THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 271, No. 40, Issue of October 4, pp. 24811–24816, 1996 Printed in U.S.A. Molecular Cloning of the cDNA and Chromosome Localization of the Gene for Human Ubiquitin-conjugating Enzyme 9* (Received for publication, June 24, 1996, and in revised form, July 22, 1996) Zhao-Yi Wang‡§, Qing-Qing Qiu‡, Wolfgang Seufert¶i, Takahiro Taguchi**, Joseph R. Testa**, S. A. Whitmore‡‡, David F. Callen‡‡, Douglas Welsh§, Thomas Shenk§ §§, and Thomas F. Deuel‡§¶¶ From the ‡Department of Medicine, Beth Israel Hospital and the Harvard Medical School, Boston, Massachusetts 02215, the §Department of Molecular Biology, Princeton University, Princeton, New Jersey 08560, the ¶Institut für Genetik und Mikrobiologie, Universität München, 80638 München, Germany, the **Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and the ‡‡Department of Cytogenetics and Molecular Genetics, Women & Children’s Hospital, North Adelaide S.A. 5006, Australia The ubiquitin-dependent protein degradation system has been recognized as a complete enzymatic pathway that is responsible for the selective degradation of abnormal and shortlived proteins (1, 2). In this process, ubiquitin is covalently linked to target proteins prior to their degradation through the combined action of three classes of proteins, the ubiquitinactivating enzyme (E1),1 ubiquitin-conjugating enzymes (E2), and in some cases, the ubiquitin-protein ligases (E3) that are believed to be important in substrate recognition. E1 catalyzes the ATP-dependent formation of a thioester bond between the C-terminal glycine of ubiquitin and the active site cysteine of * This work was supported in part by the National Institutes of Health (to T. F. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U66818 and U66867. i Heisenberg Fellow at the Deutsche Forschungsgemeinschaft. §§ Investigator of the Howard Hughes Medical Institute and an American Cancer Society Professor. ¶¶ To whom correspondence should be addressed. Tel.: 617-667-1227; Fax: 617-667-1276. 1 The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; kb, kilobase(s); Mb, megabase(s); FISH, fluorescence in situ hybridization. E1. “Activated” ubiquitin is then transferred to the active site cysteine of an E2 enzyme which itself, or in conjunction with an E3 protein, catalyzes the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and a lysine e-amino group in the protein targeted for degradation by the 20 S proteosome complex. The diversity and multiple protein-protein interactions of the ubiquitin-dependent proteolytic system suggests that a high degree of regulation is required to achieve the specificity needed to control the fate of the different proteins that are degraded through ubiquitination. It is likely that ubiquitin-conjugating (E2) enzymes may contribute to the determination of which groups of protein are targeted for selective degradation. The ubiquitin-conjugating enzymes (E2s) are a family of proteins characterized by a highly conserved catalytic site containing an invariant active site cysteine (3). In yeast Saccharomyces cerevisiae, at least 10 different E2s have been identified that are involved in essential cellular processes such as DNA repair, cell cycle control, and stress responses (4), suggesting that E2 enzymes may be key players in establishing the diversity of the ubiquitin-proteolytic system. Although genetic analysis in yeast has revealed that ubiquitin conjugation is essential to cell viability (5, 6), only a few cellular targets of the ubiquitin protein ligase system have been identified. These include cyclin B and the yeast transcription factor MATa2 (7, 8). Recently, the ubiquitin-dependent proteolytic system has also been shown to actively participate in the degradation of p53 (9), in the degradation and processing of cystic fibrosis transmembrane conductance regulator (10, 11), and in the processing of NF-kB (12), suggesting more widespread roles of this system. The Wilms’ tumor suppressor gene encodes a zinc finger DNA-binding protein (13, 14) that functions in transient transfection assays as a transcriptional repressor of several growthrelated genes such as insulin-like growth factor II (15), insulinlike growth factor IR (16), platelet-derived growth factor A-chain (17), colony stimulating factor 1 (18), and transforming growth factor b1 (19). Previously, we identified two separate domains (residues 85–179 and 250 –266) of WT1 that function independently as transcriptional repressors (20, 21). When expressed independently of its zinc finger domain, the proximal repressor domain (amino acids 85–179) relieved the repressor activity of wild type WT1 in a dose-dependent manner (22), suggesting the presence of an interactive protein with WT1 in vivo. In order to identify and clone the gene encoding this interactive protein, we have used the yeast two-hybrid system with the proximal repressor domain of WT1 as “bait.” We report here the isolation and characterization of cDNA clones that encode the human homolog of the yeast ubiquitin-conju- 24811 Downloaded from http://www.jbc.org/ by guest on May 31, 2020 We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms’ tumor gene product (WT1) in a yeast twohybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays. The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human gene fully complements yeast that have a temperaturesensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9. Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested. A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system. 24812 Molecular Cloning and Chromosome Localization of the cDNA for hUBC9 gating enzyme (yUBC9) that specifically interacts with the proximal repressor domain of WT1 in the yeast two-hybrid system and with WT1 in co-immunoprecipitation and glutathione S-transferase assays. The results suggest that UBC9 may be involved in the transcription regulation mediated by WT1. MATERIALS AND METHODS b-Galactosidase activity Fusion partner Vector alone 1WT (85-179) 1WT (250-266) 1Lamin C a Vector a ND 0.3 6 0.2 0.2 6 0.2 0.3 6 0.1 hUBC9 0.2 6 0.1 57.9 6 16.2 0.3 6 0.2 1.5 6 0.5 ND, not determined. Fluorescence in Situ Hybridization (FISH)—Metaphase chromosome spreads from normal human lymphocytes were prepared according to the method of Fan et al. (25). Chromosomal hybridization and detection of immunofluorescence were carried out as described previously (26). The chromosome preparations were stained with both diamidino-2phenylindole and propidium iodide and observed by fluorescence microscopy. Chromosome Mapping by Somatic Cell Hybrid—A panel of mouse/ human somatic cell hybrids containing breakpoints on the distal tip of the short arm of chromosome 16 (28) were digested with HindIII, transferred to GeneScreen Plus (DuPont NEN), and probed with 32Plabeled 1.1-kb hUBC9 cDNA. Controls used were human, mouse, and the hybrid CY18 which has chromosome 16 as the only human chromosome. RESULTS To identify the WT1 interactive protein previously described (22), a human placental cDNA/Gal4 transcriptional activation domain fusion library was screened in the yeast two-hybrid system with the proximal repressor domain of WT1 as bait. Eleven of 2 3 106 clones screened were strongly positive in the yeast two-hybrid screen, and seven of these clones encoded portions of the same protein. Each of the encoded proteins of the 7 positive clones interacted strongly with the bait but failed to interact with the LexA DNA-binding domain alone, with lamin C, or with the second repressor domain of WT1 (residues 250 –260) (Table I). These data suggest that the interaction of the protein products of the 7 related positive clones with the proximal repressor domain of WT1 is specific. To obtain full-length cDNAs, a placental cDNA library was probed with one of the positive clones. Eight independent clones with inserts of 0.6, 1.1, and 1.8 kb were isolated. The 1.8-kb and 1.1-kb cDNAs were fully sequenced. The two cDNA clones share the initial 15 base pairs upstream of the translation start site and the entire coding as well as 39-untranslated regions. However, the two cDNA clones differ in the more 59-untranslated regions, presumably the result of alternative splicing. The longest open reading frame of each clone predicts a protein product of 157 amino acids (Fig. 1A). To establish that the cDNAs encoded protein, each of the cDNA clones was tested by in vitro transcription/translation. Each cDNA encodes a protein that migrated identically in SDS-polyacrylamide gels with a mobility consistent with the predicted 157-amino acid protein products (data not shown). The predicted amino acid sequence of the two cDNA clones was compared with protein sequences deposited in GenBankTM. A 56% identity was established between the predicted Downloaded from http://www.jbc.org/ by guest on May 31, 2020 Yeast Two-hybrid Screening—The proximal repressor domain of human WT1 (residues 85–179) was coupled with the LexA DNA binding domain (pLexADB/WT-N) to serve as bait. To construct pLexADB/ WT-N, the cDNA fragment encoding residues 85–179 of WT1 was obtained by digesting pSGWT-N (21) with XbaI, blunt-ending it with the Klenow fragment and then digestion with EcoRI, and cloning the fragment into pStop116 digested with EcoRI and SmaI. pStop116 was modified from plasmid BTM116 (constructed by Drs. Paul Baily and Stanley Fields) by introducing stop codons into each of the three reading frames within the polylinker region. Yeast strain L40 was transformed with pLexADB/WT-N and subsequently with the Gal4 activation domain/human placenta cDNA library (Clontech). Positive clones were sequenced using dideoxynucleotide diphosphates and Sequenase 2.0. A lgtII human placenta cDNA library was provided by Dr. Evan Sadler (Washington University, St. Louis) and screened as described (23). Two million yeast transformants were screened. Sixty-five positive clones were identified in initial screening, and eleven of these clones were confirmed as positive. The 11 positive clones were further tested for specificity with plasmids encoding hybrids of the LexADB with Lamin C and with the second negative regulatory domain of WT1 (residues 250 –266). The pLexADB/WT-N alone failed to activate transcription of reporter genes containing LexA binding sites in yeast. b-Galactosidase assay (24) was used to test the interactive specificity of the DNA-binding domain fusion partners with the vector alone and with the vector containing the Gal4 activation domain fused with cDNA from positive clones. The plasmids that scored positive in these assays were reintroduced to yeast and to quantify b-galactosidase activity shown in Table I as a further test of specificity. Glutathione S-Transferase Assay—To construct GST-hUBC9, pBShUBC9 was digested with EcoRI and subcloned into the EcoRI site of the pGEX-KG vector (29). GST-hUBC9 was expressed and purified from Escherichia coli strain DH5a. Extracts from 2 3 106 (human embryonic kidney) cells transfected with cytomegalovirus promoter-driven expression vectors encoding full-length and the WT1D1– 84, WT1D1–294, and WT1D297– 429 deletion mutants of WT1 (22) were incubated with Sepharose beads containing 2–3 mg of GST and GST-hUBC9 in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml antipain) for 2–3 h at room temperature. After extensive washing with lysis buffer and lysis buffer with 0.5 M NaCl, sample were boiled in SDS-PAGE loading buffer for analysis in 15% SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose membranes, immunoblotted with polyclonal anti C-terminal and N-terminal WT1 antibodies WT(C-19) and WT 180 (Santa Cruz), and visualized with an alkaline phosphatase-conjugated secondary antibody. Co-immunoprecipitation/Western Blot Analysis—An expression vector encoding the influenza virus hemagglutinin (HA)-tagged hUBC9 was constructed by cloning the 1.1-kb EcoRI fragment of hUBC9 into the EcoRI site of expression vector pGCN in frame with HA tag. The cell lysates were prepared from 293 cells co-transfected with WT1 and HA-tagged hUBC9 plasmids and immunoprecipitated with anti-WT1 (WTC-19) and an anti-Gal4 DNA-binding domain antibody as control. The Western blot analysis was performed with anti-HA monoclonal antibody. Northern and Southern Blot Analysis—The human tissue Northern blot was obtained from Clontech and probed with the full-length 1.1-kb hUBC9 cDNA according to the manufacturer’s recommendation. Human genomic DNA was purchased from Promega, digested with different restriction enzymes as indicated, and separated on a 1% agarose gel. Southern blots were performed as described (23) with the 1.1-kb cDNA as probe. Yeast Complementary Assay—Strain W9432 (MATa, ubc9-D1::TRP1, pSE362 (ARS1, CEN4, HIS3)-ubc9-1) carries a replacement of the genomic yUBC9 coding sequence by the TRP1 marker and a plasmidborne copy of the yUBC9-1 allele (1.5-kb XbaI-SspI fragment) (10) and is otherwise to Trp-303. The human UBC9 cDNA (1.1-kb EcoRI fragment) and the yeast UBC9 gene (0.6-kb EcoRI-XbaI fragment) were fused to the GAL1 promoter in vectors p416GAL1 (ARSH4, CEN6, URA3) and pSE936 (ARS1, CEN4, URA3), respectively. TABLE I Binding specificity of hUBC9 to the negative regulatory domain of WT1 in the yeast two-hybrid system Residues 85-179 of human WT1 were coupled with the LexA DNA binding domain (pLexADB/WT-N) to serve as a bait for screening in yeast. Two million yeast transformants were screened, and 65 positive clones were identified in initial screening. After secondary screening, the 11 positive clones were tested for specificity with plasmids encoding hybrids of the LexA DB with lamin C and with a second negatively regulatory domain of WT1 (250-266). pLexADB/WT-N alone failed to activate transcription of reporter genes containing LexA binding sites in yeast. b-Galactosidase activity units are shown for the DNA-binding domain fusion partner co-expressed with the vector alone and with the vector containing Gal4 activation domain fused with hUBC9. The plasmids recovered were reintroduced to yeast to recheck specificity and for quantitation of b-gal actosidase activity shown. Molecular Cloning and Chromosome Localization of the cDNA for hUBC9 24813 Downloaded from http://www.jbc.org/ by guest on May 31, 2020 FIG. 1. A, full-length nucleotide and predicted amino acid sequences of two cDNA clones encoding a protein interactive with the WT1 repressor domain. The vertical line from cytosine 792 of the longer and cytosine 73 of the shorter form indicates the putative splice site. The longest reading frame of the cDNAs predicts a protein of 157 amino acids. B, comparison of predicted amino acid sequences of hUBC9 and of the yUBC9 (GCG program, Madison, WI). The conserved active site cysteines are boxed. amino acid sequence of our clones and yeast ubiquitin-conjugating enzyme 9 (yUBC9), a nuclear protein that is required for cell growth and that is involved in the degradation of S- and M-phase cyclins (6). The predicted protein product contains the active site cysteine residue (residue 93 boxed, Fig. 1B) necessary for thioester formation and the ubiquitin conjugating activity of all E2 enzymes. To establish that our clone encodes a functional UBC9 homolog, the 1.1-kb human cDNA was expressed in a yeast carrying a temperature-sensitive (ts) yUBC9 mutation (6). The human UBC9 homolog fully restored normal growth to the yeast strain carrying the yUBC9 ts mutant at the otherwise nonpermissive temperature (Fig. 2). This result indicates that the cDNA we isolated encodes a biologically active human homolog of yUBC9 that is structurally and functionally highly conserved with yUBC9. We have designated our gene as human (h) UBC9. We compared the predicted amino acid sequence of hUBC9 with other E2 enzymes. Identification in regions of amino acid sequence were established within a number of other ubiquitinconjugating enzymes, some of which are shown in Fig. 3. The UBCs from human, pea, yeast S. cerevisiae, and Arabidopsis thaliana retain a striking degree of amino acid identity in several domains between the UBC9 and UBC4 families (Fig. 3), suggesting perhaps some degree of functional overlap among UBC9 and UBC4. To further establish the specificity of the interaction of hUBC9 with the N-terminal repressor domain of WT1 that was revealed in the yeast two-hybrid screening, glutathione S-transferase (GST) capture assays were used. Human UBC9 was expressed as a GST-fusion protein, coupled to glutathione- 24814 Molecular Cloning and Chromosome Localization of the cDNA for hUBC9 FIG. 2. Human UBC9 complements the loss of yeast UBC9 gene function. Yeast strain W9432 carrying the temperature-sensitive ubc9-1 allele was transformed with control vectors (row 1, p416GAL1; row 4, pSE936) or constructs expressing either a human UBC9 cDNA (row 2) or the yeast UBC9 gene (row 3). To compare growth of these strains, cells were spotted in a dilution series on galactose-containing plates and incubated for 3.5 days at the permissive temperature (23 °C) or 2 days at the restrictive temperature (34 °C). Sepharose beads, and incubated with extracts from 293 cells that express the full-length (or fragments) of WT1. Eluates were analyzed by Western blots with a rabbit anti-WT1 polyclonal antisera. Repeated experiments demonstrated that GST-hUBC9 “captures” WT1 from lysates of 293 cells (Fig. 4A). In control experiments, the GST-hUBC9 fusion protein failed to capture the C-terminal DNA-binding domain of WT1 that lacks the N-terminal amino acid repressor domain used as bait in the yeast two-hybrid screening. GST alone failed to capture WT1 (Fig. 4A), and GST-hUBC9 failed to capture proteins from control 293 cells that did not express WT1 (Fig. 4A). These data support the specificity of the interaction between the N-terminal repressor domain of WT1 and hUBC9. Co-immunoprecipitation also was used to establish the specificity of the interactions of hUBC9 and WT1. hUBC9 was fused at its amino terminus with an influenza (flu) hemagglutinin (HA) “tag,” the flu-“tagged” hUBC9 was co-expressed with WT1 in 293 cells, and extracts of co-expressing cells were immunoprecipitated with anti-WT1 polyclonal antisera and washed extensively. The immunoprecipitates were separated on SDSpolyacrylamide gels, blotted, and analyzed in Western blots with an anti flu-tag monoclonal antibody. The flu-tagged hUBC9 was identified in lysates from cells that co-expressed WT1 and flu-tagged hUBC9 (Fig. 4B). The flu-tagged hUBC9 was not observed in the same lysates when a polyclonal antiGal4 antiserum was substituted for the anti-WT1 antisera in the immunoprecipitation step, and immunoprecipitate of lysates of cells that expressed hUBC9 alone (without WT1) did not contain proteins recognized by anti-HA antibody (Fig. 4B). Taken together, the results of the yeast two-hybrid screening, GST fusion protein capture assay, and co-immunoprecipitation strongly support the conclusion that the N-terminal repressor domain of WT1 and hUBC9 interact specifically both in vivo and in vitro. Northern blots from different human tissues were used to identify the tissues that expresses hUBC9. Transcripts of 4.4, 2.4, and 1.3 kb were identified in each of the tissues that were examined (Fig. 5A). Heart and smooth muscle have higher levels of hUBC9 transcript relative to the other tissues analyzed, whereas the 2.4-kb mRNA isoform is relatively less expressed in kidney, suggesting that the hUBC9 is ubiquitously expressed but at different levels in different tissues. To determine the chromosomal locus of hUBC9, we first probed blots of human genomic DNA with the 1.1-kb cDNA fragment and observed single hybridization bands in BglII and BamHI digests (Fig. 5B), suggesting that hUBC9 is a singlecopy gene. The human UBC9 was then mapped in human Downloaded from http://www.jbc.org/ by guest on May 31, 2020 FIG. 3. A multiple sequence alignment of the Arabidopsis UBC9 sequence, the yeast UBC9 and UBC4 sequences, and the human UBC9 and UBC4 sequences. The sequences were aligned using the PILEUP algorithm. PILEUP is part of the Wisconsin Sequence Analysis Package. An amino acid is placed in the consensus sequence if it is located at that position in four of the five aligned sequences. FIG. 4. The interaction of WT1 and hUBC9. A, in vitro interactions of WT1 and hUBC9. The full-length hUBC9 cDNA was fused in-frame with the GST and expressed as the GST-hUBC9 fusion protein. GST and GST-hUBC9 fusion proteins were bound to glutathioneSepharose beads. Extracts from 293 cells that expressed WT1 and deletion mutants of WT1, WT1D1– 84, WT1D297– 429, and WT1D1–294, were incubated with GST and GST-fusion protein bound to Sepharose beads for 2–3 h. Following washing with lysis buffer, beads were eluted with SDS gel loading buffer and separated by 15% SDS-PAGE and transferred to nitrocellulose filters for immunoblotting with antibodies against N- and C-terminal portions of WT1 and visualized by fluorography. B, co-immunoprecipitation of WT1 and hUBC9 from 293 cells expressing WT1 and HA-hUBC9. Extracts of 293 cells were first immunoprecipitated with either anti-WT1 (WTC-19) or a nonspecific antibody (anti-Gal4 DNA binding domain), separated on 15% SDS-PAGE, blotted, and probed with anti-HA tag monoclonal antibody. Molecular Cloning and Chromosome Localization of the cDNA for hUBC9 24815 FIG. 6. Chromosomal localization of hUBC9 probe by FISH on human metaphase chromosomes. Note specific hybridization of fluorescein-labeled probe to subband 16p13.3. The photograph represents a computer-enhanced, merged image (arrow), and diamidino-2-phenylindole-stained chromosomes. DISCUSSION FIG. 5. A, Northern blot of hUBC9 in different human tissues. A human tissue Northern blot (Clontech) was probed with the 1.1-kb hUBC9 cDNA. Each lane contained 2 mg of poly(A)1 RNA from heart (H), brain (B), placenta (Pl), lung (L), smooth muscle (SM), kidney (K), and pancreas (Pa). The b-actin cDNA was used to probe the same blot as control. The size markers are indicated on the left side of the blot. B, Southern blot analysis of the hUBC9 gene. 10 mg of human genomic DNA were digested with HindIII (H), EcoRI (E), BglII (Bg), or BamHI (B), and DNA fragments were separated by electrophoresis on 1% agarose gel. The blot was probed with the 1.1-kb hUBC9 cDNA probe. metaphase chromosomes by FISH. The hUBC9 probe hybridized on the short arm of human chromosome 16 in 22 of 26 metaphase spreads examined. Among 94 fluorescent signals on all chromosomes, 55 (58.5%) were located on band 16p13. Most signals localized distally at subband 16p13.3 (Fig. 6). A mouse/ human somatic cell panel with average distance between breakpoints of 1.2 Mb and a potential resolution of 1 Mb was used to further fine map the gene. Major bands of approximately 20 kb and 8.5 kb and mouse bands of 9.5 kb, 6 kb, and 3.2 kb were observed in the Southern blots. The human band of The data strongly suggest that the interactive protein previously identified (22) that modulates WT1 repressor activity is hUBC9. This conclusion is based upon the specificity of the interaction of the N-terminal but not the C-terminal repressor domain of WT1 with hUBC9, the failure of lamin C to interact with WT1 in the yeast two-hybrid system, and upon the specificity of the interaction of hUBC9 and WT1 demonstrated in co-immunoprecipitation and GST capture assays. The data also indicate significant identity of primary structure and retention of the invariant active site cysteine residue between the predicted amino acid sequence of our cDNA clones and yUBC9. Our cDNA clone functionally complements yeast carrying a temperature-sensitive UBC9 gene mutant, indicating that hUBC9 is fully functional as an E2 enzyme in yeast and that we have cloned the human homolog of the yUBC9. Human UBC9 retains a high degree of amino acid identity with other UBCs that have been cloned and is most closely related to the UBC4 family. The potential importance of the highly conserved domains that we have identified among UBCs is unclear, but these domains may be important in selective interactions with specific target proteins. The ability of our cDNA to fully rescue yeast that contain a temperature-sensitive mutant is a striking example of functional conservation from yeast to human and suggests a highly important role of the UBC9s throughout evolution. In yeast, UBC9 is involved in cell cycle progression. Yeast UBC9 is required for degradation of S- and M- phase cyclins and for viability (6). In this context, it is important to note that WT1 blocks cell cycle progression and that this block is relieved by expression of exogenous cyclin E and CDK2 as well as cyclin D1/CDK4 (27). Because of the striking conservation of primary structure and because hUBC9 fully complements yeast with yUBC9 temperature-sensitive mutations to allow normal progression of yeast through the cell cycle, we suggest that hUBC9 may have a similar role in eukaryotic cells. The results also suggest that the interaction of WT1 with hUBC9 may be important in the cell cycle block imposed by WT1. Human hUBC9 is an extraordinarily conserved protein in evolution from yeast to man. Whereas its precise role in human cells is unknown, the requirement of UBC9 in yeast for normal cell cycle progression and the specific interactions with WT1 suggest important roles of hUBC9 in mammalian cells and Downloaded from http://www.jbc.org/ by guest on May 31, 2020 20 kb was present on chromosome 16 and was present in the hybrids CY189 and CY193 but absent from the hybrids CY14, CY192, and additional hybrids with more proximal breakpoints of CY200, CY193 to CY189, indicating that hUBC9 maps between (data not shown). 24816 Molecular Cloning and Chromosome Localization of the cDNA for hUBC9 raise the possibility of important roles of ubiquitination in regulation of transcription. Acknowledgments—We thank Drs. Kendall Blumer, Andre Shaw, and Daniel Link for help in establishing the yeast two-hybrid system. We also thank Dr. Nobuo Horikoshi for helpful advice. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 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R., Nierman, W. C., Auffray, C., and Sutherland, G. R. (1995) Genomics 29, 503–511 29. Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262–267 Molecular Cloning of the cDNA and Chromosome Localization of the Gene for Human Ubiquitin-conjugating Enzyme 9 Zhao-Yi Wang, Qing-Qing Qiu, Wolfgang Seufert, Takahiro Taguchi, Joseph R. Testa, S. A. Whitmore, David F. Callen, Douglas Welsh, Thomas Shenk and Thomas F. Deuel J. Biol. Chem. 1996, 271:24811-24816. doi: 10.1074/jbc.271.40.24811 Access the most updated version of this article at http://www.jbc.org/content/271/40/24811 Alerts: • When this article is cited • When a correction for this article is posted This article cites 28 references, 8 of which can be accessed free at http://www.jbc.org/content/271/40/24811.full.html#ref-list-1 Downloaded from http://www.jbc.org/ by guest on May 31, 2020 Click here to choose from all of JBC's e-mail alerts