From the Intramural Research Support Program, SAIC
Frederick, NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, § NIA, National Institutes of
Health, Gerontology Research Center, Baltimore, Maryland 21224, and
the ¶ Department of Biochemistry, University of Utah School of
Medicine, Salt Lake City, Utah 84132
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ABSTRACT |
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The inactivation of the prototype NF-B
inhibitor, I
B
, occurs through a series of ordered processes
including phosphorylation, ubiquitin conjugation, and
proteasome-mediated degradation. We identify valosin-containing protein
(VCP), an AAA (ATPases associated with a
variety of cellular activities) family member, that
co-precipitates with I
B
immune complexes. The ubiquitinated
I
B
conjugates readily associate with VCP both in vivo
and in vitro, and this complex appears dissociated from
NF-
B. In ultracentrifugation analysis, physically associated VCP and
ubiquitinated I
B
complexes sediment in the 19 S fractions, while
the unmodified I
B
sediments in the 4.5 S fractions deficient in
VCP. Phosphorylation and ubiquitination of I
B
are critical for
VCP binding, which in turn is necessary but not sufficient for I
B
degradation; while the N-terminal domain of I
B
is required in all
three reactions, both N- and C-terminal domains are required in
degradation. Further, VCP co-purifies with the 26 S proteasome on
two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and
functional link between I
B
and the 26 S proteasome and play an
important role in the proteasome-mediated degradation of I
B
.
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INTRODUCTION |
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Transcription factor NF-B is involved in a large variety of
processes, such as cell growth, transcriptional regulation, immune, inflammatory, and stress responses (reviewed in Refs. 1-4). NF-
B is
a homo- or heterodimer consisting of various combinations of the family
members, including NF
B1 (p50 and precursor p105), c-Rel, RelA,
NF
B2 (p52 and precursor p100), RelB, and Drosophila proteins Dorsal and Dif. Unlike many other transcription factors that
are localized in the nucleus, the NF-
B dimeric factor is sequestered
in the cytoplasm of most cells through binding to a family of inhibitor
proteins, I
B. In response to extracellular stimuli, the inhibitors
are partially or entirely degraded, thus liberating the DNA-binding
dimer for translocation to the nucleus. The I6B family contains
I
B
, I
B
, I
B
, Bcl-3, the precursor proteins p105 and
p100, and the Drosophila protein Cactus. All members of the
I
B family contain 5-8 ankyrin motifs, thought to be involved in
protein-protein interactions. It has been shown that when the precursor
protein p105 is involved as the inhibitor, the processing from p105 to
the active p50 occurs through the ubiquitin-dependent
proteasome (Ub-Pr)1 pathway,
which degrades the C-terminal ankyrin-containing domain of p105 (5,
6).2 For the prototype
complex that contains p50, p65, and I
B
, upon stimulation the
entire NF-
B complex becomes hyperphosphorylated. The induced
phosphorylation of I
B
does not lead to its immediate dissociation
from the complex; rather, it signals for rapid I
B
degradation,
thus liberating the active p50·p65 dimer for translocation to the
nucleus (7-15). We and others showed that the degradation of I
B
is sensitive to proteasome inhibitors and is
ubiquitin-dependent. Recently, it was further shown that
signal-induced phosphorylation precedes I
B
degradation and
targets I
B
to the Ub-Pr pathway (7-15). It was proposed that the
inactivation of I
B
occurs through a series of ordered processes
including phosphorylation, ATP-dependent multiple ubiquitin
conjugation, and proteasome-mediated proteolysis. However, the link
between ubiquitination and proteasome-mediated degradation remains
unclear.
The extralysosomal, energy-dependent Ub-Pr pathway is a
major mechanism used to regulate many critical cellular proteins that must be rapidly destroyed for normal growth and metabolism (reviewed in
Refs. 16-21). The rapidly growing list of the substrates for the Ub-Pr
pathway includes mitotic cyclins, G1 cyclins,
cyclin-dependent kinase inhibitors p27, Sic1 protein,
proto-oncogene products p53, c-Myc, c-Jun, and c-Mos, NF-B
inhibitors, yeast MAT
2 transcription factor, major
histocompatibility complex molecules, and others. The Ub-Pr proteolytic
pathway is ATP-dependent and present in both cytoplasm and
nucleus. The pathway consists of two distinct, sequential steps. The
target protein is first conjugated with multiple ubiquitin (Ub)
molecules that mark the substrate for destruction. The Ub-tagged target
is then translocated to (probably with the help of molecular
chaperones) and degraded by a large protease complex with an apparent
sedimentation coefficient of 26 S. The 26 S proteasome is a
multisubunit complex, consisting of a central cylinder-shaped 20 S
multicatalytic proteinase core and a 19 S cap-like regulatory complex
attached to each end of the cylinder. The terminal cap structure
consists of at least 18 distinct subunits with molecular masses of
35-110 kDa and has ATPase and ubiquitin conjugate binding activities.
It is presumed that ATP hydrolysis by the 19 S complex provides the
energy needed for the chaperoning and unfolding of substrates degraded
within the proteasome cylinder.
VCP (22-24), the mammalian homolog of the cell division cycle protein Cdc48p in yeast and p97 in Xenopus, is a member of a recently identified AAA family (reviewed in Ref. 25). The family members are characterized by having ATPase domain(s) with striking sequence similarities and having ring structures consisting of homooligomers. Despite the high sequence and structural homology, these proteins unexpectedly are implicated in a large variety of seemingly unrelated biological functions. These functions reviewed in Ref. 25 include control of cell division cycle, T cell activation (23, 24), membrane fusion (26-29), and vesicle-mediated transport, peroxisome assembly and biogenesis, gene expressions, and the Ub-Pr degradation pathways (30). Furthermore, at least six family members have been identified as subunits of the 26 S proteasome (subunits 4, 6, 7, 8, 10, and TBP-1) (31-36), and it is likely that other family proteins will fill this function as well (31, 37). The apparent paradox between the striking sequence homology and the large diversity of functions in this family suggests that these proteins have a basic and critical role in cellular function, and the involvement in many other functions may be indirect.
During the course of studying the molecular mechanisms involved in
IB
degradation, we detected VCP physically associated with
I
B
complexes both in vivo and in vitro. In
this report, we demonstrate that VCP is involved in the
proteasome-mediated degradation of I
B
, and VCP is co-purified
with the 26 S proteasome. Consistent with this hypothesis, VCP indeed
has in vitro ATPase activity and an apparent sedimentation
coefficient of 19 S (Ref. 24 and this study), the same as that of the
regulatory complex of the 26 S proteasome. We propose that physical
association of VCP with Ub-tagged I
B
targets I
B
to the
Ub-Pr pathway.
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MATERIALS AND METHODS |
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Cell Lines and Culture Conditions-- Two interchangeably used human B cell lines, DB (38) and CA46 (39), were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin.
Immunoprecipitation (IP) and Western (Immuno-) Blot Analyses-- Both analyses were performed as described previously (9, 40, 41) with minor modifications. Cells were labeled with 0.1 mCi/ml (1000 Ci/mmol) [35S]methionine/cysteine (ICN) at a density of 5 × 106/ml for approximately 16 h, washed twice with phosphate-buffered saline, and lysed in RIPA buffer (20 mM Tris/HCl, pH 7.6, 2 mM EDTA, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100) containing protease inhibitors (1% aprotinin, 70 µg/ml phenylmethanesulfonyl fluoride, 40 µg/ml Tos-Phe-CH2Cl, 5 µg/ml Tos-Lys-CH2Cl, 5 µg/ml leupeptin). The lysates were clarified by centrifugation at 12,000 × g for 30 min and incubated with antisera. The immune complexes were collected with Protein A-Sepharose beads, washed with RIPA buffer, boiled, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electrophoretically transferred onto polyvinylpyrrolidone membranes, and analyzed by autoradiography. IPs shown in Figs. 1A and 2B were conducted in Buffer I (1% Nonidet P-40, 1% bovine serum albumin in phosphate-buffered saline) containing protease inhibitors, 100 µM calpain inhibitor I, and 10 mM iodoacetamide, and the precipitates were washed with Buffer I without bovine serum albumin. For reimmunoprecipitation experiments, the immune complexes isolated from the first IP were boiled in RIPA containing 1% SDS, diluted, and subjected to the second IP. For immunoblot analysis, equal amounts of protein or immune complexes were resolved by SDS-PAGE and transferred onto membranes. The membrane was blocked, washed, and incubated with antiserum (typically at 1:3,000), followed by reaction with peroxidase-conjugated anti-rabbit immunoglobulin serum (Boehringer Mannheim), and developed by the enhanced chemiluminescence detection system (ECL) (Amersham Corp.). When serial blotting analyses were performed, previous reactivity was stripped off from the membrane.
Antisera--
All antisera used in this study are polyclonal
rabbit sera. Anti-IB
-1 and anti-I
B
-2 (40, 9) were
independently generated against synthetic peptide corresponding to
residues 300-317 of the human I
B
sequence (42). Anti-I
B
-3
(SC-847) and -5 (SC-371) were purchased from Santa Cruz Biotechnology,
Inc., and anti-I
B
-4 (06-494) was purchased from Upstate
Biotechnology, Inc. Anti-VCP-1 and anti-VCP-2, raised against peptides
of residues 792-806 and 20-40 of murine VCP (23), respectively, were
kindly provided by L. Samelson (NIH). Anti-VCP-3, -4, -5, -6, -7, and
-8 were generated against glutathione S-transferase
(GST)-VCP (full-length) and residues 792-806, 721-734, 184-197,
240-253, and 167-180 of murine VCP (23), respectively. Anti-Ub
polyclonal antisera were purchased from either Sigma or Biogenesis or
obtained from M. Rechsteiner (University of Utah, Salt Lake City, Utah)
(43). Antisera to subunits 4 and 5 and to the 26 S proteasome were
kindly provided by M. Rechsteiner (43, 44).
Protein Sequencing-- Well resolved protein bands were sliced from Coomassie Blue-stained gel and subjected to an in-gel partial V8 digestion (45). The proteolytic fragments were resolved by 15% SDS-PAGE, transferred onto a polyvinylpyrrolidone membrane, and stained with Coomassie Blue. The well separated bands were sliced and subjected to N-terminal peptide sequencing using an ABI 494A sequencer.
In Vitro Association--
IB
(7) or VCP (23) was
synthesized in a coupled in vitro transcription and
translation reaction from a rabbit reticulocyte lysate system (Promega)
in the presence of [35S]cysteine (for I
B
) or
[35S]methionine (for VCP). GST-I
B
expression
plasmid was constructed by inserting the EcoRI fragment of
I
B
(7) into pGEX-4T-2 vector (Pharmacia Biotech Inc.) (9). The
GST-VCP (24) or GST-I
B
fusion proteins were prepared according to
the manufacturer (Pharmacia). Glutathione-Sepharose beads containing
GST or GST fusion proteins were mixed with 35S-labeled
in vitro translated proteins or unlabeled B cell lysates in
a total volume of 225 µl of binding buffer (35 mM Tris,
pH 7.6, 50 mM NaCl, 0.1% Nonidet P-40, 0.5 mM
dithiothreitol including protease inhibitors). After incubation for
2 h at 4 °C and three washes with binding buffer, the bound
products were analyzed by SDS-PAGE followed by Western transfer,
autoradiography, and immunoblotting.
Glycerol Gradient Sedimentation Centrifugation-- Velocity sedimentation centrifugation was carried out in 10-40% glycerol gradients in a total volume of 13 ml of 12 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM CaCl2, 1 mM MgCl2. Freshly prepared CA46 cell lysate in a volume of 200 µl was loaded onto the gradient and centrifuged at 4 °C, in an SW41 rotor at 36,000 rpm for 16 h. Fractions of 0.5 ml were collected and analyzed by immunoblotting or IP. Protein markers were centrifuged in separate tubes and included thyroglobulin (19 S), immunoglobulin G (7 S), and bovine serum globulin (4.5 S).
Proteasome Purification--
Cytosolic fractions were prepared
by lysing cells in a hypotonic buffer (5 mM Hepes, pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) on ice for 10 min. The lysates were
clarified by low speed centrifugation for 10 min. The supernatant was
further centrifuged at 100,000 × g for 1-2 h, and the
supernatant, S100, was stored in aliquots at 70 °C.
Proteasome-enriched (Pr+), and proteasome-depleted
(Pr
) fractions were extracted from CA46 cells as
described by Palombella et al. (5). The highly purified (26 S-1) and the partially purified (26 S-2) proteasomes were isolated from
human red blood cells as described (46).
IB
Mutants--
Deletion mutants of I
B
encoding
amino acids 37-317, and 1-242 were constructed as described by
Brockman et al. (10). The deletion mutant encoding amino
acids 23-317 was constructed by polymerase chain reaction using
specific oligonucleotide primers (5
-GGGAAACTTCTCGTCCGCGCCATGCGGCTACTGGACGACCGC-3
and
5
-GGTCTAGATCATAACGTCAGACGCTGGCCT-3
) and the wild-type I
B
cDNA (7) as a template. Amplified product was purified and cloned
into pCRII vector (TA cloning kit, Invitrogen). The site-specific
mutants, S32A/S36A and K21R/K22R, were kindly provided by D. Ballard (12).
In Vitro Assays--
The wild-type and mutants of IB
were
synthesized in reticulocyte lysate-based in vitro
transcription/translation system (Promega) in the presence of
[35S]cysteine and used as the substrates in the Ub-Pr
degradation assay (9). S100 was extracted from CA46 cells (8) and used as enzyme source. Master reaction mixture containing 5 µl of
substrate, 8 µg of dialyzed ubiquitin (Sigma), 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 four tubes on ice. At various time points, 50 µg of S100 was
added to individual tubes to start the reaction at 37 °C. The final
volume in each reaction was adjusted to 50 µl. All of the reactions
were simultaneously terminated by boiling and analyzed by SDS-PAGE
followed by Western transfer and autoradiography. The ubiquitinated
I
B
(Ub-I
B
) conjugates were generated by slightly modified
Ub-conjugation assays (6, 8), which were essentially the same as the
degradation assay except that 1 µg/ml okadaic acid, 1 mM
ATP
S, and 100 µM calpain inhibitor I were included
in the reaction, and the reactions were carried out for 90 min.
Two-dimensional Gel Electrophoresis-- Two-dimensional gel electrophoresis analysis was carried out as described previously (44). The highly purified 26 S proteasome isolated from human red blood cells was resolved by two-dimensional isoelectric focusing followed by SDS-gel electrophoresis.
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RESULTS |
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VCP and High Mr Ub-IB
Are Co-immunoprecipitated
in Vivo--
IP performed on cell lysates containing active NF-
B
detected I
B
, p50, p65, and an unidentified 90-kDa cellular
protein (p90) (Refs. 9 and 40; Fig.
1A, top,
lanes 1-4). p90 was detected in complexes precipitated by
I
B
antisera from various sources and was not detected by the
preimmune serum (Ref. 9; Fig. 1C, top,
lanes 1 and 6) or the anti-I
B
immune serum
preincubated with the immunogenic peptide (40). When the p90-containing
I
B
complex was subjected to the in vitro Ub-Pr
degradation assay, unlike I
B
molecules, p90 was resistant to
degradation (9). To identify p90, I
B
immune complexes were
resolved on SDS-polyacrylamide gel, and the p90 band was visualized by
Coomassie Blue staining, excised, and subjected to N-terminal peptide
sequencing. Although the N terminus of p90 was blocked, peptide
sequencing of two of the V8 proteolytic fragments of p90 revealed a
total of 20 residues identical to those of the murine
valosin-containing protein, VCP (23) (Fig. 1B). IP followed
by immunoblotting with either C terminus-specific anti-VCP-1 or N
terminus-specific anti-VCP-2, kindly provided by L. Samelson (National
Institutes of Health, Bethesda, MD), confirmed that the p90 was VCP
(Fig. 1A, bottom). VCP was co-precipitated with
I
B
in nondenatured lysate (Fig. 1C, lanes
2), but not in boiled lysate (lanes 7), indicating that VCP physically associates with an I
B
-containing complex. When the
reciprocal IP was performed, however, virtually no I
B
was detected in VCP immune complexes (lanes 3, 5,
8, and 10). Although a protein with a similar
size to that of I
B
was detected in VCP-2 immune complexes (Fig.
1C, top, lanes 5 and 10),
it was not I
B
because it did not react with the antiserum
specific to the C terminus of I
B
(bottom, lanes
5 and 10). The lack of 36-kDa I
B
in the anti-VCP
IP was initially unexpected. However, since VCP is resistant to
degradation in Ub-Pr assays (9) and is an ATPase, a common
characteristic of molecular chaperones, we hypothesize that VCP is a
molecular chaperone that only associates with I
B
molecules that
have been modified by ubiquitination. Therefore, the 36-kDa I
B
would not be detected in VCP complexes. Based on this hypothesis, two
predictions were made: 1) in addition to 36-kDa I
B
and VCP,
anti-I
B
antiserum should precipitate high
Mr Ub-I
B
, and 2) anti-VCP IP should also
detect high Mr Ub-I
B
molecules. With
respect to the first prediction, we previously demonstrated that
anti-I
B
-1 and -2 detected high Mr
Ub-I
B
proteins in a ladderlike pattern (9). To further
substantiate the specificity, we subjected the I
B
immune
complexes (Fig. 1A, lanes 1-4) to second IPs
using various antisera specific to I
B
(lanes 5-8) or
to Ub (lane 10). As predicted, both IPs detected abundant
high Mr Ub-I
B
molecules but not the c-Rel
proteins co-precipitated with I
B
(the 80-kDa band in lanes
1-4, indicated by a small arrow on the
left). By contrast, I
B
IP followed by VCP
reimmunoprecipitation did not detect similar high
Mr proteins (lane 9). Predominant
species with molecular masses of 90 and 85 kDa were noted. Although the
majority of p90 precipitated by anti-I
B
was indeed VCP, as shown
in the second IP with anti-VCP (lane 9), a minor portion of
p90 co-migrating with VCP was Ub-I
B
, because it was
reprecipitated by both anti-I
B
(lanes 5-8) and anti-Ub (lane 10) but not reactive to anti-VCP in the
subsequent immunoblot (bottom panel).
|
|
Ub-IB
Physically Associates and Co-sediments with VCP in the
19 S Fractions, while the Unmodified I
B
Sediments in Fractions
Deficient in VCP--
To further characterize the association of VCP
with Ub-I
B
, ultracentrifugation-fractionation analyses were
performed (Fig. 3, A and
B). Human CA46 lymphoma cells were lysed, and the lysate was
separated by 10-40% glycerol density gradient centrifugation and
fractionated. The odd-numbered fractions were analyzed by SDS-PAGE
followed by immunoblotting with anti-VCP-2 immune serum (Fig.
3A, part a). The majority of VCP (fractions
7-15) migrated similarly to the marker thyroglobulin (data not
shown) with an apparent sedimentation coefficient of 19 S, consistent
with the previous report (24). Since thyroglobulin has a molecular mass of 650 kDa, it is compatible with the previous findings that VCP and
other ATPase family proteins consist of homohexamers, reminiscent of the oligomeric nature of molecular chaperones. When the same fractions were analyzed by blotting with preimmune (part b)
or anti-I
B
-1 immune serum (part c), only the high
Mr I
B
and not the unmodified I
B
co-sedimented with VCP in the 19 S fractions (fractions
7-13). As in the previous experiments, we also detected a
prominent 85-kDa (and less reproducibly, a 90-kDa)
anti-I
B
-reactive protein, representing a major species of
Ub-I
B
(also see Fig. 3B) in CA46 cells. It was noted
that the peak fractions of VCP (fractions 7-9) did not
exactly correspond to the peak fractions of the high
Mr I
B
(fractions 11-13).
However, it is likely that VCP may exist in different populations, some
of which can bind substrates other than Ub-I
B
; thus, the peak
fractions of VCP and I
B
may not be identical. Contrary to the
high Mr I
B
, the unmodified 36-kDa I
B
was detected only in the later fractions (fractions 17-19)
virtually lacking VCP. Furthermore, anti-I
B
-1 IP followed by
anti-VCP-2 immunoblotting (part d) showed that VCP
physically associated with the high Mr I
B
,
and these complexes sedimented in the 19 S fractions (fractions
7-13), whereas the unmodified I
B
did not associate with VCP
and sedimented in fractions deficient in VCP.
|
NF-B p50·p65 Complexes Are Not Associated with VCP--
It
has been reported that Ub-I
B
remained bound to NF-
B in
vitro (8) but dissociated from NF-
B in vivo (14).
Thus, it is possible that ubiquitination per se is not
sufficient to dissociate I
B
from NF-
B complexes, whereas VCP,
which is lacking in that specific in vitro assay, may serve
as a molecular chaperone that releases Ub-I
B
from NF-
B
in vivo. We examined whether the VCP-bound Ub-I
B
conjugates were free from or bound to NF-
B in vivo and
whether exogenously added VCP could bind to the purified NF-
B
complexes (Fig. 3C). Immune complexes precipitated by
various antisera reactive to different regions of p50 and p65 did not contain detectable levels of VCP (one such example was shown in lanes 1 and 4 for p50 and p65, respectively) or
Ub-I
B
(data not shown). Further incubation of NF-
B
precipitates with VCP did not result in detectable binding of VCP to
NF-
B (lanes 2 and 5) or diminution
(lanes 3 and 6) of the input free VCP (lane 10). By contrast, I
B
immune complexes contained significant amounts of VCP (lane 7), and almost all of the added VCP
could further bind to the complexes (lanes 8 and
9). These results suggest that NF-
B p50·p65 immune
complexes do not contain detectable levels of VCP (Fig. 3C)
or Ub-I
B
conjugates (Ref. 14; data not shown) in vivo.
The NF-
B·Ub-I
B
complex detected in vitro by Chen
et al. (8) is probably an unstable intermediate that is
readily dissociated by VCP in vivo (see model in Fig.
9).
Ubiquitinated IB
Binds to VCP in Vitro--
To further
characterize the VCP-I
B
association, we performed in
vitro binding assays using GST-I
B
and GST-VCP full-length fusion proteins. VCP (Fig. 4, lane
1) and I
B
(lane 4) were in vitro
translated in the presence of [35S]methionine/cysteine in
rabbit reticulocyte lysates and incubated with glutathione-Sepharose
beads containing either GST or GST fusion proteins. Translated VCP and
I
B
bound to GST-I
B
(lane 3) and GST-VCP
(lane 7; also see "Discussion"), respectively, but not
to the control GST (lanes 2 and 6).
PhosphorImager (Molecular Dynamics) scanning showed that about 5-10%
of the input VCP was bound to GST-I
B
beads, and 15% of the input
I
B
bound to GST-VCP beads. A longer exposure showed a smeary
pattern at the high Mr region in lanes
4 and 7 (data not shown), suggesting that high Mr species of I
B
, presumably the
Ub-I
B
conjugates, were present in the in vitro
translated product and could associate with GST-VCP. The reticulocyte
lysate is known to contain enzymes required for ubiquitin conjugation;
therefore, it is capable of supporting ubiquitination of I
B
in
the in vitro translation reaction. To demonstrate the
binding between the high Mr Ub-I
B
and VCP,
we performed Ub conjugation reactions (8, 16) on the translated I
B
to increase the yield of Ub-I
B
conjugates (lane
5) and then used the products in binding assays (lanes
8 and 9). Similar to the long exposure of lane
7, a smeary pattern above and including I
B
was detected in
VCP-bound I
B
(lane 9), and scanning analysis showed an
approximately 40% binding efficiency, significantly higher than that
of the non-Ub-enriched preparations. Subsequent anti-I
B
(lanes 10-13) and anti-Ub (lanes 14-17)
immunoblotting confirmed that these high Mr
proteins were Ub-I
B
conjugates. The dissimilar patterns observed
in lanes 13 and 17 could result from the
difference in the number of epitopes recognized by different antibodies. The high Mr Ub-I
B
molecules
have many more Ub-specific epitopes than I
B
-specific epitopes and
therefore should have a stronger anti-Ub reactivity. More importantly,
VCP probably also bound to ubiquitinated proteins other than
Ub-I
B
in the assay mixture, resulting in a higher reactivity in
lane 17 than in lane 13. The immunoreactivity was
specific to Ub-I
B
, because GST-VCP alone was not reactive to
either antiserum (data not shown). Furthermore, when B cell lysates
were used in the binding assays (lanes 18-21), abundant
high Mr Ub-I
B
conjugates also specifically bound to VCP (lanes 19 and 21). Notably, among
the VCP bound Ub-I
B
proteins, a major 85-kDa species was again
detected in cell lysates and in in vitro translated I
B
(marked by dots in lanes 13, 17, 19, and 21; also see Figs. 1-3), representing a
prominent species of Ub-I
B
that bound to VCP.
|
Phosphorylation and Ubiquitination of IB
Are Critical for VCP
Association, and both N- and C-terminal Domains of I
B
Are
Required for I
B
Degradation--
The N-terminal serines 32 and
36 (7, 10, 11, 15) and lysines 21 and 22 (12, 13, 15) have been shown
to be the major signal-induced phosphorylation and ubiquitination
sites, respectively. To demonstrate the physiological significance of VCP in the I
B
degradation pathway, we studied how I
B
mutations that blocked the upstream events, specifically
phosphorylation and ubiquitination, affected the VCP association (Fig.
5A) and the subsequent
degradation (Fig. 5B; summarized in Fig. 5C). We subjected wild-type and mutant I
B
to Ub conjugation reactions (Fig. 5A, lanes 2-7) before performing VCP
binding assays (lanes 8-21). In agreement with the previous
reports, the constructs with intact phosphorylation and ubiquitination
sites were further ubiquitinated (lanes 2 and 5),
and no significant ubiquitination was detected in I
B
with mutated
phosphorylation sites (S32A/S36A, lane 6), mutated
ubiquitination sites (K21R/K22R, lane 7), or I
B
lacking both sites (37-317, lane 4). Interestingly, the
mutant I
B
-(23-317), although missing the major ubiquitination
sites Lys21 and Lys22, was detectably
ubiquitinated (lane 3). This result agrees with a similar
finding by DiDonato et al. (15) and suggests that other Lys
residues in the molecule could accept Ub. The subsequent binding
experiments revealed that while all of the constructs capable of
ubiquitination could bind to VCP (lanes 9, 11,
13, and 17), the ubiquitination-defective mutants
all failed to bind VCP (lanes 15, 19, and
21). Taken together, the data indicate that phosphorylation
and ubiquitination of I
B
are critical for VCP association, and
I
B
ubiquitination is necessary for VCP binding (see Fig.
5C).
|
The Level of VCP Correlates with the Proteolytic Activity in the
Ub-Pr Assay--
To further demonstrate the functional involvement of
VCP in IB
degradation at a biochemical level, we correlated the
proteolytic activity (Fig. 6A)
with the level of VCP (Fig. 6C) contained in purified cell
lysates. Pr+ and Pr
fractions were isolated
from the cytosolic S100 fraction of the human B cell line, and an equal
amount of protein from each fraction was resolved by SDS-PAGE and
analyzed by immunoblotting. Preimmune serum showed no reactivity (Fig.
6B), whereas anti-VCP-2 immune serum clearly detected VCP in
Pr+, S100, and cytosolic fractions but not in the
Pr
fraction (Fig. 6C). The in vitro
Ub-Pr degradation assay was used to determine the
Ub-dependent proteolytic activity of each fraction. The
proteolytic activity measured by the disappearance of the substrate
I
B
(Fig. 6A) at 30 and 60 min correlated with the
level of VCP (Fig. 6C), being the highest in Pr+
(lanes 1), the lowest in Pr
(lanes
2), and higher in the S100 fraction (lanes 3) than in the crude cytosol (lanes 4).
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VCP Antibodies Deplete Proteasome Activity, While Adding Back
Immunopurified VCP Complexes Restores the Proteolytic Activity--
We
further determined if depletion of VCP (Fig. 6D) resulted in
a loss of proteasome activity (Fig. 6E). VCP-containing
complexes were removed from the S100 fraction by subjecting it to
repeated cycles of anti-VCP-1 IPs. After the third IP, virtually no VCP could be further precipitated (Fig. 6D, lane 5).
When the VCP-depleted lysate was used as the enzyme source in the
in vitro degradation assay, no IB
-degrading activity
was detected, as shown by the undiminished level of substrate that
remained at the end of the assay (Fig. 6E, lane
5). In contrast, when the VCP complexes obtained from each of the
first three IP cycles were added back to the VCP-depleted lysate, the
I
B
-degrading activity was restored, as shown by the loss of the
input substrate at the end of the assay (Fig. 6E,
lanes 2-4). These results demonstrate that VCP is necessary
for I
B
degradation and can be a functional component of the Ub-Pr
pathway involved in I
B
degradation.
VCP Co-migrates with Subunit 2 of the Proteasome in Biochemically Purified 26 S Proteasome and Is Co-immunoprecipitated with the 26 S Proteasome-- The 26 S proteasome can be resolved into many subunits by SDS-PAGE (Refs. 31, 32, and 48; Fig. 7B, part a; Fig. 8A, lane 6). Based on the observation that the molecular mass of VCP is similar to that of S2 of the purified 26 S proteasome, we determined the presence of VCP in 26 S proteasome preparations (Fig. 7). Partially purified (Fig. 7A, lanes 1 and 5) and highly purified (lanes 2 and 6) 26 S proteasomes isolated from human red blood cells (46) were compared with the Pr+ fraction isolated from a human B cell line (lanes 3 and 7). Coomassie Blue staining (lanes 1-3) and anti-VCP-1 immunoblotting (lanes 5-7) revealed that VCP was present in all three preparations (lanes 5-7), and VCP co-migrated with S2 of the purified proteasome (lanes 1-2; for the designation of S2, see Fig. 7B and Refs. 31, 32, and 48). To demonstrate the specificity of this analysis, Pr+ was immunoblotted with preimmune (lane 8), anti-VCP-2 (lane 9), and anti-VCP-1 (lane 10) sera and was shown to only be reactive to immune sera. To further resolve the proteins co-migrating with S2, highly purified 26 S proteasomes (as in Fig. 7A, lane 2) from human red blood cells were subjected to two-dimensional gel electrophoresis analysis (Fig. 7B). The stained filter showed a major protein with a pI around 5.1 (marked by an arrow in part a) and a more basic triplet that co-migrated to the S2 position; immunoblotting confirmed that VCP was the more acidic species (marked by an arrow in part b). The pI values of S2, cellular VCP, and in vitro translated VCP were all estimated to be between 5 and 6 (data not shown), consistent with the reported pI of 5.5 for the Xenopus counterpart, p97 (49).
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DISCUSSION |
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In this study, we identified a cellular ATPase, VCP, that
physically associated with Ub-IB
before the proteasome-mediated degradation of I
B
. This association was detected by more than one
type of I
B
antiserum (Fig. 1A) in a variety of cells,
including human B cell lines (this study), activated HeLa cells,
activated T lymphocytes, and proteasome inhibitor-treated cell lines
(data not shown). These results suggest that the physical complexing is
a general phenomenon and not a cell line- or an antibody-specific observation. We demonstrated that VCP was physically associated with 26 S proteasome by biochemical and immunological analyses, thus providing
a physical and functional link between I
B
modification and
proteolysis. We propose the following model shown in Fig. 9. In response to stimulation, the
inhibitor I
B
is hyperphosphorylated by a kinase and
polyubiquitinated. The Ub-I
B
conjugate physically associates with
VCP, which displaces the NF-
B dimer, thus releasing the dimer for
translocation into the nucleus. The Ub-I
B
conjugate is then
transferred to the 26 S proteasome, unfolded, transported into the
proteolytic core, and degraded. Meanwhile, Ub and VCP molecules are
released and recycled.
|
In this model, two non-mutually exclusive roles, as a molecular
chaperone that transfers the Ub-IB
to the proteasome and/or as a
physical component of the 26 S proteasome, are proposed for VCP. The
hypothesis of VCP being a molecular chaperone was first suggested by
our observation that when the p90(VCP)-containing I
B
complexes
were subjected to the in vitro degradation assay, only
I
B
and not p90(VCP) was degraded (9). This is consistent with the
nature of a molecular chaperone, which should be recycled and not
consumed after the degradation of the substrates. In addition, VCP is
an ATPase and forms an oligomeric ring structure (24, 49); both are
common properties of molecular chaperones. One important characteristic
of a molecular chaperone is its ready association with specific
substrates. If VCP is truly a molecular chaperone in Ub-Pr-mediated
degradation of I
B
, it should preferentially bind the Ub-tagged
I
B
. The in vivo IP and co-sedimentation experiments clearly showed such a substrate specificity, in that 1) I
B
IP detected physically associated VCP and high Mr
Ub-I
B
in addition to the 36-kDa I
B
; 2) serial IPs using VCP
followed by I
B
antisera showed that VCP immune complexes
contained high Mr Ub-I
B
but not 36 kDa-I
B
; 3) VCP and the high Mr Ub-I
B
co-sedimented in the 19 S fractions; 4) 36-kDa I
B
sedimented in
the 4.5 S fractions lacking VCP; 5) in the 19 S pool, VCP and
Ub-I
B
were co-precipitated in the same complex; and 6) in the 4.5 S pool, the majority of I
B
was not ubiquitinated and was free
from VCP. The in vitro binding experiments also showed a
ready association of VCP with Ub-I
B
. When cell lysates were used
as the source of I
B
, only Ub-I
B
and not the 36-kDa I
B
bound to VCP, consistent with the in vivo analysis.
Furthermore, when in vitro translated I
B
constructs
with various mutations were used in the assays (Fig. 5, A
and C), a perfect correlation between I
B
ubiquitination and VCP binding was detected; all I
B
variants
capable of ubiquitination could bind VCP, and all constructs defective
in ubiquitination could not. It was noted that when, and only when, the
in vitro translated I
B
was used in the in
vitro binding assays, in addition to Ub-I
B
, the 36-kDa
I
B
was also detected in VCP complexes (Figs. 4 and
5A). It is presently unclear whether the 36-kDa I
B
genuinely binds to VCP in vitro or is detected as a
consequence of rapid deubiquitination under the specific experimental
condition. The recent identification of a Ub isopeptidase, a component
of the 26 S proteasome, that quickly removes Ub chains to regenerate the substrate (50) supports the latter contention. In addition, the
preference for the substrates with short Ub chains, commonly found in
in vitro conditions, by this isopeptidase is consistent with
our findings. It is also formally possible that this 36-kDa I
B
is
hyperphosphorylated but not ubiquitinated. Although not detected
in vivo, this form of I
B
may bind to VCP in
vitro. Interestingly, we frequently detected binding of VCP to an
85-kDa Ub-I
B
in cell lysates (Figs. 1-4) and in Ub-I
B
generated from the Ub conjugation reaction (Fig. 4). In addition, we
also detected prominent Ub-I
B
species with
Mr of 120 and 90 kDa (Figs. 1A, 2B, and 4). Consistently, Ub-I
B
species with similar
sizes were also detected in other studies (8, 14, 15). These
Ub-I
B
molecules probably represent major polyubiquitinated
species that are preferentially bound by VCP. In summary, VCP readily
binds Ub-I
B
conjugates, requires I
B
ubiquitination for
binding, and clearly demonstrates a binding preference for them over
the unmodified I
B
in vivo, consistent with the
property of a molecular chaperone.
Chen et al. (8) showed that Ub-IB
was still bound to
NF-
B in vitro, while Roff et al. (14) showed
that Ub-I
B
was dissociated from p50-containing NF-
B in
vivo. The discrepancy between the two findings suggests the
existence of an in vivo-specific molecular chaperone that
displaces the NF-
B from Ub-I
B
. We identified VCP as the
candidate chaperone to fulfill this function. Our data demonstrating
VCP binding to I
B
but not p50·p65 complexes is consistent with
the model that following I
B
ubiquitination, VCP binds the
Ub-I
B
conjugates and releases NF-
B to allow for its nuclear
translocation. It is noted that, contradictory to the findings of Roff
et al. (14), DiDonato et al. (15) reported that
Ub-I
B
remained bound to the p65-containing complexes in cells
treated with proteasome inhibitor. At present, we do not have a
definitive explanation for this discrepancy. However, both studies were
performed in proteasome inhibitor-treated cells, in which unusually
high amounts of Ub-I
B
could be accumulated. It is conceivable
that when the level of Ub-I
B
exceeds the level of available VCP,
presumably as in the latter but not in the former study,
Ub-I
B
-p65 complexes can be detected.
To demonstrate the involvement of VCP in the signaling pathway of
IB
degradation, both mutant and biochemical studies were performed. We examined how the phosphorylation- and/or
ubiquitination-defective I
B
mutants affected the VCP binding and
the ultimate degradation (summarized in Fig. 5C). A perfect
correlation between ubiquitination and VCP binding was detected,
supporting the model that VCP binding requires the upstream
ubiquitination. Interestingly, the mutant 37-317, lacking both
phosphorylation and ubiquitination sites, was defective in VCP binding;
whereas the mutant 23-317, lacking the major ubiquitination sites, was
capable of ubiquitin conjugation and VCP binding. This result suggested
two points. First, other lysine residues in the I
B
molecule could
also accept Ub, which then signaled for VCP binding. This notion is
consistent with another report (15) and is supported by studies
reporting that the Ub-conjugating apparatus has the capacity to utilize
alternative lysines when the primary acceptors are disrupted (51, 52). However, if alternative Lys residues can accept Ub molecules, it is
unclear why the mutant K21R/K22R was defective in ubiquitination and
VCP binding, although consistent with other reports. Second, the
recognition of Ub by VCP is probably not highly site-specific. Ub
attached to Lys residues other than the major sites Lys21
and Lys22 can also be efficiently recognized by VCP, at
least in our in vitro binding assays. Presently, we do not
know how VCP recognizes Ub-I
B
conjugates and whether VCP can
recognize Ub chains attached to all of the nine Lys residues in
I
B
. Nevertheless, this finding further substantiated the
requirement of ubiquitination for VCP binding.
The in vitro degradation assays showed that not only were
the mutants defective in VCP binding unable to be degraded, but several
mutants capable of binding VCP were also resistant to degradation (Fig.
5C). These results support the model that while binding with
VCP is necessary for degradation, it is not sufficient. In agreement
with a previous study (47), we also found both N- and C-terminal
domains of IB
were required for degradation. This is consistent
with the findings that the C-terminal acidic PEST (Pro-Glu-Ser-Thr)
sequences are often associated with rapid protein turnover and
frequently found in Ub-Pr substrates. Moreover, the N- and the
C-terminal domains must be present in a cis relationship, suggesting
that a successful degradation requires the presence of a complete
protein domain and that intramolecular conformation of the I
B
protein may be important in the process.
All of our data are compatible with both roles proposed for VCP, one
being a molecular chaperone with a distinct hexameric structure and
the other being a component of the 26 S proteasome. Biochemically, VCP
co-purifies with the 26 S proteasome; immunologically, VCP
co-immunoprecipitates with subunits of the proteasome, all supporting
the "component" role. Interestingly, the VCP antisera raised
against synthetic oligopeptides co-precipitated the high Mr Ub-IB
but not the proteasome
components. On the other hand, anti-VCP-3, which was raised against a
full-length VCP fusion protein, readily co-precipitated the proteasome
subunits, but only co-precipitated low levels of Ub-I
B
. These
data along with the serial IP experiments (data not shown) seem to
suggest that there are at least two populations of VCP in the cell;
while the peptide-specific VCP antisera may recognize the
proteasome-free VCP, anti-VCP-3 may react more readily with the
proteasome-bound VCP. It is conceivable that VCP may exist in a state
of equilibrium between a mobile form, which presumably functions as a
molecular chaperone, and a relatively immobile form that assembles on
the top of the 26 S proteasome. Functionally, VCP may hydrolyze ATP (24) to provide the energy needed for the unfolding of the modified I
B
molecule and its entry into the 20 S proteolytic core.
Analogous dual activities have been demonstrated in VCP relatives, ClpA and ClpX in Escherichia coli. Both proteins appear to
exhibit intrinsic ATP-dependent chaperone activities (53)
and are supposed to be part of the regulatory structure on top of the
cylinder-shaped Clp protease (54). These notions also bear similarities
with a recent report that Ub chain-binding protein Mcb1, subunit 5a, exists in two populations, 26 S proteasome-bound and -free forms (55).
Although VCP co-purifies with the 26 S proteasome and co-migrates with
S2 of the 19 S cap complex on SDS-gels, it is not identical to S2.
Recently, an unrelated protein named TRAP-2 (56), corresponding to the
more basic triplet of S2 on our two-dimensional gel (Fig. 7B), has been identified as S2 by protein sequencing. It is
possible that there is more than one protein constituting S2. This
biochemical heterogeneity observed in a protein resolved by SDS-PAGE to
apparent homogeneity is precedented by the finding that S5 of the 26 S proteasome consists of two unrelated proteins, S5a and S5b (43, 44).
Alternatively, the regulatory 19 S cap may not be a single homogeneous
entity, and the VCP complex may represent an alternative version of the
19 S cap that confers specificity to certain substrates such as
IB
. Thus, subunit composition and the functional capability of
the 26 S proteasome may vary with physiological conditions.
VCP has been highly conserved during evolution and found in every
tissue examined to date. The paradox present in the high sequence
homology and the variety of functions suggests a basic and critical
role for VCP/Cdc48p/p97. Recently, cell cycle progression, cytokine-induced signal transduction pathways, and endoplasmic reticulum degradation were reported to be regulated by the Ub-Pr pathways (16-30, 57, 58). By identifying the involvement of VCP in
IB
degradation through the Ub-Pr pathway, we may provide a
potential link between the 26 S proteasome and other Ub-Pr substrates, thus offering an explanation for the paradox. In conjunction with our
findings, it would be interesting to examine whether VCP is also
involved in other Ub-Pr-mediated processes in the context of
proteasomes.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. L. Samelson and M. Rechsteiner for providing the critical reagents and making valuable suggestions about the manuscript. We thank Dr. D. Ballard for site-specific mutants; Dr. R. Fisher and Y. Kim for protein sequencing; Dr. J.-J. Lin for graphics; and Drs. M. Beckwith, H. F. Kung, H. Young, and J.-J. Lin for reviewing the manuscript.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed. Tel. 301-846-1478;
Fax: 301-846-6107; E-mail: licc{at}ncifcrf.gov.
1
The abbreviations used are: Ub-Pr,
ubiquitin-dependent proteasome; Ub, ubiquitin;
Ub-I, ubiquitinated I
B
; IP, immunoprecipitation; RIPA,
radioimmune precipitation; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; ATP
S, adenosine
5
-O-(thiotriphosphate); VCP, valosin-containing
protein.
2 C.-C. H. Li, unpublished observations.
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REFERENCES |
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