NUB1, a NEDD8-interacting Protein, Is Induced by Interferon and Down-regulates the NEDD8 Expression*

Katsumi Kito, Edward T. H. Yeh, and Tetsu KamitaniDagger

From the Department of Cardiology, University of Texas M. D. Anderson Cancer Center and Division of Molecular Medicine, University of Texas-Houston Health Science Center, Houston, Texas 77030

Received for publication, January 31, 2001, and in revised form, March 16, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NEDD8, a ubiquitin-like protein, covalently conjugates to cullin family members. It appears to control vital biological events through its conjugation to cullins. To study how this conjugation pathway is regulated, we performed yeast two-hybrid screening by using NEDD8 as a bait and isolated a cDNA fragment encoding a potent down-regulator of the NEDD8 expression. Here, we report this novel regulator, NUB1 (NEDD8 Ultimate Buster-1). NUB1 is composed of 601 residues with a calculated 69.1-kDa molecular mass. It is an interferon-inducible protein and predominantly localized in the nucleus. The NUB1 message is specifically expressed in adult human testis, ovary, heart, and skeletal muscle tissues and is developmentally down-regulated in mouse embryos. In biochemical analysis, we found that NUB1 overexpression leads to severe reduction of NEDD8 monomer and NEDD8 conjugates in cells. This reduction is not due to down-regulation of NEDD8 transcription, but due to post-transcriptional mechanism. As expected from this activity, overexpression of NUB1 had a profound growth-inhibitory effect on U2OS cells. Thus, NUB1 is a strong down-regulator of the NEDD8 expression and appears to play critical roles in regulating biological events, including cell growth.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NEDD8 is a highly conserved 81-amino acid protein that shares 60% identity and 80% homology with ubiquitin. Expression of the NEDD8 message is highly restricted to the heart and skeletal muscle in adult human tissues (1) and is developmentally down-regulated in mouse embryos (1, 2). NEDD8 and its yeast homologue, Rub1 (3, 4), belong to an expanding family of ubiquitin-like proteins that includes UCRP (5), sentrin-1/SUMO1 (6, 7), sentrin-2 (8), and sentrin-3 (9). These proteins share a common distinction; the mature form is always translated in precursor form, with one or more amino acids following a Gly-Gly dipeptide that forms the C terminus of the mature protein (10). In the NEDD8-conjugation process, the C-terminal tail of the precursor protein is cleaved off by a C-terminal hydrolase, such as UCH-L3 (11). The mature form has been shown to conjugate to a large number of nuclear proteins (1). The pathway of NEDD8 conjugation is thought to be catalyzed by three enzymes, termed E1 (NEDD8-activating), E2 (NEDD8-conjugating), and E3 (NEDD8-ligating), in a manner analogous to ubiquitination and sentrinization (10, 12, 13).

All known NEDD8 targets in mammalian cells are cullin (Cul)1 family members, including Cul-1, -2, -3, -4A, -4B, and -5 (14, 15). Human Cul-1 is a major component of ubiquitin ligase, known as an SCF complex that catalyzes the ubiquitination of Ikappa Balpha , beta -catenin, and p27 (Kip1) (16-18). Interestingly, the NEDD8 conjugation to Cul-1 is required for the ubiquitin ligase activity of the SCF complex containing Cul-1 (19, 20). Thus, NEDD8 conjugation seems to be involved in many important biological functions, including NFkappa B signaling and cell-cycle regulation by p27, and must be strictly regulated. However, the regulation system of NEDD8-conjugation is still unclear, with the exception of the recent discovery of USP21, a novel isopeptidase for NEDD8-conjugated proteins (21).

To define the unknown regulators of NEDD8 conjugation, the yeast two-hybrid system was applied in this study. From library screening, we isolated a cDNA clone encoding a novel NEDD8-interacting protein, NUB1. Here, we report NUB1 as a strong down-regulator of the NEDD8 expression.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines, Culture Conditions, and Interferon Treatment-- We purchased the following human cell lines from American Type Culture Collection (Manassas, VA): rectal adenocarcinoma SW837, neuroblastoma SK-N-SH, malignant melanoma SK-MEL28, myeloid leukemia U937, Burkitt lymphoma Raji, T-cell leukemia Jurkat, chronic myelogenous leukemia K562, promyelocytic leukemia HL60, human embryonic kidney 293, osteosarcoma U2OS, renal cell carcinoma 786-0, and cervical adenocarcinoma HeLa. COS-M6 cells were a generous gift from Dr. Steve Goldring of Harvard Medical School. SW837, SK-N-SH, SK-MEL28, human embryonic kidney 293, U2OS, 786-0, HeLa, and COS-M6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. U937, Raji, Jurkat, K562, and HL60 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. Human interferon-beta (IFN-beta ) was purchased from Calbiochem (La Jolla, CA).

Antibodies-- Mouse monoclonal antibody 16B12 (Covance; Richmond, CA) is an antibody to the peptide sequence YPYDVPDYA of influenza hemagglutinin (HA). Rabbit anti-human NUB1 antiserum was generated by immunization with a GST fusion protein of NUB1 corresponding to amino acids 432-601 (NUB1432-601). Rabbit polyclonal anti-actin antibody (specific for the C-terminal actin fragment) was purchased from Sigma.

Plasmid Construction and Transfection-- To express proteins tagged with epitope at the N terminus in mammalian cells, plasmid vectors pcDNA3/HA-N (7) and pcDNA3/RH-N (8) were used as described previously (1). The human cDNAs used in this study have been described previously: ubiquitin (7), NEDD8 (1), sentrin-1 (7), and Ubc12(C111S) (22). These cDNAs were inserted into the aforementioned plasmid vectors. The sequence of each cDNA was confirmed by automated DNA sequencing. Plasmids were transfected into mammalian cells using FuGENE 6 (Roche Molecular Biochemicals). The transfected cells were processed for immunostaining, Western blotting, or Northern blotting 20 h after transfection.

Yeast Two-hybrid Screening-- Yeast strain L40 was purchased from Invitrogen (Carlsbad, CA). Prey vector pGAD10 was purchased from CLONTECH (Palo Alto, CA). The bait plasmid pHybLex/HA-NEDD8-GG (11) was transformed into L40 using the lithium acetate method (6). The transformants were plated on YPD medium containing adenosine and Zeocin (YPAD/Zeo) and selected for 2 days at 30 °C. The L40 clone carrying pHybLex/HA-NEDD8-GG was cultured in YPAD/Zeo medium and sequentially transformed with 500 µg of human heart cDNA (CLONTECH) fused to GAL4 DNA-activating domain vector, pGAD10. The transformed cells were incubated for 6 days at 30 °C on selection plates (Ura-, Lys-, His-, Leu-, and Zeocin+). The positive colonies were picked and replated on selection plates (Ura-, Lys-, His-, Leu-, and Zeocin+) and assayed for beta -galactosidase activity on filter papers as described in the protocol of CLONTECH.

Domain Search by Research Tools in Web Sites-- Domain search of NUB1 was performed by using several research tools as described below (all three programs are available via the World Wide Web). Coiled coil regions and ubiquitin-associated (UBA) domains were determined by the SMART program. Bipartite nuclear localization signal (NLS) was determined by the ProfileScan program. PEST sequence was determined by the PESTfind program.

Immunoabsorption-- To demonstrate specificity of the immunoreactivity to NUB1, rabbit antiserum against GST-NUB1432-601 was preabsorbed with either GST or GST-NUB1432-601 and used for Western blot analysis as a primary antibody. For this preabsorption, 1 ml of diluted anti-NUB1 antiserum (1:1000) was incubated overnight with GST or GST-NUB1432-601 fusion protein-coated beads. After the incubation, the beads were removed by centrifugation. The supernatant was filtered, diluted to 1:10,000 with 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 0.1% Tween 20, containing 1% skim milk, and used for Western blot analysis.

Western Blotting-- Protein samples were treated at 45 °C for 1 h in 150 µl of 2% SDS treating solution containing 5% beta -mercaptoethanol. After SDS-polyacrylamide gel electrophoresis, Western blotting was performed using the protocol provided with the ECL detection system (Amersham Pharmacia Biotech). As secondary antibodies, horseradish peroxidase (HRP)-conjugated antibodies against mouse IgG or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

Immunostaining-- Immunocytochemical staining was performed by the avidin-biotin-HRP complex (ABC-HRP) method (23), using the Vectastain ABC kit system (Vector, Burlingame, CA). Transfected HeLa cells on a coverslip were fixed in 3.7% paraformaldehyde solution for 20 min and permeabilized in 0.1% Triton X-100 for 10 min at room temperature. After washing with PBS, the fixed cells were incubated with PBS containing 0.1% H2O2 for 10 min to quench endogenous peroxidase activity and then washed with PBS. The cells were incubated for 10 min with PBS containing 5% horse serum for blocking, followed by additional incubation with anti-HA antibody (16B12) for 30 min at 37 °C. After rinsing with PBS, the cells were incubated with biotinylated anti-mouse IgG for 30 min at 37 °C, washed with PBS, and treated with the ABC reagent (avidin-biotin-HRP complex) for 30 min at 37 °C. Finally, the enzymatic disclosing procedure was performed as reported previously (23).

Northern Blot Analyses-- To study the level of NUB1 message in various human tissues and mouse embryos, Northern blotting was performed. Fragments of human NUB1 and NEDD8 cDNAs were cut out from pcDNA3/RH-NUB1 or pcDNA3/HA-NEDD8 (1), respectively. The fragments were labeled with [alpha -32P]dCTP by a Megaprime labeling kit (Amersham Pharmacia Biotech). The radioactive probe was hybridized with two human multiple tissue Northern blots and a mouse embryo multiple tissue Northern blot purchased from CLONTECH.

To examine message induction by human IFN-beta , Northern blotting was performed using nonradioactive probes. HeLa cells were cultured with or without human IFN-beta (Calbiochem) in 6-cm dishes, and total RNA was extracted using TRIzol (Life Technologies, Inc.). Equal amounts of RNA (16 µg) were resolved on a 1% agarose gel containing formaldehyde and transferred to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech), followed by alkaline-cross-linking. As probes, fragments (400 bp) of NUB1 and GAPDH cDNAs were amplified by polymerase chain reaction and purified by a Qiagen II gel extraction kit (Qiagen Inc.). These fragments were labeled with alkaline phosphatase by cross-linker using the AlkPhos Dilect system (Amersham Pharmacia Biotech). After prehybridization for 1 h and hybridization with alkaline phosphatase-labeled probe overnight at 55 °C, the blots were washed five times. The signal was detected by the chemiluminescent method using CDP-Star (Amersham Pharmacia Biotech).

To detect the message level of epitope-tagged protein in transfectants, Northern blot analysis was performed using a 32P-labeled oligo encoding epitope tag as a probe. Total RNA was extracted using TRIzol (Life Technologies, Inc.) from COS-M6 cells transfected with the indicated plasmids. Twenty micrograms of total RNA was subjected to electrophoresis on 1% agarose-formaldehyde gels and blotted onto a Hybond N+ membrane (Amersham Pharmacia Biotech). The probes for HA tag (39 mer oligo: GATCCGCTAGCGTAATCCGGAACATCGTATGGGTACATA) and RGS-His tag (36 mer oligo: GATCCGTGATGGTGATGGTGATGCGATCCTCTCATA) were radiolabeled with [gamma -32P]ATP using T4 polynucleotide kinase. The probe for beta -actin (2.0-kb human cDNA) was radiolabeled with [alpha -32P]dCTP using the Megaprime DNA labeling system (Amersham Pharmacia Biotech). The probes were hybridized to the membrane in ExpressHyb hybridization solution (CLONTECH) and washed in 0.1× SSC and 0.1% SDS. Autoradiography was performed at -80 °C. The membrane was repeatedly hybridized with other probes after removing the previous probe.

Cell Growth Assay-- Cell growth assay was performed as described previously (22, 24). U2OS cells (1.0 × 105) were plated in a 6-cm dish and transfected by FuGENE 6 (Roche) with 5 µg of control empty pcDNA3 vector, pcDNA3/RH-NUB1, or pcDNA3/RH-Ubc12(C111S). After 24 h, the cells were washed twice with PBS and incubated with fresh medium containing 10% fetal calf serum and 0.6 mg/ml G418. The medium was changed every 2 days. Nine days after transfection, drug-resistant cells were harvested and counted. The data were analyzed for statistical differences by Fisher's protected least significant difference method.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screening-- To identify NEDD8-interacting proteins, we screened ~1 × 106 primary library transformants as reported previously (11). A total of 450 colonies grew on the selection plates, 18 of which stained positive when tested for beta -galactosidase expression. Subsequent analysis of DNA sequencing indicated that 7 of the 18 clones encoded human UCH-L3 (11). In addition, 1 of the 18 clones turned out to be a human homologue of mouse BS4 (GenBank accession no. U27462.1).

Structure of NUB1, a Human Homologue of Mouse BS4-- Using yeast two-hybrid screening, we isolated 3,113 bp of cDNA from a human heart cDNA library (Fig. 1A). This cDNA encodes a predicted 69.1-kDa protein of 601 amino acids. Multiple termination codons were found in the other two reading frames. The ATG initiation codon was contained within a nearly perfect Kozak consensus sequence, which is necessary for efficient translation (25). A presumptive polyadenylation signal was found 28 bp upstream from the 3' end of the cDNA. A BLAST search of the entire data base through the National Center for Biotechnology Information (Bethesda, MD) showed that this predicted protein was 76.9% identical to mouse BS4 in amino acid sequence, indicating that the protein is a human homologue of mouse BS4. Since this protein negatively regulates the NEDD8 expression as described below, we designated this protein as NUB1 (NEDD8 Ultimate Buster-1), and its cDNA sequence was submitted to GenBank (accession no. AF300717). Although the cDNA sequence of mouse BS4 was registered in GenBank, we could not find any publication on its biological function in the Medline data base. Thus, the function of proteins NUB1 and BS4 was totally unknown.


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Fig. 1.   Structure of NUB1 molecule. A, nucleotide and predicted protein sequence of NUB1. The presumptive polyadenylation signal AATAAA is underlined. The initiation codon fits within the Kozak consensus sequence GCC(A/G)CCATGG, in 7 of 10 bases (indicated by dots). The termination codon TAA is indicated by an asterisk. The nucleotide sequence was determined on both strands by automated sequencing. The nucleotide sequence has been submitted to the GenBankTM data base and assigned accession no. AF300717. The predicted amino acid sequence is given below the nucleotide sequence in single-letter code. B, schematic representation of deduced domains. CC, the coiled coil domain; UBA, the ubiquitin-associated domain; NLS, the nuclear localization signal; PEST, PEST sequence.

To predict the protein function, we searched consensus sequences or domains in NUB1 by using several research tools in Web sites. As shown in Fig. 1B, there were two coiled coil regions at the N-terminal side of NUB1. The first coiled coil region was located from Leu-36 to Ala-67. The second coiled coil region was located from Val-155 to Thr-203. At the C-terminal side of NUB1, there were two UBAs, a bipartite NLS and a PEST sequence. The first UBA domain was located from Asp-376 to Asn-413. The second UBA domain was located from Ser-477 to His-514. The UBA domain has been reported to occur in subsets of the ubiquitin-conjugation enzymes (E2), ubiquitin ligases (E3), and deubiquitinating enzymes (26). Between these two UBA domains, a bipartite NLS was located from Arg-414 to Arg-431. Finally, a PEST sequence (27) was located from His-514 to His-568. Based on the domain search data, we predicted that NUB1 was a nuclear protein and its turnover was rapid because of the PEST motif.

Detection of Endogenous NUB1 Expression in Various Cell Lines-- To characterize protein expression of NUB1 in human cell lines, rabbit polyclonal antiserum specific for NUB1 was generated. The expression of NUB1 was surveyed in 12 different human cell lines and COS cells by Western blotting. The antiserum was preabsorbed with either GST (Fig. 2, upper panel) or GST-NUB1 (data not shown) to demonstrate specificity of the immunoreactivity to NUB1. As shown in the upper panel of Fig. 2, a 69-kDa band specific for NUB1 was strongly observed in SK-N-SH, Raji, K562, and 786-0 cells (arrowhead). A moderate signal was detected in SW837, 293, HeLa, and COS cells. In SK-MEL28, Jurkat, and HL60 cells, the band was very weak. Although the 69-kDa band was undetectable in U937 and U2OS cells, a longer exposure allowed us to detect the faint band. In contrast to this blotting, the antiserum preabsorbed with GST-NUB1 could not detect anything (data not shown). Thus, our antiserum specifically detected endogenous NUB1, and its expression level varied widely among cell lines.


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Fig. 2.   Western blot analysis of NUB1 expression in human cell lines and COS cells. Total cell lysates were analyzed by Western blotting using rabbit antiserum against GST-NUB1432-601 preabsorbed with either GST (upper panel) or GST-NUB1432-601 (data not shown) as a primary antibody. To demonstrate an equal loading amount of total cell lysates, Western blotting using anti-actin antibody was also performed (lower panel). Molecular size markers are shown in kilodaltons (kDa).

IFN-induced Expression of NUB1 Protein-- The sequence information on BS4, a mouse homologue of human NUB1, was deposited to GenBank on June 21, 1995, and assigned to accession number U27462.1. Although the title of the data base entry was "BS4: an interferon-inducible gene with novel regulatory properties," BS4 has not been published in the literature so far. To confirm whether NUB1 is an INF-inducible protein, HeLa, 293, and U2OS cells were cultured with human IFN-beta and the induction of NUB1 was examined by Western blotting. As shown in the upper panel of Fig. 3A, treatment with 250 units/ml IFN-beta for 16 h induced expression of the NUB1 protein in HeLa cells (~10-fold) (lane 1 versus lane 2) and 293 cells (~5-fold) (lane 3 versus lane 4) but not in U2OS cells (lane 5 versus lane 6). Thus, we found that NUB1 is an IFN-inducible protein and that HeLa cells are very sensitive to this induction. Next, we examined the dose dependence of NUB1 protein induction by using HeLa cells treated for 16 h with various concentrations of IFN-beta . As shown in the upper panel of Fig. 3B, NUB1 protein was moderately expressed in the untreated cells (lane 1), and it could be induced with IFN-beta in a dose-dependent manner (lanes 2-6). Furthermore, the time course of NUB1 induction was examined by using HeLa cells treated with 250 units/ml IFN-beta for various times. As shown in the upper panel of Fig. 3C, the expression level of NUB1 protein was not changed by 2 h after treatment (lanes 1-3), whereas it increased with time from 4 h to 16 h after treatment (lanes 4-6). Taken together, these data indicated that the NUB1 protein can be induced by IFN-beta in a dose- and time-dependent manner. In addition to IFN-beta , IFN-gamma was also examined for its effects on induction of NUB1. In HeLa cells, IFN-gamma could induce NUB1 protein as well as IFN-beta (data not shown).


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Fig. 3.   NUB1 protein induction by human IFN-beta . A, IFN-mediated induction of NUB1 protein in HeLa (lanes 1 and 2), 293 (lanes 3 and 4), and U2OS cells (lanes 5 and 6). Cells were treated with 0 (lanes 1, 3, and 5) or 250 units/ml (lanes 2, 4, and 6) of human IFN-beta for 16 h. Total cell lysates were prepared and analyzed by Western blotting using anti-NUB1 antiserum (upper panel) or anti-actin antibody (lower panel). B, dose dependence of IFN-mediated induction of NUB1 protein. HeLa cells were treated for 16 h with human IFN-beta at concentrations of 0 (lane 1), 0.4 (lane 2), 2 (lane 3), 10 (lane 4), 50 (lane 5), or 250 units/ml (lane 6). Total cell lysates were prepared and analyzed by Western blotting using anti-NUB1 antiserum (upper panel) or anti-actin antibody (lower panel). C, time course for IFN-mediated induction of NUB1 protein. HeLa cells were treated with 250 units/ml human IFN-beta for 0 h (lane 1), 1 h (lane 2), 2 h (lane 3), 4 h (lane 4), 8 h (lane 5), or 16 h (lane 6). Total cell lysates were prepared and analyzed by Western blotting using anti-NUB1 antiserum (upper panel) or anti-actin antibody (lower panel).

Tissue-specific Expression and Developmental Down-regulation of NUB1 Message-- To determine the expression of NUB1 message in human tissues, Northern blot analyses were performed using 32P-labeled human NUB1 cDNA as probe. As shown in Fig. 4A (upper panel), 3.5 kb of NUB1 message was weakly detected in testis, ovary, heart, and skeletal muscle. In all other tissues, the message signal was much weaker or undetectable. In addition, a 2.3-kb smaller band was strongly detected in the testis but not in the other tissues. As shown in the middle panel, NEDD8 message was detected on the identical blot. Interestingly, NEDD8 message was enriched in the ovary, heart, and skeletal muscle. This tissue specificity is similar to that of NUB1. Since NEDD8, which interacts with NUB1, was originally isolated as a developmentally down-regulated message in the mouse brain, we also examined the expression of NUB1 message in developing mouse embryos. As shown in the upper panel of Fig. 4B, 3.0-, 2.5-, and 2.0-kb NUB1 messages were detected. The 3.0-kb message was strongest, the 2.5-kb message was moderate, and the 2.0-kb message was weakest. These NUB1 messages were strongest in the day 7 mouse embryo and were markedly decreased in the day 11, day 15, and day 17 embryos (upper panel). In contrast, the strength of the NEDD8 message peaked in the day 11 mouse embryo and was markedly decreased in the day 15 and day 17 embryos (middle panel). Thus, messages of NUB1 and NEDD8 were developmentally down-regulated differently.


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Fig. 4.   Northern blot analysis of NUB1. A, expression of NUB1 mRNA in a variety of human tissues. B, expression of NUB1 mRNA in mouse embryos of different developmental stages. Samples of poly(A)+ RNA (2 µg) from indicated sources were run on a denaturing gel, transferred to a nylon membrane, and hybridized with a 32P-labeled cDNA fragment of human NUB1 (upper panel), NEDD8 (middle panel), or beta -actin (lower panel). Open arrows indicate isoforms of NUB1 message. RNA size markers are shown in kilobases (kb). C, IFN-mediated induction of NUB1 mRNA. HeLa cells were treated for 16 h with human IFN-beta at concentrations of 0 (lane 1), 0.4 (lane 2), 2 (lane 3), 10 (lane 4), 50 (lane 5), or 250 units/ml (lane 6). Total RNA samples were extracted and analyzed by Northern blotting using a fragment of NUB1 cDNA as a probe (upper panel). The blot was subsequently reprobed by GAPDH as a control (lower panel). The NUB1 or GAPDH message is indicated by an arrowhead. The positions of 28 and 18 S RNAs are indicated on the left-hand margin.

IFN-induced Expression of NUB1 Message-- We defined whether IFN regulates the transcription of NUB1. HeLa cells were treated for 16 h with various concentrations of IFN-beta , and the level of NUB1 message was analyzed by Northern blotting (Fig. 4C, upper panel). The NUB1 message could not be detected in IFN-beta -untreated cells (lane 1), whereas it was induced by 0.4 units/ml IFN-beta (lane 2) and increased in a dose-dependent manner (lanes 2-6). Thus, IFN-beta up-regulates the transcription of NUB1.

Subcellular Localization of NUB1-- The subcellular localization of NUB1 was determined. HeLa cells were transfected with a plasmid containing an insert of HA-NUB1 cDNA. As controls, we transfected an expression plasmid alone or a plasmid containing an insert of HA-USP21 (21) or HA-NEDD8 cDNA. The cells were fixed, permeabilized, and stained with anti-HA antibody. As shown in Fig. 5, HA-USP21 could be detected in both the cytosol and the nucleus. HA-NUB1 and HA-NEDD8 were mostly restricted to the nucleus. This nuclear localization of NUB1 is consistent with the fact that NUB1 has an NLS in its sequence (Fig. 1B).


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Fig. 5.   Immunocytochemical localization of NUB1. Hela cells were transfected with empty vector (Control), plasmid with a HA-USP21 cDNA insert (USP21), plasmid with a HA-NUB1 cDNA insert (NUB1), or plasmid with a HA-NEDD8 cDNA insert (NEDD8). The transfected HeLa cells were fixed, permeabilized, and immunostained with anti-HA antibody. The bar indicates 20 µm.

Specific Reduction of NEDD8 Monomer and Its Conjugates by Overexpression of NUB1-- The yeast two-hybrid assay showed the interaction between NEDD8 and NUB1, implying that NUB1 may regulate the NEDD8-conjugation pathway. To examine this hypothesis, COS cell coexpression assay was used. HA-tagged NEDD8 was co-expressed in COS cells with RH-tagged NUB1 or a control plasmid. As further controls, HA-tagged ubiquitin or sentrin-1 was co-expressed with empty vector or RH-tagged NUB1. As shown in Fig. 6, when HA-NEDD8 was expressed with empty vector (lane 1), we clearly detected a 6.5-kDa band of unconjugated HA-NEDD8 and high molecular mass bands of NEDD8-conjugated proteins. This NEDD8 expression pattern was identical to our previous result (1). When HA-NEDD8 was co-expressed with RH-NUB1 (lane 2), we could not detect both the unconjugated and conjugated forms of NEDD8 except for a 80-kDa band. This result suggested that NUB1 negatively regulates the protein expression of NEDD8 monomer and its conjugates. In contrast, when RH-NUB1 was co-expressed with HA-ubiquitin (lane 4) or HA-sentrin-1 (lane 6), the overexpression of NUB1 did not lead to reduction of the expression of ubiquitin or sentrin-1. Thus, the down-regulation by NUB1 was specific to NEDD8.


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Fig. 6.   Specific reduction of NEDD8 by NUB1 overexpression. In COS cells, HA-tagged NEDD8 was co-expressed with empty vector (lane 1) or RH-tagged NUB1 (lane 2). Total cell lysates were prepared from transfectants and analyzed by Western blotting using anti-HA antibody to detect HA-NEDD8 and proteins conjugated with HA-NEDD8. As controls, HA-ubiquitin (lanes 3 and 4) or HA-sentrin-1 (lanes 5 and 6) was also co-expressed with empty vector or RH-NUB1 and subjected to Western blot analysis. Nonspecific bands are indicated by an asterisk. Molecular size markers are shown in kilodaltons.

Effect of NUB1 on Transcription of NEDD8 Message-- Although we showed the inhibitory effect of NUB1 on expression of the NEDD8 protein, the molecular mechanism was unclear. At which step did NUB1 act as an inhibitor? To address this question, we examined the effect of NUB1 on expression of the NEDD8 message. As shown in Fig. 7, overexpression of RH-NUB1 did not reduce the expression level of HA-NEDD8 message (lane 2 versus lane 3, upper panel). This result suggested that NUB1 inhibits NEDD8 expression post-transcriptionally.


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Fig. 7.   Effect of NUB1 on NEDD8 transcription. COS cells were transfected with plasmids to express message(s) of empty vector (lane 1), HA-NEDD8 (lane 2), or HA-NEDD8 plus RH-NUB1 (lane 3). Total RNA was prepared from the transfected COS cells and analyzed by Northern blotting. The message of HA-NEDD8 was detected by 32P-labeled HA-oligo (upper panel) and the message of RH-NUB1 was detected by 32P-labeled RH-oligo (middle panel). As a control, the beta -actin message was also detected (lower panel).

Inhibitory Effect of NUB1 on Cell Growth-- We previously reported two down-regulators of the NEDD8-conjugation pathway. The first down-regulator is USP21, which has isopeptidase activity with dual specificity for ubiquitin- and NEDD8-conjugated proteins. The second down-regulator is a dominant-negative UBC12 mutant that sequesters NEDD8 and inhibits NEDD8 conjugation. These proteins inhibit U2OS cell growth (21, 22). We asked whether NUB1 has a similar biological property. As shown in Fig. 8, overexpression of NUB1 inhibited cell growth up to 83% as compared with control. UBC12(C111S), a dominant-negative UBC12 mutant, also inhibited cell growth, up to 89%.


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Fig. 8.   Growth-inhibitory effect of NUB1 on U2OS cells. U2OS cells were plated in a 6-cm dish and transfected with control empty pcDNA3 vector, pcDNA3/RH-NUB1, or pcDNA3/RH-Ubc12(C111S). After 24 h, the cells were washed and incubated with fresh medium containing 0.6 mg/ml G418. The medium was changed every 2 days. Nine days after transfection, drug-resistant cells were harvested and counted. The data were statistically analyzed, and p values were calculated by Fisher's protected least significant difference method. *, p < 0.0001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IFNs are a group of cytokines with pleiotropic cellular effects. One prominent effect of IFNs is their potent antimitogenic action, which can be observed on both malignant and nonmalignant cells of many different origins. To explain this antimitogenic effect, multiple mechanisms have been proposed (28). In this study, we found three important properties of NUB1. First, IFN induces NUB1. Second, NUB1 negatively regulates the NEDD8 expression. Third, NUB1 inhibits cell growth. These findings suggest that treatment with IFN causes NUB1 induction, resulting in inhibition of the NEDD8 expression and affecting cell growth. Thus, NUB1-mediated inhibition of cell growth may be one of the mechanisms of the antimitogenic effect of IFNs.

Cullin family members are known as NEDD8 targets in mammalian cells (15). The cullin family is composed of Cul-1, -2, -3, -4A, -4B, and -5 (14). These cullins assemble multiprotein complexes, called SCF or SCF-like complexes, which have enzymatic activity of E3 ubiquitin ligase (10). Cul-1 assembles an SCF complex to catalyze the ubiquitination of p21 (CIP1/WAF1), cyclin D proteins, beta -catenin, p27 (KIP1), and Ikappa Balpha (29-34). Cul-2 assembles an SCF-like complex and is involved in the ubiquitination of hypoxia-inducible factor-1alpha (HIF1alpha ) (35). Cul-3 has been shown to target cyclin E for ubiquitination and control S phase in mammalian cells (36). Thus, cullin family members play important roles in many biological events. Recently, several groups reported that NEDD8 conjugation to Cul-1 is required for the E3 ubiquitin ligase activity of the SCF complex (19, 20, 31). These reports imply that the ubiquitin ligase activity of all SCF and SCF-like complexes is controlled by conjugation of NEDD8 to cullins. Since the NEDD8 expression is down-regulated by NUB1, the expression level of NUB1 may control many biological events, including cell growth, NFkappa B signaling, and biological responses to hypoxia.

With this study, we showed that overexpression of NUB1 leads to reduction of NEDD8 monomer and its conjugates. This reduction probably results from the down-regulation of NEDD8 expression by NUB1. Since NUB1 does not affect NEDD8 expression at message level, the reduction appears to be caused by a post-transcriptional mechanism. Further studies are required to define the mechanism.

    ACKNOWLEDGEMENTS

We thank Hung Phi Nguyen and Dr. Hiroyoshi Wada for technical and editorial assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01 DK56298-02 (to T. K.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF300717.

Dagger To whom correspondence should be addressed: Dept. of Cardiology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 449, Houston, TX 77030. Tel.: 713-500-6667; Fax: 713-500-0626; E-mail: tkamitani@mdanderson.org.

Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M100920200

    ABBREVIATIONS

The abbreviations used are: Cul, human cullin; IFN, interferon; HA, hemagglutinin epitope; RH, RGS-poly(His); HRP, horseradish peroxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UCH, ubiquitin C-terminal hydrolases; UBA, ubiquitin-associated domain; NLS, nuclear localization signal; SCF, Skp1-Cullin-F-box; kb, kilobase(s); bp, base pair(s); PBS, phosphate-buffered saline; GST, glutathione S-transferase; E1, activating enzyme; E2, conjugating enzyme; E3, ligating enzyme.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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