Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition
2009; Elsevier BV; Volume: 8; Issue: 10 Linguagem: Inglês
10.1074/mcp.m900079-mcp200
ISSN1535-9484
AutoresVittoria Matafora, Alfonsina D’Amato, Silvia Mori, Francesco Blasi, Angela Bachi,
Tópico(s)Protein Degradation and Inhibitors
ResumoMany cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity. Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity. Targeting of proteins by conjugation of Small Ubiquitin-like MOdifier (SUMO) 1The abbreviations used are:SUMOsmall ubiquitin-like modifierArg0[12C6,14N4]arginineArg10[13C6,15N4]arginineAos1/Uba2SUMO-activating enzyme subunit 1/2 (SAE1/SAE2)E3SUMO ligasehnRNPheterogeneous nuclear ribonucleoproteinIPIInternational Protein IndexLys0[12C6,14N2]lysineLys8[13C6,15N2]lysinep160p160 Myb-binding protein 1ASILACstable isotope labeling by amino acids in cell cultureUbc9ubiquitin-conjugating enzyme 9nLCnano-LCLTQlinear trap quadrupolePANTHERProtein Analysis through Evolutionary Relationships.1The abbreviations used are:SUMOsmall ubiquitin-like modifierArg0[12C6,14N4]arginineArg10[13C6,15N4]arginineAos1/Uba2SUMO-activating enzyme subunit 1/2 (SAE1/SAE2)E3SUMO ligasehnRNPheterogeneous nuclear ribonucleoproteinIPIInternational Protein IndexLys0[12C6,14N2]lysineLys8[13C6,15N2]lysinep160p160 Myb-binding protein 1ASILACstable isotope labeling by amino acids in cell cultureUbc9ubiquitin-conjugating enzyme 9nLCnano-LCLTQlinear trap quadrupolePANTHERProtein Analysis through Evolutionary Relationships. is a key mechanism for regulating many cellular processes (1Zhang F.P. 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SUMO proteins are ubiquitously expressed throughout the eukaryotic kingdom. Yeast, Caenorhabditis elegans, and Drosophila melanogaster carry a single SUMO gene, whereas plants and vertebrates have several SUMO genes (5Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1315) Google Scholar). In particular, humans express four distinct SUMO family members: SUMO-1, SUMO-2, SUMO-3, and SUMO-4 (7Guo D. Li M. Zhang Y. Yang P. Eckenrode S. Hopkins D. Zheng W. Purohit S. Podolsky R.H. Muir A. Wang J. Dong Z. Brusko T. Atkinson M. Pozzilli P. Zeidler A. Raffel L.J. Jacob C.O. Park Y. Serrano-Rios M. Larrad M.T. Zhang Z. Garchon H.J. Bach J.F. Rotter J.I. She J.X. Wang C.Y. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes.Nat. Genet. 2004; 36: 837-841Crossref PubMed Scopus (325) Google Scholar, 8Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (646) Google Scholar). SUMO-1 is an 11.6 kDa protein. It shares about 47% homology with SUMO-2 and SUMO-3 that, on the contrary, differ from each other only by three amino-terminal residues and form a distinct subfamily known as SUMO-2/-3 (9Lapenta V. Chiurazzi P. van der Spek P. Pizzuti A. Hanaoka F. Brahe C. SMT3A, a human homologue of the S. cerevisiae SMT3 gene, maps to chromosome 21qter and defines a novel gene family.Genomics. 1997; 40: 362-366Crossref PubMed Scopus (102) Google Scholar). Despite the low sequence homology, SUMO-1 and SUMO-2/-3 share a similar protein size, tertiary structure, and a carboxyl-terminal diglycine motif (10Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1358) Google Scholar, 11Martin S. Wilkinson K.A. Nishimune A. Henley J.M. Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction.Nat. Rev. Neurosci. 2007; 8: 948-959Crossref PubMed Scopus (166) Google Scholar). At the cellular level, different amounts of free SUMO-1 and SUMO-2/-3 are present. The majority of SUMO-1 in fact is conjugated to substrates, whereas the conjugation of SUMO-2/-3 is strongly induced in response to various stresses (10Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1358) Google Scholar). Finally SUMO-1 and SUMO-2/-3 serve distinct functions as they modify different target proteins (5Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1315) Google Scholar). Unlike SUMO-1, SUMO-2, and SUMO-3, which are ubiquitously expressed (7Guo D. Li M. Zhang Y. Yang P. Eckenrode S. Hopkins D. Zheng W. Purohit S. Podolsky R.H. Muir A. Wang J. Dong Z. Brusko T. Atkinson M. Pozzilli P. Zeidler A. Raffel L.J. Jacob C.O. Park Y. Serrano-Rios M. Larrad M.T. Zhang Z. Garchon H.J. Bach J.F. Rotter J.I. She J.X. Wang C.Y. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes.Nat. Genet. 2004; 36: 837-841Crossref PubMed Scopus (325) Google Scholar), SUMO-4 isoform has yet to be characterized. It seems to be expressed mainly in the kidney, lymph nodes, and spleen, but its role still remains unclear because its mature form has never been reported in vivo (7Guo D. Li M. Zhang Y. Yang P. Eckenrode S. Hopkins D. Zheng W. Purohit S. Podolsky R.H. Muir A. Wang J. Dong Z. Brusko T. Atkinson M. Pozzilli P. Zeidler A. Raffel L.J. Jacob C.O. Park Y. Serrano-Rios M. Larrad M.T. Zhang Z. Garchon H.J. Bach J.F. Rotter J.I. She J.X. Wang C.Y. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes.Nat. Genet. 2004; 36: 837-841Crossref PubMed Scopus (325) Google Scholar, 12Owerbach D. McKay E.M. Yeh E.T. Gabbay K.H. Bohren K.M. A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation.Biochem. Biophys. Res. Commun. 2005; 337: 517-520Crossref PubMed Scopus (173) Google Scholar). Several SUMO targets are known; they are mostly nuclear proteins presenting a consensus acceptor site: ΨKXE (in which Ψ is an aliphatic branched amino acid and X is any amino acid) (5Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1315) Google Scholar). The mutation of this site abolishes sumoylation of substrates and is commonly used to understand the biological implication of the substrate modification. Also SUMO-2/-3 present a conserved lysine in this motif, and they form polymeric SUMO chains (13Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 14Matic I. van Hagen M. Schimmel J. Macek B. Ogg S.C. Tatham M.H. Hay R.T. Lamond A.I. Mann M. Vertegaal A.C. In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy.Mol. Cell. Proteomics. 2008; 7: 132-144Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). SUMO-1, however, lacks this consensus site and is not thought to form chains even if recent studies demonstrate that SUMO-1 can be linked to the end of a poly-SUMO-2/-3, terminating the chain (11Martin S. Wilkinson K.A. Nishimune A. Henley J.M. Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction.Nat. Rev. Neurosci. 2007; 8: 948-959Crossref PubMed Scopus (166) Google Scholar). Recently two different extensions of the simple consensus SUMO acceptor site have been identified. These motifs share a negative charge next to the basic SUMO consensus site: one involves a phosphorylated (p) Ser and a Pro residue (ΨKXEXXpSP), and the other contains a negatively charged amino acid close to the acceptor Lys residue (5Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1315) Google Scholar). Although many targets contain the above mentioned motifs, there are examples of substrates that do not contain these acceptor sites. The presence of a phosphorylated residue in the motif indicates that regulatory mechanisms, which can enhance or decrease the sumoylation of specific targets, may occur at the level of the target itself (15Hietakangas V. Anckar J. Blomster H.A. Fujimoto M. Palvimo J.J. Nakai A. Sistonen L. PDSM, a motif for phosphorylation-dependent SUMO modification.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 45-50Crossref PubMed Scopus (376) Google Scholar). Indeed sumoylation often acts in coordination with other post-translational modifications like acetylation, methylation, and ubiquitination (16Zhao J. Sumoylation regulates diverse biological processes.Cell. Mol. Life Sci. 2007; 64: 3017-3033Crossref PubMed Scopus (183) Google Scholar). As discussed above, SUMO proteins are similar in three-dimensional structures. Although they do not display high sequence homology, they share the same structure of ubiquitin and a common conjugation mechanism. In fact like ubiquitination sumoylation also requires the formation of an isopeptide bond between the carboxyl-terminal Gly residue of the modifier protein and the ε-amino group of a Lys residue in the acceptor protein (5Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1315) Google Scholar). The enzymatic cascade that mediates SUMO conjugation is similar to that of ubiquitin. The immature precursor is first processed by a specific carboxyl-terminal hydrolase that exposes the diglycine motif, and then mature SUMO proteins are activated by an ATP-dependent heterodimer of SUMO-activating enzyme subunit 1 (SAE1) and SAE2. The above dimer transfers the activated SUMO protein to the ubiquitin-conjugating enzyme 9 (Ubc9) through a transesterification reaction. Ubc9 usually acts together with an E3 ligating enzyme that catalyzes SUMO conjugation to the substrate. In contrast to the ubiquitin pathway in which an E3 enzyme is essential for conjugation, SUMO modification just requires Ubc9, which is able to bind directly to the SUMO consensus sequence and substrates, aligning them for conjugation (5Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1315) Google Scholar, 10Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1358) Google Scholar, 11Martin S. Wilkinson K.A. Nishimune A. Henley J.M. Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction.Nat. Rev. Neurosci. 2007; 8: 948-959Crossref PubMed Scopus (166) Google Scholar). Despite the similarity between SUMO and ubiquitin, the molecular consequences of these two modifications are distinct (17Ulrich H.D. Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest.Trends Cell Biol. 2005; 15: 525-532Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 18Pickart C.M. Fushman D. Polyubiquitin chains: polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (814) Google Scholar). In some cases, such as IκBα modification, SUMO plays an antagonistic role to ubiquitin, competing for the same lysine (19Desterro J.M. Rodriguez M.S. Hay R.T. SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation.Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). In other cases, as for NFκB essential modulator/IκB kinase γ, SUMO and ubiquitin are conjugated in a sequential manner in response to a toxic stress; in further cases SUMO may regulate protein localization, stabilizing substrate, independently from ubiquitination as for Smad4 (20Huang T.T. Wuerzberger-Davis S.M. Wu Z.H. Miyamoto S. Sequential modification of NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by genotoxic stress.Cell. 2003; 115: 565-576Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 21Shimada K. Suzuki N. Ono Y. Tanaka K. Maeno M. Ito K. Ubc9 promotes the stability of Smad4 and the nuclear accumulation of Smad1 in osteoblast-like Saos-2 cells.Bone. 2008; 42: 886-893Crossref PubMed Scopus (23) Google Scholar, 22Lee P.S. Chang C. Liu D. Derynck R. 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We analyzed the subcellular distribution of sumoylated proteins in HeLa cells upon MG132 treatment and identified SUMO-1 targets by mass spectrometric techniques. Moreover we measured the effect of MG132 on target modification by stable isotope labeling by amino acids in cell culture (SILAC), and we demonstrated that, upon proteasome inhibition, the amount of SUMO-1 species increases and accumulates in nucleolar structures (28Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4475) Google Scholar, 29Ong S.E. Foster L.J. Mann M. Mass spectrometric-based approaches in quantitative proteomics.Methods. 2003; 29: 124-130Crossref PubMed Scopus (380) Google Scholar, 30Ong S.E. Mann M. Stable isotope labeling by amino acids in cell culture for quantitative proteomics.Methods Mol. Biol. 2007; 359: 37-52Crossref PubMed Google Scholar). This enrichment of SUMO-1 allowed the detection of sumoylated targets at endogenous levels, although usually the abundance of sumoylated proteins is relatively low in the cell, and they are difficult to detect. Based on these data, we focused our attention on the nucleolar compartment and identified nucleolar sumoylated proteins that accumulate after proteasome inhibition. The analysis of such proteins strongly indicates that sumoylation is involved in the regulation of nucleolar dynamics. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 100 units/ml penicillin and streptomycin (Invitrogen). Stable isotope labeling was carried out essentially as described previously (28Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4475) Google Scholar) using [12C6,14N4]arginine (referred to as Arg0), [12C6,14N2]lysine (referred to as Lys0), [13C6,15N4]arginine (referred to as Arg10), and [13C6,15N2]lysine (referred to as Lys8) (Cambridge Isotope Laboratories, Cambridge, MA). Arg0-labeled cells in experiments I and II or Arg0, Lys0-labeled cells in experiment III were treated with 10 µm MG132 in DMSO overnight; Arg10-labeled cells or Arg10, Lys8-labeled cells were treated with DMSO as control. The labeled cells, for each experiment, were mixed in a 1:1 ratio (3 × 107 cells each). HeLa cells were grown on sterile 13-mm coverslips and then treated with 10 µm MG132 or DMSO overnight. A time course was performed, treating HeLa cells with 10 µm MG132 for 1, 6, and 12 h using DMSO as control. Comparison of SUMO-1 staining under several stresses was carried out using 0.2 µm actinomycin D (Sigma-Aldrich) for 12 h, 10 µg/ml cycloheximide (Sigma-Aldrich) for 12 h, 1 µm Velcade (bortezomib) (Millennium Pharmaceuticals, Cambridge, MA) for 12 h, 1 mm H2O2 (Sigma-Aldrich) for 1 h, and 10 µm MG132 for 12 h. Cells were fixed with PBS, 3.0% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.2% Triton X-100, 300 mm sucrose, 20 mm Hepes, pH 7.4, 50 mm NaCl, 3 mm MgCl2 for 3 min at 4 °C. HeLa cells were incubated with the indicated antibodies in blocking buffer (0.2% bovine serum albumin in PBS) for 1 h at 37 °C, rinsed with PBS, incubated with purified Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G antibodies, rinsed, and mounted with Immuno-Fluore Mounting Medium (ICN Biomedicals, Costa Mesa, CA). The nuclei were visualized by Hoechst 33258 (Sigma-Aldrich) staining for 3 min at room temperature, and then after several washes with PBS, the nucleoli were stained with 0.66 mm pyronin Y (Sigma-Aldrich). Fluorescence was visualized with an inverted fluorescence microscope (DM IRBE; Leica, Wetzlar, Germany) and captured with a TCS-NT argon/krypton confocal laser microscope (Leica). The plasmid encoding His-SUMO-1 wild type was obtained by cloning SUMO-1 cDNA, kindly donated from Ronald Hay as pGEX-SUMO-1 construct (31Desterro J.M. Thomson J. Hay R.T. Ubc9 conjugates SUMO but not ubiquitin.FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (302) Google Scholar), into pet28a vector. The plasmid encoding Ubc9, obtained by cloning Ubc9 cDNA into pet23a vector, and the plasmids encoding His-Aos1 in pet28a and Uba2 in pet11d were kind gifts from Frauke Melchior (32Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). His-SUMO-1 protein was purified by Ni2+ beads (Qiagen, Valencia, CA) according to the manufacturer's procedure. Aos1-Uba2 complex was purified as described previously (32Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). The following antibodies were used: anti-SUMO-1 monoclonal antibody (21C7 from Zymed Laboratories Inc.), anti-SUMO-1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-nucleophosmin monoclonal antibody was kindly provided by Emanuela Colombo (33Cordell J.L. Pulford K.A. Bigerna B. Roncador G. Banham A. Colombo E. Pelicci P.G. Mason D.Y. Falini B. Detection of normal and chimeric nucleophosmin in human cells.Blood. 1999; 93: 632-642Crossref PubMed Google Scholar), anti-ubiquitin monoclonal antibody (Santa Cruz Biotechnology), anti-lamin A/C monoclonal antibody (Santa Cruz Biotechnology), anti-α-tubulin monoclonal antibody, anti-FLAG monoclonal antibody (both from Sigma-Aldrich), and anti-histone H3 polyclonal antibody (Abcam, Cambridge, MA). HeLa cells were separated into cytoplasmic, nuclear, nucleoplasmic, and nucleolar fractions using a previously published protocol (34Bush H. Muramatsu M. Adams H. Steele W.J. Liau M.C. Smetana K. Isolation of nucleoli.Exp. Cell Res. 1963; 24: 150-163Crossref Scopus (28) Google Scholar, 35Andersen J.S. Lam Y.W. Leung A.K. Ong S.E. Lyon C.E. Lamond A.I. Mann M. Nucleolar proteome dynamics.Nature. 2005; 433: 77-83Crossref PubMed Scopus (927) Google Scholar, 36Leung A.K. Trinkle-Mulcahy L. Lam Y.W. Andersen J.S. Mann M. Lamond A.I. NOPdb: Nucleolar Proteome Database.Nucleic Acids Res. 2006; 34: D218-D220Crossref PubMed Scopus (91) Google Scholar). Purified nucleoli were lysed in 4% SDS, 50 mm Tris, pH 8.0 and then diluted to reconstitute RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mm Tris, pH 8.0) supplemented with 50 mmN-ethylmaleimide and protease inhibitor mixture. Nucleolar SUMO-1 target proteins were immunoprecipitated using anti-SUMO-1 monoclonal antibody that was incubated with protein G-Sepharose 4 Fast Flow beads (GE Healthcare/Amersham Biosciences) for 1 h at 4 °C. The antibody was then linked to protein G by 3,3′-dithiobis(sulfosuccinimidylpropionate) cross-linker (Pierce) according to the manufacturer's instructions. Nucleolar lysates were incubated with the antibody bound to protein G beads at 4 °C overnight. After extensive washing with RIPA buffer, immunoprecipitates were eluted in non-reducing Laemmli buffer and then in reducing buffer. SUMO-1 proteins were separated by 10% SDS-PAGE, stained with Coomassie Brilliant Blue (Bio-Rad), and excised in 24 slices for LC-MS/MS analysis. Concerning immunoblotting experiments, proteins separated by SDS-PAGE were subsequently transferred onto nitrocellulose membranes (GE Healthcare/Amersham Biosciences). These membranes were incubated with specific antibodies as indicated. For p160 Myb-binding protein 1A immunoprecipitation, NIH 3T3 cells were infected using a p160-FLAG retrovirus as described before (37Díaz V.M. Mori S. Longobardi E. Menendez G. Ferrai C. Keough R.A. Bachi A. Blasi F. p160 Myb-binding protein interacts with Prep1 and inhibits its transcriptional activity.Mol. Cell. Biol. 2007; 27: 7981-7990Crossref PubMed Scopus (42) Google Scholar). Then NIH 3T3 cell nuclear extracts (38Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar) were adjusted to IBB buffer (10 mm Tris-HCl, pH 8, 0.2% Nonidet P-40, 150 mm NaCl) and precleared with protein G-Sepharose beads for 1 h at 4 °C. The clarified supernatants were incubated with M2 anti-FLAG affinity resin (Sigma-Aldrich) overnight at 4 °C. The beads were rinsed several times with IBB buffer, resuspended in Laemmli buffer, heated at 85 °C, and centrifuged at 10,000 × g. The in vitro reaction was performed on HeLa extracts (38Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar) as follows. 1.3 mg of HeLa nuclear extract and 6 mg of HeLa cytosolic extract were incubated with 100 µg of His-SUMO-1 previously bound to Ni2+ beads (Qiagen), 30 µg of Ubc9, 0.5 units/ml inorganic pyrophosphatase, and 10 mm ATP in sumoylation buffer (10 mm MgCl2, 0.1 mm DTT, 50 mm Tris-HCl, pH 7.5) for 1 h at room temperature (39Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. In vitro SUMO-1 modification requires two enzymatic steps, E1 and E2.Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (181) Google Scholar). The reaction mixture was incubated in the absence of SUMO-1 as a control. The sumoylation reactions were stopped by adding 10 mm N-ethylmaleimide and 50 mm imidazole in sumoylation buffer. After exhaustive washings, the His-SUMO-1-conjugated proteins were eluted from beads with 500 mm imidazole in 50 mm Tris-HCl, 150 mm NaCl. Proteins were separated by 10% SDS-PAGE, stained by silver staining (40Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7736) Google Scholar), and excised in 34 slices for LC-MS/MS analysis. For histones, a mixture of calf thymus total histones (1 µg) or purified histone H3 (1 µg) (Sigma-Aldrich) was incubated in the presence (or absence as control) of His-SUMO-1 (1 µg), Ubc9 (10 ng), Aos1/Uba2 (150 ng), 0.5 units/ml inorganic pyrophosphatase, and 10 mm ATP in sumoylation buffer for 1 h at room temperature. Mass Spectrometry analysis was performed using a hybrid quadrupole time-of-flight mass spectrometer (API QStar PULSAR, PE-Sciex, Toronto, Canada) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). A total of 5 µl of trypsin-digested sample was injected in a capillary chromatographic system Agilent 1100 Series equipped with a Nano Pump, Iso Pump, and Degasser (Agilent, Santa Clara, CA). Peptide mixtures were separated on a 10-cm fused silica capillary (75-µm inner diameter and 360-µm outer diameter; Proxeon Biosystems) filled with Reprosil-Pur C18 3-µm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) using a pressurized "packing bomb." Peptides w
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