The Elongin B Ubiquitin Homology Domain
IDENTIFICATION OF ELONGIN B SEQUENCES IMPORTANT FOR INTERACTION WITH ELONGIN C*

Christopher S. BrowerDagger §, Ali Shilatifard, Timothy Matherparallel , Takumi KamuraDagger **, Yuichiro TakagiDagger Dagger , Dewan HaqueDagger **, Annemarie Treharne§§, Stephen I. Foundling§§, Joan Weliky ConawayDagger §**¶¶, and Ronald C. ConawayDagger ||

From the Dagger  Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, the § Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, the  Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104, the parallel  Program in Cardiovascular Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, the Dagger Dagger  Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, ** Howard Hughes Medical Institute, and the §§ School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mammalian Elongin B is a 118-amino acid protein composed of an 84-amino acid amino-terminal ubiquitin-like domain and a 34-amino acid carboxyl-terminal tail. Elongin B is found in cells as a subunit of the heterodimeric Elongin BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling complexes. As part of our effort to understand how the Elongin BC complex regulates the activity of Elongin A, we are characterizing Elongin B functional domains. In this report, we show that the Elongin B ubiquitin-like domain is necessary and sufficient for interaction with Elongin C and for positive regulation of Elongin A transcriptional activity. In addition, by site-directed mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaction with Elongin C. Finally, we observe that both the ubiquitin-like domain and carboxyl-terminal tail are conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that efficiently substitute for mammalian Elongin B in reconstitution of the transcriptionally active Elongin ABC complex, suggesting that the carboxyl-terminal tail performs an additional function not detected in our assays.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Elongin B is a subunit of the heterodimeric Elongin BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor Elongin A (1, 2). Subsequently the Elongin BC complex was identified as a component of both the multiprotein von Hippel-Lindau tumor suppressor complex (3, 4), where it appears to function in tumor suppression (5) and regulation of expression of hypoxia-inducible genes (6-8), and the suppressor of cytokine signaling-1 complex, where it may function at least in part to regulate the stability of the suppressor of cytokine signaling-1 protein (9).

Previous studies from our laboratory have shown that Elongin B and C perform distinct functions in regulation of Elongin A transcriptional activity (10). Elongin C is capable of binding directly to a site in the Elongin A elongation activation domain and inducing Elongin A transcriptional activity in vitro in the absence of Elongin B (2, 10, 11). Elongin B does not directly affect Elongin A activity in the absence of Elongin C; instead, Elongin B appears to bind directly to Elongin C and to promote its interaction with Elongin A.

Molecular cloning of the mammalian Elongin B gene revealed that it encodes a 118-amino acid protein composed of an ~84 residue NH2-terminal ubiquitin-like domain fused to an ~34 residue COOH-terminal tail (2). The ubiquitin family comprises a diverse collection of proteins that fall into at least three classes. One class is composed of ubiquitin, which is conjugated by E2/E3 ubiquitin ligases to a variety of proteins involved in signal transduction and cell cycle control and targets them for degradation by the proteosome (12, 13). A second class is composed of a small set of ribosomal proteins that contain NH2-terminal ubiquitin moieties, which are proteolytically removed following incorporation of these proteins into ribosomes (14, 15). A third class includes Elongin B and a growing number of other ubiquitin-like proteins. Among the members of this class are NEDD8/Rub1p, SUMO, and Rad23, which have roles in cell cycle control, nucleocytoplasmic transport, and DNA repair, respectively (16-19).

As part of our effort to understand the structure of the Elongin BC complex and its mechanism of action in regulation of Elongin A transcriptional activity, we are carrying out a systematic structure-function analysis of each of the Elongin subunits. In this report, we demonstrate that the Elongin B ubiquitin-like domain is necessary and sufficient for interaction of Elongin B with Elongin C and for regulation of Elongin A activity. In addition, by site-directed mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaction with Elongin C and that is evolutionarily conserved in Elongin B homologs from D. melanogaster and C. elegans.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Unlabeled ultrapure ribonucleoside 5'-triphosphates were purchased from Pharmacia Biotech Inc. [alpha -32P]CTP (>650 Ci/mmol) was obtained from Amersham Corp. Ni2+-nitrilotriacetic acid-agarose (Ni2+-agarose)1 was from Invitrogen. Placental ribonuclease inhibitor (RNasin) and acetylated bovine serum albumin were from Promega. Guanidine hydrochloride (sequanal grade) was purchased from Pierce. Phenylmethylsulfonyl fluoride and polyvinyl alcohol (average molecular weight 30,000-70,000) were obtained from Sigma. Phenylmethylsulfonyl fluoride was dissolved in dimethyl sulfoxide to 1 M. Polyvinyl alcohol was dissolved in water to 20% (w/v) and centrifuged or filtered through a 0.2-µm filter prior to use.

DNA Template for Transcription-- pDN-AdML plasmid DNA (20) was isolated from Escherichia coli using the Triton-lysozyme method (21). Plasmid DNA was banded twice in CsCl-ethidium bromide density gradients, precipitated with ethanol, and dissolved in TE buffer (20 mM Tris-HCl (pH 7.6), and 1 mM EDTA). A restriction fragment prepared by digestion of pDN-AdML DNA with EcoRI and NdeI was used as template in transcription reactions. The fragment was purified from 1.0% low melting temperature agarose gels using GELase (Epicentre Technologies) according to the manufacturer's instructions. After phenol-chloroform extraction and ethanol precipitation, purified DNA fragments were resuspended in TE buffer.

Preparation of RNA Polymerase II and Initiation Factors-- RNA polymerase II (22) and TFIIH (rat delta , TSK DEAE 5-PW fraction (23)) were purified from rat liver nuclear extracts as described. Recombinant yeast TBP (24) and rat TFIIB (rat alpha  (25)) were expressed in E. coli and purified as described. Recombinant TFIIE was prepared as described (26), except that the 56-kDa subunit was expressed in BL21(DE3)-pLysS. Recombinant TFIIF was purified as described previously from E. coli strain JM109(DE3) infected with M13pET-Rap30 and M13pET-Rap74 (27).

Assay of Run-off Transcription-- ~200 ng of wild type or mutant Elongin B proteins were mixed with ~40 ng of wild type rat Elongin A and ~200 ng of rat Elongin C-(Delta 41-50) and diluted ~2 fold to a final volume of 50 µl with 40 mM Hepes-NaOH (pH 7.9), 100 mM KCl, 50 µM ZnSO4, and 10% (v/v) glycerol. After 90 min on ice, the mixtures were dialyzed for 2 h at 4 °C against 40 mM Tris-HCl (pH 7.9), 40 mM KCl, 0.1 mM EDTA, and 10% (v/v) glycerol. Elongin complexes were then assayed in 60-µl reaction mixtures as follows. Preinitiation complexes were assembled by preincubation of ~10 ng of the EcoRI to NdeI fragment from pDN-AdML, ~10 ng of recombinant TFIIB, ~10 ng of recombinant TFIIF, ~7 ng of recombinant TFIIE, ~40 ng of rat TFIIH, ~20 ng of TBP, ~0.01 unit of RNA polymerase II, and 8 units of RNasin in 20 mM Hepes-NaOH (pH 7.9), 20 mM Tris-HCl (pH 7.9), 60 mM KCl, 2 mM DTT, 0.5 mg/ml bovine serum albumin, 2% (w/v) polyvinyl alcohol, and 3% (v/v) glycerol for 30 min at 28 °C. 10 µl of dialyzed Elongin complexes were then added to each reaction mixture. Transcription was initiated by addition of 7 mM MgCl2 and 50 µM ATP, 50 µM GTP, 10 µM CTP, 2 µM UTP, and 10 µCi of [alpha -32P]CTP. After incubation of reaction mixtures for 12 min at 28 °C, run-off transcripts were analyzed by electrophoresis through 6% polyacrylamide gels containing 7.0 M urea. Transcription was quantitated using a Molecular Dynamics PhosphorImager.

Construction of Elongin B Mutants-- Elongin B mutants were constructed by oligonucleotide-directed mutagenesis (28) of M13mpET-Elongin B (2) with the Muta-Gene M13 in vitro mutagenesis kit (Bio-Rad) and confirmed by dideoxy DNA sequencing with the fmol DNA Sequencing System (Promega). Mutagenic oligonucleotides included 15 nucleotides from the parental rat Elongin B sequence on either side of the site of mutation.

Expression of Elongin B Mutants and Elongin Subunits in Mammalian Cells-- cDNAs encoding full-length Elongin B and Elongin B mutants containing NH2-terminal c-Myc epitope tags were subcloned into the pcDNA3.1 vector (Invitrogen). Full-length Elongin C containing an NH2-terminal HSV epitope tag was subcloned into the pCI-neo vector (Promega). 24 h after transfection of 293T cells, cells were collected and lysed in ice-cold buffer containing 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 1 mM DTT, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml peptstatin A, and 5 µg/ml aprotinin. Lysates were then centrifuged at 10,000 × g for 20 min at 4 °C.

Immunoprecipitation and Western Blotting-- Anti-c-Myc epitope antibodies were from Roche Molecular Biochemicals and anti-HSV epitope antibodies were from Novagen. To immunoprecipitate Elongin B and associated proteins from 293T cell lysates, the lysates were incubated with anti-c-Myc antibody for 1 h at 4 °C and then with protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C. Protein A/G beads were washed two times in buffer containing 40 mM Hepes-NaOH (pH 7.9), 500 mM NaCl, 1 mM DTT, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol and once in 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 1 mM DTT, 10% (v/v) glycerol. Immunoprecipitated proteins or proteins in total cell lysates were analyzed by electrophoresis through 13.5% SDS-polyacrylamide gels and transferred to polyvinylidine difluoride membranes (Millipore) and vizualized by Western blotting using the chemiluminescense reagent (NEN Life Science Products Inc.).

Expression and Purification of Elongin B Mutants and Elongin Subunits-- NH2-terminal histidine-tagged, wild type rat Elongin A was overexpressed in E. coli using a pET16b expression vector (Novagen) and purified as described (29). Untagged wild type rat Elongin C and NH2-terminal histidine-tagged wild type rat Elongin C, rat Elongin C-(Delta 41-50), wild type rat Elongin B, and rat Elongin B mutants were overexpressed in E. coli using the M13mpET bacteriophage expression system (27) and purified as described below. Briefly, 100-ml cultures of E. coli strain JM109(DE3) were grown at 37 °C to an A600 of 0.6 in Luria broth. Cells were infected with the appropriate M13mpET bacteriophage expression vectors at a multiplicity of infection of 10-20. After an additional 2 h at 37 °C, cells were induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside, and cultures were incubated an additional 3 h. Cells were harvested by centrifugation at 2000 × g for 10 min at 4 °C. The cell pellets were resuspended in 7 ml of 20 mM Tris-HCl (pH 8.0), 1 mg/ml lysozyme, and 10 mM imidazole (pH 8.0) and incubated for 30 min on ice. After one cycle of freeze-thaw, the suspensions were centrifuged at 100,000 × g for 35 min. Inclusion bodies were solubilized by resuspension in 7 ml of ice-cold 6 M guanidine hydrochloride, 40 mM Tris-HCl (pH 8.0), 0.5 M KCl, 10 mM imidazole (pH 8.0), and 0.5 mM phenylmethylsulfonyl fluoride, and the resulting suspensions were clarified by centrifugation at 100,000 × g for 35 min. All histidine-tagged proteins were further purified by Ni2+-agarose chromatography in guanidine hydrochloride as described (29).

Expression and Purification of D. melanogaster and C. elegans Elongin B Proteins-- A TBLASTN search of the GenBank EST data base identified potential Elongin B homologs from D. melanogaster and C. elegans. EST yk121b3 encoding the potential C. elegans Elongin B homolog was obtained from Y. Kohara (National Institute of Genetics, Mishima, Japan). EST LD16189 encoding the potential Drosophila Elongin B homolog was obtained from Genome Systems, Inc. The entire open reading frames of the Drosophila and C. elegans proteins were overexpressed in E. coli with NH2-terminal histidine tags using the M13mpET bacteriophage expression system (27).

Assay of Elongin BC Complex Formation-- ~2 µg of histidine-tagged wild type rat Elongin B or rat Elongin B mutants were mixed with ~2 µg of untagged wild type rat Elongin C and diluted 20-fold with 40 mM Hepes-NaOH (pH 7.9), 100 mM KCl, 50 µM ZnSO4, and 10% (v/v) glycerol. After 30 min on ice, the mixtures were dialyzed for 2 h at 4 °C against 40 mM Hepes-NaOH (pH 7.9), 0.1 mM EDTA, 100 mM KCl, and 10% (v/v) glycerol. Following dialysis, the mixtures were incubated for 1 h at 4 °C with ~20 µl of Ni2+-agarose pre-equilibrated in dialysis buffer containing 10 mM imidazole (pH 8.0) and then centrifuged for 20 s at 2000 rpm in a Micromax microcentrifuge (International Equipment Co.). Following centrifugation, the supernatants containing unbound proteins were collected, and the Ni2+-agarose was washed 3 times by resuspension in 500 µl of 40 mM Hepes-NaOH (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 10% (v/v) glycerol, and 40 mM imidazole (pH 8.0) and centrifugation for 20 s at 2000 rpm. Finally, bound material was eluted with 300 µl of the same buffer containing 300 mM imidazole (pH 8.0). Aliquots of each fraction were analyzed by 13.5 or 18% SDS-polyacrylamide gel electrophoresis, and the proteins were visualized by silver staining.

Assay of Elongin ABC Complex Formation-- ~6 µg of histidine-tagged wild type rat, C. elegans, or D. melanogaster Elongin B or rat Elongin B mutants were mixed with ~45 µg of wild type, histidine-tagged rat Elongin A and ~6 µg of wild type, histidine-tagged rat Elongin C and diluted 5-fold with 40 mM Hepes-NaOH (pH 7.9), 100 mM KCl, 50 µM ZnSO4, and 10% (v/v) glycerol. After 90 min on ice, the mixtures were dialyzed for 2 h at 4 °C against 40 mM Tris-HCl (pH 7.9), 40 mM KCl, 0.1 mM EDTA, and 10% (v/v) glycerol. Following dialysis, the mixtures were centrifuged at 60,000 × g for 15 min at 4 °C. The resulting supernatants were applied to TSK SP-NPR columns (35 × 4.6 mm, Toso-Haas) pre-equilibrated in 40 mM Hepes-NaOH (pH 7.9), 100 mM KCl, 1 mM DTT, and 10% (v/v) glycerol and fractionated using a SMART microchromatography system (Pharmacia) at 8 °C. The columns were eluted at 0.6 ml/min with a 9-ml linear gradient from 100 to 900 mM KCl in 40 mM Hepes-NaOH (pH 7.9), 1 mM DTT, and 10% (v/v) glycerol. Aliquots of each column fraction were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis, and the proteins were visualized by silver staining.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Evolutionary Conservation of Elongin B-- We previously described cloning of rat and human Elongin B cDNAs (2). Characterization of these cDNAs revealed that Elongin B is composed of an ~84 amino acid NH2-terminal ubiquitin-like domain fused to an ~34-amino acid COOH-terminal tail. The NH2-terminal ubiquitin-like domain can be modeled as ubiquitin (2), a compact globular structure containing a 3.5 turn alpha -helix (alpha 1), a short 310 helix, and a 5-stranded mixed beta -sheet (30).

As an initial step in our analysis of the structure and function of Elongin B, we identified and characterized potential D. melanogaster and C. elegans Elongin B homologs. Comparison of the amino acid sequences of mammalian Elongin B and the potential Drosophila and C. elegans Elongin B homologs indicates that they are highly conserved (Fig. 1). According to the BESTFIT program of GCG, the rat and Drosophila proteins are 55% identical and 78% similar; the rat and C. elegans proteins are 39% identical and 62% similar; and the Drosophila and C. elegans proteins are 36% identical and 56% similar. A TBLASTN search of the S. cerevisiae data base revealed no S. cerevisiae open reading frames with significant sequence similarity to mammalian Elongin B, even though the S. cerevisiae genome includes an open reading frame that encodes a 100-amino acid protein that exhibits significant sequence similarity to mammalian Elongin C (29, 31).


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Fig. 1.   Evolutionary conservation of Elongin B. The alignment of rat and human Elongin B with D. melanogaster and C. elegans Elongin B homologs was determined with the MACAW program (31) using the BLOSUM 80 score table. Positions of identical residues conserved in at least three of the four Elongin B proteins are indicated in the alignment by dark shading, and the positions of similar residues conserved in at least three of the four Elongin B proteins are indicated by light shading. Positions of predicted Elongin B structural elements are shown above the alignment, and positions of ubiquitin structural elements identified in the crystal structure (30) are shown below the alignment. Ub, ubiquitin; alpha , alpha -helix; beta , beta -strand.

To determine whether the potential Drosophila and C. elegans Elongin B homologs possess activities similar to those of mammalian Elongin B, we investigated their abilities to substitute for rat Elongin B in reconstitution of the Elongin complex. In previous studies, we showed that the transcriptionally active Elongin ABC complex could be reconstituted by recombining individual Elongin subunits purified from rat liver (1, 32) or by refolding bacterially expressed Elongin subunits purified from guanidine hydrochloride-solubilized inclusion bodies (10). Formation of the Elongin ABC complex can be assayed by ion exchange HPLC using TSK SP-NPR (2, 29). Elongin BC complexes and free Elongin B and C flow-through TSK SP-NPR at low ionic strength, whereas Elongin ABC complexes bind tightly to TSK SP-NPR and elute with ~0.3 M KCl.

To investigate the abilities of the potential Drosophila and C. elegans Elongin B homologs to assemble with mammalian Elongin A and C into chromatographically isolable ternary complexes, the Drosophila and C. elegans proteins were expressed in E. coli, purified from guanidine-solubilized inclusion bodies, refolded together with bacterially expressed rat Elongin A and C, and subjected to TSK SP-NPR HPLC. As shown in Fig. 2A, like rat Elongin B, both the potential Drosophila and C. elegans Elongin B homologs are capable of assembling with rat Elongin A and C to form ternary complexes that can be isolated by TSK SP-NPR HPLC; neither the Drosophila nor C. elegans proteins bound to TSK SP-NPR in the absence Elongin A (data not shown).


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Fig. 2.   Characterization of D. melanogaster and C. elegans Elongin B homologs. Panel A, Drosophila and C. elegans Elongin B homologs were assayed for their abilities to form Elongin ABC complexes by TSK SP-NPR HPLC as described under "Experimental Procedures." L, load; FT, flow-through. Panel B, the indicated Elongin B molecules were assayed for Elongin transcriptional activity as described under "Experimental Procedures."

To determine whether the potential Drosophila and C. elegans Elongin B homologs function similarly to mammalian Elongin B in transcription, we assayed them for their abilities to promote activation of Elongin A transcriptional activity by Elongin C. We previously showed that Elongins B and C play different roles in activation of Elongin A activity (10). Elongin C functions as the inducing ligand and activates Elongin A by binding to a site in the Elongin A elongation activation domain. Although Elongin B is not essential for activation of Elongin A by Elongin C, it promotes interaction of Elongin C with Elongin A and, in so doing, increases both the yield and stability of the functional Elongin complex.

To devise an assay for Elongin transcriptional activity with the strongest possible dependence on Elongin B, we took advantage of an Elongin C mutant (Elongin C-(Delta 41-50)) (29) that does not bind stably to Elongin A in the absence of Elongin B (data not shown) and that does not detectably activate Elongin A transcriptional activity unless Elongin B is present (Fig. 2B). In these experiments, Elongin complexes were assembled with rat Elongin A and Elongin C-(Delta 41-50) in the absence of Elongin B or in the presence of either rat Elongin B or the potential Drosophila or C. elegans Elongin B homologs. The Elongin complexes were then assayed for their abilities to stimulate the rate of accumulation of run-off transcripts synthesized by RNA polymerase II from the AdML promoter in a purified basal transcription system reconstituted with the general initiation factors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. As shown in Fig. 2B, like rat Elongin B, the potential Drosophila and C. elegans Elongin B homologs were capable of strongly promoting activation of Elongin A by Elongin C-(Delta 41-50). Taken together, the results of both binding and transcription assays argue that the Drosophila and C. elegans proteins are homologs of mammalian Elongin B.

The Elongin B Ubiquitin-like Domain Is Sufficient for Elongin B Function in Vitro-- To determine whether the NH2-terminal Elongin B ubiquitin-like domain, the COOH-terminal tail, or both are required for Elongin B function, a series of NH2-terminal, COOH-terminal, and internal deletion mutants of rat Elongin B were constructed (Fig. 3A), expressed in E. coli, purified from guanidine hydrochloride-solubilized inclusion bodies, and assayed for their abilities to assemble into Elongin BC and ABC complexes and to promote activation of Elongin A by Elongin C-(Delta 41-50).


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Fig. 3.   The Elongin B ubiquitin-like domain is necessary and sufficient for Elongin B activity In Vitro. Panel A, Elongin B deletion mutants. Panel B, wild type rat Elongin B and Elongin B mutants were assayed for their abilities to form Elongin BC complexes by Ni2+-agarose chromatography as described under "Experimental Procedures." Panel C, Elongin B deletion mutants were assayed for their abilities to form Elongin ABC complexes by TSK SP-NPR HPLC as described under "Experimental Procedures." L, load; FT, flow-through. Panel D, Elongin B deletion mutants were assayed for transcriptional activity as described under "Experimental Procedures".

To assess the abilities of Elongin B deletion mutants to assemble with Elongin C into Elongin BC complexes, we assayed rat Elongin B mutants containing NH2-terminal histidine tags for their abilities to retain untagged wild type rat Elongin C on Ni2+-agarose. In these experiments, individual wild type Elongin B or Elongin B deletion mutants were refolded together with Elongin C and subjected to Ni2+-agarose chromatography. Unbound and bound protein fractions were collected, and equivalent amounts of each fraction were analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 3B, untagged Elongin C does not bind to Ni2+-agarose, but is retained on the resin in the presence of histidine-tagged wild type Elongin B. Elongin C was quantitatively retained on Ni2+-agarose only by the COOH-terminal deletion mutant Elongin B-(1-84), which is the only deletion mutant with an intact ubiquitin-like domain. Elongin C was not retained on Ni2+-agarose by any Elongin B mutants with deletions of the NH2-terminal portion of the ubiquitin-like domain, and only very small amounts of Elongin C were retained on Ni2+-agarose by Elongin B mutants with deletions of the COOH-terminal portion of the ubiquitin-like domain. Taken together, these results argue that the Elongin B ubiquitin-like domain is necessary and sufficient for stable interaction with Elongin C in vitro.

To assess the abilities of the Elongin B deletion mutants to support formation of functional Elongin ABC complexes, we assayed Elongin B mutants for their abilities (i) to form Elongin ABC complexes isolable by TSK SP-NPR HPLC and (ii) to promote activation of Elongin A by Elongin C-(Delta 41-50). In experiments investigating the abilities of Elongin B deletion mutants to support formation of the Elongin ABC complex, individual wild type Elongin B and Elongin B deletion mutants were refolded together with bacterially expressed wild type rat Elongins A and C and subjected to TSK SP-NPR HPLC. In experiments investigating the abilities of Elongin B deletion mutants to promote activation of Elongin A by Elongin C-(Delta 41-50), bacterially expressed wild type rat Elongin A and Elongin C-(Delta 41-50) were refolded together in the presence or absence of wild type rat Elongin B or Elongin B deletion mutants. Elongin complexes were then assayed for their abilities to stimulate the rate of accumulation of full-length run-off transcripts synthesized from the AdML promoter by RNA polymerase II and purified initiation factors TBP, TFIIB, TFIIE, TFIIF, and TFIIH.

As shown in Fig. 3, C and D, Elongin B-(1-84), which includes the entire ubiquitin-like domain and binds stably to Elongin C (Fig. 3B), was also capable of assembling with Elongin A and C into an isolable Elongin ABC complex and of strongly promoting activation of Elongin A transcriptional activity by Elongin C-(Delta 41-50) mutant. In contrast, Elongin B deletion mutants lacking as few as 10 amino acids from the NH2 terminus of the ubiquitin-like domain did not form Elongin ABC complexes and did not promote activation of Elongin A by Elongin C-(Delta 41-50). Elongin B-(1-74), Elongin B-(1-64), and Elongin B-(Delta 58-66), which have deletions in the COOH terminus of the ubiquitin-like domain, did not promote detectable activation of Elongin A by Elongin C-(Delta 41-50), but did form Elongin ABC complexes. Thus, the entire Elongin B ubiquitin-like domain is necessary and sufficient for promoting activation of Elongin A transcriptional activity by Elongin C-(Delta 41-50). Elongin B residues 58-84 of the ubiquitin-like domain are not essential for formation of Elongin ABC complexes, although deletion mutants lacking these residues are severely impaired in their abilities to form Elongin BC complexes. Elongin B-(Delta 58-66), Elongin B-(1-64), and Elongin B-(1-74) lack portions of predicted helix 2, the extended surface loop, and/or predicted beta -sheet 5, regions of the protein that would be important for maintaining a ubiquitin-like tertiary structure (30, 33-35). Our results raise the possibility that contacts between Elongin B and the Elongin AC complex may contribute to proper folding of the Elongin B ubiquitin-like domain by compensating for loss of the predicted surface loop, portions of helix 2, or predicted beta -sheet 5.

Mutagenesis of the Elongin B Ubiquitin-like Domain-- Although ubiquitin is similar in sequence to Elongin B, ubiquitin neither binds detectably to Elongin C nor promotes activation of Elongin A transcriptional activity by Elongin C (2), suggesting that Elongin B sequences differing from those of ubiquitin are important for Elongin B function. As discussed above, the Elongin B ubiquitin-like domain can be modeled as ubiquitin. Notable features of the Elongin B model include its striking conservation of the ubiquitin hydrophobic core and conservation of the hydrophobic character of residues corresponding to ubiquitin surface residues Phe-4 and Leu-71. The Elongin B model also predicts Elongin B features not shared by ubiquitin; these include two additional hydrophobic surface residues Phe-15 and Phe-25, a prominent basic surface patch composed predominantly of residues Arg-8, Arg-9, His-10, and Lys-11, and an extended surface loop that falls between residues 62 and 70 and accommodates a 7-amino acid insertion in the ubiquitin-like domain.

To explore the relationship between Elongin B and ubiquitin and define in more detail Elongin B residues critical for its function, we investigated the activities of a collection of additional Elongin B mutants that were constructed either by replacing predicted Elongin B surface residues with those from the corresponding positions of ubiquitin, mutating the predicted Elongin B surface hydrophobic residues, or mutating Elongin B residues within the predicted basic patch. These mutants, which contained mutations spanning the entire ubiquitin-like domain, were tested for their abilities, (i) to assemble into isolable Elongin BC complexes in cells and (ii) to assemble into Elongin BC and ABC complexes in vitro and to promote activation of Elongin A by Elongin C-(Delta 41-50) in vitro. To assay formation of Elongin BC complexes in cells, c-Myc-tagged Elongin B and Elongin B mutants were coexpressed with HSV-tagged Elongin C in 293T cells and tested for their abilities to interact with one another by coimmunoprecipitation with an anti-c-Myc antibody. Formation of Elongin BC and ABC complexes and activation of Elongin transcription activity in vitro were assayed as described above. Results of these experiments can be summarized as follows.

(i) Of the Elongin B mutants tested, only three exhibited reduced ability to form Elongin BC complexes in cells (Fig. 4). These mutants, R29A, G33D/I34K/L35E/K36G, and EloB(Delta 31-40), also failed to assemble into isolable Elongin BC and ABC complexes in vitro and to promote activation of Elongin A by Elongin C-(Delta 41-50) (Fig. 5). R29A, G33D/I34K/L35E/K36G, and EloB(Delta 31-40) mutations are confined to a short region in the COOH-terminal portion of predicted helix 1 and in the predicted turn between helix 1 and beta -strand 3. Notably, the R29A and GILK to DKEG mutations change Elongin B residues to the corresponding residues from ubiquitin. Additional ubiquitin-like mutants, including F15T, D17E, K19E/E20P, Y45I/K46F/D47A/D48G/Q49K, D52E, K55R, G76H, and the mutant in which Ala-81 and Asp-82 were mutated to LRGG, were capable of binding Elongin C in vivo (Fig. 4), suggesting that differences between Elongin B and ubiquitin in helix 1 and in the turn immediately following may be sufficient to account for the inability of ubiquitin to bind Elongin C. 


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Fig. 4.   Interaction of Elongin B mutants with Elongin C in mammalian cells. 293T cells were transiently transfected in 35-mm wells with 1 µg of wild type or mutant c-Myc-Elongin B and 1 µg of HSV-Elongin C expression vectors as described under "Experimental Procedures." Cell lysates containing approximately equivalent amounts of wild type Elongin B or Elongin B mutants were immunoprecipitated with anti-c-Myc antibodies (IP: myc-EloB), except that ~30% less Elongin B R29A was present in the cell lysate used for immunoprecipitation of that mutant. The concentrations of Elongin B proteins in cell lysates were determined by densitometry of appropriate exposures of Western blots. Elongin B and Elongin C proteins were detected in both total lysate and immunoprecipitated fractions by Western blot using anti-c-Myc (WB: myc-EloB) and anti-HSV (WB: HSV-EloC) antibodies, respectively.


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Fig. 5.   Analysis of Elongin B mutants with ubiquitin-like substitutions. Panel A, wild type rat Elongin B and Elongin B mutants were assayed for their abilities to form Elongin BC complexes by Ni2+-agarose chromatography as described under "Experimental Procedures." Panel B, Elongin B mutants were assayed for their abilities to form Elongin ABC complexes by TSK SP-NPR HPLC as described under "Experimental Procedures." L, load; FT, flow-through. Panel C, Elongin B mutants were assayed for transcriptional activity as described under "Experimental Procedures."

(ii) Of the ubiquitin-like mutants that were capable of binding Elongin C in vivo, none assembled into Elongin BC complexes in vitro, suggesting that the mutations interfere with proper folding of Elongin B in vitro (Fig. 5A). Several of these mutants, however, were able to form Elongin ABC complexes and/or to promote activation of Elongin A by Elongin C-(Delta 41-50) (Fig. 5, A and B). The mutant Y45I/K46F/D47A/D48G/Q49K, in which residues located at the COOH terminus of beta -strand 3 and in the turn immediately following were mutated to the corresponding residues of ubiquitin, was able to form an Elongin ABC complex and to promote activation of Elongin A by Elongin C-(Delta 41-50) as well as or better than wild type Elongin B. Another mutant, constructed by replacing Elongin B residues Lys-19 and Glu-20 with the predicted corresponding ubiquitin residues Glu-18 and Pro-19, which occupy a position in the turn between the second beta -sheet and the first alpha -helix, failed to form isolable Elongin BC or ABC complexes in vitro, but did promote activation of Elongin A by Elongin C-(Delta 41-50). A third mutant K11G, formed isolable Elongin ABC complexes in vitro, but did not promote activation of Elongin A by Elongin C-(Delta 41-50).

(iii) The single Elongin B point mutations R8N, R9G, H10S, and K11N, which decrease the net positive charge in the predicted basic surface patch and which alter residues in a predicted turn between beta -strands 1 and 2, had no detectable effect on the ability of Elongin B to form Elongin BC (Fig. 6A) and ABC complexes in vitro (Fig. 6B) or to promote activation of Elongin A transcriptional activity A by Elongin C-(Delta 41-50) (Fig. 6C). Consistent with these observations, R9G could be immunoprecipitated from cell lysates with Elongin C (Fig. 4). An additional mutant, K11G, could also be immunoprecipitated from cell lysates with Elongin C (Fig. 4); however, when assayed for activity in vitro, this mutant was found to assemble into ABC complexes but not to promote activation of Elongin A by C-(Delta 41-50) (Fig. 5, B and C). K11G was also unable to assemble into isolable Elongin BC complexes in vitro, suggesting that its failure to promote activation of Elongin A by C-(Delta 41-50) might be due to improper folding of the protein purified from inclusion bodies.


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Fig. 6.   Analysis of Elongin B mutants with point mutations in the predicted basic patch. Panel A, wild type rat Elongin B and Elongin B mutants were assayed for their abilities to form Elongin BC complexes by Ni2+-agarose chromatography as described under "Experimental Procedures." Panel B, Elongin B deletion mutants were assayed for their abilities to form Elongin ABC complexes by TSK SP-NPR HPLC as described under "Experimental Procedures." L, load; FT, flow-through. Panel C, Elongin B mutants were assayed for transcriptional activity as described under "Experimental Procedures."

(iv) Mutations of predicted Elongin B surface hydrophobic residues Phe-4, Phe-15, Phe-25, and Phe-62, which fall within predicted beta -strand 1, beta -strand 2, at the NH2 terminus of predicted helix 1, and in predicted helix 2, respectively, had different effects on Elongin B function (Fig. 7). Elongin B point mutants F4N, F15N, and F25N could assemble into isolable Elongin BC and ABC complexes in vitro and promote activation of Elongin A by Elongin C-(Delta 41-50), although Elongin B mutant F15N was less active than wild type Elongin B in promoting activation of Elongin A. Elongin B point mutant F62N, which contains a mutation at the NH2 terminus of the predicted Elongin B surface loop not present in ubiquitin, assembled into Elongin BC and ABC complexes, but did not promote activation of Elongin A by Elongin C-(Delta 41-50). Finally, F15T, which was constructed by replacing Elongin B residue Phe-15 with the predicted corresponding ubiquitin residue Thr-14, formed isolable Elongin BC complexes in cells but did not assemble into detectable Elongin BC or ABC complexes in vitro and did not promote activation of Elongin A by Elongin C-(Delta 41-50), suggesting that the F15T mutation might interfere with proper folding of Elongin B in vitro.


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Fig. 7.   Analysis of Elongin B mutants with mutations of predicted surface hydrophobic residues. Panel A, wild type rat Elongin B and Elongin B mutants were assayed for their abilities to form Elongin BC complexes by Ni2+-agarose chromatography as described under "Experimental Procedures." Panel B, Elongin B deletion mutants were assayed for their abilities to form Elongin ABC complexes by TSK SP-NPR HPLC as described under "Experimental Procedures." L, load; FT, flow-through. Panel C, Elongin B mutants were assayed for transcriptional activity as described "Experimental Procedures."


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we have investigated Elongin B sequences required for its interaction with Elongin C. Mammalian Elongin B is a 118-amino acid protein composed of an ~84 amino acid ubiquitin-like domain fused to an ~34 amino acid COOH-terminal tail (2). The Elongin B ubiquitin-like domain shares ~30% sequence identity with ubiquitin. Other ubiquitin-like proteins include NEDD8 and its yeast homologue Rub1p, SUMO, and Rad23, which have roles in cell cycle control, nucleocytoplasmic transport, and DNA repair, respectively (16-19). These ubiquitin-like proteins exhibit a variable degree of similarity with ubiquitin, ranging from more than 50% sequence identity (NEDD8 and Rub1p) to less than 20% identity (SUMO). Despite the variation in their primary sequences, ubiquitin, NEDD8, Rub1p, and SUMO have very similar tertiary structures (33-35). Although a crystal structure of Elongin B has not yet been reported, Elongin B can be modeled as ubiquitin (2).

Analysis of the functions of Elongin B mutants has revealed that the Elongin B ubiquitin-like domain is sufficient for its interaction with Elongin C in vivo and in vitro and for reconstitution of the transcriptionally active Elongin complex in vitro. Furthermore, we observe that neither the predicted surface hydrophobic residues nor residues that make up the predicted basic surface patch are critical for reconstitution of functional Elongin complexes in vitro. We did, however, identify three mutations, R29A, G33D/I34K/L35E/K36G, and Delta 31-40, which fall within the ubiquitin-like domain and significantly reduce the interaction of Elongin B with Elongin C in vivo and in vitro. Notably, these mutations are confined to a short region in the COOH-terminal portion of predicted alpha -helix 1 and in the predicted turn between helix 1 and beta -strand 3. Although we cannot rule out the possibility that these mutations affect the overall conformation of Elongin B, our results are consistent with the model that the COOH terminus of predicted alpha -helix 1 and the predicted turn between helix 1 and beta -strand 3 may form a surface that interacts directly with Elongin C or may be important for maintaining Elongin B in a conformation that can interact with Elongin C. Two of the inactivating mutations, R29A and G33D/I34K/L35E/K36G, change Elongin B residues to the corresponding residues from ubiquitin. Sequence differences between Elongin B and ubiquitin in this region may be sufficient to account for the inability of ubiquitin to bind Elongin C, since eight additional ubiquitin-like mutations located throughout the ubiquitin-like domain had no significant effect on the interaction of Elongin B with Elongin C in cells.

Finally, by characterizing Drosophila and C. elegans Elongin B homologs that efficiently replace mammalian Elongin B in reconstitution of the transcriptionally active Elongin ABC complex, we show that both the Elongin B ubiquitin-like domain and COOH-terminal tail have been highly conserved during evolution. Although we have not yet identified a function for the Elongin B tail, it is noteworthy that it includes a PXXP motif that is a potential target for binding by SH3 domain proteins (36). Efforts to identify cellular proteins that interact with the Elongin B tail are underway.

    ACKNOWLEDGEMENTS

We thank Y. Kohara for providing the EST encoding C. elegans Elongin B and K. Jackson of Molecular Biology Resource Facility at the Warren Medical Research Foundation for oligonucleotide synthesis.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM41628.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¶¶ Associate Investigator of the Howard Hughes Medical Institute.

|| To whom correspondence should be addressed. Tel.: 405-271-1950; Fax: 405-271-1580.

    ABBREVIATIONS

The abbreviations used are: Ni2+-agarose, Ni2+-nitrilotriacetic acid-agarose; RNasin, placental ribonuclease inhibitor; AdML, adenovirus 2 major late; EST, expressed sequence tag; HPLC, high pressure liquid chromatography; HSV, herpes simplex virus; TF, transcription factor.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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