Mass Spectrometric Analysis of Lysine Ubiquitylation Reveals Promiscuity at Site Level

Abstract

The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation shows promiscuity at the site level, as evidenced by low evolutionary conservation of ubiquitylation sites across eukaryotic species. Among lysine modifications a significant overlap (20%) between ubiquitylation and acetylation at site level highlights extensive competitive crosstalk among these modifications. This site-specific crosstalk is not prevalent among cell cycle ubiquitylations. Between SUMOylation and ubiquitylation the preferred interaction is through mixed-chain conjugation. Overall these data provide novel insights into the site-specific selection and regulatory function of lysine ubiquitylation. The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation shows promiscuity at the site level, as evidenced by low evolutionary conservation of ubiquitylation sites across eukaryotic species. Among lysine modifications a significant overlap (20%) between ubiquitylation and acetylation at site level highlights extensive competitive crosstalk among these modifications. This site-specific crosstalk is not prevalent among cell cycle ubiquitylations. Between SUMOylation and ubiquitylation the preferred interaction is through mixed-chain conjugation. Overall these data provide novel insights into the site-specific selection and regulatory function of lysine ubiquitylation. The proteins in a eukaryotic cell are subject to a large variety of post-translational modifications (PTMs) 1The abbreviations used are:PTMspost-translational modificationsHCDhigher-energy collisional activation dissociationLTQlinear trap quadropoleLC-MSliquid chromatography mass spectrometryESIelectrospray ionizationU2OSHuman osteosarcoma cellsHEK293Human Embryonic Kidney 293 cells.1The abbreviations used are:PTMspost-translational modificationsHCDhigher-energy collisional activation dissociationLTQlinear trap quadropoleLC-MSliquid chromatography mass spectrometryESIelectrospray ionizationU2OSHuman osteosarcoma cellsHEK293Human Embryonic Kidney 293 cells., which greatly extend the diversity of the proteome and play critical roles in regulating cellular functions (1.Yang X.J. Multisite protein modification and intramolecular signaling.Oncogene. 2005; 24: 1653-1662Crossref PubMed Scopus (222) Google Scholar). Among the estimated 200 different PTMs, phosphorylation, methylation, acetylation, and ubiquitylation are some of the most widespread and well characterized modifications (2.Mann M. Jensen O.N. Proteomic analysis of post-translational modifications.Nat. Biotechnol. 2003; 21: 255-261Crossref PubMed Scopus (1629) Google Scholar). Ubiquitylation refers to the covalent attachment of ubiquitin, a 76-residue polypeptide that is highly conserved among eukaryotes, via an isopeptide bond to the ε-amino group of lysines in proteins. The attachment of one or more ubiquitin moieties plays a central regulatory mechanism in eukaryotic cells, and regulates numerous cellular processes, including protein degradation, signal transduction, DNA repair, and cell division, as well as the control of stability, function, and intracellular localization of a wide variety of proteins (3.Ciechanover A. The ubiquitin-proteasome proteolytic pathway.Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1597) Google Scholar). post-translational modifications higher-energy collisional activation dissociation linear trap quadropole liquid chromatography mass spectrometry electrospray ionization Human osteosarcoma cells Human Embryonic Kidney 293 cells. post-translational modifications higher-energy collisional activation dissociation linear trap quadropole liquid chromatography mass spectrometry electrospray ionization Human osteosarcoma cells Human Embryonic Kidney 293 cells. Formation of the covalently linked ubiquitin-protein conjugates requires three enzymatic steps. First the formation is catalyzed by an ubiquitin-activating enzyme (E1) and then transferred from E1 to an ubiquitin-conjugating enzyme (E2). These two enzymes cooperate to transfer one ubiquitin moiety to a lysine residue in the target protein through a thio-esterification reaction, with the help of an ubiquitin ligase (E3) (4.Scheffner M. Nuber U. Huibregtse J.M. Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade.Nature. 1995; 373: 81-83Crossref PubMed Scopus (743) Google Scholar). The process of protein ubiquitylation can be highly dynamic and reversible, evidenced by an estimated 600 potential E3 ubiquitin ligases and some 80–90 deubiquitylating enzymes encoded by the human genome (5.Hussain S. Zhang Y. Galardy P.J. DUBs and cancer: the role of deubiquitinating enzymes as oncogenes, non-oncogenes and tumor suppressors.Cell Cycle. 2009; 8: 1688-1697Crossref PubMed Scopus (205) Google Scholar, 6.Love K.R. Catic A. Schlieker C. Ploegh H.L. Mechanisms, biology and inhibitors of deubiquitinating enzymes.Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (180) Google Scholar, 7.Reyes-Turcu F.E. Ventii K.H. Wilkinson K.D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.Ann. Rev. Biochem. 2009; 78: 363-397Crossref PubMed Scopus (1063) Google Scholar). These numbers illustrate the widespread use of substrate-specific ubiquitylation as an important regulatory principle in cell biology. This is further supported by the increasing number of scientific reports, which identify defects in ubiquitin-dependent signaling pathways as being involved in multiple human diseases (8.Petroski M.D. The ubiquitin system, disease, and drug discovery.BMC Biochem. 2008; 9 (Suppl 1): S7Crossref PubMed Scopus (148) Google Scholar, 9.Paul S. Dysfunction of the ubiquitin-proteasome system in multiple disease conditions: therapeutic approaches.Bioessays. 2008; 30: 1172-1184Crossref PubMed Scopus (107) Google Scholar, 10.Singhal S. Taylor M.C. Baker R.T. Deubiquitylating enzymes and disease.BMC Biochem. 2008; 9 (Suppl 1): S3Crossref PubMed Scopus (71) Google Scholar). Despite great biological and clinical interest, the knowledge of specific lysine ubiquitylation sites is still very limited. Liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the key technology for large-scale identifications of various PTMs such as phosphorylation (11.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2810) Google Scholar, 12.Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10762-10767Crossref PubMed Scopus (1251) Google Scholar), acetylation (13.Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. 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A to protein Biotechnol. 2003; 21: PubMed Scopus Google and to sites in human cells J. of ubiquitin an for proteins by mass 2007; PubMed Scopus Google Scholar, P. analysis of ubiquitination in cells by mass 2008; PubMed Scopus Google Scholar, F. Y. J. Y. Y. J. Y. B. J. Proteomic analysis of proteins in cell 2008; 8: PubMed Scopus Google Scholar, J. M. Proteomic analysis of proteins from human cells by and mass 2005; PubMed Scopus Google Scholar). a for using a specific a total of ubiquitylation sites analysis of lysine ubiquitination by ubiquitin Biotechnol. PubMed Scopus Google Scholar). These numbers are still the total number of ubiquitylation on the number of in the ubiquitin system, and the in protein ubiquitylation. that with the of a of high-resolution mass Orbitrap which for of at very mass and at low using the higher-energy collisional activation dissociation technology J.V. J. Nielsen M.L. P. Taylor M. M. A. Mann M. S. A linear trap Orbitrap with very 2009; Full Text Full Text PDF PubMed Scopus Google Scholar), an of lysine ubiquitylation be analysis very and a of the human greatly the number of in ubiquitylation sites and ubiquitin Human osteosarcoma cells and cells were in and cell ubiquitin, these cell were with ubiquitin, and with were then for of and to S. C. F. J. J. and of at the sites of DNA are by of cell biology. 2005; PubMed Scopus (222) Google Scholar). highly of cell in were in complexes were in in and on The with into and for mass were with and to mass were on a liquid chromatography system from to a with a and in a with with a from to in The from the into the mass data analysis with as J. Mann M. identification mass and protein Biotechnol. 2008; PubMed Scopus Google supported by as the for by a and of the human identify and ubiquitylation sites in human we a human osteosarcoma cell This for and of proteins from cell by of the that ubiquitin into cellular proteins in cell at a level ubiquitin, not the ubiquitylation of proteins and ubiquitin, the in the ubiquitin a of biological with ubiquitin, such as of on protein in to and The of proteins were on and that of a used in mass the used for site-specific identification of protein ubiquitylation M.L. M. J. Mann M. ubiquitination in mass 2008; PubMed Scopus Google Scholar). with in to in were and by of LTQ Orbitrap Velos. The mass of in which are at low and mass J.V. Macek B. Mortensen P. A. S. Mann M. mass on an Orbitrap mass via mass into a 2005; Full Text Full Text PDF PubMed Scopus Google for in identifications M.L. protein identification using in mass 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). 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S. ubiquitination of and by the Biol. 24: PubMed Scopus Google Scholar). we 5756 putative proteins with identification requires a for ubiquitin we an sequence of for proteins a site-specific ubiquitylation the sequence to be of Despite large of which from the of proteins a overlap in ubiquitylation sites among cell This overlap is the overlap to ubiquitylation and in a of H.L. S.A. K.R. F.E. B. M. P. S.J. M. P. C. and in identifications by liquid mass PubMed Scopus Google Scholar). the protein identification of ubiquitylation a overlap for the ubiquitin substrates in that is and and that ubiquitylation is a widespread with an in to phosphorylation and The most function of ubiquitin is proteins for The ubiquitin signal for process is a of ubiquitin linked through M. of the proteolytic J. PubMed Scopus Google Scholar). of the ubiquitin lysine residues such as A. S. 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S. of lysine 9 a site for PubMed Scopus Google Scholar). that proteins are in a as PTMs to a regulatory involved in for the of an the Biol. 2006; 8: PubMed Scopus Google Scholar). report one ubiquitylation site on and one on in ubiquitylation of we a protein the residues and the ubiquitylation site to as a to that ubiquitylation plays a role in the of in lysine ubiquitylation sites is for proteins and these illustrate the and of and that the ubiquitylation sites are of in The selection of target lysine residues by the ubiquitin system is to be on lysine and not the sequence E3 ubiquitin Rev. Biochem. 2009; 78: PubMed Scopus Google Scholar, M.D. of attachment the of 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, of requires site-specific Natl. Acad. Sci. U. S. 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This a more of lysine ubiquitylation with PTMs and lysine residues low evolutionary with a analysis that of ubiquitylation sites were conserved in more of eukaryotic the lysine residue which shows conservation This is in to the evolutionary conservation of the ubiquitin system across eukaryotic species. ubiquitylation shows promiscuity at the site level and E3 ligases targets lysine residues for ubiquitylation and not to a sequence This has in an in system lysine sites are In data that the lysine ubiquitylation is not a the ubiquitin in the ubiquitin process the of and E3 enzymes Rev. Biochem. PubMed Scopus Google Scholar), through the of E3 or through a by between and E3 A. A. of cell by and from Natl. Acad. Sci. U.S.A. 2008; 105: PubMed Scopus Google Scholar). is that E3 ligases can and is a E3 ligase that and to proteins specific residues to the A. A. The signal in a Full Text PDF PubMed Scopus Google Scholar, A. A. In of a protein is a function of PubMed Scopus Google Scholar, B. 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J.M. of inhibitors by the 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, and proteolytic of by the 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). that cell cycle target proteins for of lysine residues protein the of among cell cycle regulating proteins be In total we identify lysine sites on the biological process cell cycle proteins Among these ubiquitylation sites and and two ubiquitylation sites are to be on and on with the large overlap the the cell cycle overlap is This is that the cell cycle is the interaction of the (13.Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular 2009; PubMed Scopus Google Scholar). Overall these data that by acetylation plays an important and role in protein the between ubiquitylation and SUMOylation is well and through at the lysine residue J. M. J. Mann M. The ubiquitin-proteasome system is a key of the 2008; Full Text Full Text PDF PubMed Scopus Google Scholar), or through for the lysine J.M. R.T. modification of Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. between and ubiquitin on is by of the 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). SUMOylation sites in the A between SUMOylation and ubiquitylation sites is not for of the preferred interaction between these two modifications we at ubiquitylation sites in the sequence is or and is amino attachment of the protein to the Biol. PubMed Scopus Google Scholar). of lysine ubiquitylation sites located in that between ubiquitylation and be In lysine SUMOylation sites on are as ubiquitylation targets in that signaling interaction between SUMOylation and ubiquitylation through mixed-chain conjugation. for the site-specific and competitive be of SUMOylation and ubiquitylation with to biological function A. and functions of Rev. Biol. 2003; PubMed Scopus Google Scholar). The mixed-chain between SUMOylation and ubiquitylation is to the of ubiquitin to ubiquitin we estimated the of ubiquitylation sites that is the most ubiquitin on by and These that is the major site for M. J. Macek B. R.T. Mann M. In identification of human sites by mass spectrometry and an in to in 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). is the residue to be the of lysine the total of to that mixed-chain between and ubiquitin, through is the biological of mixed-chain interaction between SUMOylation and ubiquitylation to be more lysine ubiquitylation has the a number of ubiquitylation sites and specific the ubiquitin system still to be Here we a large-scale identification of lysine ubiquitylation and that in ubiquitylation In total we identify 753 sites to 471 the of lysine in human proteins by as with and data for the scientific to ubiquitin and proteins. the process by which ubiquitin is to lysine residues still is to in the ubiquitylation This sequence specific on lysine residues and amino for which large-scale mass analysis is an of data a on the ubiquitin system which specific lysine residues are targeted for ubiquitylation lysine residues are that lysine ubiquitylation sites are and not to unique sequence from these that of ubiquitylation sites sequence of lysine residues is not data that of lysine ubiquitylation conservation of lysine residues between eukaryotic is not ubiquitin lysines are through using sequence or site conservation is a to ubiquitylation the is very and is to between ubiquitin sites and of ubiquitylation of by the mass analysis to be the most for large-scale identification of lysine ubiquitylation lysine residues are targets of PTMs data provide an to site-specific of various lysine modifications. a and overlap between lysine ubiquitylation and Lysine acetylation is to dynamic regulatory through with a variety of PTMs, and proteins are key of various signaling the scientific in competitive with lysine ubiquitylation is into cellular by the of large ubiquitylation data that of are to be that competitive between these two PTMs is The biological of such is by that among cell cycle proteins the is of ubiquitylation sites are to be and of key the cell cycle is of the is that ubiquitin-dependent and in a and of protein by of lysine residues is not cell cycle In to acetylation we interaction between ubiquitylation and identify novel ubiquitylation sites located on various and site-specific analysis that interaction between SUMOylation and ubiquitylation through mixed-chain conjugation. the role of is in the has that mixed-chain not target proteins for J. M. J. Mann M. The ubiquitin-proteasome system is a key of the 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The large number of ubiquitylation sites on proteins that mixed-chain is more In data provide an extensive into human lysine ubiquitylation with novel the of lysine ubiquitylations. that lysine ubiquitylation is an extensive with modifications to signaling that are for cell The large of lysine ubiquitylation sites be through post-translational modification such as F. S. J. Olsen J.V. Macek B. M. Mann M. site and evolutionary and of Biol. 2007; 8: PubMed Scopus Google Scholar). The data is at Ubiquitylation of the for and of the with