BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*

The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCFβ-TrCP1 and SCFβ-TrCP2 are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCFβ-TrCP1/2 interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCFβ-TrCP1/2 substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for β-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that β-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases. The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCFβ-TrCP1 and SCFβ-TrCP2 are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCFβ-TrCP1/2 interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCFβ-TrCP1/2 substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for β-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that β-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases. More than 600 putative ubiquitin E3 ligases are encoded in the human genome (1.Varshavsky A. The ubiquitin system, an immense realm.Annu. Rev. Biochem. 2012; 81: 167-176Crossref PubMed Scopus (218) Google Scholar, 2.Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6880) Google Scholar). Although a number of these proteins are known to play critical roles in human health (1.Varshavsky A. The ubiquitin system, an immense realm.Annu. Rev. Biochem. 2012; 81: 167-176Crossref PubMed Scopus (218) Google Scholar, 2.Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6880) Google Scholar, 3.Scheffner M. Staub O. HECT E3s and human disease.BMC Biochem. 2007; 8: S6Crossref PubMed Scopus (78) Google Scholar, 4.Jiang Y.H. Beaudet A.L. Human disorders of ubiquitination and proteasomal degradation.Curr. Opin. Pediar. 2004; 16: 419-426Crossref PubMed Scopus (72) Google Scholar), the specific biological functions–and substrates–of most E3s remain poorly characterized. The identification of E3 substrates has been difficult in part because: (1) ligase - substrate interactions are often of low affinity (generally in the high nm to microMolar (μM) range) and/or of a transient nature; (2) many substrates are subjected to rapid proteasomal degradation and are therefore not available for detection; (3) the human ubiquitome is extremely complex, and; (4) many substrate proteins are localized to poorly soluble cellular compartments, making their isolation and identification by standard immunoprecipitation (IP)-based techniques extremely challenging (1.Varshavsky A. The ubiquitin system, an immense realm.Annu. Rev. Biochem. 2012; 81: 167-176Crossref PubMed Scopus (218) Google Scholar, 2.Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6880) Google Scholar, 3.Scheffner M. Staub O. HECT E3s and human disease.BMC Biochem. 2007; 8: S6Crossref PubMed Scopus (78) Google Scholar, 4.Jiang Y.H. Beaudet A.L. Human disorders of ubiquitination and proteasomal degradation.Curr. Opin. Pediar. 2004; 16: 419-426Crossref PubMed Scopus (72) Google Scholar). Methods such as protein chip (5.Persaud A. Rotin D. Use of proteome arrays to globally identify substrates for E3 ubiquitin ligases.Methods Mol. Biol. 2011; 759: 215-224Crossref PubMed Scopus (6) Google Scholar) and yeast two-hybrid screening (6.Rual J.F. Venkatesan K. Hao T. Hirozane-Kishikawa T. Dricot A. Li N. Berriz G.F. Gibbons F.D. Dreze M. Ayivi-Guedehoussou N. Klitgord N. Simon C. Boxem M. Milstein S. Rosenberg J. Goldberg D.S. Zhang L.V. Wong S.L. Franklin G. Li S. Albala J.S. Lim J. Fraughton C. Llamosas E. Cevik S. Bex C. Lamesch P. Sikorski R.S. Vandenhaute J. Zoghbi H.Y. Smolyar A. Bosak S. Sequerra R. Doucette-Stamm L. Cusick M.E. Hill D.E. Roth F.P. Vidal M. Towards a proteome-scale map of the human protein-protein interaction network.Nature. 2005; 437: 1173-1178Crossref PubMed Scopus (2284) Google Scholar) have been used to identify a limited number of E3-substrate interactions. However, these methods are not conducted in live mammalian cells, and may not be generally applicable for the identification of substrates of the hundreds of unique multi-protein E3 complexes (e.g. SCF, APC, VHL, etc.) or the identification of E3-substrate interactions that are dependent on specific types of post-translational modifications. Several putative inhibitors of apoptosis substrates were identified using the recently described NEDDylator technique (7.Zhuang M. Guan S. Wang H. Burlingame A.L. Wells J.A. Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.Mol. Cell. 2013; 49: 273-282Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), in which the E2 protein UBC12 (UBE2M) is fused to the E3 ligase of interest, allowing for the conjugation of the ubiquitin-like protein NEDD8 to substrates. Global protein stability profiling (8.Yen H.C. Elledge S.J. Identification of SCF ubiquitin ligase substrates by global protein stability profiling.Science. 2008; 322: 923-929Crossref PubMed Scopus (151) Google Scholar) and quantitative mass spectrometry methods (9.Hor S. Ziv T. Admon A. Lehner P.J. Stable isotope labeling by amino acids in cell culture and differential plasma membrane proteome quantitation identify new substrates for the MARCH9 transmembrane E3 ligase.Mol. Cell. Proteomics. 2009; 8: 1959-1971Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) have also been used successfully to identify E3 targets. However, because of their cost and/or complexity, and the challenges posed by the extremely large size of the human ubiquitome, these methods have not been widely adopted to date. Proximity-based biotinylation, or BioID 1The abbreviations used are:BioIDproximity-based biotinylationCHXcyclohexamideEDMDEmery-Dreifuss Muscular Dystrophy. 1The abbreviations used are:BioIDproximity-based biotinylationCHXcyclohexamideEDMDEmery-Dreifuss Muscular Dystrophy. (10.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar), is a new method developed for the characterization of protein-protein interactions in living cells. Briefly, a protein of interest is fused in-frame with an E. coli biotin conjugating enzyme mutant (BirA R118G, or BirA*). The BirA* moiety a affinity for the K. D. and the in the E. coli biotin PubMed Scopus Google from BirA* and with - on in cell proteins be affinity using and identified using mass spectrometry are with be used to localized to poorly soluble cellular (10.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar, D. E. E. M. M. D. S. B. J. L. and with in Biol. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, N. T. interactions identify of Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. K. The and of are by proteins and protein Biol. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, A. K. M. ligase identifies proteins proximal to a of and Cell PubMed Scopus Google Scholar, A.L. G. A. T. H. interaction of the of 2013; PubMed Scopus Google Scholar). method not that protein-protein interactions be and/or transient may also be We that BioID may be to ubiquitin E3 ligase substrates and Muscular Dystrophy. Muscular Dystrophy. The human and are proteins amino and and as substrate of SCF complexes M. M. A to the ubiquitin SCF and 2004; PubMed Scopus Google Scholar). A number of β-TrCP substrates have been these ligases in biological of the Hippo, and N. of by ubiquitination and degradation of the Rev. 2012; PubMed Scopus Google Scholar, The Biol. 2013; PubMed Scopus Google Scholar, B. Li L. K. Wang Guan A by and stability PubMed Scopus Google Scholar, D. M. by the proteins and the of Rev. 2008; 8: PubMed Scopus Google Scholar). β-TrCP targets the or of is for SCFβ-TrCP1/2 and the of these proteins D. M. by the proteins and the of Rev. 2008; 8: PubMed Scopus Google Scholar, P. P. Elledge S.J. The ligase with in and and ubiquitination in PubMed Scopus Google Scholar). However, SCFβ-TrCP1/2 substrates or which are to turnover D. M. by the proteins and the of Rev. 2008; 8: PubMed Scopus Google Scholar). A that β-TrCP has not been we that BioID on with the proteasome inhibitor many of the previously substrates and of mass spectrometry, we identify and validate a number of new these well-studied E3 ligases to new biological The method used here is and should be broadly applicable for the identification of substrates for many were using to and are in were with and and the using ligase The mutant by and by 2007; PubMed Scopus Google Scholar) using and the in BioID (10.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar) as we described previously D. E. E. M. M. D. S. B. J. L. and with in Biol. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). human and were by and our in the or were of were for in with and 50 biotin were with inhibitor were and the with and were were in of inhibitor for to The for were with for were by with 50 and with The the and were in and of the of or were with and biotin as were with in and by for Cell were The cell and of 50 and inhibitor were on for subjected to for to a and were The for with were by for and with of to a were with and with by the with of The and the for to a and were in and of the and were from fused from and with conducted using a to on a with a mass A in the using a of to the most were for of for using standard were in the such that of the a size were from for protein were to the using D. M. Burke R. D. P. for rapid 2008; PubMed Scopus Google Scholar), using R. proteins with mass 2004; PubMed Scopus Google Scholar) Human a of and an of with to for of as a were using the a for Mol. Biol. PubMed Scopus Google Scholar) the G. Zhang J. B. C. A. K. A. T. B. M. for mass interaction PubMed Scopus (151) Google Scholar). identified with a of to were with H. B. A. D. D. M. of affinity spectrometry 2011; 8: PubMed Scopus Google Scholar, G. G. Zhang J. H. and in of Proteomics. PubMed Scopus Google Scholar). were used for of a BioID conducted on the of BioID conducted on cells, and from a BioID conducted on an were conducted on and were conducted on with MG132, as The were to the for for IP-MS were IP-MS of proteins in cells, in the presence and of The were to the for data are available and have been to the are available for the a for Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) of for Based on and biological for in the presence and of were as high protein a were in the presence of in the of of the the in from with and using as putative substrates a and in BioID and IP-MS a and BioID and IP-MS are presented as for as were on of unique biological for protein a of in the presence and of the of are of for of the in the presence the of to for to by were as by not and/or not identified as a high with the technique and substrate in substrate in are in here to be β-TrCP and are in with were with nm nm nm or nm T. Kim M. S. T. M. A. of the of by and degradation Cell. Biol. 2011; PubMed Scopus Google Scholar) using to and by and were subjected to a with or substrate in or using to were and were to a for and of 50 to in were and to an and of on a were to and in or were in and used as were with and in with were with and using substrate were using using or to the for the is presented as a of the for that or cell were on with for and in with were in in for in with or for and were used and and for the were with of in for with were with were using a on an and were using the and using and were by an or or an are in Stable cell were as described in A.L. spectrometry to mammalian and 2012; PubMed Scopus Google Scholar). The and were from the number and with or with the or or stress were with or in and for with to were and stress were with of for were with in with were in a The used to the number of or stress and were for protein to the and and were for a and and size and for and stress used to the from with or with nm were for were as with a of were a and were were conducted for and were conducted for stress with or were and using and the used for the and a of and stress and cells. or were with or using the were with for and for to with were and in with were for in and to an for and the cell Cell were in inhibitor 50 for on an and for of the to as and the with on an were for and with were and to to or were with using as to for the (1) (2) (3) MG132, with and were in with were for in of and to an 50 of were for were for and were in 50 inhibitor and for were with and with on an were for and with were and to to or were a were with with or for were in and in and also the stress the used in Cell were for and protein for and were used in or were a a of to the for to protein were with the of in of in to of in and for of and in to and the to the were nm and - for were and the for to the of the cell and standard with a in SCFβ-TrCP1 and SCFβ-TrCP2 target substrates for 26S We using the BioID interacting as of the SCF should be with biotin to a in the presence and of MG132, substrates should be proteasome is in in the A semiquantitative mass spectrometry approach be to identify these putative substrates. have that β-TrCP P. P. Elledge S.J. The ligase with in and and ubiquitination in PubMed Scopus Google Scholar, M. R. H. C. B. R. P. The protein with and in the Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. M. M. N. K. A. K. of J. PubMed Scopus Google Scholar, E. D.S. M. The human protein with the and the stability of PubMed Scopus Google Scholar, C. S. the ubiquitination of Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), and our BioID that more (e.g. SCF and previously substrates than a protein not A fused to the of and and and these proteins were in cells. cell were for The were in a and protein were by the of biotin to the culture were using a by and treatment to the of proteins localized to poorly soluble or with or proteins were using a proteins were with and the identified using mass spectrometry cells, cell the and an protein were subjected to the to for proteins and that with the or the G. Zhang J. B. C. A. K. A. T. B. M. for mass interaction PubMed Scopus (151) Google Scholar) system, data were with the R. proteins with mass 2004; PubMed Scopus Google Scholar), and the subjected to A. E. R. A for proteins by mass PubMed Scopus Google Scholar) the data were subjected to of H. B. A. D. D. M. of affinity spectrometry 2011; 8: PubMed Scopus Google Scholar, G. G. Zhang J. H. and in of Proteomics. PubMed Scopus Google Scholar) to identify SCFβ-TrCP1/2 interacting partners. identified with a and a are in unique were identified as high SCFβ-TrCP1/2 using technique known of the SCF and a number of previously substrates in the presence of MG132, previously SCFβ-TrCP1/2 substrates etc.) were identified with the known SCFβ-TrCP1/2 and were with as for proteins that are not in to proteasome The in in to treatment biological of the protein and for and we the previously SCFβ-TrCP1/2 as substrate the these a in the and/or BioID and the BioID method to a standard a standard also conducted on in the presence and of and the proteins identified using high were identified and proteins a substrate of which were identified with a in BioID and previously substrates and substrate interacting were identified with proteins with a substrate were that were not also identified in BioID and Together, these data that BioID a to standard for the identification of E3 targets. with many of the proteins a substrate in the BioID and IP-MS data are in cell Wnt, and specific - of which are with soluble cellular and not previously with β-TrCP - were in the BioID data and and identified to the new putative a number of to a of biological and localized to were as or proteins in cells. were with to protein and protein were in the presence (1) a and T. Kim M. S. T. M. A. of the of by and degradation Cell. Biol. 2011; PubMed Scopus Google (2) a of a β-TrCP and; (3) a Although the the the protein on or the nuclear proteins by G. Identification and characterization of a nuclear Full Text PDF PubMed Scopus Google Scholar) However, as the known substrate P. S. M. A. degradation of the in a Cell. Biol. 2011; PubMed Scopus Google Scholar) in to the substrate 12 were β-TrCP and the protein a substrate not to be in to β-TrCP protein is therefore to be a substrate interacting than a β-TrCP of by β-TrCP nuclear membrane were with or and conducted using with of and with the cell of with and or Together, these that BioID be used as an to standard IP-MS or techniques for the identification of in BioID not data for the of IP-MS a substrate also identified many substrate and is a β-TrCP substrate P. P. Elledge S.J. The ligase with in and and ubiquitination in PubMed Scopus Google Scholar, M. R. H. C. B. R. P. The protein with and in the Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. M. M. N. K. A. K. of J. PubMed Scopus Google Scholar, E. D.S. M. The human protein with the and the stability of PubMed Scopus Google Scholar) identified with high in our the of is and by the The Biol. 2013; PubMed Scopus Google Scholar). The β-TrCP which and also a substrate in our BioID data these proteins are not targets However, these are with a that is by the of an P.J. S. T. H. of degradation in an 2012; Full Text Full Text PDF PubMed Scopus Google BioID is a labeling is by treatment we may to of and of the is therefore important to that a substrate that is part of a protein to of the substrate and interacting partners. the in which a substrate is of the be to identify the The of and is of proteins localized to the nuclear and localized to the nuclear proteins with nuclear and with Together, the and proteins are critical for the maintenance of nuclear and A. the nuclear the of the reveal 2013; PubMed Scopus Google Scholar). an in and a in protein by of SCFβ-TrCP1/2 also with in nuclear the of with the by nuclear membrane as using in and with these of transient also with nuclear membrane and and by of Together, these data suggest that an important in the maintenance of nuclear membrane and reveal a the of and the maintenance of nuclear membrane be to the and nuclear membrane the with are to previously in from Muscular in nuclear such as and S. E. P. S. L. S. M. in and J. Cell Biol. 2012; PubMed Scopus Google Scholar). many of the new targets identified by BioID are in and translational control. and are of the complex, which the of to K. of PubMed Scopus Google Scholar) and an important in with an putative target identified here - J. G. and an in 2013; PubMed Scopus Google Scholar). of these proteins to processing poorly in and proteins and with Biol. PubMed Scopus Google Scholar). to the of in the of were in live or of the and or have that or of number A. E. the of Rev. Mol. Cell Biol. 2007; 8: PubMed Scopus Google Scholar). with our that and are in to β-TrCP number in with as with with the not to be a on as of or on the number of stress of here with the and be to in the of these data β-TrCP in known as of C. S. P. Zhang D. of a of cells.J. Cell Biol. PubMed Scopus Google Scholar) is a of the protein targets to the is on in to protein stress and D. in cellular stress and in and 2007; Scholar). the which the a with and to the to protein D. in cellular stress and in and 2007; Scholar). to protein targets to to low of C. S. P. Zhang D. of a of cells.J. Cell Biol. PubMed Scopus Google Scholar). of is critical for the maintenance of cellular as the and Zhang D. D. an for in mammalian 2009; PubMed Scopus Google Scholar), and a stress C. S. P. Zhang D. of a of cells.J. Cell Biol. PubMed Scopus Google Scholar). to β-TrCP in a in a putative that be by SCFβ-TrCP1/2 P. P. Elledge S.J. The ligase with in and and ubiquitination in PubMed Scopus Google Scholar). The in were to and the of the mutant protein characterized. to that in to β-TrCP a than the is the mutant protein with than the protein the of the to β-TrCP or with were the and proteins by of of the mutant were However, of as with Together, these data suggest that is a β-TrCP substrate and that represents a β-TrCP in or proteins were in the presence and of or that stress by with protein or of in the protein in to stress the mutant also to a The of protein is critical for the of in to protein to that previously in D. B. E. C. R. P. T. S. is for the protein and in Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), of the mutant protein to as by cell in the presence of of Together, our data validate as an and reveal an important for E3 ligase in the of the of the ubiquitin represents a in cell The identification of E3 ligase substrates has been in part because: (1) ligase - substrate interactions are often of (generally in the high nm to microMolar (μM) range) and/or transient in nature; (2) many substrates are subjected to rapid proteasomal degradation, and therefore be using standard (3) the human ubiquitome is extremely complex, and therefore difficult to and; (4) many substrate proteins are localized to poorly soluble cellular compartments, making their isolation and identification by standard techniques we that BioID with mass spectrometry, conducted on in the presence and of MG132, be used for the identification of E3 substrates. on the well-studied proteins and we identified a number of targets with new substrate β-TrCP in biological important of for the characterization of protein-protein interactions is that and cell be to protein-protein interactions or transient E3-substrate and proteins in a BioID a biotin to protein the maintenance of protein-protein interactions is not for their and techniques may be used to proteins from poorly soluble cellular that are often not by The technique presented here is complementary to standard IP-MS and is to be broadly applicable for E3 substrate a of the complex, the new β-TrCP substrate an important in the maintenance of nuclear integrity, nuclear and nuclear membrane P. The and human 2011; PubMed Scopus Google Scholar). in interacting have been with a by and and from nuclear and by the presence of S. T. S. M. M. nuclear in Mol. 2007; 16: PubMed Scopus Google Scholar, A. J. M. C. T. M. S. M. A. M. K. G. in from with and 2004; PubMed Scopus Google Scholar, C. E. C. C. J. M. D. B. of A in the an nuclear to that in from with and Cell PubMed Scopus Google Scholar). are with our in with Although the and have not been to these proteins for the with have been in than of P. The and human 2011; PubMed Scopus Google Although also be to the of β-TrCP in the of nuclear membrane and our data reveal a critical E3 ligase and of a β-TrCP is the E3 ligase for which the NFκB in the is by the in to or types of cellular the is the NFκB to the to in the data reveal previously β-TrCP and proteins in the cellular stress response. play important roles in the of and and turnover is in to cellular stress and with Biol. PubMed Scopus Google Scholar, G. N. of with stress Biol. 2013; PubMed Scopus Google Scholar). are also in to many types of protein stress and D. in cellular stress and in and 2007; Scholar). β-TrCP may therefore an important hub for the proteome in to cellular in to in the NFκB the of the or degradation of in with a global in a rapid and to the proteome in to be on the of in the cellular stress response. our in were on the identification of β-TrCP substrates M. R. S. D. A identifies substrates of the ubiquitin PubMed Scopus Google Scholar, D. K. of the Cell. Biol. PubMed Scopus Google Scholar), more standard IP-MS and affinity the of widely many of the β-TrCP were the number of β-TrCP substrates and well-studied E3 ligase in new biological the of BioID as a complementary approach for the identification of E3 targets in living cells. We for for cells, and we are extremely to and for with our mass spectrometry data to the in the in of and the in and with

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.