerase-1 is a component of the oncogenic T-cell factor-4/β-catenin complex.Gastroenterology. 2005; 128: 1919-1936Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 18Idogawa M. Masutani M. Shitashige M. et al.Ku70 and poly(ADP-ribose) polymerase-1 competitively regulate β-catenin and T-cell factor-4-mediated gene transactivation: possible linkage of DNA damage recognition and Wnt signaling.Cancer Res. 2007; 67: 911-918Crossref PubMed Scopus (62) Google Scholar, 26Sato S. Idogawa M. Honda K. et al.β-Catenin interacts with the FUS proto-oncogene product and regulates pre-mRNA splicing.Gastroenterology. 2005; 129: 1225-1236Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar indicating the authenticity of the results. We noticed that a large number of NPC components were coimmunoprecipitated with anti-TCF-4 antibody (see Supplementary Table S3> online at www.gastrojournal.org). Nucleoporin (Nup) is a generic name for the structural proteins of the NPC.27Rout M.P. Aitchison J.D. The nuclear pore complex as a transport machine.J Biol Chem. 2001; 276: 16593-16596Crossref PubMed Scopus (223) Google Scholar Ran is a nuclear GTP-binding protein required for bidirectional transport of proteins and ribonucleoproteins across the NPC.27Rout M.P. Aitchison J.D. The nuclear pore complex as a transport machine.J Biol Chem. 2001; 276: 16593-16596Crossref PubMed Scopus (223) Google Scholar, 28Koepp D.M. Silver P.A. A GTPase controlling nuclear trafficking: running the right way or walking RANdomly?.Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar RanGAP1 resides at the cytoplasmic periphery of the NPC29Mahajan R. Delphin C. Guan T. et al.A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar and regulates the hydrolysis of RanGTP.28Koepp D.M. Silver P.A. A GTPase controlling nuclear trafficking: running the right way or walking RANdomly?.Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar RanBP2/Nup358 is the SUMO E3 ligase of RanGAP1.29Mahajan R. Delphin C. Guan T. et al.A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 30Reverter D. Lima C.D. Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex.Nature. 2005; 435: 687-692Crossref PubMed Scopus (383) Google Scholar RanBP2 is necessary for localization of SUMO-conjugated RanGAP1 to the cytoplasmic face of the NPC.29Mahajan R. Delphin C. Guan T. et al.A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar RanBP2, Ubc9, RanGAP1, and SUMO1 have been reported to form a stable protein complex.30Reverter D. Lima C.D. Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex.Nature. 2005; 435: 687-692Crossref PubMed Scopus (383) Google Scholar, 31Tatham M.H. Kim S. Jaffray E. et al.Unique binding interactions among Ubc9, SUMO, and RanBP2 reveal a mechanism for SUMO paralog selection.Nat Struct Mol Biol. 2005; 12: 67-74Crossref PubMed Scopus (115) Google Scholar The identity of these proteins was confirmed by immunoblotting using available antibodies. β-Catenin, RanBP2, RanGAP1, Ubc9, histone H4, and various Nups were present in the immunoprecipitate with the anti-TCF-4 antibody but not with control IgG (Figure 1A). Conversely, TCF-4 protein was detected in the immunoprecipitates with anti-RanBP2, anti-RanGAP1, anti-Ubc9, anti-histone H4, anti-SUMO1, and anti-Nup antibodies but not with control IgG (Figure 1B). To examine whether RanBP2 has E3-ligase activity for TCF-4, we reconstituted the sumoylating complex in vitro using recombinant Aos1/Uba2 (SUMO E1-activating enzyme), Ubc9 (SUMO E2-conjugating enzyme), and His6-tagged SUMO1[GG] (Figure 2).32Melchior F. Schergaut M. Pichler A. SUMO: ligases, isopeptidases and nuclear pores.Trends Biochem Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 33Seeler J.S. Dejean A. Nuclear and unclear functions of SUMO.Nat Rev Mol Cell Biol. 2003; 4: 690-699Crossref PubMed Scopus (587) Google Scholar The SUMO1 precursor is processed by a C-terminal hydrolase to produce the mature form with exposure of the C-terminal Gly-Gly residues (SUMO1[GG]) that is required for the conjugation of SUMO1 to substrates.21Rangasamy D. Wilson V.G. Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein.J Biol Chem. 2000; 275: 30487-30495Crossref PubMed Scopus (55) Google Scholar RanBP2ΔFG retains the E3 ligase activity and substrate recognition but lacks the RING finger-like domain that shows homology with other SUMO E3 ligases.34Pichler A. Gast A. Seeler J.S. et al.The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar The conjugation of SUMO1 was monitored by detection of slowly migrating bands in immunoblots with anti-TCF-4, anti-SUMO1, and anti-GST antibodies. GST-tagged RanBP2ΔFG was autosumoylated in the presence of SUMO1[GG] (Figure 2A, lane 5, anti-SUMO1 and anti-GST). TCF-4 was sumoylated in the presence of SUMO1 and RanBP2ΔFG (Figure 2A, lane 7, anti-TCF-4), and the degree of its sumoylation increased in a time-dependent manner (Figure 2B). RanGAP1 is a known substrate of RanBP2 E3-ligase.31Tatham M.H. Kim S. Jaffray E. et al.Unique binding interactions among Ubc9, SUMO, and RanBP2 reveal a mechanism for SUMO paralog selection.Nat Struct Mol Biol. 2005; 12: 67-74Crossref PubMed Scopus (115) Google Scholar The inclusion of RanGAP1 slightly inhibited the sumoylation of TCF-4 (Figure 2C, lane 4), probably because of competition between the 2 substrate proteins. Addition of SUMO-specific proteases SENP1 and SENP234Pichler A. Gast A. Seeler J.S. et al.The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 35Hang J. Dasso M. Association of the human SUMO-1 protease SENP2 with the nuclear pore.J Biol Chem. 2002; 277: 19961-19966Crossref PubMed Scopus (196) Google Scholar inhibited the sumoylation of TCF-4 (Figure 2D, lanes 6 and 7). We next examined whether endogenous TCF-4 protein is sumoylated in vivo by RanBP2. HCT-116 (Figure 3A, left) and DLD1 (Figure 3A, right) cells were transfected with SUMO1[GG], RanBP2, and SENP1 or SENP2 cDNA and analyzed by immunoprecipitation and immunoblotting. Expression of SUMO1 and RanBP2 induced the sumoylation of TCF-4 (Figure 3A, lane 3, anti-TCF-4), and the sumoylation was diminished by SENP2 (Figure 3A, lane 5). SENP2 has been shown to bind to Nup153, which is localized to the nucleoplasmic face of the NPC.35Hang J. Dasso M. Association of the human SUMO-1 protease SENP2 with the nuclear pore.J Biol Chem. 2002; 277: 19961-19966Crossref PubMed Scopus (196) Google Scholar SENP1 inhibited the sumoylation of TCF-4 in vitro (Figure 2D), but not in vivo (Figure 3A, lane 4), probably because SENP1 is not
Referência(s)