SUMO-1/Ubc9 Promotes Nuclear Accumulation and Metabolic Stability of Tumor Suppressor Smad4

Tumor suppressor Smad4/DPC4 is a central intracellular signal transducer for transforming growth factor-β (TGF-β) signaling. We recently reported that transcriptional potential of Smad4 was regulated by SUMOylation in transfected HeLa cells (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), but the precise mechanism and function of Smad4 SUMOylation in TGF-β signaling remain to be elucidated. Here, we describe the regulation of TGF-β signaling by SUMOylation through the control of Smad4 metabolic stability and subcellular localization. We found that SUMO-1 overexpression strongly increases Smad4 levels, while inhibition of SUMOylation by small interfering RNA (siRNA)-mediated knockdown of the E2 enzyme Ubc9 reduces endogenous Smad4 levels. Concomitantly, SUMO-1 overexpression enhances and Ubc9 knockdown reduces levels of intranuclear Smad4, growth inhibitory response, as well as transcriptional responses to TGF-β. Comparison of wild type and mutant forms of Smad4 for SUMOylation, ubiquitination, and half-life allows the conclusion that SUMO-1 modification serves to protect Smad4 from ubiquitin-dependent degradation and consequently enhances the growth inhibitory and transcriptional responses of Smad4. Tumor suppressor Smad4/DPC4 is a central intracellular signal transducer for transforming growth factor-β (TGF-β) signaling. We recently reported that transcriptional potential of Smad4 was regulated by SUMOylation in transfected HeLa cells (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), but the precise mechanism and function of Smad4 SUMOylation in TGF-β signaling remain to be elucidated. Here, we describe the regulation of TGF-β signaling by SUMOylation through the control of Smad4 metabolic stability and subcellular localization. We found that SUMO-1 overexpression strongly increases Smad4 levels, while inhibition of SUMOylation by small interfering RNA (siRNA)-mediated knockdown of the E2 enzyme Ubc9 reduces endogenous Smad4 levels. Concomitantly, SUMO-1 overexpression enhances and Ubc9 knockdown reduces levels of intranuclear Smad4, growth inhibitory response, as well as transcriptional responses to TGF-β. Comparison of wild type and mutant forms of Smad4 for SUMOylation, ubiquitination, and half-life allows the conclusion that SUMO-1 modification serves to protect Smad4 from ubiquitin-dependent degradation and consequently enhances the growth inhibitory and transcriptional responses of Smad4. The strength and intensity of TGF-β 1The abbreviations used are: TGF-β, transforming growth factor-β; SUMO-1, small ubiquitin-like modifier-1; RNAi, RNA interference; siRNA, small interfering RNA; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; SBE, Smad-binding element; FITC, fluorescein isothiocyanate; MTS, 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NES, nuclear export sequence; NLS, nuclear localization sequence; N, nuclear; C, cytoplasmic; PAI-1, plasminogen activator inhibitor-1; DAPI, 4′,6-diamidino-2-phenylindole. signaling require a tight control of the activity of each signaling component, including the central signal transducing Smad proteins. Tumor suppressor Smad4/DPC4 is the common mediator for TGF-β signaling by forming a complex with R-Smads in response to ligand stimulation (2Dennler S. Goumans M.J. Ten Dijke P. J. Leukoc. Biol. 2002; 71: 731-740PubMed Google Scholar, 3Massagué J. Nat. Rev. Mol. Cell. 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Cell Biol. 1999; 1: 472-478Crossref PubMed Scopus (297) Google Scholar, 11Xu J. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4820-4825Crossref PubMed Scopus (172) Google Scholar, 12Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (427) Google Scholar, 13Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar). Ubiquitination, the covalent attachment of ubiquitin to proteins, predominantly serves to target proteins for their degradation by proteasomes (14Ciechanover A. Orian A. Schwartz A.L. Bioessays. 2000; 22: 442-451Crossref PubMed Scopus (702) Google Scholar). Interestingly, a number of ubiquitin-related proteins are also present in eukaryotic cells (15Hochstrasser M. Nat. Cell Biol. 2000; 2: E153-E157Crossref PubMed Scopus (368) Google Scholar). These proteins, including the small ubiquitin-like modifier-1 (SUMO-1), utilize a conjugation system that is similar to ubiquitination (16Hay R.T. Trends Biochem. Sci. 2001; 26: 332-333Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (653) Google Scholar, 18Yeh E.T. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (418) Google Scholar). In contrast to ubiquitination, SUMO-1 modifications of target proteins do not promote their degradation, but modulate the subcellular localization or biological activities of targets (19Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 20Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar, 21Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (957) Google Scholar, 22Kamitani T. Kito K. Nguyen H.P. Wada H. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 26675-26682Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 23Lin X. Sun B. Liang M. Liang Y.-Y. Gast A. Hildebrand J. Brunicardi F.C. Melchior F. Feng X.-H. Mol. Cell. 2003; 11: 1389-1396Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 24Freiman R. Tjian R. Cell. 2003; 112: 11-17Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 25Verger A. Perdomo J. Crossley M. EMBO Rep. 2003; 4: 137-142Crossref PubMed Scopus (375) Google Scholar). Smad4 is the central mediator for signaling of TGF-β superfamily, and thus it is important to study the regulation of Smad4. Recently, we and another group identified Smad4 as a substrate of SUMOylation pathway (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 33Lee P.S. Chang C. Liu D. Derynck R. J. Biol. Chem. 2003; 278: 27853-27863Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Here we further elucidate the mechanism of how the SUMOylation regulates Smad4 activity under physiological conditions. We have found that RNA interference (RNAi)-mediated silencing of the human Ubc9 gene disrupts Smad4 SUMOylation, decreases Smad4 stability, reduces Smad4 accumulation in the nucleus, and consequently blocks TGF-β signaling. Thus, SUMOylation of tumor suppressor Smad4 provides a novel mechanism to control TGF-β antiproliferative signaling. Plasmids, Cell Lines, and Transfections—Mammalian expression plasmids for epitope (HA, FLAG, and His)-tagged Smad4, Ubc9, and SUMO were described previously (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 26Feng X.-H. Liang Y.-Y. Liang M. Zhai W. Lin X. Mol. Cell. 2002; 9: 133-143Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 27Feng X.-H. Lin X. Derynck R. EMBO J. 2000; 19: 5178-5193Crossref PubMed Scopus (348) Google Scholar). Human HeLa cells and mink lung epithelial Mv1Lu (RI-14 line) cells were grown and transfected as described previously (26Feng X.-H. Liang Y.-Y. Liang M. Zhai W. Lin X. Mol. Cell. 2002; 9: 133-143Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 27Feng X.-H. Lin X. Derynck R. EMBO J. 2000; 19: 5178-5193Crossref PubMed Scopus (348) Google Scholar). Ni-NTA Precipitation and Western Blot—Ni-NTA precipitation and Western blot analysis were essentially as described previously (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Briefly, HeLa cells were transfected with expression plasmids for His-tagged Smad4 and FLAG-tagged SUMO1 and harvested in guanidinium lysis buffer (6 m guanidinium HCl, 0.1 m NaPO4, 0.01 m Tris·Cl, pH 8). His-tagged Smad4 was retrieved from the lysates by using Ni-NTA beads (Qiagen), followed by standard gel electrophoresis and Western blotting analysis using anti-Smad4 (Santa Cruz Biotechnology) and anti-FLAG (Sigma) antibody. Transcription Reporter Assays—Transfections, TGF-β treatment, and reporter assays were carried out as described previously (27Feng X.-H. Lin X. Derynck R. EMBO J. 2000; 19: 5178-5193Crossref PubMed Scopus (348) Google Scholar). Generally, HeLa cells at 25–30% confluence were transfected with expression plasmids for Smads and/or reporter plasmids. Reporter plasmid SBE-Luc contains the luciferase gene under control of the Smad-binding elements (SBE) (28Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). The amount of transfected DNA was always the same by adding vector DNA, whenever needed. 40–45 h after transfection, cells were treated with 200 pm TGF-β for 12 h. Cells were then harvested for measurement of luciferase and β-galactosidase activities. All assays were done in triplicate and all values were normalized for transfection efficiency against β-galactosidase activity. Immunofluorescence—HeLa and RI-14 cells, untransfected or transfected as specified in the text and figure legends (Figs. 1, C and E, and 3C), were grown on cover slips, fixed with cold methanol, and blocked with 2% bovine serum albumin in phosphate-buffered saline, pH 7. Cells were then stained with anti-Smad4 or anti-His monoclonal antibodies, followed with FITC-conjugated anti-mouse antibody (Sigma), and visualized under a Zeiss Axioplan II microscope.Fig. 3Gene silencing of Ubc9 reduces Smad4 SUMOylation and decreases Smad4 stability. A, Ubc9 siRNA down-regulated Ubc9 expression and Smad4 SUMOylation. HeLa cells were tranfected with Ubc9 siRNA as described under “Experimental Procedures.” Smad4-SUMO conjugates were similarly analyzed as described in the legend to Fig. 1B. Endogenous Ubc9 and β-actin were detected using anti-Ubc9 and anti-actin antibody, respectively. WCL, whole cell lysates. B, Ubc9 siRNA reduced the steady state level of endogenous Smad4. Endogenous Ubc9, Smad4, Smad2/3, and β-actin were detected using appropriate antibodies in Western blots. C, Ubc9 siRNA inhibited TGF-β-induced nuclear accumulation of endogenous Smad4. Smad4 was visualized using FITC-conjugated anti-Smad4 antibody. DAPI indicates the nuclear staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pulse-Chase and Ubiquitination Assay—HeLa cells were transfected with His-tagged wild type, K113R/K159R or R100T mutant. 48 h later, cells were pulsed for 30 min with 400 μCi ml–1 35Smethionine/cysteine, and then chased in regular medium supplemented with non-radioactive methionine/cysteine for varying time periods, as indicated in Fig. 2A. Cell lysates were harvested and subjected to Ni-NTA precipitation. The precipitated Smad4 proteins were analyzed on SDS-PAGE and visualized by autoradiography. For ubiquitination assay, we also performed precipitation of Smad4 from lysates of the HeLa cells, which was co-transfected with HA-tagged ubiquitin. Precipitated Smad4 were subjected to SDS-PAGE, and its ubiquitination was recognized by immunoblotting with anti-HA (ubiquitin) antibody. RNA Interference—The target sequence of the Ubc9 siRNA was CAAAAAATCCCGATGGCAC (Dharmacon Research), which corresponds with nucleotides 86–104 downstream from initiation codon of the human Ubc9 coding region. siRNA was transfected into HeLa cells using LipofectAMINE 2000 (Invitrogen), as described previously (29Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8160) Google Scholar). For immunofluorescence, 48 h upon siRNA transfection, cells were treated with TGF-β for 1 h, fixed with ice-cold methanol, and immunostained with FITC-conjugated anti-Smad4 antibody (B8, Santa Cruz) to visualize the endogenous Smad4. For reporter assays, SBE-luc reporter plasmid was cotransfected with siRNA. h later, cells were treated with TGF-β for 12 h and harvested for luciferase For HeLa cells were transfected with plasmid DNA, as specified in the upon DNA transfection, the same cells were transfected with Ubc9 siRNA. h later, cells were and cell lysates were used to Ubc9 in anti-Ubc9 Western blot or further to Smad4 SUMOylation. Cell cells were transfected with siRNA as described h, 1 cells were then into each well of a attachment to the well cells were treated with or TGF-β for or of cell was then carried out using was to the cells, which were then to the cell for h. The of cells was at in a The TGF-β-induced inhibition of cell was by the inhibition SUMOylation of Smad4 in the and by of Smad4 signaling that Smad4 is in cells (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The modification on in the of Smad4, and X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). the subcellular of Smad4 SUMOylation, we the in SUMOylation of Smad4 in the nuclear export sequence or the nuclear localization sequence Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). SUMOylation and subcellular localization of Smad4 are shown in Fig. 1, and C, respectively. Smad4 mutant was in the nucleus and its SUMOylation was In conjugation of SUMO1 to the which was in the not be detected In we the SUMOylation of wild type Smad4 in the or of nuclear export B. the level of SUMOylation of Smad4 Thus, that Smad4 SUMOylation in the studies also to the nucleus as SUMOylation M.S. C. Hay R.T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), SUMOylation in the Smad4 is in the nucleus in the We that TGF-β on SUMOylation of Smad4 We then how TGF-β regulated SUMOylation of Smad4. of 200 pm TGF-β to the medium the level of SUMO1 modification of Smad4 treatment, the Smad4-SUMO the at 12 h, the level to or level Thus, the the that the in Smad4 SUMOylation be to the TGF-β-induced of Smad4 into the nucleus, where SUMOylation SUMOylation TGF-β-induced of Smad4 regulation of Smad4 activity is its nuclear in response to TGF-β. Smad4 also Fig. and is the nucleus and Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). In we SUMOylation Smad4 subcellular accumulation in RI-14 from are shown in Fig. Smad4 is a we the subcellular localization of Smad4 into nuclear or nuclear the or a the nucleus and In the of TGF-β were of cells that wild type Smad4 in the nucleus and of cells in the or in TGF-β of cells Smad4 in the nucleus, while were in the or the that TGF-β a nuclear of Smad4 We also the of SUMO1 expression on subcellular of Smad4. In the of TGF-β SUMO1 a on Smad4 nuclear localization. In with cells of cells Smad4 in the SUMO1 of TGF-β-induced nuclear accumulation of Smad4, with of cells nuclear localization of Smad4. of and nuclear of Smad4 also similar not SUMO expression on Smad4 nuclear in the of TGF-β, that SUMO1 to Smad4 and/or stability in the of SUMOylation and of Smad4 which degradation of SUMO1 not to that SUMOylation proteins by for the same for ubiquitination (19Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). the of SUMOylation we that the steady state level of Smad4 is in the of but overexpression of SUMO1 the steady state level of Smad4 (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). the metabolic stability of Smad4, we performed We the stability of wild type, K113R/K159R and a Smad4 mutant with that previously shown to degradation J. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4820-4825Crossref PubMed Scopus (172) Google Scholar). The half-life of wild type Smad4 was found to be h, the level of the mutant upon h of The R100T mutant a half-life of h. were in not These that or also as a for Smad4 Thus, the SUMOylation on or of the same Smad4 from conclusion is with the reduced ubiquitination on the K113R/K159R mutant X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google and ubiquitination on the R100T mutant 11Xu J. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4820-4825Crossref PubMed Scopus (172) Google Scholar). is of of which SUMOylation ubiquitination through the (19Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). a of was found to promote Smad4 degradation M. X. Y. S. L. X. EMBO Rep. 2002; PubMed Scopus (140) Google Scholar). overexpression of the degradation of K113R/K159R mutant not that ubiquitin or factors are for Smad4 degradation through the and/or a of SUMOylation in Smad4 stability, we out to the R100T mutant have a or in SUMOylation. shown in Fig. the R100T mutant to be while the wild type was in HeLa cells that the of mutant be for its to be In of the that the R100T mutant nuclear localization with in response to TGF-β J. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4820-4825Crossref PubMed Scopus (172) Google Scholar), its of SUMOylation be to a that SUMO modification on the SUMOylation or blocks ubiquitination on Smad4, the of SUMOylation thus to the ubiquitination and degradation of the R100T mutant and of ubiquitination at and the metabolic stability of the that the mutant a half-life the R100T mutant similar was reported recently P.S. Chang C. Liu D. Derynck R. J. Biol. Chem. 2003; 278: 27853-27863Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). SUMOylation as mechanism for cells to TGF-β growth inhibitory control in Smad4 SUMOylation the of Endogenous Smad4 described that SUMO modification a in Smad4 activity. reporter assays that overexpression of SUMO1 and Ubc9 TGF-β-induced transcriptional responses (1Lin X. Liang M. Liang Y.-Y. Brunicardi F.C. Melchiors F. Feng X.-H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). in a we to the of endogenous Ubc9 expression using Ubc9 is the E2 enzyme for modification by of SUMO inhibition of its expression SUMOylation of its We used a siRNA that targets to the human Ubc9 coding and its in silencing Ubc9 expression in HeLa 48 h after transfection with the siRNA, HeLa cells a level of endogenous Ubc9 in with the control (Figs. and B, and In expression of β-actin (Figs. and B, and of HeLa cells with not the level of Ubc9 not We then Ubc9 expression the SUMOylation of Smad4 and the of SUMOylation on Smad4 stability. that of Ubc9 siRNA in the steady state level of Smad4 in transfected HeLa cells the steady state level of endogenous Smad4 upon siRNA transfection and Smad4 was present in the nucleus and and a and TGF-β a of Smad4 in the nucleus, a image under of Ubc9 expression with siRNA the of TGF-β on Smad4 of and were the same with or the siRNA Ubc9 for TGF-β-induced and Endogenous its responses by gene transcription involved in growth regulation and extracellular matrix We then the of Ubc9 gene silencing on TGF-β gene responses and cell We the SBE-luc reporter (28Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). of siRNA of TGF-β-induced SBE-luc reporter expression in HeLa cells as well as cells not the of Ubc9 or siRNA on SBE-luc activity of the Ubc9 expression also the of endogenous target gene of TGF-β as In HeLa cells, TGF-β the of 1 and with Ubc9 siRNA to the and the level of β-actin of TGF-β and/or siRNA TGF-β also of epithelial of HeLa cells be inhibited by TGF-β, to growth inhibition not HeLa cells be transfected using siRNA, we the growth inhibitory response in cells in the or of Ubc9 siRNA. of TGF-β (for or a inhibition on the of HeLa cells treated with transfection In the growth inhibitory response of TGF-β was in Ubc9 cells, which growth inhibition by TGF-β. the HeLa cells a in to cells in the of TGF-β stimulation not that of Ubc9 expression using SUMOylation and consequently the on Smad4 activity in growth Smad4 TGF-β in further the of Ubc9 in Smad4 we overexpression of Ubc9 TGF-β signaling in and human Ubc9 but the target sequence in the human and Ubc9 we that Ubc9 for human by shown in Fig. overexpression of the Ubc9 gene the of TGF-β-induced SBE-luc reporter of Ubc9 Smad4 and of TGF-β be to the of TGF-β response by Smad4. we transfected HeLa cells with Smad4 or the mutant. is that expression of Smad4 or mutant the silencing of Ubc9 siRNA strongly that the in TGF-β signaling by Ubc9 siRNA is not to the of SUMO In study the function of SUMOylation in TGF-β signaling. SUMOylation on the growth inhibitory and transcriptional of Smad4, at to in Smad4 stability. SUMOylation to with ubiquitination at and SUMOylation by Ubc9 expression of the endogenous Smad4 and the growth inhibitory and transcriptional of Smad4. In with is the of a mutant which to be by SUMO and the that SUMOylation be important that Smad4 from degradation in SUMOylation of Smad4 in the The of Smad4 SUMOylation on TGF-β signaling also be to inhibition of its nuclear export by SUMOylation. The SUMOylation on and which the of Smad4, the of the with the export Fig. on the of SUMOylation, stability, and nuclear of Smad4. on the mechanism of Smad4 nuclear and export into the of SUMOylation in signaling We Melchior for We are to for Smad4 Vogelstein for for the cell and for B.