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Sumoylation of Smad4, the Common Smad Mediator of Transforming Growth Factor-β Family Signaling

2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês

10.1074/jbc.m301755200

1083-351X

Pierre S.W. Lee, Chenbei Chang, Dong Liu, Rik Derynck,

Kruppel-like factors research

Transforming growth factor-β (TGF-β) and TGF-β-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-β family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-β signaling through Smads. Transforming growth factor-β (TGF-β) and TGF-β-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-β family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-β signaling through Smads. Cellular processes, like cell proliferation, differentiation, and apoptosis, are elaborated by intracellular proteins that are regulated by signaling pathways (1Pawson T. Saxton T.M. Cell. 1999; 97: 675-678Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Individual mediators often respond to multiple pathways and participate in multiprotein complexes of varying composition, thus requiring an exquisitely sensitive, versatile and often rapid regulation of protein function, beyond the control at the level of gene expression (2Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1904) Google Scholar). Intracellular signaling mediators, including those that participate in transcription, are often subject to post-translational modifications that alter the activity and specificity, subcellular localization, stability, and/or interactions with other macromolecules, and are major determinants of a function of the protein (3Parekh R.B. Rohlff C. Curr. Opin. Biotechnol. 1997; 8: 718-723Crossref PubMed Scopus (53) Google Scholar, 4Brivanlou A.H. Darnell Jr., J.E. Science. 2002; 295: 813-818Crossref PubMed Scopus (511) Google Scholar). One class of modifications involves addition of a small chemical group to amino acids, e.g. through phosphorylation, acetylation, ADP-ribosylation, or methylation, whereas the second class involves attachment of larger macromolecules, such as polypeptides in the case of ubiquitylation and sumoylation, or polysaccharides. A variety of enzymes mediate polypeptide modifications and thereby often display substrate specificity, whereas complementary enzyme sets remove these modifications, thus illustrating the reversible nature of post-translational modification processes (5Yeh E.T. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (420) Google Scholar). This dynamic nature and the observations that a single signaling mediator is often subject to multiple types of modification illustrate the plasticity and the combinatorial control that regulates the functions of the signaling effectors. Ubiquitylation results in attachment of one or several 76-amino acid long ubiquitin polypeptides to Lys residues of target proteins (6Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3380) Google Scholar). This process requires the sequential actions of a ubiquitin-activating E1 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; SUMO-1, small ubiquitin-related modifier-1; TGF-β, transforming growth factor-β; aa, amino acid(s); HA, hemagglutinin; bFGF, basic fibroblast growth factor; Pipes, 1,4-piperazinediethanesulfonic acid. enzyme, conjugating E2 enzyme, and E3 ligase. Ubiquitin is activated by the E1 enzyme in an ATP-dependent manner and transferred as a thiol ester intermediate to one of several E2-conjugating enzymes. Covalent attachment of ubiquitin to a target protein is then mediated by one of the many E3 ligases that define substrate specificity. Polyubiquitylated proteins are usually targeted for proteasomal degradation (6Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3380) Google Scholar). Sumoylation involves the covalent attachment of SUMO, a ubiquitin-related polypeptide, to a Lys residue (7Hay R.T. Trends Biochem. Sci. 2001; 26: 332-333Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). However, unlike ubiquitylation, sumoylation has not been reported to target a substrate for degradation. As with ubiquitylation, sequential activities of E1, E2, and E3 enzymes are involved in sumoylation, but the identities of the enzymes are different. The SUMO activating enzyme E1, a heterodimer of Aos1 and Uba2, transfers activated SUMO to the E2-conjugating enzyme Ubc9 that directly catalyzes the isopeptide bond formation between the C-terminal glycine residue of the activated SUMO to Lys residue of the target. PIAS family members have been implicated as E3 SUMO ligases, although their roles in the sumoylation of the diverse substrates need to be further defined (8Jackson P.K. Genes Dev. 2001; 15: 3053-3058Crossref PubMed Scopus (202) Google Scholar). For example, RanBP2 can serve as E3 ligase for the HDAC4 deacetylase and Sp100 (9Kirsh O. Seeler J.S. Pichler A. Gast A. Muller S. Miska E. Mathieu M. Harel-Bellan A. Kouzarides T. Melchior F. Dejean A. EMBO J. 2002; 21: 2682-2691Crossref PubMed Scopus (266) Google Scholar, 10Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar), whereas ARIP3 is involved in sumoylation of the androgen receptor (11Kotaja N. Karvonen U. Janne O.A. Palvimo J.J. Mol. Cell. Biol. 2002; 22: 5222-5234Crossref PubMed Scopus (355) Google Scholar, 12Nishida T. Yasuda H. J. Biol. Chem. 2002; 277: 41311-41317Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). As with other post-translational modifications, sumoylation is thought to be a reversible and dynamic process. Sumoylation can regulate the function of a protein by changing its subcellular localization, protein-protein interactions, and/or stability, depending on the identity of the protein. For example, SUMO-1 modification targets the PML protein to nuclear bodies (13Muller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (581) Google Scholar), and the homeodomain-interacting protein kinase 2 into nuclear speckles (14Kim Y.H. Choi C.Y. Kim Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12350-12355Crossref PubMed Scopus (143) Google Scholar). Sumoylation negatively impacts the transcription activities of the transcription factors Sp3 (15Sapetschnig A. Rischitor G. Braun H. Doll A. Schergaut M. Melchior F. Suske G. EMBO J. 2002; 21: 5206-5215Crossref PubMed Scopus (228) Google Scholar), LEF-1 (16Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (466) Google Scholar), and androgen receptor (12Nishida T. Yasuda H. J. Biol. Chem. 2002; 277: 41311-41317Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and may also affect their subnuclear distribution, whereas other studies indicate a positive effect of sumoylation on transcription factor activity such as heat shock transcription factor-1 (17Hong Y. Rogers R. Matunis M.J. Mayhew C.N. Goodson M.L. Park-Sarge O.K. Sarge K.D. Goodson M. J. Biol. Chem. 2001; 276: 40263-40267Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Signaling from the cell surface can also be directly impacted by sumoylation, as illustrated in the case of IκB, which sequesters NFκB in the cytoplasm. Tumor necrosis factor-α-induced phosphorylation of IκB allows for its ubiquitination, thus targeting it for proteasomal degradation and enabling nuclear translocation of NFκB, whereas sumoylation of the same Lys in IκB stabilizes it against degradation (18Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar). This dynamic balance between sumoylation and ubiquitination of IκB can then regulate the gene expression of the cell response mediated by NFκB. Taken together, the effects of sumoylation on the function of the protein appear to depend on the substrate and, unlike ubiquitylation, which frequently targets substrate for proteasomal degradation, are not predictable. TGF-β and TGF-β-related proteins are key regulators of cell proliferation and differentiation. They regulate development from nematodes and flies to mammals (6Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3380) Google Scholar), and play important roles in tumor progression (19Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1974) Google Scholar). The central signaling pathway from TGF-β-activated cell surface receptors to alterations in gene expression involves a small class of signaling effectors, the Smads, as key mediators of a response of the cell to TGF-β (20Attisano L. Wrana J.L. Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1138) Google Scholar). Thus, following ligand binding to type II/type I receptor complexes, the activated type I receptors phosphorylate the C-terminal two serines of receptor-activated Smads, e.g. Smad2 and Smad3 in response to TGF-β and Smad1, -5, and -8 upon stimulation by bone morphogenetic proteins. The phosphorylated Smads are then released from the receptors to form a heterotrimeric complex with Smad4 as a common component of all Smad signaling pathways, activated by TGF-β family members (20Attisano L. Wrana J.L. Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1138) Google Scholar). The Smad complexes enter the nucleus where they interact at the promoter with other transcription factors and co-regulators to regulate gene expression. In this pathway, Smad4 acts as a central coactivator of all receptor-activated Smads, presumably through its ability to stabilize the interaction of receptor-activated Smads with the essential coactivator cAMP-response element-binding protein/p300 (21Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (451) Google Scholar) and its ability to recruit yet another coactivator, SMIF1 (22Bai R.Y. Koester C. Ouyang T. Hahn S.A. Hammerschmidt M. Peschel C. Duyster J. Nat. Cell Biol. 2002; 4: 181-190Crossref PubMed Scopus (78) Google Scholar). In addition to C-terminal phosphorylation, the activation of the receptor-activated Smads is also regulated by other phosphorylations, e.g. in response to mitogen-activated protein kinase signaling (23Blanchette F. Rivard N. Rudd P. Grondin F. Attisano L. Dubois C.M. J. Biol. Chem. 2001; 276: 33986-33994Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and is counteracted by inhibitory Smad6 and -7 (24Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (873) Google Scholar, 25Nakao 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 (1572) Google Scholar). Whereas phosphorylation plays a central role in the activation of the Smad pathway, recent studies have revealed an additional level of regulation of Smad signaling through ubiquitylation. Smurf1 and Smurf2, two Hect family E3 ubiquitin ligases, target receptor-activated Smads for ubiquitylation and proteasomal degradation, thus decreasing their availability (26Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (693) Google Scholar, 27Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (428) Google Scholar). In addition, ubiquitylation and consequent degradation of ligandactivated Smad2 and Smad3 in the nucleus may be involved in the termination of their functions in transcriptional regulation (28Lo R.S. Massague J. Nat. Cell Biol. 1999; 1: 472-478Crossref PubMed Scopus (297) Google Scholar). In contrast to the receptor-activated Smads, Smad4 levels are not regulated through ubiquitin-mediated degradation. However, some mutations, found in human tumors, destabilize Smad4 via ubiquitylation and consequent degradation (29Xu J. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4820-4825Crossref PubMed Scopus (172) Google Scholar, 30Maurice D. Pierreux C.E. Howell M. Wilentz R.E. Owen M.J. Hill C.S. J. Biol. Chem. 2001; 276: 43175-43181Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The role of Smad4 as the central mediator of signaling by all receptor-activated Smads in response to all TGF-β family members, and its shuttling between cytoplasm and nucleus (31Pierreux C.E. Nicolas F.J. Hill C.S. Mol. Cell. Biol. 2000; 20: 9041-9054Crossref PubMed Scopus (231) Google Scholar) suggests that some mechanism may be in place to allow for stabilization and re-utilization during signaling. In this report, we show that among the Smads known to be involved in TGF-β signaling, Smad4 is the only one that is preferentially subjected to sumoylation. We identified two sumoylation sites in Smad4, and sumoylation was enhanced in the presence of the conjugating enzyme Ubc9 and PIAS family E3 ligases. Sumoylation by the PIASy E3 ligase resulted in redistribution of Smad4 to subnuclear speckles that colocalized with SUMO-1 and PIASy. Replacement of the sumoylation target lysines with arginine, or increased sumoylation, enhanced the protein stability and increased transcription in mammalian cells and in Xenopus embryos. These results suggest a role for Smad4 sumoylation in the regulation of TGF-β family signaling through Smads. Plasmids—Expression plasmids for N-terminal FLAG-tagged Smad1, -2, -3, -4, -6, and -7 have been described (32Zhang Y. Musci T. Derynck R. Curr. Biol. 1997; 7: 270-276Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 33Choy L. Skillington J. Derynck R. J. Cell Biol. 2000; 149: 667-682Crossref PubMed Scopus (245) Google Scholar). Additionally, plasmids encoding Smad4 truncation mutants, i.e. pRK5F-Smad4N (aa 1–140), Smad4NL (aa 1–300), and Smad4C (aa 294–552) have also been described (32Zhang Y. Musci T. Derynck R. Curr. Biol. 1997; 7: 270-276Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Plasmids encoding Smad4 with amino acid substitutions were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, and mutations were confirmed by nucleic acid sequencing. The pRK5-based expression plasmid for N-terminal FLAG-tagged SUMO-1 expressed the first 97 amino acids of SUMO-1 was kindly provided by Dr. Y. Zhang (Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health). The expression plasmids for Ubc9, PIASy, and ARIP3, i.e. pcDNA3-Ubc9, CMV-T7-PIASy, and pFlag-CMV2-ARIP3 (39Carvalho T. Seeler J.S. Ohman K. Jordan P. Pettersson U. Akusjarvi G. Carmo-Fonseca M. Dejean A. J. Cell Biol. 1995; 131: 45-56Crossref PubMed Scopus (246) Google Scholar, 62Mulder K.M. Cytokine Growth Factor Rev. 2000; 11: 23-35Crossref PubMed Scopus (386) Google Scholar, 64Engel M.E. McDonnell M.A. Law B.K. Moses H.L. J. Biol. Chem. 1999; 274: 37413-37420Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar) were generously provided by Dr. R. T. Hay (University of St. Andrews), Dr. R. Grosschedl (University of Munich), and Drs. O. A. Janne and J. J. Palvimo (University of Helsinki), respectively. Plasmids pRK5-β-gal and (SBE)4-lux were also previously described (34Feng X.H. Filvaroff E.H. Derynck R. J. Biol. Chem. 1995; 270: 24237-24245Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 35Zawel 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 (892) Google Scholar, 36Carcamo J. Weis F.M. Ventura F. Wieser R. Wrana J.L. Attisano L. Massague J. Mol. Cell. Biol. 1994; 14: 3810-3821Crossref PubMed Google Scholar). Cell Culture, Transfections, and Reporter Assays—COS-1, HeLa, and MDA-MB-468 cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 μg/ml streptomycin sulfate, 100 units/ml penicillin G. COS-1 and HeLa cells were transfected using LipofectAMINE (Invitrogen) and MDA-MB-468 cells were transfected with FuGENE 6 (Roche Diagnostics) according to the manufacturer's instructions. In transcription reporter assays, MDA-MB-468 cells were seeded onto 6-well culture dishes and 2 μg of DNA, including 0.5 μg of (SBE)4-luc luciferase reporter plasmid, 0.1 μg of pRK5-β-gal, and other expression plasmids, as required, were used for each transfection. The total amount of DNA was kept constant by addition of pRK5 DNA. 12–16 h after transfection, cells were treated with or without 10 ng/ml TGF-β in Dulbecco's modified Eagle's medium containing 0.2% fetal bovine serum for 20 h. At the end of stimulation, cells were washed twice in cold phosphate-buffered saline and processed for luciferase or β-galactosidase enzyme assays as described (37Qing J. Zhang Y. Derynck R. J. Biol. Chem. 2000; 275: 38802-38812Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Cell Lysis, Immunoprecipitation, and Western Blotting Analysis— Cells were washed four times in phosphate-buffered saline and lysed in RIPA lysis buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris, pH 8.0), supplemented with 20 mm N-ethylmaleimide (Calbiochem, 34115), protease inhibitor mixture (Sigma, P8340), and 5% glycerol. In co-immunoprecipitation experiments cells were lysed in RIPA buffer without the ionic detergents. Cell lysates were cleared by centrifugation at 20,000 × g at 4 °C for 30 min. The resulting supernatant was subjected to immunoprecipitation with anti-FLAG M2-agarose (Sigma, A2220) overnight at 4 °C. The agarose beads were washed five times in lysis buffer and the bound proteins were eluted in SDS-PAGE loading buffer. The samples were subjected to SDS-PAGE, followed by Western transfer and immunoblotting as described (38Lee P.S. Wang Y. Dominguez M.G. Yeung Y.G. Murphy M.A. Bowtell D.D. Stanley E.R. EMBO J. 1999; 18: 3616-3628Crossref PubMed Scopus (253) Google Scholar). Mouse monoclonal anti-FLAG M2 antibody (Sigma, F3165), anti-Myc tag 9E10 antibody (Covance, MMS-150P), anti-HA tag HA11 antibody (Covance, MMS-101R), anti-GMP-1 (SUMO-1) antibody (Zymed Laboratories Inc.), and rabbit polyclonal anti-Smad4 antibody H-552 (Santa Cruz, sc-7154) were used at 1 μg/ml in Western blotting. Peroxidase-linked anti-mouse (NA931) and anti-rabbit (NA9340) secondary antibodies and ECL detection reagents (RPN2106) were purchased and used according to the manufacturer's instructions (Amersham Biosciences). Pulse-Chase Analysis of Smad4 Stability—Transfected COS cells were starved in methionine- and cysteine-free medium containing 0.2% dialyzed fetal bovine serum for 2 h. The cells were then labeled with 0.25 μCi/ml [35S]EXPRESS protein labeling mix (PerkinElmer Life Sciences, NEG072) in the same medium for 1 h. At the end of the labeling, the cells were washed three times in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum before incubating in the same medium for various times, as needed. Where indicated, lactacystin (Calbiochem, number 426100) was used at 30 μm during the pulse-chase. Cells were harvested and processed as above. The cell lysates were subjected to anti-FLAG immunoprecipitation for isolation of exogenous Smad4, and immunoprecipitates were analyzed by SDS-PAGE. Smad4 was visualized by autoradiography and analyzed by densitometry. Immunofluorescence—HeLa cells were grown on coverslips and transfected with the indicated plasmid DNA. 24 h after transfection cells were kept in Dulbecco's modified Eagle's medium containing 0.2% fetal bovine serum for 3 h and subsequently treated with 5 ng/ml TGF-β for 40 min. Cells were then permeabilized with 0.5% Triton X-100 in CSK buffer (10 mm Pipes, pH 6.8, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 2 mm EDTA) for 2 min on ice, and fixed in 3.7% paraformaldehyde in CSK buffer for 5 min at 37 °C (39Carvalho T. Seeler J.S. Ohman K. Jordan P. Pettersson U. Akusjarvi G. Carmo-Fonseca M. Dejean A. J. Cell Biol. 1995; 131: 45-56Crossref PubMed Scopus (246) Google Scholar). Cells were rinsed twice in TBS (20 mm Tris, pH 8, 150 mm NaCl) and then blocked in 3% bovine serum albumin in TBS containing 0.03% Triton X-100 for 1 h at room temperature. After incubation with a primary antibody in blocking solution, the cells were washed in blocking solution five times before incubating with 1:500 diluted secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG (A11029) and Alexa Fluor 594 goat anti-mouse IgG (A-11034) from Molecular Probes) for 1 h. After washings cells were stained in 300 nm 4,6-diamidino-2-phenylindole (Sigma, 32670) in TBS for 5 min, followed by brief washes in TBS. The samples were finally mounted in FluorSave Reagent (Calbiochem, 345789) and examined by fluorescence microscopy. Mesoderm Marker Induction in Xenopus Embryos—cRNAs encoding Smad4 (wild-type and mutants) and SUMO-conjugating enzyme Ubc9 and β-catenin were synthesized in vitro, using Ambion mMessage Machine kit. Capped RNAs were injected into both animal poles of two-cell stage embryos. The doses of cRNAs used were indicated in the legend to Fig. 7. The ectodermal explants (animal caps) of injected embryos were dissected at blastula stages (stage 9), and total RNA was extracted from these caps at gastrula stages (stage 11). For treatment with basic fibroblast growth factor (bFGF), the animal caps were dissected at stage 9 and incubated in buffer containing 100 ng/ml bFGF. The caps were harvested at stage 11 as other samples. Reverse transcription-PCR was performed using the primers as described previously (40Chang C. Wilson P.A. Mathews L.S. Hemmati-Brivanlou A. Development. 1997; 124: 827-837Crossref PubMed Google Scholar). Smad4 Is Sumoylated—Proteins that are substrates of ubiquitin modification are often modified by sumoylation, raising the possibility that the TGF-β Smads could be targeted for sumoylation. Furthermore, in a yeast two-hybrid screen for interaction proteins we found that Smad4, but not Smad3, had an affinity for SUMO-1 (data not shown). We therefore evaluated whether Smad3 and Smad4, expressed in transfected cells, were sumoylated. In the presence of coexpressed Myctagged SUMO-1, but not in its absence, a fraction of Smad4 reacted in Western blotting with the anti-Myc antibody (Fig. 1A, top panel). Its apparent size was compatible with the addition of a single SUMO-1 polypeptide to Smad4, and corresponded to the upper edge of the total Smad4 band, detected by Western blotting (Fig. 1B, lower panel). Smad3 did not display reactivity for Myc-tagged SUMO-1 under these conditions. Although many gene expression responses of TGF-β are mediated by Smad3 and Smad4 (41Itoh S. Itoh F. Goumans M.J. Ten Dijke P. Eur. J. Biochem. 2000; 267: 6954-6967Crossref PubMed Scopus (458) Google Scholar), Smad2 and Smad1 can also be activated by TGF-β (42Macias-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 43Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (938) Google Scholar), and Smad6 and Smad7 act as inhibitory Smads for the TGF-β response (25Nakao 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 (1572) Google Scholar, 41Itoh S. Itoh F. Goumans M.J. Ten Dijke P. Eur. J. Biochem. 2000; 267: 6954-6967Crossref PubMed Scopus (458) Google Scholar). We therefore evaluated whether any of these Smads are targeted by sumoylation. As shown in Fig. 1B, only Smad4, and not the other Smads tested, was sumoylated under this condition. Lysines 159 and 113 Are Major Sumoylation Sites in Smad4 —To localize the site of sumoylation within Smad4, we expressed several Smad4 segments, either in the presence or absence of coexpressed SUMO-1. Immunoprecipitation followed by Western detection of SUMO-1 indicated that the NL segment, corresponding to amino acids 1–300 and containing the MH1 domain and linker segment, was sumoylated (Fig. 2A). Smad4C, i.e. amino acids 266–552 containing the MH2 domain, was not detectably sumoylated. Smad4N, i.e. amino acids 1–140 corresponding to the MH1 domain, showed only a minimal level of immunoreactivity for Myc-SUMO-1 (Fig. 2A). Direct Western blotting of the cell lysates for Smad4 detected small fractions of Smad4, Smad4N, and Smad4NL (Fig. 2A, lower panel) with the same mobilities as the sumoylated forms that were detected using the anti-Myc antibody for SUMO-1 (Fig. 2A, upper panel). SUMO conjugation occurs on lysine (K) residues within a general minimal consensus sequence ψKXE, in which ψ is a large hydrophobic residue (7Hay R.T. Trends Biochem. Sci. 2001; 26: 332-333Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Only 1 lysine within such sequence context, i.e. Lys-159 within the VKDE sequence, is present in Smad4 and is located within the Smad4NL, but not the Smad4N segment. Within the Smad4NL sequence are two other lysines, i.e. Lys-51 and Lys-106, that are located within a KXE sequence that lack a preceding hydrophobic residue. To examine which lysine is sumoylated, we individually replaced each of these three lysines within Smad4NL by an arginine (R). As shown in Fig. 2B, the K51R and K106R mutants were sumoylated to a similar extent as the normal Smad4NL. In contrast, the K159R mutation strongly reduced, yet did not abolish the sumoylation. This result suggests that Lys-159 is a major sumoylation site. The triple mutation of Smad4NL, resulting in replacement of all three lysines with arginines further reduced the reactivity in Western blotting for SUMO to background level. This result suggests that Lys-51 and/or Lys-106 can serve as targets for sumoylation enzymes, albeit with a much lower efficiency than Lys-159. We also tested the effect of these lysine mutations on sumoylation of full-length Smad4 (Fig. 2C). These results confirmed Lys-159 as the major sumoylation site, because the K159R mutation nearly abolished sumoylation. In contrast, mutation of Lys-106 exerted only a minor decrease, and mutation of Lys-51 had no effect. We also replaced Lys-392, which is located within the CKGE sequence in the carboxyl MH2 domain of Smad4, by Arg. This mutation also had only a minimal, if any, effect on Smad4 sumoylation (Fig. 2C), consistent with the lack of detectable sumoylation of Smad4C (Fig. 2A). Finally, an alternate, less common sumoylation target sequence, ψKXD, has also been described (44Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (328) Google Scholar). Lys-122, within an LKCD sequence, was therefore mutated and the effect on Smad4 sumoylation was assessed. As shown in Fig. 2C, the K122R mutation did not detectably decrease the level of Smad4 sumoylation. We also examined the sumoylation status of double lysine mutants, K51R/L159R and K122R/L159R and found that they had a low level of sumoylation like K159R (Fig. 2C). While this paper was under review, Lin et al. (45Lin X. Liang M. Liang Y.Y. Brunicardi F.C. Melchior F. Feng X.H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) reported that Smad4 is sumoylated on Lys-113, in addition to Lys-159. We therefore verified the possible sumoylation of this site (Fig. 2D). As evaluated in the presence of SUMO-1, mutation of Lys-113 to Arg reduced sumoylation of Smad4.