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J Biol Chem, Vol. 273, Issue 52, 35201-35207, December 25, 1998
From the The c-rel proto-oncogene product,
c-Rel, belongs to the Rel/NF- The proto-oncogene c-rel
is a member of the rel/NF- The extralysosomal, energy-dependent ubiquitin-proteasome
(Ub-Pr)1 pathway is a major
mechanism used to regulate the turnover of many cellular proteins,
which include highly abnormal proteins, short lived and long lived
proteins (for recent reviews, see 14-19). The pathway, present in both
the cytoplasm and nucleus, consists of two distinct and sequential
steps. The target protein is first conjugated with multiple ubiquitin
molecules and then translocated to and degraded by the large 26 S
proteasome (reviewed in Ref. 20). The list of Ub-Pr pathway substrates
has been growing rapidly. Prominent examples include proto-oncogene
products, transcription factors, cell cycle regulators, cytokine and
growth factor receptors, major histocompatibility complex class I
molecules, the cystic fibrosis transmembrane conductance regulator, and others.
Activation of NF- Cell Lines and Chemicals--
MT-2, HUT102, and 81-66 are
HTLV-I-infected human T-cell lines (28). Jurkat is a T-cell line
established from a patient with T-cell leukemia (America Type Culture
Collection). DB and CA46 are B-lymphoma cell lines (24). All human T
and B cells were cultured in RPMI 1640 medium supplemented with 10%
fetal calf serum, and antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin). DT95, an avian IgM-producing mature B cell line (29), was cultured in Dulbecco's modified Eagle's medium supplemented with 5% bovine calf serum, 5% chicken serum, and antibiotics. NIH3T3
and monkey COS cells were cultured in Dulbecco's modified Eagle's
medium with 10% fetal calf serum and antibiotics. Proteasome inhibitors, lactacystin (LC) and LLnL (Calpain inhibitor I;
N-acetyl-L-leucinyl-L-leucinyl-norleucinal) were purchased from Corey Laboratory (Harvard University) and Sigma, respectively.
N-p-tosyl-L-phenylalanine
chloromethyl ketone (TPCK),
N- Western Blot and Immunoprecipitation Analyses--
For Fig.
1a, the cytoplasmic and nuclear fractions were extracted as
described previously (30). For Fig. 2b, Jurkat cells were
lysed with Nonidet P-40 buffer (20 mM Tris/HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA)
containing protease inhibitors (1% aprotinin, 50 µM
LLnL, 70 µg/ml phenylmethanesulfonyl fluoride, 40 µg/ml TPCK, 5 µg/ml TLCK, 5 µg/ml leupeptin) and 10 mM iodoacetamide. The Nonidet P-40-soluble and -insoluble fractions were further analyzed
by immunoprecipitations and Western blot. For Western analysis, equal
amounts of protein (assayed with the BCA kit; Pierce) or fractions
isolated from equal number cells were resolved by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred onto
polyvinylpyrrolidone membranes. The membrane was blocked, washed,
incubated with antiserum (typically at 1:1000) followed by reacting
with peroxidase-conjugated anti-rabbit immunoglobulin serum (Boehringer
Mannheim), and developed by the enhanced chemiluminescence detection
system (Amersham Pharmacia Biotech). For immunoprecipitation analysis,
cells were lysed in radioimmune precipitation buffer (20 mM
Tris/HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1%
deoxycholate, 1% Triton X-100) or in other specified buffers
containing protease inhibitors and 10 mM iodoacetamide. The
lysates were clarified by centrifugation at 12,000 × g
for 30 min and then incubated with individual antiserum in the presence
or absence of the competing peptide (2 µg for each µl of
antiserum). The immune complexes were collected with Protein
A-Sepharose beads, washed with radioimmune precipitation buffer,
boiled, resolved by SDS-PAGE, transferred onto membranes, and further analyzed.
Expression Plasmids--
The human c-Rel expression plasmid
pRC/CMV-h-rel, kindly provided by N. Rice (NCI-Frederick Cancer
Research and Development Center), was used in both in vivo
and in vitro expressions. All of the expression plasmids
containing C-terminally truncated avian c-rel were
constructed using pc-rel2 (31) as the template. Synthetic oligonucleotide adapters (Table I, column 5) containing stop codons
(underlined) were inserted between specific restriction sites in
c-rel coding sequence (columns 3 and 4) and the
BssHII site of pc-rel2, thus replacing a portion of
c-rel coding sequence. For pc-rel Antisera--
The previously published anti-(c-Rel 1135) and
anti-(c-Rel 265) sera were generated against peptides corresponding to
residues 493-509 and 573-587 of human c-Rel, respectively (32). Other human c-Rel antisera, purchased from Santa Cruz Biotechnology, Inc.
(catalog no. sc-70) and Upstate Biotechnology, Inc. (catalog no.
06-421), were raised against peptides corresponding to murine c-Rel
residues 152-176 and human c-Rel residues 573-587, respectively. Anti-ubiquitin immune sera were purchased from Sigma and Dako, Inc.
Avian c-Rel antisera V1, V2, and V3 (33) were generated against
residues 115-292, 293-458, and 425-437 of avian c-Rel, respectively.
In Vitro Assays--
Human c-Rel, avian c-Rel, and C-terminally
truncated c-Rel Transfection--
Transient transfection was carried out using
the SuperFect Transfection kit (Qiagen) according to the instructions.
The coding sequence of chicken c-rel (35) was cloned as a
XhoI-BssHII fragment from pc-rel2 between
XhoI and MluI sites of mammalian expression vector pCI-neo (Promega). An aliquot of 5 µg of DNA was used to transfect 4 × 105 NIH3T3 or COS cells seeded in each
60-mm dish. After transfection for 2.5 h, cells were washed and
grown in fresh medium for 40 h. Cells were then either treated
with inhibitors or used for the pulse-chase experiments.
High Molecular Weight c-Rel Proteins Are Present in HTLV-I-infected
Cells and in B-lymphoma Cells--
We and others previously showed
that NF- High Molecular Weight c-Rel Are Ubiquitinated c-Rel
Conjugates--
To confirm that the high Mr
anti-c-Rel-reactive proteins were indeed the ubiquitinated c-Rel,
serum-starved Jurkat cells were stimulated with serum, which induced
the expression of c-Rel, in the presence of a proteasome-specific
inhibitor, LC (Fig. 2a) (17).
The cell lysates were immunoprecipitated with c-Rel-specific antiserum,
and the immune complexes were analyzed by SDS-PAGE followed by
immunoblotting with either c-Rel antiserum (lanes 1-3) or Ub antiserum (lanes 4-6). As
in Fig. 1b, high Mr c-Rel-specific reactivity in a ladder-like pattern was detected after inhibitor treatment for 2 h (lane 3). The similar high
Mr reactivity detected in anti-Ub immunoblotting
(lane 6) further demonstrated that these high
Mr proteins are the bona fide ubiquitin-c-Rel
conjugates. Similar results were also obtained using other proteasome
inhibitors including MG132 and LLnL (also known as ALLN or Calpain
inhibitor I) (data not shown).
We also treated Jurkat cells with the inhibitor LLnL, and measured the
steady state level of c-Rel in Jurkat cells (Fig. 2b). Interestingly, while the high Mr c-Rel proteins
expectedly accumulated during the inhibitor treatment, these high
Mr c-Rel proteins were detected mainly in the
detergent-insoluble fractions (lanes 6-10). This
result suggests that c-Rel is constantly ubiquitinated and that the
ubiquitinated c-Rel tends to accumulate in the noncytoplasmic fractions.
Inhibition of Proteasome Activity Blocks Degradation of c-Rel and
Promotes Accumulation of Ub-c-Rel Conjugates--
To demonstrate that
the normal turnover of c-Rel is regulated by the Ub-Pr pathway, we
treated cells with LLnL to block the proteasome activity and measured
the stability of c-Rel. In cells expressing high levels of NF- c-Rel Is Degraded by the Ub-Pr Pathway in in Vitro Assays--
To
further study the Ub-Pr regulation of c-Rel, a cell-free, in
vitro Ub-Pr degradation assay was used (22, 24). In this assay,
human c-Rel was in vitro transcribed and translated in the
presence of [35S]methionine and used as the substrate.
The degradation can be demonstrated using c-Rel synthesized in either a
reticulocyte lysate (Fig. 4,
lanes 1-6) or a wheat germ (data not shown)
translation system, or using endogenous c-Rel immunoprecipitated from
cell lysates (data not shown). This in vitro degradation of
c-Rel is Ub-Pr pathway-specific because c-Rel was not degraded in
assays performed in the presence of proteasome inhibitors, lactacystin (lanes 7-9), LLnL (lanes
10-12), MG132 (data not shown), or apyrase (an ATP remover)
(data not shown). By contrast, TPCK (lanes
13-15), TLCK (lanes 16-18), and weak
bases (data not shown), inhibitors of chymotrypsin-like proteases,
trypsin-like proteases, and lysosomes, respectively, did not inhibit
the degradation. These results strongly suggest that the degradation of
c-Rel is mediated by the proteasome pathway in the in vitro
system.
C-terminal Sequence of c-Rel Plays an Important Role in
Degradation--
In addition to the full-length 80-kDa c-Rel, a
smaller protein with an apparent molecular mass of 45 kDa (p45) was
also readily degraded in the in vitro assay (Fig. 4,
lanes 1-6; Fig. 5,
lanes 1-4). Presumably, p45 is either a c-Rel
proteolytic fragment generated from the translation product in the
rabbit reticulocyte system, or a smaller c-Rel translation product
resulted from an internal initiation. To characterize p45, we
immunoprecipitated the in vitro reactions (Fig. 5,
lanes 1-4) with antisera specific to either the
N-terminal (lanes 5-8) or the C-terminal
(lanes 9-12) sequence of c-Rel. The C-terminal
specific antiserum precipitated both the full-length and p45 c-Rel,
whereas the N-terminal specific antiserum only precipitated the
full-length c-Rel, indicating that p45 lacks the N-terminal sequence.
This result suggests that the N-terminal domain of c-Rel is not
required in the in vitro degradation of c-Rel, and the
C-terminal domain may play a more important role in mediating c-Rel
degradation. Similar experiments were also performed in avian c-Rel,
and the same conclusion was obtained (data not shown).
To examine the importance of the C-terminal sequences, a series of
C-terminally truncated avian c-rel expression plasmids were
constructed (Table I and Fig.
6a), and used to translate the
corresponding c-Rel proteins in vitro (Fig. 6b).
When these 35S-labeled c-Rel variants were tested in the
in vitro degradation assays (Fig.
7a), interestingly, mutants
with truncations larger than 118 amino acids displayed nearly complete
resistance to degradation. PhosphorImager scanning (Molecular Dynamics,
Inc. Sunnyvale, CA) revealed that about 80, 82, 100, and 100% of the
input substrates remained at the end of the assay for mutants lacking
the C-terminal 171, 231, 275, and 313 amino acids, respectively. In
contrast, only 5, 10, and 25% of the input c-Rel remained for mutants
missing the C-terminal 16, 55, and 118 amino acids, respectively (Fig. 7b). Since c-Rel and v-Rel share sequence similarities only
at the N-terminal 118 amino acids, these results seem to suggest that
the region between residues 118 and 171 of c-Rel, i.e. the c-Rel-specific sequence immediately following the c-Rel/v-Rel homology
domain, plays an important role in mediating c-Rel degradation. To
determine whether the resistance to degradation resulted from a
differential capability of ubiquitination, in vitro
ubiquitin conjugation assays were performed (Fig. 7c).
Although ubiquitinated c-Rel could be detected in all mutants, wild
type, Certain inducers, e.g. bacterial LPS, activate NF- We recently identified a cellular ATPase, the 97-kDa valosin-containing
protein (VCP), that co-purifies with I A feature shared by many Ub-Pr substrates is that the proteins normally
function within multimeric complexes or by interacting with other
macromolecules within the cells. Through the physical association with
other cellular proteins, the substrate is protected from the
proteasome. Recent work suggests that the association of retinoblastoma
protein or adenovirus transforming protein protects E2F-1 from
degradation by the Ub-Pr pathway (38, 39). This raises the possibility
that the regions of the protein targeted for ubiquitination or
degradation are those involved in protein-protein interactions. After
the dissociation of the multimeric complexes, this region is exposed
and targeted for degradation. Indeed, c-Rel is almost always physically
associated with other NF- Many substrates of the Ub-Pr pathway contain the acidic PEST sequences,
which are defined as the regions enriched in proline, glutamate,
serine, and threonine, and are uninterrupted by positively charged
residues (43). The precise mechanism of how a PEST sequence is
recognized by the Ub-Pr system is not known. It could be recognized by
a component of the proteasome or by kinases that function upstream of
the proteolysis. Since PEST sequences are rich in serine/threonine phosphorylation sites, it is also possible that phosphorylation of them
may induce conformational changes that unmask regions required for
proteolysis. We and others previously demonstrated that the C-terminal
PEST sequences of I Our in vitro studies showed that C-terminal sequences of
c-Rel are important for ubiquitination and degradation (Fig. 7). All of
the C-terminal deletion mutants displayed a certain degree of
resistance to in vitro degradation. Interestingly, the
region between residues 118 and 171 of c-Rel, i.e. the
c-Rel-specific sequence immediately following the c-Rel/v-Rel homology
domain, appeared to play an important role in mediating ubiquitin
conjugation and the subsequent degradation. However, based on the low
levels of conjugates detected in Phosphorylation and dephosphorylation have frequently been found
coupled with the Ub-Pr pathways. For example, the degradation of cyclin
and I We thank R.-M. Dai for technical assistance;
Dr. N. Rice for human c-rel expression vector; and Drs. F. Ruscetti, H.-F. Kung, D. Derse, and D. Ferris for reviewing the manuscript.
*
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.
¶
Supported by Council for Tobacco Research Project 4163 and by
NCI, National Institutes of Health, Public Health Service Grant CA33192.
**
To whom correspondence should be addressed. Tel.: 301-846-1478;
Fax: 301-846-6641; E-mail: licc{at}ncifcrf.gov.
The abbreviations used are:
Ub-Pr, ubiquitin-proteasome; Ub, ubiquitin; HTLV-I, human T-cell lymphotrophic
virus; LC, lactacystin; LLnL, N-acetyl-L-leucinyl-L-leucinyl-norleucinal; TPCK, N-p-tosyl-L-phenylalanine
chloromethyl ketone; TLCK, N-
Degradation of Proto-oncoprotein c-Rel by the
Ubiquitin-Proteasome Pathway*
,
,**
Intramural Research Support Program, SAIC
Frederick, NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, the § Department of Microbiology
and Institute for Cellular and Molecular Biology, University of Texas,
Austin, Texas 78712, and the NIA, National Institutes of Health,
Gerontology Research Center, Baltimore, Maryland 21224
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B transcription factor family, which
regulates a large variety of cellular functions. The activation of
NF-B involves the degradation of the inhibitor, IB, through the
ubiquitin-proteasome (Ub-Pr)-mediated pathway. Here we report that the
turnover of c-Rel is also regulated by the Ub-Pr pathway, thus adding
another level of complexity to the regulation of NF-B. High
molecular weight ubiquitinated c-Rel conjugates are detected in cells
and accumulate in cells treated with proteasome inhibitors. In a
cell-free in vitro degradation assay, c-Rel is degraded
specifically through the Ub-Pr pathway. N-terminally truncated c-Rel is
readily degraded, implying the dispensability of N-terminal sequence;
in contrast, a series of deletion mutants missing C-terminal sequences
display a reduced susceptibility to the degradation. Interestingly, the
sequence between residues 118 and 171 of c-Rel, i.e. the
region immediately following the c-Rel/v-Rel homology domain, appears
to play an important role in mediating ubiquitin conjugation and the
subsequent degradation. Together with our previous study showing an
elevated tumorigenic potential for C-terminally truncated mutants, our data suggest that the C-terminal domain of c-Rel plays an important role in mediating c-Rel degradation and growth control.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B transcription factor family
(for recent reviews, see Refs. 1-6). NF-B is highly regulated and
is activated by a large variety of stimuli, such as cytokines, growth
factors, oxidants, UV irradiation, infections, and physical or
oxidative stress. NF-B plays a pivotal role in the regulation of
many genes involving immune and inflammatory responses, healing and
regeneration processes, and embryogenesis. This family of proteins
share sequence homology in the N-terminal Rel homology region, which is
responsible for DNA binding, dimerization, and inhibitor association.
c-Rel is expressed at high levels in hematopoietic cells and regulates
the expression of different genes in B and T cells that are crucial for
cell division, apoptosis, and immune functions. Although
c-rel knockout mice develop normally, they exhibit defects
in lymphocyte proliferation, humoral immunity, and interleukin-2
expression (7). In most cells, Rel/NF-B proteins serve an
antiapoptosis function, and overexpression of c-Rel results in cellular
transformation (reviewed in Ref. 8). However, in developing chicken
embryos and in chicken bone marrow cells, high levels of c-Rel
expression are associated with programmed cell death (9). Despite these
seemingly contradictory reports, regulation of c-Rel expression
undoubtedly plays an important role in maintaining normal growth and
development. Alterations or amplification of the rel locus
have been found in human lymphomas (reviewed in Ref. 10), and viral
transduction of c-rel resulted in v-rel, the
transforming gene of the reticuloendotheliosis virus, strain T (REV-T)
(4, 11). REV-T is a highly virulent retrovirus that induces a fatal
lymphoid leukemia in galliform birds within 7-10 days after infection.
The v-Rel protein differs structurally from c-Rel in that v-Rel lacks
the N-terminal two amino acids and the C-terminal 118 amino acids that
are normally present in c-Rel. Instead, v-Rel has viral env
sequences at both termini and contains multiple amino acid
substitutions and small in-frame deletions in the middle (11, 12).
Recently, transgenic mice overexpressing v-Rel were shown to develop
aggressive T-cell lymphoma/leukemia, further demonstrating the
oncogenic potential of v-Rel in mammals (13).
B also involves the Ub-Pr degradation pathway. In
the absence of inducers, the NF-B dimeric transcription factor is
sequestered in the cytoplasm through physical association with a member
of the IB family of inhibitor proteins. In response to extracellular
stimuli, IB
(the best studied inhibitor) is phosphorylated,
ubiquitinated, and degraded by the proteasome, thus releasing the
NF-B dimer for translocation into the nucleus. NF-B inhibitors,
including p105 (21, 22), p100, IB (23-25), IB
(26), and
IB
(27), were shown to be subject to Ub-Pr-mediated degradation.
In this study, we demonstrate that the degradation of the NF-B
family protein, c-Rel, also involves the Ub-Pr pathway, and C-terminal
sequence of c-Rel plays an important role in the degradation of
c-Rel.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-p-tosyl-L-lysine chloromethyl
ketone (TLCK), and iodoacetamide (an isopeptidase inhibitor) were
purchased from Sigma.
118, stop codon was
introduced by oligonucleotide site-directed mutagenesis, in which the
mutagenic oligonucleotide (5'-CGTGAACATGTAGACCAATGAC-3') and the
MutaGene in vitro mutagenesis kit (Bio-Rad) were used. Sequences around the insertion sites and sequences subjected to site-directed mutagenesis were verified by sequencing. Plasmids designated pc-relX (X indicates the number of deleted amino acids) were used in the in vitro transcription and translation for
further analyses.
X proteins were synthesized with
[35S]methionine in a reticulocyte lysate-based in
vitro transcription/translation system (Promega). The
35S-labeled proteins were purified away from the free
[35S]methionine by concentrating in Centricon 30 (Amicon)
and used as the substrates in the in vitro degradation and
conjugation assays (24, 34), in which S100 extracted from human B cell line CA46 or avian DT95 was used as the enzyme source. For degradation assays, master reaction mixture containing substrate (1 µl/time point), dialyzed Ub (6 µg/time point), 12 mM Tris-HCl, pH
7.5, 60 mM KCl, 3.5 mM MgCl2, 5 mM CaCl2, 1 mM dithiothreitol, and 1 mM ATP was prepared and aliquoted into separate tubes on
ice. At each time point, 50 µg of S100 was added to individual tubes to start the reaction at 37 °C, and the final volume of each
reaction was adjusted to 50 µl. All of the reactions were
simultaneously terminated by boiling in SDS-sample buffer, analyzed by
SDS-PAGE followed by Western transfer and autoradiography. For
conjugation assays, reactions were carried out as in degradation assays
except that okadaic acid (1 µg/ml), proteasome inhibitor LLnL (100 µM), and isopeptidase inhibitor iodoacetamide (10 mM) were included (34).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B is constitutively activated and c-Rel is highly induced in
both HTLV-I-infected T cells and B-lymphoma cells (28, 36, 37).
Immunoblotting analyses showed that, in addition to the 80-kDa
full-length c-Rel, high Mr proteins were
detected in both the cytoplasmic and the nuclear lysates of
HTLV-I-infected cells (Fig.
1a). These high Mr proteins were reactive to a number of c-Rel
antisera (one such experiment is shown in Fig. 1a,
lanes 1-6) but not to the corresponding preimmune sera (data not shown) or the immune sera preincubated with
the competing peptides (lanes 7 and
8). To further characterize these c-Rel proteins, cell
lysates from a B cell lymphoma cell line were immunoprecipitated with
c-Rel-specific antiserum in the absence (Fig. 1b,
lane 1) or presence (lane
2) of the competing peptide, and the immune complexes were
separated and analyzed by immunoblotting with c-Rel antiserum.
Reactivities in a ladder-like pattern, indicative of multiubiquitin
conjugates, were detected only in the former (lane
1), suggesting that c-Rel may be subject to ubiquitin
modification.
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Fig. 1.
Detection of high Mr
c-Rel proteins in cells. a, HTLV-I-infected cells,
MT-2, HUT102, and 81-66, were fractionated to nuclear (N)
and cytoplasmic (C) fractions (29). An equal amount (80 µg) of protein from each fraction was resolved by SDS-PAGE,
Western-transferred onto membrane, and immunoblotted with anti-c-Rel
1135 serum (lanes 1-6). As a control, lysates of
81-66 cells were immunoblotted with the same antiserum in the presence
of competing peptide (lanes 7 and 8).
In lanes 7 and 8, the residual 80-kDa
reactivity is a result of incomplete competition, and the undiminished
reactivity detected slightly larger than 60 kDa is nonspecific
(ns). The positions of the full-length 80-kDa and the
truncated 64- and 60-kDa c-Rel-specific reactivity are indicated on the
left. The mobilities of prestained molecular weight
standards (in kDa) are marked between lanes 6 and
7. b, human DB B-lymphoma cells were lysed, and
the lysates were immunoprecipitated with anti-c-Rel 1135 serum in the
absence (lane 1) or presence (lane
2) of the competing peptide. The immune complexes were
subjected to SDS-PAGE, Western transfer, and immunoblotting with
anti-c-Rel 1135 serum. The position of the full-length 80-kDa c-Rel is
indicated. The mobilities of prestained molecular mass standards (in
kDa) are marked on the right.
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Fig. 2.
Identification of ubiquitinated c-Rel
proteins and detection of ubiquitinated c-Rel in Nonidet P-40-insoluble
fractions in proteasome inhibitor-treated cells. a, human
Jurkat T cells were cultured in growth medium with 0.5% serum for
16 h and then stimulated with 15% serum in the presence of LC for
0, 0.5, and 2 h. Cells were lysed, and the lysates were
immunoprecipitated with anti-(c-Rel 1135) serum. The immune complexes
were separated into two aliquots, and each was analyzed by SDS-PAGE,
Western transfer, and immunoblotting (IB) with either
anti-c-Rel 1135 (lanes 1-3) or anti-Ub
(lanes 4-6) serum. The full-length c-Rel and the
ubiquitinated c-Rel (Ubn-c-Rel) are
indicated. The heavy bands at the
bottom of the gel represent Ig reactivities. b,
Jurkat cells were treated with proteasome inhibitor, LLnL, for up to
20 h. The cells were lysed, and the lysates were separated to
Nonidet P-40-soluble and -insoluble fractions. The cleared Nonidet
P-40-soluble lysates were immunoprecipitated with anti-c-Rel serum
(Upstate Biotechnology) (lanes 1-5), separated
on SDS-gel with the Nonidet P-40-insoluble fractions (lanes
6-10), and Western blotted with c-Rel antiserum (Santa Cruz
Biotechnology).
B,
e.g. B cells and certain T cells, c-Rel is often associated
with other NF-B or IB family proteins, which may provide c-Rel
physical protections against proteosomal degradation. To avoid this
physical protection, we transiently overexpressed avian c-Rel in NIH3T3
cells, which have a low NF-B endogenous expression, treated the
cells with LLnL, and analyzed the total cell lysates. Similar to Fig.
2, immunoblot using c-Rel-specific antiserum revealed that the 80-kDa
c-Rel was stabilized and that the high Mr
Ub-c-Rel conjugates accumulated in a time-dependent fashion
(Fig. 3a). Furthermore, we
overexpressed avian c-Rel in monkey COS cells and performed pulse-chase
experiments in the presence or absence of the proteasome inhibitor,
LLnL (Fig. 3b). Cells collected from each time point were
lysed by boiling in SDS-containing buffer, and the lysates were
immunoprecipitated with c-Rel antiserum. Although c-Rel is relatively
stable with an estimated half-life of slightly longer than 9 h in
untreated cells (lanes 2-7), the half-life of
c-Rel in LLnL-treated cells is considerably longer than 9 h
(lanes 8-13). It was noted that a decrease of
c-Rel was detected at 20 h (lane 12) in
treated cells, similar to that in the untreated cells (lane
6). However, this decrease was largely due to the cell death
resulting from the long period of inhibitor treatment. More than 50%
of the cells were dead after 20 h of LLnL treatment (data not
shown). Importantly, the level of c-Rel in inhibitor-treated cells is
significantly higher than that in the untreated cells at every time
point (compare lanes 8-13 with lanes
2-7, respectively), indicating that LLnL is capable of
stabilizing c-Rel and inhibiting the normal degradation of c-Rel.
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Fig. 3.
Accumulation of ubiquitinated c-Rel
conjugates in cells treated with proteasome inhibitors.
a, NIH3T3 cells were transiently transfected with plasmid
expressing avian c-rel and treated with LLnL for up to
16 h. Cell lysates from each time point were separated by SDS-PAGE
and Western blotted with a combination of c-Rel antisera (V1, V2, and
V3). The unmodified c-Rel was indicated. b, COS cells were
transfected with plasmid expressing avian c-rel,
pulse-labeled with [35S]methionine/cysteine for 2 h
in the absence or presence of LLnL and then chased in fresh media in
the absence (lanes 2-7) or presence of LLnL
(lanes 8-13) for the indicated periods of time.
Cells were lysed by boiling in radioimmune precipitation buffer
containing 0.5% SDS; the lysates were then diluted and
immunoprecipitated with anti-c-Rel sera (combination of V1 and sc-70).
Lane 1 represents the same immune complex
isolated from control COS cells.
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Fig. 4.
Proteasome-specific degradation of c-Rel in
in vitro assays. 35S-Labeled in
vitro translated human c-Rel protein was subjected to the
cell-free in vitro degradation assay without inhibitors
(lanes 1-6) or with one of the following
inhibitors, 10 µM LC, 50 µM LLnL, 100 µM TPCK, and 100 µM TLCK. All reactions
were terminated at the indicated times (in min) by boiling in SDS gel
loading buffer and analyzed by SDS-PAGE, Western transfer, and
autoradiography. The mobility of the full-length c-Rel is
indicated.
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Fig. 5.
In vitro degradation of N-terminally
truncated 45-kDa c-Rel. The labeled human c-Rel was subjected to
the in vitro degradation assay as described in Fig. 4. The
reaction at each time point was separated into three parts; one part
was used for gel analysis (lanes 1-4), and the
other two parts were boiled in radioimmune precipitation buffer
containing 0.5% SDS and then diluted and immunoprecipitated with the
N-terminal-specific anti-c-Rel serum (sc-70) (lanes
5-8), or the C-terminal-specific anti-(c-Rel 265) serum
(lanes 9-12).
16, 55, and 118 c-Rel variants appeared to have
significantly higher conjugation efficiency than 171, 231,
275, and 313. These results are consistent with the degradation
data and are compatible with the notion that the sequences between
residues 118 and 171 of c-Rel may be the major ubiquitination sites
or/and important for recognition by the ubiquitination machinery.
Construction of expression vectors for synthesis of C-terminally
truncated variants of c-Rel
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Fig. 6.
Schematic diagram of wild type and
C-terminally truncated c-Rel mutants and the corresponding in
vitro translation products. a, the Rel homology
region (RHR) consists of two domains, D1 and D2, and
contains five loops that contact DNA directly (black
boxes). The region of c-Rel sufficient and necessary for
inhibition of DNA binding by I B is shown (INHIBITION BY
IB). The C terminus of c-Rel is composed of three different
subregions: the positively charged domain (waved
line pattern) containing the nuclear localization
signal (NLS) and transactivation domains I and II. The
protein structures of the C-terminally truncated mutant Rel proteins
derived from c-Rel are shown below. b, wild-type
avian c-Rel and C-terminally truncated c-Rel mutants were synthesized
with [35S]methionine in a coupled in vitro
transcription/translation reticulocyte system. The translation products
were resolved by SDS-PAGE and then transferred onto membrane and
visualized by autoradiography. The mobilities of the prestained
molecular mass standards (in kDa) are indicated on the
left.
View larger version (55K):
[in a new window]
Fig. 7.
In vitro degradation and in vitro
conjugation assays of the wild type and C-terminally truncated
avian c-Rel proteins. a, wild-type and C-terminally
truncated mutants of c-Rel were analyzed by in vitro
degradation assays as described under "Materials and Methods."
Degradation is assessed by the reduction of the input substrate.
b, the substrates remained at the end of the degradation
assays were determined by PhosphorImager analysis, and plotted as
percentage of the input substrates. c, wild-type and
C-terminally truncated mutants of c-Rel were analyzed by in
vitro conjugation assays as described under "Materials and
Methods." Conjugation is assessed by the increase of high molecular
weight forms of the input substrates.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B
with biphasic kinetics, an early transient phase and a late persistent phase (1-6). In the early phase, rapid degradation of IB and concomitant nuclear translocation of the activated NF-B are detected within minutes after stimulation. It is transient, because the induced
NF-B activity up-regulates the synthesis of IB, which subsequently inhibits the NF-B activity itself. The late phase activation constitutes a prolonged induction of NF-B over hours and
even days after stimulation. This prolonged activation results mainly
from the degradation of IB and an elevated level of the transcription factor. Activation of both early and late phases has been
shown to be regulated, at least in part, by the Ub-Pr pathway.
Specifically, when cells are stimulated, the IB molecules undergo
site-specific phosphorylation and ubiquitination and then are degraded
by the 26 S proteasome. The released dimeric NF-B enters the nucleus
and induces expression of the target genes. In the present study, we
report that in addition to IB molecules, the transcription factor
c-Rel is also regulated by the Ub-Pr pathway. It is not likely that the
c-Rel contained in the latent NF-B-IB trimeric complex is
degraded along with IB in the early phase induction. Otherwise, a
significant diminution of c-Rel would be detected, and a concomitant
nuclear translocation of c-Rel would not take place. Our results show
that when Jurkat cells were treated with the proteasome inhibitors,
Ub-c-Rel conjugates accumulated in a time-dependent manner.
This was detected in the presence (Fig. 2a) and the absence
(Fig. 2b) of the extracellular inducers. We conclude that
the basal turnover of c-Rel is mediated by the Ub-Pr pathway, and this
level of regulation most likely is involved in the late phase of
NF-B activation.
B immune complexes and the
26 S proteasome (34). IB-VCP binding requires the phosphorylation
and ubiquitination of IB and in turn is required for the
degradation of IB. Because VCP was readily detected in IB
immune complexes, but not in c-Rel or RelA immunoprecipitates, we
hypothesize that the binding of VCP to the ubiquitinated IB disrupts the NF-B-IB trimeric complex, thus liberating the NF-B dimer. The VCP-bound IB is then transferred to the
proteasome for degradation (34). Our failure to detect VCP in c-Rel
immune complexes suggests that binding to VCP is not a prerequisite for Ub-Pr-mediated degradation of c-Rel. However, we could not completely rule out the possibility that this is because Ub-c-Rel conjugates tend
to be detergent-insoluble (Fig. 2b), resulting in a low
abundance of Ub-c-Rel in the lysates. In addition, this could result
from low sensitivity of the available antisera to recognize the
ubiquitin-modified c-Rel. The observations that Ub-IB molecules
are found mostly in the cytoplasm, whereas Ub-c-Rel conjugates are
mainly detected in Nonidet P-40-insoluble noncytoplasmic fractions, may
suggest a different location for c-Rel degradation. It appears that
although both IB and c-Rel are regulated by Ub-Pr pathways, there
may exist significant differences between the molecular mechanisms involved.
B components, through which c-Rel is
stabilized and exhibits a relatively long half-life (15 h in DB cells;
data not shown). However, when c-Rel is transiently expressed in NIH3T3
(data not shown) or COS (Fig. 3b) cells, which have a low
level of endogenous NF-B, the half-life is reduced. Similarly, it
was reported that the inhibitor IB has a short half-life when
expressed in its free state but is significantly stabilized when
complexed with RelA or c-Rel (40-42).
B are required for its degradation (24, 44,
45). C-terminally truncated IB is resistant to the Ub-Pr
degradation and acts as a dominant mutant. Moreover, the C-terminal
PEST sequence of NF-B-1 p105 also plays an important role in its
processing; it was shown by MacKichan et al. (46) that
phosphorylation of the PEST sequences of p105 up-regulates the
proteolytic processing of p105 into the active p50. Examination of
human c-Rel sequence (47) revealed that the C-terminal region of the
molecule is highly enriched in PEST sequences. In the C-terminal 80 amino acids, no arginine or lysine is found, and 30 out of 80 residues
are PEST sequences. A similar observation was also made in avian c-Rel
protein (35, 48).
275 and 313, this region is
probably not the absolute requirement for conjugation. The biological
significance of a C-terminal deletion of c-Rel was first documented by
the observation that removal of 55 C-terminal amino acids of c-Rel significantly enhanced its ability to transform splenic cells in
vitro (35, 49). In an attempt to determine the in vivo evolution of c-rel oncogenic potential, we previously
inserted c-rel into an REV-T-based retroviral vector and
infected 1-day-old chicks (50). All birds developed tumors, and all
cell lines established from the tumors expressed c-Rel proteins that
lacked C-terminal sequences. These truncated proteins are probably
responsible for both in vivo and in vitro cell
proliferation and are selected for their oncogenic potential. Our
present finding of an elevated resistance to degradation in
C-terminally truncated c-Rel mutants seems to suggest a correlation
between oncogenic transformation and a reduced susceptibility to the
Ub-Pr-mediated degradation. Our preliminary data showing a much reduced
in vitro degradation of v-Rel (data not shown) is consistent
with this contention. In agreement with these findings, deletion of the
PEST sequences in c-Fos resulted in oncogenic v-Fos, which has a much
reduced susceptibility to Ub-Pr-mediated degradation (51).
B requires an upstream phosphorylation event, and the
phosphorylation of c-Jun targets the degradation of c-Fos in AP-1
complexes (51, 52). On the other hand, phosphorylation of Ser3 of c-Mos
prevents its degradation (53). Presently, we do not know whether
phosphorylation of c-Rel is required for the Ub-Pr degradation. It has
been reported that c-Rel is highly phosphorylated immediately after
NF-B stimulation (32, 54), and a protein kinase A phosphorylation
site toward the C-terminal end of the Rel homology domain is essential
for c-Rel activation (55). It would be interesting to determine whether
the phosphorylation-defective c-Rel mutants are regulated by the Ub-Pr pathway.
ACKNOWLEDGEMENTS
FOOTNOTES
-p-tosyl-L-lysine chloromethyl
ketone; PAGE, polyacrylamide gel electrophoresis; VCP, valosin-containing protein.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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