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• W2023120014 abstract "The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown (Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPΦP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as ∼80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases. The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown (Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPΦP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as ∼80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases. A growing body of evidence has implicated members of the families of Cullin 2 (Cul2)- and Cullin 5 (Cul5)-containing E3 ubiquitin ligases in regulation of diverse cellular processes including cell signaling, growth, and differentiation (reviewed in Ref. (1Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 9-20Crossref PubMed Scopus (1652) Google Scholar). Cul2 and Cul5 ubiquitin ligases each include the heterodimeric Elongin BC complex, composed of the 112-amino acid Elongin C protein, which is similar in sequence to the Skp1-Cullin1-F box ubiquitin ligase subunit Skp1, and the 118-amino acid Elongin B protein, which includes an N-terminal ubiquitin-like domain and a 34-amino acid C-terminal extension. In the context of Cul2 and Cul5 ubiquitin ligases, the Elongin BC complex functions as an adaptor that links a BC-box substrate recognition subunit to either a Cul2-Rbx1 or a Cul5-Rbx2 module that uses the RING finger domains of the related Rbx1 or Rbx2 proteins to recruit and activate an E2 ubiquitin-conjugating enzyme for ubiquitination of substrates.The founding member of the Cul2 family of E3 ubiquitin ligases is the von Hippel-Lindau (VHL) 3The abbreviations used are: VHLVon Hippel-LindauMudPITmultidimensional protein identification technologySOCSsuppressor of cytokine signalingPCMTprotein-l-isoaspartate carboxylmethyltransferase. 3The abbreviations used are: VHLVon Hippel-LindauMudPITmultidimensional protein identification technologySOCSsuppressor of cytokine signalingPCMTprotein-l-isoaspartate carboxylmethyltransferase. tumor suppressor complex (2Pause A. Lee S. Worrell R.A. Chen D.Y.T. Burgess W.H. Linehan W.M. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2156-2161Crossref PubMed Scopus (429) Google Scholar, 3Lonergan K.M. Iliopoulos O. Ohh M. Kamura T. Conaway R.C. Conaway J.W. Kaelin W.G. Mol. Cell. Biol. 1998; 18: 732-741Crossref PubMed Scopus (327) Google Scholar, 4Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (662) Google Scholar), which functions at least in part to regulate hypoxia-inducible transcription by controlling the levels of hypoxia-inducible factor transcription factors (5Maxwell P.H. Wiggener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4071) Google Scholar, 6Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (544) Google Scholar). The founding members of the Cul5 family of E3 ubiquitin ligases are the Src homology 2 domain-containing suppressors of cytokine signaling (SOCS) proteins (7Kamura T. Burian D. Yan Q. Schmidt S.L. Lane W.S. Querido E. Branton P.E. Shilatifard A. Conaway R.C. Conaway J.W. J. Biol. Chem. 2001; 276: 29748-29753Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), which negatively regulate Jak signal transducers and activators of transcription signaling by inhibiting the activities and reducing the levels of Jak or receptor tyrosine kinases (8Endo T. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1221) Google Scholar, 9Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-928Crossref PubMed Scopus (1128) Google Scholar, 10Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1793) Google Scholar). Sequence comparison of the VHL and SOCS-box proteins revealed that they each include a degenerate, ∼12-amino acid sequence motif with consensus sequence [STP]LXXX[CSA]XXXΦ. This motif, which is referred to as the BC-box, is required for binding to the Elongin BC complex. Solution of a cocrystal structure of the VHL-Elongin BC complex revealed that binding of Elongin BC to the BC-boxes of VHL (11Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (681) Google Scholar), SOCS2 (12Bullock A.N. Debreczeni J.E. Edwards A.M. Sundstrom M. Knapp S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7637-7642Crossref PubMed Scopus (131) Google Scholar), and SOCS4 (Protein Data Bank code 2izv) is governed by interaction of an invariant leucine at the N terminus of the BC-box with a hydrophobic pocket created by residues in the C-terminal half of Elongin C. The ubiquitin-like domain of Elongin B interacts with a short N-terminal Elongin C region, while the Elongin B C-terminal extension can be seen to make limited contacts with SOCS2 and SOCS4 outside of the BC-box (12Bullock A.N. Debreczeni J.E. Edwards A.M. Sundstrom M. Knapp S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7637-7642Crossref PubMed Scopus (131) Google Scholar). To date, more than 30 different BC-box proteins have been identified using bioinformatic or biochemical approaches, and a number of these have been shown to function as components of Cul2 and Cul5 ubiquitin ligases.The initial observation that some BC-box proteins assemble into Cul2 ubiquitin ligases whereas others assemble into Cul5 ubiquitin ligases raised questions about how specificity governing recruitment of Cul2 or Cul5 is determined. Kamura et al. (13Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar) recently proposed that assembly of Elongin BC-box proteins into Cul5 or Cul2 ubiquitin ligases can be determined by the presence of specific motifs, termed “Cullin-boxes” located downstream of the BC-box. The Cul5-box corresponds to the C-terminal portion of the canonical SOCS-box defined by Hilton et al. and has the consensus sequence ΦXXLPΦPXXΦXX(Y/F) (L/I) (13Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar, 14Hilton D.J. Richardson R.T. Alexander W.S. Viney E.M. Willson T.A. Sprigg N.S. Starr R. Nicholson S.E. Metcalf D. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 114-119Crossref PubMed Scopus (609) Google Scholar). Structure-function studies of several SOCS-box proteins suggested that the central LPΦP might play a particularly important function as a determinant of Cul5 binding (13Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar). Although a region downstream of the BC-box is clearly needed for interaction with Cul2, it has been difficult to define a specific sequence motif that correlates with Cul2 binding because only three BC-box proteins, VHL, LRR1/PPIL5, and FEM1b, that assemble into Cul2 ubiquitin ligases had been identified when this study was initiated. In addition, assembly of Cul5 ubiquitin ligases containing primate lentiviral Vif proteins has been shown to depend critically on a conserved zinc binding motif located upstream of the BC-box (15Mehle A. Thomas E.R. Rajendran K.S. Gabuzda D. J. Biol. Chem. 2006; 281: 17259-17265Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 16Yu Y. Xiao Z. Ehrlich E.S. Yu X. Yu X.F. Genes Dev. 2004; 18: 2867-2872Crossref PubMed Scopus (270) Google Scholar, 17Luo K. Ehrlich E. Xiao Z. Zhang W. Ketner G. Yu X.F. FASEB J. 2007; 21: 1742-1750Crossref PubMed Scopus (43) Google Scholar).To define in more detail the primary structural determinants governing assembly of Cul2 and Cul5 ubiquitin ligases, we have purified and characterized a larger collection of BC-box proteins and performed structure-function analyses of sequence motifs responsible for their interactions with Cul2 or Cul5. Here we describe the results of these experiments, which have provided new insights into the nature of the Cul5-box and which have identified a consensus Cul2-box present in a large fraction of BC-box proteins that assemble into Cul2 ubiquitin ligases.EXPERIMENTAL PROCEDURESAntibodies—Mouse monoclonal anti-FLAG M2 antibody was obtained from Sigma, herpes simplex virus tag monoclonal antibody from Novagen, c-Myc monoclonal antibody from Roche Applied Science, rabbit polyclonal anti-Cul-5 (H300) from Santa Cruz Biotechnology, rabbit polyclonal anti-Cul-2 from Zymed Laboratories, and a mouse monoclonal antibody against Elongin C/SIII p15 from BD Transduction Laboratories.Generation and Growth of 293 and M1 Cell Lines Stably Expressing FLAG-tagged Proteins—Full-length cDNAs encoding Elongin B or Elongin BC-associating proteins were obtained from the American Type Culture Collection, subcloned with N-terminal FLAG epitope tags into pcDNA5/FRT (Invitrogen) or the retroviral vector pMXs-neo (18Kitamura T. Koshino Y. Shibata F. Oki T. Nakajima H. Nosaka T. Kumagai H. Exp. Hematol. 2003; 31: 1007-1014Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar), and stably introduced either into Flp-In™ 293 cells (Invitrogen) using the Invitrogen Flp-in™ system or into M1 cells by retroviral infection. 293 cell lines were grown on plates or in roller bottles in Dulbecco's modified Eagle's medium with 5% glutamax and 10% fetal bovine serum. M1 cells were grown in flasks or in roller bottles with the same medium as the 293 cells.Transient Transfections—Human embryonic kidney 293T cells grown to ∼50% confluence in 10-cm plates were transiently transfected with pcDNA5/FRT constructs encoding full-length BC-box proteins or various mutants using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions. Cells were collected 48 h after transfection and processed using the high salt extraction protocol described below. Mutagenesis to generate BC-box, Cul2-box, or Cul5-box mutants was performed using the QuikChange® II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.Anti-FLAG-Agarose Chromatography—To prepare protein samples for mass spectrometry, 293 cells were grown to 70-80% confluence in 4-5 15-cm plates or 1-2 roller bottles. M1 cells were grown in 1-2 roller bottles to a density of ∼106 cells/ml. Cells in plates or roller bottles were washed with phosphate-buffered saline and then lysed by resuspension in lysis buffer (1 ml/plate or 3 ml/roller bottle). Two different protocols were used, a low salt extraction (0.15 m NaCl), low speed centrifugation or a high salt extraction (0.42 m NaCl), high speed centrifugation method. In the low salt extraction, low speed centrifugation method, cells are lysed in 40 mm HEPES-NaOH, pH 7.9, 0.15 m NaCl, 1.5 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, and 0.2% Triton X-100, and the cell lysate is spun at 14,000 × g for 30 min in a Beckman-Coulter 5417R refrigerated microfuge. In the high salt extraction, high speed centrifugation method, cells are lysed in 20 mm HEPES-NaOH, pH 7.9, 0.42 m NaCl, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, and 0.2% Triton X-100, and the cell lysate was spun for 30 min at 40,000 × g in a Beckman-Coulter Optima XL-100K refrigerated ultracentrifuge in a Ti 70.1 rotor. For both methods, supernatants were mixed with anti-FLAG (M2)-agarose beads (Sigma), equilibrated in 10 mm HEPES-NaOH, pH 7.9, 0.2% Triton X-100, 0.3 m NaCl, 10 mm KCl, 1.5 mm MgCl2, and protease inhibitor mixture (Sigma catalogue number P8340) in a ratio of 100 μl of packed beads/3 ml of supernatant and gently rocked overnight at 4 °C. The beads were washed four times with a 50-fold excess of the same buffer. Proteins were eluted by incubation for 30 min at 4 °C with one packed bead volume of 10 mm HEPES-NaOH, pH 7.9, 0.05% Triton X-100, 0.1 m NaCl, 1.5 mm MgCl2, protease inhibitor mixture, and 0.2 mg/ml FLAG peptide (Sigma). Beads were removed by centrifugation and the elution was repeated an additional two times.Mass Spectrometry and Data Analysis—Identification of proteins in purified samples was accomplished by MudPIT (multidimensional protein identification technology) (19Washburn M.P. Wolters D. Yates III, J.R. Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4054) Google Scholar, 20Wolters D. Washburn M.P. Yates J.R. Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1557) Google Scholar). Trichloroacetic acid-precipitated proteins were reduced, alkylated, and digested with endoproteinase LysC and trypsin (Roche Applied Science). Peptide mixtures were applied to a three-phase microcapillary column (21McDonald W.H. Ohi R. Miyamoto D.T. Mitchison T.J. Yates J.R. Int. J. Mass Spectrom. 2002; 219: 245-251Crossref Scopus (266) Google Scholar) and eluted into an LCQ Deca-XP ion trap mass spectrometer equipped with a nano-liquid chromatography electrospray ionization source (ThermoFinnigan) as described (22Florens L. Washburn M.P. Methods Mol. Biol. 2006; 328: 159-175PubMed Google Scholar).SEQUEST (23Eng J.K. McCormick A.L. Yates III, J.R. J. Am. Soc. Mass. Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5367) Google Scholar) was used to match tandem mass spectra to 82242 amino acid sequences consisting of 40877 Homo sapiens protein sequences downloaded from the National Center for Biotechnology (2006-03-03 release), 177 usual contaminants, 102 custom sequences for epitope-tagged proteins, as well as randomized versions of each non-redundant protein entry to estimate false discovery rates. Spectra/peptide matches were sorted, filtered, and compared using DTASelect/CONTRAST (24Tabb D.L. McDonald W.H. Yates III, J.R. J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1126) Google Scholar). The chosen selection criteria led to false discovery rates of at most 0.6% (supplemental tables). Normalized spectral abundance factors were calculated as described (25Zybailov B. Mosley A.L. Sardiu M.E. Coleman M.K. Florens L. Washburn M.P. J. Proteome Res. 2006; 5: 2339-2347Crossref PubMed Scopus (805) Google Scholar).RESULTSIdentification and Characterization of New Members of the Families of Cul2- and Cul5-containing Ubiquitin Ligases—As described above, a major impediment to efforts to define sequence elements that support recruitment of Cul2-Rbx1 rather than Cul5-Rbx2 modules to certain ubiquitin ligases was lack of a sufficient number of known Cul2-containing ubiquitin ligases for derivation of a consensus Cul2-box. At the time this work began, only the VHL tumor suppressor protein and the LRR1 and FEM1B proteins were known to contain functional Cul2-boxes (13Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar). Consequently, we sought to identify additional BC-box proteins that contain functional Cul2-boxes.To identify new members of the BC-box family of proteins, we generated human embryonic kidney Flp-In™ 293 and murine lymphoblastic (myeloid leukemia) M1 cell lines stably expressing Elongin B with an N-terminal FLAG epitope tag. FLAG-Elongin B-associating proteins were purified by anti-FLAG-agarose immunoaffinity chromatography from cell lysates prepared from ten independent 293 cell preparations and seven independent M1 cell preparations. Immunopurified Elongin B-associating proteins were then identified by MudPIT mass spectrometry. In a MudPIT mass spectrometry experiment, a mixture of proteins is digested into peptides without prior separation by one- or two-dimensional polyacrylamide gel electrophoresis. Peptides are then fractionated by two-dimensional chromatography and identified by tandem mass spectrometry. As a control for the specificity of our immunoaffinity purifications, anti-FLAG-agarose eluates from cell lysates of parental 293 and M1 cells not expressing a FLAG-tagged protein were also analyzed by mass spectrometry.As expected, anti-FLAG-agarose eluates from FLAG-Elongin B-expressing 293 and M1 cells contained Elongin B and Elongin C, Cul2, Cul5, Rbx1, and Rbx2. In addition, these eluates contained a collection of additional proteins (supplemental Tables S1-S4).Twenty of these had been previously reported to be BC-box proteins based on data from either biochemical or bioinformatic studies, and about half of these had been shown to be able to assemble into Cul2 or Cul5 ubiquitin ligases (Fig. 1, protein names in bold text). In many of the remaining proteins, we identified short sequences that could be aligned with the BC-boxes of previously described Elongin BC-binding proteins (Fig. 1), suggesting that most of them are likely to bind directly to Elongins B and C and may function as substrate recognition subunits of Cul2 or Cul5 ubiquitin ligases. These new BC-box proteins could be classified into several groups based on the presence of specific structural domains. In addition to new members of the ankyrin repeat-containing and leucine-rich repeat (LRR)-containing families of BC-box proteins, these included proteins containing PCMT (protein-l-isoaspartate carboxylmethyltransferase) domains, KELCH repeats, ZSWIM (SWI2/SNF2 MuDR zinc finger) domains, armadillo repeats, tetratricopeptide repeats, and two proteins containing both LXXLL nuclear receptor binding motifs and domains resembling serine/threonine protein kinase catalytic domains (S_TKc), none of which had previously been found in BC-box proteins (Fig. 2).FIGURE 2Domain organization of some Cul2-box and Cul5-box proteins. The BC-box, Cul2-box, and Cul5-box are represented as black, gray, and hatched boxes, respectively. LRR, leucine-rich repeats; ANK, ankyrin repeats; ARM, armadillo; TPR, tetratricopeptide repeats; K, KELCH domain; PCMT, protein-l-isoaspartate carboxylmethyltransferase; RAB, Rab-like GTPase domain; SII, transcription factor SII-like domain; SH, Src homology 2 phosphotyrosine binding domain; SW, SWIM (SWI2/SNF2 MuDR) zinc fingers; TUB, Tubby-like domain; LXXLL, LXXLL nuclear receptor binding motifs; SPRY, SP1a and ryanodine receptor domain; S_TKc, serine/threonine protein kinase catalytic domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the putative BC-boxes found in Elongin B-associating proteins were functional, selected candidates were tested for their abilities to bind to the Elongin BC complex when coexpressed with Elongins B and C in 293 cells. As shown in Fig. 3, FLAG-tagged NRBP1 (nuclear receptor-binding protein 1), APPBP2 (amyloid precursor protein-binding protein 2), ZYG11BL, and PCMTD2 all coimmunoprecipitated with Elongins B and C, as did the Elongin BC-binding proteins VHL and FEM1B. Confirming that the putative BC-boxes were important for binding of each protein to Elongins B and C, mutation of key residues in the BC-box motifs of VHL, PCMTD2, FEM1B, ZYG11BL, APPBP2, and NRBP1 interfered with binding of Elongins B and C to each protein (Fig. 3).FIGURE 3BC-box mutations interfere with binding of newly identified Elongin BC-associating proteins to Elongins B and C. Human embryonic kidney 293T cells were co-transfected with pcDNA5 constructs encoding c-Myc-tagged Elongin B, herpes simplex virus-tagged Elongin C, and FLAG-tagged wild type or mutant versions of the indicated proteins. Cells were lysed using the low salt extraction, low speed centrifugation protocol described under “Experimental Procedures.” Proteins were immunoprecipitated using anti-FLAG-agarose, eluted from anti-FLAG beads with FLAG peptide, and subjected to Western blotting with anti-FLAG, anti-c-Myc, or anti-herpes simplex virus antibodies. IP, immunoprecipitation; WB, Western blotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the newly identified BC-box proteins assemble into Cul2- or Cul5-containing ubiquitin ligases, we generated 293 cell lines stably expressing Cul2, Cul5, or selected candidate proteins with N-terminal FLAG tags and purified their associated proteins from cell lysates by anti-FLAG-agarose chromatography. Copurifying proteins were identified by MudPIT mass spectrometry. VHL and FEM1B were known to interact with Cul2 (2Pause A. Lee S. Worrell R.A. Chen D.Y.T. Burgess W.H. Linehan W.M. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2156-2161Crossref PubMed Scopus (429) Google Scholar, 3Lonergan K.M. Iliopoulos O. Ohh M. Kamura T. Conaway R.C. Conaway J.W. Kaelin W.G. Mol. Cell. Biol. 1998; 18: 732-741Crossref PubMed Scopus (327) Google Scholar, 13Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar), whereas ASB1 and WSB1 interact with Cul5 (7Kamura T. Burian D. Yan Q. Schmidt S.L. Lane W.S. Querido E. Branton P.E. Shilatifard A. Conaway R.C. Conaway J.W. J. Biol. Chem. 2001; 276: 29748-29753Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Accordingly, cell lines expressing FLAG-tagged VHL, FEM1B, ASB1, and WSB1 were used as controls in these experiments. The results are summarized in Fig. 4 and in supplemental Table S5.FIGURE 4BC-box proteins and their associated Cullins. MudPIT analyses of immunopurified material from Flp-In™ 293 cell lines stably expressing FLAG-tagged Elongin BC-associating proteins. The numbers shown correspond to the number of spectra detected in MudPIT runs. Detailed supporting data are provided in supplemental Table S5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All of the FLAG-tagged proteins analyzed copurified with Elongins B and C. As expected, FLAG-VHL and FLAG-FEM1B copurified with Cul2, whereas the SOCS-box proteins FLAG-ASB1 and FLAG-WSB1 copurified with Cul5. In addition, ASB3, which like ASB1 is a member of the SOCS-box protein family, copurified with Cul5. Of the additional Elongin BC-box proteins that were expressed in 293 cells as FLAG-tagged proteins, PCMTD2 copurified with Cul5 and APPBP2, KLHDC2, KLHDC3, and ZYG11BL copurified with Cul2. Consistent with our observation that ZYG11BL is associated with Cul2, Vasudevan et al. (26Vasudevan S. Starostina N.G. Kipreos E.T. EMBO Rep. 2007; 8: 279-286Crossref PubMed Scopus (40) Google Scholar) recently reported that ZYG11 family members are components of Cul2-containing ubiquitin ligases. Neither FLAG-Muf1, FLAG-Elongin A, nor FLAG-NRBP1 copurified with Cul2 or Cul5. Muf1 was recovered, however, by anti-FLAG-agarose chromatography of lysates from 293 cells stably expressing FLAG-Cul5, as were TULP4 (Tubby-like 4), PCMTD1, and several previously known SOCS-box proteins (supplemental Table S5). Notably, although no Cullin protein copurified with full-length FLAG-Elongin A, two different Elongin A mutants containing functional BC-box motifs but lacking the highly conserved SII-like N-terminal domain copurified with Elongins B and C and with Cul5 (Fig. 4), consistent with our previous observation that a potential Elongin A-containing ubiquitin ligase can be reconstituted in insect cells with Elongins B and C, Cul5, and Rbx1 (7Kamura T. Burian D. Yan Q. Schmidt S.L. Lane W.S. Querido E. Branton P.E. Shilatifard A. Conaway R.C. Conaway J.W. J. Biol. Chem. 2001; 276: 29748-29753Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and suggesting that the Elongin A N terminus may negatively regulate the assembly of Elongin A into a ubiquitin ligase.Characterization of Sequence Motifs Governing Interaction with Cul2 or Cul5—The Cul5-box corresponds to the C-terminal portion of the canonical SOCS-box defined by Hilton et al. (14Hilton D.J. Richardson R.T. Alexander W.S. Viney E.M. Willson T.A. Sprigg N.S. Starr R. Nicholson S.E. Metcalf D. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 114-119Crossref PubMed Scopus (609) Google Scholar) and has the consensus sequence ΦXXLPΦPXXΦXX(Y/F) (L/I) (13Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar). In all previously described SOCS-box proteins, the Cul5-box is located immediately C-terminal to the BC-box. Comparison of newly identified BC-box proteins that assemble with Cul5 revealed that they all included an easily recognizable Cul5-box. Surprisingly, however, the Cul5-box in two of them, PCMTD1 and PCMTD2, is located ∼90 amino acids downstream of the BC-box.Although sequences downstream of the BC-box have been shown to be needed for interaction with Cul2, it has been difficult to define a specific sequence motif that correlates with Cul2 binding (13Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar) because only a limited number of BC-box proteins that assemble into Cul2 ubiquitin ligases had been identified prior to this study. In an effort to identify such a sequence motif, we generated a multiple sequence alignment of VHL, MED8, PPIL5 (LRR1), and FEM1B with Cul2-interacting BC-box proteins ZYG11BL, APPBP2, KLHDC2, and KLHDC3 from several species. Also included in the alignment were additional members of the leucine-rich r" @default.
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• W2023120014 title "Characterization of Cullin-box Sequences That Direct Recruitment of Cul2-Rbx1 and Cul5-Rbx2 Modules to Elongin BC-based Ubiquitin Ligases" @default.
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