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
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ABSTRACT |
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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.
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 I 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.
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- 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 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%
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 [
To examine message induction by human IFN-
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 [ 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.
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
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.
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.
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- 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.
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- 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).
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.
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.
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%.
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, 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
,
-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 NF
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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IFN-
) was
purchased from Calbiochem (La Jolla, CA).
, Lys
, His
,
Leu
, and Zeocin+). The positive colonies were
picked and replated on selection plates (Ura
,
Lys
, His
, Leu
, and
Zeocin+) and assayed for
-galactosidase activity on
filter papers as described in the protocol of
CLONTECH.
-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.
-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.
, Northern blotting was
performed using nonradioactive probes. HeLa cells were cultured with or
without human IFN-
(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).
-32P]ATP using T4 polynucleotide kinase. The
probe for
-actin (2.0-kb human cDNA) was radiolabeled with
[
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
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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.
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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).
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-
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-
. 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-
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-
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-
in a dose- and
time-dependent manner. In addition to IFN-
, IFN-
was
also examined for its effects on induction of NUB1. In HeLa cells, IFN-
could induce NUB1 protein as well as IFN-
(data not
shown).
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Fig. 3.
NUB1 protein induction by human
IFN- . 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-
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-
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-
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).
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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 -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-
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.
, and the level of NUB1
message was analyzed by Northern blotting (Fig. 4C,
upper panel). The NUB1 message could not be detected in IFN-
-untreated cells (lane 1),
whereas it was induced by 0.4 units/ml IFN-
(lane
2) and increased in a dose-dependent manner
(lanes 2-6). Thus, IFN-
up-regulates the transcription of NUB1.
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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.
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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.
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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
-actin message was also detected (lower
panel).
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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
-catenin, p27
(KIP1), and I
B
(29-34). Cul-2 assembles an SCF-like complex and
is involved in the ubiquitination of hypoxia-inducible factor-1
(HIF1
) (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, NF
B signaling, and biological responses to hypoxia.
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ACKNOWLEDGEMENTS |
---|
We thank Hung Phi Nguyen and Dr. Hiroyoshi Wada for technical and editorial assistance.
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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.
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
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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.
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