The DNA Binding Activities of Smad2 and Smad3 Are Regulated by Coactivator-mediated Acetylation
2006; Elsevier BV; Volume: 281; Issue: 52 Linguagem: Inglês
10.1074/jbc.m607868200
ISSN1083-351X
AutoresMaria Simonsson, Meena Kanduri, Eva Grönroos, Carl‐Henrik Heldin, Johan Ericsson,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoPhosphorylation-dependent activation of the transcription factors Smad2 and Smad3 plays an important role in TGFβ-dependent signal transduction. Following phosphorylation of Smad2 and Smad3, these molecules are translocated to the nucleus where they interact with coactivators and/or corepressors, including p300, CBP, and P/CAF, and regulate the expression of TGFβ target genes. In the current study, we demonstrate that both Smad2 and Smad3 are acetylated by the coactivators p300 and CBP in a TGFβ-dependent manner. Smad2 is also acetylated by P/CAF. The acetylation of Smad2 was significantly higher than that of Smad3. Lys19 in the MH1 domain was identified as the major acetylated residue in both the long and short isoform of Smad2. Mutation of Lys19 also reduced the p300-mediated acetylation of Smad3. By generating acetyl-Lys19-specific antibodies, we demonstrate that endogenous Smad2 is acetylated on this residue in response to TGFβ signaling. Acetylation of the short isoform of Smad2 improves its DNA binding activity in vitro and enhances its association with target promoters in vivo, thereby augmenting its transcriptional activity. Acetylation of Lys19 also enhanced the DNA binding activity of Smad3. Our data indicate that acetylation of Lys19 induces a conformational change in the MH1 domain of the short isoform of Smad2, thereby making its DNA binding domain accessible for interactions with DNA. Thus, coactivator-mediated acetylation of receptor-activated Smad molecules could represent a novel way to regulate TGFβ signaling. Phosphorylation-dependent activation of the transcription factors Smad2 and Smad3 plays an important role in TGFβ-dependent signal transduction. Following phosphorylation of Smad2 and Smad3, these molecules are translocated to the nucleus where they interact with coactivators and/or corepressors, including p300, CBP, and P/CAF, and regulate the expression of TGFβ target genes. In the current study, we demonstrate that both Smad2 and Smad3 are acetylated by the coactivators p300 and CBP in a TGFβ-dependent manner. Smad2 is also acetylated by P/CAF. The acetylation of Smad2 was significantly higher than that of Smad3. Lys19 in the MH1 domain was identified as the major acetylated residue in both the long and short isoform of Smad2. Mutation of Lys19 also reduced the p300-mediated acetylation of Smad3. By generating acetyl-Lys19-specific antibodies, we demonstrate that endogenous Smad2 is acetylated on this residue in response to TGFβ signaling. Acetylation of the short isoform of Smad2 improves its DNA binding activity in vitro and enhances its association with target promoters in vivo, thereby augmenting its transcriptional activity. Acetylation of Lys19 also enhanced the DNA binding activity of Smad3. Our data indicate that acetylation of Lys19 induces a conformational change in the MH1 domain of the short isoform of Smad2, thereby making its DNA binding domain accessible for interactions with DNA. Thus, coactivator-mediated acetylation of receptor-activated Smad molecules could represent a novel way to regulate TGFβ signaling. Transforming growth factor β (TGFβ) 3The abbreviations used are: TGFβ, transforming growth factor β; ChIP, chromatin immunoprecipitation; EMSA, electromobility shift assay; DNAP, DNA precipitation; P/CAF, p300/CBP-associated factor; GST, glutathione S-transferase; HA, hemagglutinin; CBP, cAMP-responsive element-binding protein-binding protein. 3The abbreviations used are: TGFβ, transforming growth factor β; ChIP, chromatin immunoprecipitation; EMSA, electromobility shift assay; DNAP, DNA precipitation; P/CAF, p300/CBP-associated factor; GST, glutathione S-transferase; HA, hemagglutinin; CBP, cAMP-responsive element-binding protein-binding protein. is a member of the TGFβ superfamily of cytokines that regulate multiple cellular processes, including extracellular matrix production, cell growth, apoptosis, and differentiation. Dysfunction of TGFβ signaling has been implicated in various human disorders ranging from vascular diseases to cancer progression (for a review, see Ref. 1Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2039) Google Scholar). The effects of TGFβ are mediated through type I and type II receptors, which are transmembrane proteins possessing cytoplasmic serine/threonine kinase domains for signal propagation. TGFβ first binds to the type II receptor; the type I receptor is thereafter recruited to the receptor complex and is phosphorylated in the cytoplasmic domain by the type II receptor (2Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar). The activated type I receptor then phosphorylates the receptor-activated Smads (R-Smads; Smad2 and Smad3) in their C-terminal SSXS motif (3Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4693) Google Scholar). The activated Smads then interact with Smad4 and translocate into the nucleus were they act as transcription factors together with co-activators and co-repressors (4Feng X.H. Derynck R. Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1507) Google Scholar). A large number of transcriptional coactivators, including CBP, p300, P/CAF, and GCN5, have intrinsic acetyltransferase activities that are important for their abilities to enhance transcription (5Bannister A.J. Miska E.A. Cell. Mol. Life Sci. 2000; 57: 1184-1192Crossref PubMed Scopus (128) Google Scholar, 6Brown C.E. Lechner T. Howe L. Workman J.L. Trends Biochem. Sci. 2000; 25: 15-19Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 7Chen H. Tini M. Evans R.M. Curr. Opin. Cell Biol. 2001; 13: 218-224Crossref PubMed Scopus (150) Google Scholar, 8Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (992) Google Scholar, 9Marmorstein R. Roth S.Y. Curr. Opin. Genet. Dev. 2001; 11: 155-161Crossref PubMed Scopus (306) Google Scholar, 10Marmorstein R. Cell. Mol. Life Sci. 2001; 58: 693-703Crossref PubMed Scopus (128) Google Scholar, 11Ogryzko V.V. Cell. Mol. Life Sci. 2001; 58: 683-692Crossref PubMed Scopus (44) Google Scholar, 12Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1562) Google Scholar). Acetylation involves the transfer of the acetyl moiety from acetyl coenzyme-A to the amino group of a lysine residue of the acceptor protein. Acetylation is a dynamic process and the balance between the acetylation and deacetylation of histones has major effects on chromatin structure and transcription (for a review see Ref. 12Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1562) Google Scholar). Histones H3 and H4 are acetylated on specific lysine residues in their N-terminals, thereby relaxing the nucleosomal structure and allowing transcription. It has been demonstrated that non-histone proteins such as p53 (13Ito A. Lai C.H. Zhao X. Saito S. Hamilton M.H. Appella E. Yao T.P. EMBO J. 2001; 20: 1331-1340Crossref PubMed Scopus (433) Google Scholar), E2F (14Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (565) Google Scholar), YY1 (15Yao Y.L. Yang W.M. Seto E. Mol. Cell. Biol. 2001; 21: 5979-5991Crossref PubMed Scopus (347) Google Scholar), NFκB (16Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1027) Google Scholar), SREBP (17Giandomenico V. Simonsson M. Gronroos E. Ericsson J. Mol. Cell. Biol. 2003; 23: 2587-2599Crossref PubMed Scopus (182) Google Scholar), and Smad7 (18Gronroos E. Hellman U. Heldin C.H. Ericsson J. Mol. Cell. 2002; 10: 483-493Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) also are acetylated and that this modification affects their interactions with DNA and other proteins. Protein acetylation can also affect protein stability, because it has been demonstrated that acetylation prevents ubiquitination of the same lysine residues (18Gronroos E. Hellman U. Heldin C.H. Ericsson J. Mol. Cell. 2002; 10: 483-493Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 19Ito A. Kawaguchi Y. Lai C.H. Kovacs J.J. Higashimoto Y. Appella E. Yao T.P. EMBO J. 2002; 21: 6236-6245Crossref PubMed Scopus (441) Google Scholar, 20Zhao Q. Cumming H. Cerruti L. Cunningham J.M. Jane S.M. J. Biol. Chem. 2004; 279: 41477-41486Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 21Caron C. Boyault C. Khochbin S. Bioessays. 2005; 27: 408-415Crossref PubMed Scopus (197) Google Scholar). We have previously found that the stability of Smad7, an inhibitory Smad molecule, is regulated by reversible acetylation (22Simonsson M. Heldin C.H. Ericsson J. Gronroos E. J. Biol. Chem. 2005; 280: 21797-21803Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Alternative splicing of exon 3 in the Smad2 gene gives rise to two distinct protein isoforms (23Yagi K. Goto D. Hamamoto T. Takenoshita S. Kato M. Miyazono K. J. Biol. Chem. 1999; 274: 703-709Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The short isoform (Smad2(ΔE3)), unlike full-length Smad2 (Smad2(FL)), retains DNA-binding activity (24Dennler S. Huet S. Gauthier J.M. Oncogene. 1999; 18: 1643-1648Crossref PubMed Scopus (151) Google Scholar). The two isoforms of Smad2 are coexpressed throughout mouse development, but Smad2(FL) is the dominant isoform in most cell lines (23Yagi K. Goto D. Hamamoto T. Takenoshita S. Kato M. Miyazono K. J. Biol. Chem. 1999; 274: 703-709Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). It has been demonstrated that expression of Smad2(ΔE3), but not Smad2(FL), in Smad2-deficient mice results in viable and fertile animals (25Dunn N.R. Koonce C.H. Anderson D.C. Islam A. Bikoff E.K. Robertson E.J. Genes Dev. 2005; 19: 152-163Crossref PubMed Scopus (71) Google Scholar). These results demonstrate that Smad2(ΔE3), but not Smad2(FL), has the ability to activate all essential target genes downstream of TGFβ during development. In the current study, we demonstrate that both isoforms of Smad2, as well as Smad3, are acetylated on a specific lysine residue, Lys19, in their MH1 domains in response to TGFβ signaling. Acetylation of the short isoform of Smad2 (Smad2(ΔE3)) augments its DNA binding activity in vitro and enhances its association with target promoters in vivo. Acetylation of Lys19 also enhances the DNA binding of Smad3. Our data indicate that acetylation of Lys19 induces a conformational change in the MH1 domain of Smad2(ΔE3), thereby making its DNA binding domain accessible for interactions with DNA. Cell Culture—All tissue culture media and antibiotics were obtained from Invitrogen and Sigma. 293T, HepG2, HeLa, COS-1, and HaCaT cells were from the American Type Culture Collection. Smad2-deficient mouse embryonic fibroblasts were obtained from Anita Roberts (NCI) (26Piek E. Ju W.J. Heyer J. Escalante-Alcalde D. Stewart C.L. Weinstein M. Deng C. Kucherlapati R. Bottinger E.P. Roberts A.B. J. Biol. Chem. 2001; 276: 19945-19953Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Cells were maintained at 37 °C in Dulbecco′s modified Eagle's medium supplemented with 10% fetal calf serum, sodium pyruvate (1 mm), non-essential amino acids (1×), 50 units/ml penicillin and 50 μg/ml streptomycin, in 5% CO2. For overnight starvation, cells were incubated in Dulbecco's modified Eagle's medium supplemented with 0.5% fetal calf serum, sodium pyruvate (1 mm), non-essential amino acids (1×), 50 units/ml penicillin, and 50 μg/ml streptomycin. TGFβ, Reagents, and Antibodies—TGFβ was obtained from Peprotech EC. TSA was obtained from Sigma. Antibodies against Myc (9E10), HA (Y-11), p300 (N15), P/CAF (E8), and Gal4-DBD (RK5C1) were from Santa Cruz Biotechnology. FLAG antibodies (M5) were from Sigma and anti-acetyl lysine antibodies were from Cell Signaling Technology and Upstate Biotechnology. The monoclonal Smad2/3 antibody (cat. no. 610843) was from BD Biosciences. Rabbit polyclonal anti-acetyl-Lys19 Smad2 antisera was raised against an acetylated peptide corresponding to amino acids 15–24 in Smad2, and was affinity-purified as described (22Simonsson M. Heldin C.H. Ericsson J. Gronroos E. J. Biol. Chem. 2005; 280: 21797-21803Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The rabbit polyclonal anti-phosphorylated Smad2 antibody has been described elsewhere (27Persson U. Izumi H. Souchelnytskyi S. Itoh S. Grimsby S. Engstrom U. Heldin C.H. Funa K. ten Dijke P. FEBS Lett. 1998; 434: 83-87Crossref PubMed Scopus (335) Google Scholar). Secondary anti-mouse and anti-rabbit antibodies and protein-G Sepharose were from Amersham Biosciences. Plasmids and DNA Transfections—The expression vectors for FLAG- and FLAG-tagged Smad2(FL) and Smad3 in the mammalian expression vector pcDNA3 (Invitrogen) were generously provided by P. ten Dijke (The Netherlands Cancer Institute, Amsterdam). The short isoform of Smad2 (Smad2(ΔE3)) was generated from the corresponding Smad2(FL) construct by PCR. The expression vectors for p300, CBP and P/CAF have been described (28Itoh S. Ericsson J. Nishikawa J. Heldin C.H. ten Dijke P. Nucleic Acids Res. 2000; 28: 4291-4298Crossref PubMed Google Scholar). Point mutants in Smad2(FL), Smad2(ΔE3), and Smad3 were generated by site-directed mutagenesis (QuikChange, Stratagene). The Smad-responsive ARE-Luc and 12xCAGA-Luc promoter-reporter constructs have been described (28Itoh S. Ericsson J. Nishikawa J. Heldin C.H. ten Dijke P. Nucleic Acids Res. 2000; 28: 4291-4298Crossref PubMed Google Scholar). Transient transfections were performed using the MBS transfection kit (Stratagene). Immunoprecipitations and Immunoblotting—Cells were lysed in ice-cold lysis buffer (50 mm HEPES, pH 7.2, 150 mm NaCl, 1 mm EDTA, 20 mm NaF, 2 mm sodium orthovanadate, 1% (w/v) Triton X-100, 10% (w/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 mm sodium butyrate, and 1% aprotinin) and cleared by centrifugation. Immunoprecipitations were performed by adding the appropriate antibodies plus protein G-Sepharose beads, followed by incubation for 3 h at 4 °C. The immune complexes were washed three times with lysis buffer, once with 0.5 m NaCl and once with water. The samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Millipore). After blocking in phosphate-buffered saline with the addition of 5% bovine serum albumin, the membranes were incubated with the appropriate antibodies, washed with phosphate-buffered saline containing 0.05% Triton X-100 and incubated with horseradish peroxidase-coupled secondary antibodies. After washing, the blots were visualized with Western Blotting Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology). Luciferase and β-Galactosidase Assays—Cells were transiently transfected with promoter-reporter genes in the absence or presence of expression vectors for the indicated Smad protein, either wild-type or the indicated mutants. 24-h post-transfection, the medium was replaced with medium containing 0.5% fetal calf serum and treated in the absence or presence of TGFβ (5 ng/ml). After 36 h, luciferase activities were determined in duplicate samples as described by the manufacturer (Promega). The pCH110 vector encoding the β-galactosidase reporter gene (Amersham Biosciences) was used as an internal control for transfection efficiency. Luciferase values (relative light units, RLU) were calculated by dividing the luciferase activity by the β-galactosidase activity. The data represent the average ±S.D. of three independent experiments performed in duplicates. Electromobility Shift Assay—Total cell extracts were prepared from transiently transfected COS-1 cells using hypertonic lysis buffer (20 mm Hepes, pH 7.6, 20% (w/v) glycerol, 500 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1% (w/v) Triton X-100, 1 mm dithiothreitol, 1 mm sodium orthovanadate, and 1% aprotinin). The transfected proteins were visualized by Western blotting using anti-FLAG antibodies, followed by quantitation with a charge-coupled device camera (Fuji) and image analysis software (Aida Image Analyzer, version 3.10). Equal amounts of proteins were incubated with 1 μg of poly-(dIdC) and a 32P-labeled oligonucleotide probe containing four Smad-binding sites (4xCAGA) in hypotonic lysis buffer (20 mm Hepes, pH 7.6, 20% (w/v) glycerol, 20 mm NaCl, 10 mm MgCl2, 0.2 mm EDTA, 1% (w/v) Triton X-100, 1 mm dithiothreitol, 1 mm sodium orthovanadate, and 1% aprotinin). The samples were incubated for 15 min on ice and run on 5% polyacrylamide gels. The gels were analyzed by PhosphorImager analysis. For EMSAs with purified GST-Smad2(ΔE3) and in vitro translated Smad2(ΔE3)-MH1, the proteins were visualized by Western blotting, followed by quantitation with a charge-coupled device camera (Fuji) and image analysis software (Aida Image Analyzer, version 3.10). Equal amounts of proteins were incubated with 1 μg of poly(dIdC) and 32P-labeled 4xCAGA probe in binding buffer (50 mm Hepes, pH 7.9, 15% (w/v) glycerol, 75 mm KCl, 10 mm MgCl2, 1 mm dithiothreitol, and 10 mm spermidine). The reaction products were separated and analyzed as described above. Where indicated, GST-Smad2(ΔE3) was incubated in the presence of purified GST-P/CAF in the absence or presence of 1 mm acetyl coenzyme A in acetylation buffer (50 mm Tris-HCl, pH 8.0, 10% (w/v) glycerol, 1 mm dithiothreitol, 1 mm MgCl2, and 20 mm sodium butyrate) for 2 h prior to the EMSA. DNAP Assays—Cell lysates from transiently transfected 293T cells were precleared with streptavidin-agarose (Sigma) and subsequently used in DNA precipitation (DNAP) assays. The biotinylated double-stranded DNA was composed of a multimerized Smad-binding element (4xCAGA). DNA-bound proteins were precipitated with streptavidin-agarose for 60 min at 4 °C, washed, and detected by Western blot analysis. Chromatin Immunoprecipitation—The chromatin immunoprecipitation assays were performed as described previously (29Kurisaki K. Kurisaki A. Valcourt U. Terentiev A.A. Pardali K. Ten Dijke P. Heldin C.H. Ericsson J. Moustakas A. Mol. Cell. Biol. 2003; 23: 4494-4510Crossref PubMed Scopus (128) Google Scholar). For the analysis of transfected material, 1 × 106 COS-1 cells were transfected with 1 μg of FLAG-tagged Smad constructs, with or without constitutively active ALK5. After transfection, cells were fixed with 1% formaldehyde, sonicated, and one-fourth of the material was immunoprecipitated with 5 μg of the indicated antibody. The cross-link was reversed at 65 °C overnight, followed by proteinase K treatment. The DNA was extracted using phenol:chloroform. For analysis of endogenous Smad2, two 15-cm dishes of HeLa cells (10 × 106 cells) were used per immunoprecipitation. The PCR conditions for each target gene were optimized to remain in the linear range of amplification. The primers used to amplify the PAI-1 promoter have been described (29Kurisaki K. Kurisaki A. Valcourt U. Terentiev A.A. Pardali K. Ten Dijke P. Heldin C.H. Ericsson J. Moustakas A. Mol. Cell. Biol. 2003; 23: 4494-4510Crossref PubMed Scopus (128) Google Scholar). The primers used for the p21 promoter were (forward primer) 5′-CAT TGT GAA GCT CAG TAC CAC AA-3′ and (reverse primer) 5′-TGC TTT CAG GCA TTT CAA ATA GAC-3′. The PCR primers used for 12xCAGA-Luc reporter gene were (forward primer) 5′-ACT GCA GGT GCC AGA AC ATT-3′ and (reverse primer) 5′-GTT CCA TCT TCC AGC GGA TA-3′. The PCR products were separated by electrophoresis in 6% polyacrylamide gels, and stained by ethidium bromide. Smad2(FL), Smad2(ΔE3), and Smad3 Are Acetylated—The histone acetyltransferases CBP/p300 are able to interact with Smad2, -3, and -4, and they function as coactivators of TGFβ-induced transcription in a Smad4-dependent fashion (30Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (446) Google Scholar, 31Janknecht R. Wells N.J. Hunter T. Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (432) Google Scholar, 32Nishihara A. Hanai J.I. Okamoto N. Yanagisawa J. Kato S. Miyazono K. Kawabata M. Genes Cells. 1998; 3: 613-623Crossref PubMed Scopus (130) Google Scholar, 33Pouponnot C. Jayaraman L. Massague J. J. Biol. Chem. 1998; 273: 22865-22868Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 34Shen X. Hu P.P. Liberati N.T. Datto M.B. Frederick J.P. Wang X.F. Mol. Biol. Cell. 1998; 9: 3309-3319Crossref PubMed Scopus (183) Google Scholar). In addition, P/CAF and GCN5 have been shown to potentiate TGFβ signaling (28Itoh S. Ericsson J. Nishikawa J. Heldin C.H. ten Dijke P. Nucleic Acids Res. 2000; 28: 4291-4298Crossref PubMed Google Scholar, 35Kahata K. Hayashi M. Asaka M. Hellman U. Kitagawa H. Yanagisawa J. Kato S. Imamura T. Miyazono K. Genes Cells. 2004; 9: 143-151Crossref PubMed Scopus (39) Google Scholar). These observations prompted us to determine if Smad2(FL) and Smad3 were acetylated in vivo. When Smad2(FL) or Smad3 were transiently expressed in 293T cells, both proteins were acetylated in response to coexpression of either p300 or CBP (Fig. 1A). The acetylation of Smad2(FL) was more pronounced than that of Smad3, indicating that Smad2(FL) is a better substrate for these acetyltransferases. Smad2(FL) and Smad3 are highly similar proteins, but whereas Smad3 can bind DNA directly, Smad2(FL) is dependent on coactivators to associate with DNA. This difference between Smad2(FL) and Smad3 is caused by an additional exon (exon 3) that is inserted in front of the DNA binding domain in Smad2(FL) (24Dennler S. Huet S. Gauthier J.M. Oncogene. 1999; 18: 1643-1648Crossref PubMed Scopus (151) Google Scholar). A splice variant of Smad2, which lacks exon 3 (Smad2(ΔE3)) has been identified (23Yagi K. Goto D. Hamamoto T. Takenoshita S. Kato M. Miyazono K. J. Biol. Chem. 1999; 274: 703-709Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). This isoform of Smad2 plays an important role during embryonal development (25Dunn N.R. Koonce C.H. Anderson D.C. Islam A. Bikoff E.K. Robertson E.J. Genes Dev. 2005; 19: 152-163Crossref PubMed Scopus (71) Google Scholar). To determine if Smad2(ΔE3) was acetylated and establish if the acetylation of the R-Smads was regulated by TGFβ signaling, Smad2(FL), Smad2(ΔE3), and Smad3 were transfected together with p300 in the absence or presence of constitutively active ALK5 receptor. As illustrated in Fig. 1B, expression of ALK5 enhanced the acetylation of all the Smads tested. In vitro acetylation assays using deletion mutants of GST-Smad2(FL) and immunoprecipitated p300, indicated that the major acetylation site in Smad2(FL) resided in the N-terminal MH1 domain (data not shown). To identify the lysine residues targeted by p300-mediated acetylation, all lysine residues in the MH1 domain of Smad2(FL) were mutated to arginine, either individually or in sets of two residues, and the mutant proteins were expressed in 293T cells in the absence or presence of p300. Mutation of Lys19 and Lys20 (K19R/K20R), but not mutation of other lysine residues, blocked the acetylation of Smad2(FL) (Fig. 1C). Lys19 and Lys20 are conserved between Smad2 and Smad3 and are located just in front of a specific insert in Smad2 (Fig. 2A). To identify the specific lysine residue acetylated by p300, Lys19, and Lys20 in Smad2(FL) were mutated individually and subjected to p300-mediated acetylation following expression in 293T cells. Mutation of Lys19 blocked the p300-mediated acetylation of Smad2(FL), whereas mutation of Lys20 had no effect (Fig. 2B), suggesting that Lys19 is the preferred site for p300-mediated acetylation of Smad2(FL). Mutation of Lys19 also blocked the p300-dependent acetylation of Smad2(ΔE3) (Fig. 2C, left panel). Mutation of Lys19 also reduced the acetylation of Smad2(FL) in HepG2, COS-1, and HeLa cells (supplemental Fig. S1), suggesting that Lys19 is the major acetylated residue in these cell lines. We were unable to detect any differences in the phosphorylation or interaction with p300 between wild-type Smad2(FL) or Smad2(ΔE3) and the corresponding K19R mutants (data not shown). The p300-dependent acetylation of Smad3 was attenuated when Lys19 was mutated, whereas mutation of Lys20 had no effect (Fig. 2C, right panel), suggesting that Lys19 is acetylated also in Smad3. Smad2(FL) and Smad2(ΔE3) were also acetylated by P/CAF in vivo and the acetylation of both proteins was lost following mutation of Lys19 (Fig. 2D), suggesting that Lys19 in Smad2 is also targeted by P/CAF. We were unable to detect any acetylation of Smad3 by P/CAF under these conditions. Because Smad2(FL) and Smad2(ΔE3) were better substrates for p300, CBP, and P/CAF when compared with Smad3, we focused on Smad2 in our attempts to elucidate the functional role of acetylation of R-Smads. Lys19 in Smad2 Is Acetylated in Vivo—To determine if endogenous Smad2 was acetylated on Lys19, we generated an acetyl-Lys19-specific Smad2 antisera (AcK19). The affinity-purified antibody recognized wild-type Smad2(FL) following expression in 293T cells together with p300, whereas it failed to recognize the K19R mutant (Fig. 3A). The acetylation of Lys19 in endogenous Smad2 in HaCaT cells was enhanced following TGFβ stimulation (Fig. 3B). The acetylation was further enhanced when cells were treated with the deacetylase inhibitor TSA (compare lanes 2 and 4 in Fig. 3B), suggesting that the acetylation of Lys19 is a dynamic process regulated by deacetylases. Smad2 was also acetylated on Lys19 in response to TGFβ stimulation in HeLa cells (Fig. 3C). The acetylation of endogenous Smad2 was inhibited by the acetyltransferase inhibitor anacardic acid (Fig. 3D), confirming that the acetylation of Lys19 in Smad2 is dependent on cellular acetyltransferases. The Lys19-specific antibody also recognized Smad3 in response to p300-mediated acetylation (Fig. S2), confirming that Lys19 is acetylated in Smad3. However, we were unable to detect any acetylation of endogenous Smad3 on Lys19, confirming our observation that the acetylation of Smad3 is low compared with Smad2. Following receptor-mediated phosphorylation, Smad2 functions as a transcription factor by interacting with the promoters of Smad target genes. We used the AcK19 antibody in chromatin immunoprecipitation (ChIP) assays to determine if acetylated Smad2 was associated with the promoters of target genes in vivo. Smad2 acetylated on Lys19 was bound to the promoters of both the PAI-1 and p21 genes in HeLa cells and the occupancy increased in response to TGFβ stimulation (Fig. 3E). The recruitment of acetylated Smad2 to the PAI-1 promoter in response to TGFβ stimulation followed the same time course as the recruitment of total Smad2 (Fig. 3F). The increased association of acetylated Smad2 with the PAI-1 promoter in response to TGFβ stimulation coincided with an increased recruitment of p300 and P/CAF to the promoter (Fig. 3G). Acetylated Smad2 was also recruited to the endogenous PAI-1 and p21 promoters in HaCaT cells in response to TGFβ stimulation (supplemental Fig. S3). Thus, our results demonstrate that endogenous Smad2 is acetylated on Lys19 in response to TGFβ signaling and that the acetylated molecules are associated with the promoters of target genes in vivo. Because the AcK19 antibody also recognizes acetylated Smad3, it is possible that Smad3 contributes to the positive signals observed in the ChIP assays in Fig. 3. However, HeLa cells express very low levels of Smad3 compared with Smad2 (Fig. 3C). In addition, the acetylation of Smad3 is low compared with Smad2, suggesting that acetylated Smad2 is the major contributor to the positive ChIP signals in our experiments. Further work is required to analyze the recruitment of acetylated Smad3 to target genes in vivo. Acetylation of Lys19 Enhances the Transcriptional Activity of Smad2(ΔE3)—Smad3 and the short form of Smad2 (Smad2(ΔE3)) function as transcription factors by directly binding to DNA. However, the long form of Smad2 is unable to bind DNA and is, therefore, dependent on interactions with other DNA binding factors to function as a transcription factor. To test if the acetylation of Lys19 affected the transcriptional activity of Smad2, HepG2 cells were transfected with Smad-responsive promoter-reporter genes and Smad2(FL) or Smad2(ΔE3), either wild-type or the corresponding K19R mutants, in the absence or presence of p300. We were unable to detect any difference in transcriptional activity between wild-type Smad2(FL) and the K19R mutant on the ARE-Luc promoter-reporter gene (data not shown), indicating that the acetylation of Lys19 in the full-length form of Smad2 does not affect its transcriptional activity under these conditions. However, the transcriptional activity of Smad2(ΔE3) on the 12xCAGA-Luc promoter-reporter gene was enhanced in response to p300 expression (Fig. 4A). Importantly, the transcriptional activity of the K19R mutant of Smad2(ΔE3) was insensitive to p300 (Fig. 4A), suggesting that p300-mediated acetylation of Lys19 enhances the transcriptional activity of the short isoform of Smad2. Similar results were obtained following coexpression of Smad2(ΔE3) and P/CAF (Fig. 4B). Our data indicate that the acetylation of Lys19 in Smad2(ΔE3) is important for its ability to transactivate promoter-reporter genes. To test if this was also true for endogenous target genes, Smad2-deficient mouse embryonic fibroblasts were transfected with Smad2(ΔE3), either wild-type or the K19R mutant, in the absence or presence of p300 and the expression of the p21 gene was analyzed by RT-PCR. The expression of the p21 gene was greatly enhanced in the presence of Smad2(ΔE3) and p300, whereas p300 failed to enhance the expression of the p21 gene in the presence of the K19R mutant (Fig. 4C). Taken together, our results suggest that the acetylation of Lys19 in Smad2(ΔE3) enhances its transcriptional activity. Acetylation of Lys19 Enhances
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