From the Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115
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INTRODUCTION |
NF-
B1 is a ubiquitous
transcription factor that regulates the expression of multiple genes
involved in immune and inflammatory responses (1). Greater knowledge
about the mechanisms of NF-
B activation is therefore of major
importance for understanding human disease and may indicate new targets
for pharmacological intervention. NF-
B is a member of the Rel family
of dimeric transcription factors present in many organisms (2). The
prototype of this family is a heterodimer of a p50 (NF-
B1) and a p65
(RelA) subunit. Its activity is regulated primarily at the
posttranslational level, by two separate processes (3). The p50
subunit is generated from a relatively stable precursor, p105, which
undergoes proteolytic processing in the cytoplasm (4-6). In this
process, the C-terminal part of p105 is degraded, and the remaining
N-terminal half of the molecule serves as the p50 subunit of NF-
B.
However, in uninduced cells, the p50/p65 (NF-
B) complex is
maintained in an inactive form in the cytoplasm by the inhibitor
I
B
, which associates with p50/p65 and prevents its migration to
the nucleus (7-11). The final activation of NF-
B involves the
proteolytic destruction of I
B
(12), which is triggered by its
phosphorylation following a variety of stimuli (13, 14). The kinases
p90rsk (15, 16) and CHUK (17-19) have very recently been
shown to be involved in this process. Previous data have shown that the processing of the p105 precursor and the degradation of I
B
both require ubiquitin (Ub) conjugation to these polypeptides, leading to
their proteolytic digestion by the 26S proteasome (6, 14, 20,
21).
The Ub-proteasome system is a major pathway for degradation of
intracellular proteins in eukaryotic cells (22, 23). In this pathway,
substrates are marked for degradation by covalent attachment of poly-Ub
chain(s) (24). In this process, the Ub-activating protein, E1, utilizes
ATP to form a high energy Ub-thiol ester and then transfers the
activated Ub to an E2 (Ub carrier protein (UBC)), forming an E2-Ub
thiol ester. The Ub is then linked to the substrate in a reaction
requiring E3, a Ub-protein ligase (24, 25). Cells contain a large
number of E2s (23), each of which acts on a limited spectrum of protein
substrates (26, 27). The E3s seem to provide most of the substrate
specificity of the ubiquitination process, although only a limited
number have been identified.
Once ubiquitinated, proteins are usually rapidly degraded to small
peptides by the 26S proteasome (22). The proteolytic core of this
2000-kDa complex is the 20S proteasome (28), which is sandwiched at
each end by the 19S complex (PA700) (29, 30). The 19S complex contains
multiple activities, including an isopeptidase that catalyzes the
release of free Ub (31, 32) and several ATPases, the likely function of
which is to facilitate the unfolding of substrates and their
translocation into the 20S proteasome (33), where degradation proceeds
in a processive fashion (34).
The finding that the Ub-proteasome pathway is responsible for the
limited processing of p105 was quite surprising, because its other
known substrates undergo complete degradation. To dissect the
mechanisms of this process and to define its components, we undertook
to identify the enzymes necessary for p105 ubiquitination and p50
generation in HeLa cell extracts and to reconstitute this process with
purified proteins.
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EXPERIMENTAL PROCEDURES |
Materials--
All chemicals were of analytical grade. DE52 was
obtained from Whatman; the Bio-Scale CHT20-I column was from Bio-Rad;
the MonoQ, Superose 6, Superose 12, and Sephacryl S100 HR HiPrep 16/60 columns and [35S]methionine (SJ1515, >37 TBq/mmol) were
from Amersham Pharmacia Biotech, and iodine-125 (NEZ-033A, 629 GBq/mg)
from NEN Life Science Products. Cytoplasmic HeLa cell extracts were
kindly provided by Dr. V. J. Palombella (ProScript, Cambridge,
MA), Dr. R. Reed (Harvard Medical School) or Dr. P. A. Sharp
(Massachusetts Institute of Technology), and purified rabbit
reticulocyte E1 and E2s were provided by Dr. Z. Chen (ProScript).
Strains, Plasmids, and Recombinant Proteins--
Strains,
plasmids, and recombinant proteins were generously provided by the
following colleagues: E. coli strains expressing plant
6His-Ub and 6His-UbR48 by Dr. J. Callis (University of California, Davis) (35); strain expressing the GST-yeast Ub fusion by Dr. J. M. Huibregtse (Rutgers University) (36); plasmids encoding human UBCH5C
(37) and human E2F1 (UBCH7) (38) by Dr. A. M. Weissman (Bethesda)
and Dr. M. Scheffner (Heidelberg), respectively. UBCH5C and E2F1 were
purified by cation-exchange chromatography of the flow-through of a
DE52 column using an HiTrap SP column (Amersham Pharmacia Biotech).
Recombinant bovine E2-25K (39) was provided by Dr. C. M. Pickart
(Johns Hopkins University). The C170S form of E2-25K used here has a
serine in place of cysteine 170 (nonactive site Cys). Purified
recombinant human UBC2 and UBCH5B (also called UBC4) (40) by Drs.
R. W. King and J. M. Peters (Harvard Medical School).
Preparation of FI and FII--
HeLa cell cytoplasmic extracts
were centrifuged for 20 min at 10,000 × g (4 °C) to
remove debris. The supernatant was dialyzed against 3 mM
potassium phosphate (pH 7.0), 1 mM DTT, 10% glycerol and
loaded onto a DE52 column equilibrated with the same buffer. The
flow-through (fraction I) was collected, the column was washed with the
equilibration buffer supplemented with 20 mM KCl, and the
bound proteins (fraction II) were eluted with a buffer containing 20 mM Tris-HCl (pH 7.5), 0.5 M KCl, 5 mM MgCl2, 0.5 mM ATP, 1 mM DTT, 10% glycerol. FII was dialyzed overnight against
the same buffer without KCl. In some experiments, FI and FII were
prepared from extracts depleted of proteasomes by ultracentrifugation
at 100,000 × g for 5 h or 200,000 × g for 3 h (4 °C). In those cases, after
sedimentation of the proteasomes, MgCl2 and ATP were
omitted in the buffers used to prepare the FII.
Ubiquitin-affinity Chromatography--
Ubiquitin was covalently
bound to a CH-activated Sepharose matrix (Amersham Pharmacia Biotech),
as recommended by the manufacturer. The final concentration of
ubiquitin was about 20 mg/ml of gel. Fraction II was supplemented with
5 mM MgCl2, 2 mM ATP and mixed with
the Ub-Sepharose for 1 h at room temperature (with shaking). The
ubiquitinating enzymes were sequentially eluted as follows (41, 42): E1
with 20 mM Tris, pH 7.5, 2 mM AMP, and 2 mM NaPPi; E2s with 20 mM Tris, pH
7.5, 20 mM DTT, and 100 mM KCl; other ubiquitin-binding proteins with 50 mM Tris, pH 9.0, 1 M KCl, 2 mM DTT.
Purification of 26S Proteasomes--
Pellets obtained by
centrifugation at 200,000 × g for 3 h of the HeLa
cytoplasmic extracts were resuspended in Buffer A (50 mM
Tris, pH 7.5, 5 mM MgCl2, 0.5 mM
EDTA, 1 mM ATP, 1 mM DTT, 10% glycerol). After
centrifugation at 10,000 × g for 20 min, the
supernatant was centrifuged again at 200,000 × g for
30 min to remove polysomes. The supernatant was loaded onto a MonoQ
10/10 column equilibrated with Buffer A, and the proteins were eluted with a NaCl gradient (0-500 mM). The fractions were
assayed for activity against the fluorogenic proteasome substrate
Suc-LLVY-MCA (Bachem). The active fractions (
320 mM
NaCl) were pooled, diluted twice in Buffer A, and loaded onto a MonoQ
5/5 column. The proteins were eluted with a 200-500 mM
NaCl gradient. Fractions active against Suc-LLVY-MCA were pooled and
concentrated using a Centricon-50 (Amicon) concentrator and loaded onto
a Superose 6 column equilibrated with Buffer A containing 100 mM NaCl. 20S and 26S proteasomes were identified by their
activity against Suc-LLVY-MCA in the presence or absence of 0.02% SDS.
At this concentration, SDS greatly activates the 20S proteasomes but
inhibits the 26S proteasomes, allowing easy discrimination of the two
forms (43). Fractions containing 26S proteasomes and 20S proteasomes
were pooled separately and stored frozen at
70 °C.
Preparation of Substrates--
The p105T, p97T, and p60Tth
constructs (5) were generously provided by Dr. Tom Maniatis (Harvard
University). The proteins were translated in vitro in a
wheat germ extract using the coupled transcription/translation TNT
system of Promega, as recommended by the manufacturer. Unless specified
otherwise, the reaction mixture was diluted 3-fold after translation in
the assay buffer (20 mM Tris, pH 7.5, 50 mM
KCl, 5 mM MgCl2, 1 mM DTT), and the labeled protein was separated from the free
[35S]methionine using a Nick Spin column (Amersham
Pharmacia Biotech) equilibrated in the same buffer. The wheat germ
extract (WGE) containing the in vitro-translated p97 and
p105 used in Figs. 7 and 8 was kindly provided by Dr. M. A. Read
(ProScript).
Ubiquitination and Processing Reaction--
Unless specified
otherwise, reactions were carried out in the assay buffer (20 mM Tris, pH 7.5, 50 mM KCl, 5 mM
MgCl2, 1 mM DTT) supplemented with other
components as indicated. When the processing of p105 was studied, an
ATP-regenerating system (0.1 mg/ml creatine phosphokinase, 10 mM creatine phosphate) was added. The products were
separated by SDS-PAGE (44), and the gels were dried and analyzed using
a PhosphorImager (Molecular Dynamics) or a Fujix Bas 1000 (Fuji) and
autoradiography.
Analysis of Ub Thiol Ester Formation--
Reactions were carried
out for 10 min at 37 °C in 10 mM Tris, pH 7.6, 10 mM MgCl2, 2 mM ATP, 0.1 mM DTT in the presence of 125I-labeled Ub
(50-100 µg/ml) and 50 units/ml of inorganic pyrophosphatase. Reactions were stopped by addition of 1 volume of 2× sample buffer (120 mM Tris, pH 6.8, 4% SDS, 4 M urea, 20%
glycerol) containing or not containing 100 mM DTT. The
products were analyzed by SDS-PAGE as described above.
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RESULTS |
Ubiquitin Conjugation Is Essential for NF-
B1
Processing--
Prior studies have shown that p105 and its
C-terminally truncated forms p60 and p97 can be converted into the
NF-
B subunit p50, when expressed in cells or added to crude cell
extracts (5, 6). To learn more about this process,
35S-labeled p60, translated in WGE, was added to a
cytoplasmic extract (S100) of HeLa cells, and its fate was analyzed at
different times by SDS-PAGE and autoradiography (Fig.
1). Within minutes, very high molecular
weight forms of p60 appeared, which entered the resolving gel only
slightly. This heterogenous 35S-labeled material then
disappeared concomitantly with an increase in the mature, processed p50
(although some p50 also appeared within 5 min of p60 addition). Thus
the high molecular weight forms behave like intermediates in the
proteolytic processing of p60 and probably correspond to ubiquitinated
forms of p60.

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Fig. 1.
Ubiquitination and processing of p60 in HeLa
cell extracts. 35 µl of HeLa cell S100 extract ( 250 µg of
proteins) were mixed with in vitro-translated p60 and
incubated with 0.5 mM ATP at 30 °C in a final volume of
70 µl. Aliquots of 10 µl were removed, boiled in sample buffer, and
analyzed by SDS-PAGE and autoradiography.
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To verify this conclusion, the extract was supplemented with various
recombinant species of Ub: wild-type Ub; UbR48, a mutated form of Ub
that has a defect in Ub chain formation due to the replacement of
lysine 48 by an arginine (45) (both with a His6 tag); and
GST-Ub, a hybrid molecule in which the enzyme glutathione S-transferase has been fused to the N-terminal end of normal
Ub (36). Addition of the modified Ub species significantly altered the
events from those seen upon addition of normal Ub. With UbR48, we found
p60 conjugates of reduced size (data not shown), probably because of
the premature termination of the Ub-chain due to the K48R mutation.
Moreover, addition of UbR48 to the crude extract inhibited the
production of p50, although this inhibition was not complete, probably
due to the presence of endogenous normal Ub in this extract. These
observations are consistent with previous work showing that UbR48
inhibits p105 processing (6). It is noteworthy that the effect of UbR48
on p105 ubiquitination and processing rules out a possible explanation
of how p105 may be converted to p50: that p105 is modified by addition
of an atypical type of poly-Ub chain, which directs the protein toward
limited processing instead of complete degradation. Indeed, lysine 48 of Ub is the residue commonly used for complete degradation of proteins
by the proteasome (45).
On the other hand, in the presence of GST-Ub, bands of very high
molecular weight accumulated (data not shown, see below), which
presumably correspond to poly-GST-Ub adducts of p60 because GST-Ub is
about four times larger than normal Ub. Because the type of Ub added
determined the size of the high molecular weight derivatives of p60,
these derivatives must represent Ub-conjugated forms of the
molecule.
Furthermore, when the same extract was incubated with ATP
S, an ATP
analog that supports ubiquitination of proteins but not the proteolytic
activity of the 26S complex (46), high molecular weight ubiquitinated
forms of p60 accumulated, but p50 was not formed (data not shown). A
similar accumulation of ubiquitinated species was obtained when the
extract was depleted of proteasomes by prolonged ultracentrifugation,
before being incubated with ATP (Fig. 2).
These results confirm that the ubiquitinated species are indeed
intermediates in the production of the p50 subunit, as was proposed
previously (6, 47).

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Fig. 2.
Ubiquitination and processing of p60 in
fraction II (FII). In vitro-translated p60 (2 µl) was
incubated in a 40-µl reaction for 1 h at 30 °C in the
presence of the protein fractions indicated at the top. The
protein was then immunoprecipitated as described in Ref. 6.
S100, HeLa cell S100 extract (10 µl, 54 µg of protein);
Prot., HeLa cell S100 after removal of proteasomes by
ultracentrifugation (6) (10 µl); Prot., proteasome-rich
fraction (the pellet obtained by ultracentrifugation at 100 000 × g for 6 h) of reticulocyte extract (5 µl);
FI, HeLa cell FI (6.5 µg of protein); FII, HeLa
cell FII (28 µg of protein); Ub, bovine Ub, 5 µg. The
diamond indicates an artifactual band caused by the IgG
heavy chain in the gel.
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Isolation of the Enzymes Catalyzing p105 Ubiquitination--
To
identify the enzymes required for the formation of these Ub conjugates,
the HeLa extract was loaded onto a DE52 column (Whatman). The proteins
that did not bind (FI) were collected and, after washing the column,
the bound proteins (FII) were eluted with 0.5 M KCl. After
extensive dialysis, both fractions were assayed for their ability to
support the processing of p60 into p50. In accord with previous
findings (6) with FII from rabbit reticulocytes, we found that HeLa
FII, when supplemented with Ub, was fully competent at p50 formation
(Fig. 2). Moreover, the addition of FI did not increase the yield of
p50 (Fig. 2). Thus, it is most likely that the HeLa FII contains all of
the enzymes necessary for NF-
B1 processing. It has been reported
that FI (from reticulocytes) contains an E2 essential for p105
processing and that this E2 could also be provided in the reaction by
the WGE in which p60 (or p105) is translated (47). However, our subsequent experiments clearly showed that HeLa FII contains E2 and E3
ubiquitination enzymes supporting p60 (or p105) ubiquitination and
processing.
FII was then further fractionated to isolate the enzymes active in p60
ubiquitination. Ammonium sulfate precipitation resolved two fractions
that independently had some activity in ubiquitinating p60 (data not
shown): FIIA, which precipitated between 0 and 40% (NH4)2SO4, and FIIB, which
precipitated between 40 and 90%
(NH4)2SO4. However, mixing these
two fractions resulted in more than an additive effect in promoting p60
ubiquitination (data not shown). Thus, each of these fractions appeared
to be enriched in distinct component(s) of the ubiquitination pathway.
These components were then further purified by anion-exchange
chromatography of FIIA and FIIB, using a MonoQ column. To assay their
ability to support ubiquitination of p60, each fraction derived from
FIIA was combined with FIIB, and each from FIIB was combined with FIIA,
in the presence of E1 and GST-Ub. We used GST-Ub rather than normal Ub
because the larger conjugates formed with GST-Ub accumulate at the top
of the acrylamide gel and are easily detected.
After anion-exchange chromatography of FIIA and FIIB, two protein
fractions were obtained: A (from FIIA), which eluted at about 250 mM NaCl, and B (from FIIB), which eluted at about 100 mM NaCl. These two fractions, when mixed, supported
efficient formation of Ub conjugates when p60 or p105 was used as the
substrate (Fig. 3A).
Interestingly, the decrease in the p60 and p105 bands (which accounted
for 30% or less of the substrate added, as analyzed with a
PhosphorImager) could not account for the amount of labeled protein
accumulating as conjugates. Therefore, the bands of lower molecular
weight (most likely degradation products or products of premature
termination of translation) apparently can also be ubiquitinated by
these enzymes.

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Fig. 3.
Fraction A contains E1 and an E3, whereas
fraction B contains two E2s. A, fractions A and B are
necessary and sufficient for ubiquitination of p60 and p105. The
capacity of fractions A and B (obtained by ammonium sulfate cuts of FII
and anion-exchange chromatography) to support ubiquitination of p60
(left) or p105 (right) were assayed. These two
fractions were analyzed either alone or together with or without pure
reticulocyte E1 (20 µg/ml) and in the presence of ATP (2 mM), GST-Ub (0.35 mg/ml), and an ATP-regenerating system
(creatine phosphokinase + creatine phosphate). The samples were
incubated at 30 °C for 20 min and then analyzed by SDS-PAGE and
autoradiography. The wheat germ extracts containing the substrates were
diluted about 30-fold in each incubation. The bar indicates
the position of Ub conjugates in the gel. B, thiol ester
formation in fractions A and B. The two fractions, A and B (1 µl
each), were analyzed for their ability to form thiol esters with
125I-labeled Ub in the presence or absence of purified
reticulocyte E1 (10 µg/ml). After incubation at 37 °C for 10 min,
the samples were separated by SDS-PAGE under nonreducing
(left) or reducing (right) conditions. The
vertical bar indicates the position of high molecular weight
Ub conjugates formed with endogenous proteins.
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The poly-ubiquitination of a substrate involves the successive thiol
ester linkage of Ub to E1 and then to E2 and, in some cases, to E3 (24,
25). Using 125I-Ub, such a thiol ester adduct can be
detected after SDS-PAGE, provided that the sample is not exposed to a
reducing agent. We analyzed fractions A and B for their content of
enzymes capable of forming a thiol ester linkage with
125I-Ub. As shown in Fig. 3B, one DTT-sensitive
band of about 110 kDa was detected in fraction A after electrophoresis.
This band comigrated with the band formed when Ub was incubated with E1 purified from rabbit reticulocytes, and thus it corresponded to the
human E1. No E2 could be detected in fraction A. In fraction B, no
Ub-thiol ester could be detected, unless reticulocyte E1 was added.
With E1 present, two Ub-protein adducts of about 27.5 and 33 kDa were
evident under nonreducing conditions, but they disappeared if the
sample was boiled in the presence of DTT. These two bands therefore
must correspond to distinct ubiquitin carrier proteins (E2s) linked to
Ub by a thiol ester. Finally, mixing fractions A and B allowed
formation of the same two Ub-thiol esters, without exogenous E1
addition, and did not reveal any additional E2 (Fig. 3B). In
addition, some high molecular weight radiolabeled bands (Fig. 3B,
vertical bar) were formed that were DTT-resistant and therefore
corresponded to Ub conjugates of proteins in fractions A and B.
Neither fraction A, which contains E1, nor fraction B, which contains
two E2s, was able by itself to conjugate Ub to p60 or p105, even if
reticulocyte E1 was added (Fig. 3A). Significant conjugation
to p60 or p105 occurred only when fractions A and B were mixed. Thus,
in addition to E1, fraction A must contain another activity, presumably
an E3, that is required for the ubiquitination of p60 and p105. The
finding that all three activities were present in fractions A and B is
further evidence that FII is fully competent in p105 ubiquitination and
that no essential ubiquitination enzyme was derived from the WGE, as
had been suggested by Orian et al. (47). The activities
present in fractions A and B were then purified further using a
Superose 12 column (Amersham Pharmacia Biotech). Surprisingly, the E3
activity from fraction A, which we propose to call E3
B, was eluted
with an apparent molecular mass of
60 kDa (data not shown), which is
appreciably smaller than known E3s (see below).
Several E2s Can Function in NF-
B1 Ubiquitination--
Following
the gel filtration step, the active fraction B', derived from fraction
B, still contained two E2s able to form thiol esters with radioactive
Ub. To identify which E2 is active in p105 ubiquitination, we tested
whether fraction B' could be replaced in the reaction by individual E2s
isolated from rabbit reticulocytes FII by Ub-affinity and MonoQ
chromatography (41). Only one of the reticulocyte E2s, E2-25K, was
able to promote p60 ubiquitination in this screen (data not shown).
This E2, when incubated with 125I-Ub in presence of E1,
formed a thiol ester adduct that migrated upon electrophoresis at the
same position as the slowest-migrating E2-Ub thiol ester formed from
fraction B' (Fig. 4A). This
finding strongly suggested that E2-25K functions in p105
ubiquitination.

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Fig. 4.
E2-25K and UBCH5 are active in NF- B1
ubiquitination. A, fraction B' from HeLa cells contains an
E2 similar to rabbit reticulocyte E2-25K. The E2 fraction from HeLa
cell extract (B') (2 µl) and the purified rabbit
reticulocyte E2-25K (2 µl) were incubated in presence of
human E1 (200 ng) and 125I-labeled Ub at 37 °C for 10 min in a volume of 10 µl. The reaction mixtures were then analyzed by
SDS-PAGE under nonreducing conditions. B, recombinant
E2-25K and UBCH5B each support NF- B1 ubiquitination. P60 or p105
was incubated for 15 min at 37 °C in a final volume of 10 µl
containing human E1 (100 ng), ATP (2.5 mM), purified E3 B
(2 µl), and GST-Ub (0.35 mg/ml). Either recombinant bovine E2-25K
(380 ng), recombinant human UBCH5B (190 ng), or an equal volume of
buffer was added. The samples were analyzed by SDS-PAGE and
autoradiography.
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To investigate this possibility further, we tested whether recombinant
bovine E2-25K (39) can function in this process in a manner similar to
the E2 purified from HeLa cells. As shown in Fig. 4B,
recombinant E2-25K supported the ubiquitination of p60 and p105 when
mixed with E1, E3
B, and GST-Ub. These findings provide strong
evidence that E2-25K is the E2 supporting p105 ubiquitination and
processing in FII. The other E2 present in fraction B' corresponds to a
lower molecular mass E2 of about 18 kDa, which was eluted from the
MonoQ column at a slightly lower salt concentration than E2-25K. In
subsequent experiments, better separation of this 18 kDa E2 from
E2-25K allowed us to show that it is not involved in p105
ubiquitination (data not shown).
Although E2-25K by itself appears to account for the E2 activity
supporting p105 ubiquitination in fraction II, we tested whether
certain other recombinant E2s could also function in this reaction with
E3
B. Human UBC2 (Rad6) (40) was not active in this process (data not
shown). However, recombinant human UBCH5B (also called UBC4) (37, 40)
supported p105 ubiquitination (Fig. 4B), as could UBCH5C, an
E2 closely related to UBCH5B (Ref. 37 and see below). This result is
consistent with the observation of Orian et al. (47) that
E2s from the UBCH5 subfamily can support p105 ubiquitination, although
they used the UBCH5A isoform (37, 48). In view of the extensive
similarity of the different E2s of the UBCH5 subfamily (37), it is
likely that UBCH5A can also function in our p105 ubiquitination assay.
We did not find any E2 of the UBCH5 subfamily in our fractionation of
the HeLa extract because these proteins are found in fraction I (36).
In conclusion, in the presence of E1 and E3
B, we found that at least
two different types of E2s can efficiently catalyze the ubiquitination
of p105: one from FII, E2-25K, and a subfamily of E2s from FI, UBCH5.
The fact that fraction II contains all the enzymes necessary for p105
ubiquitination (E1, E2-25K, and E3
B), as well as the proteasome,
confirms our observation that FII is active in p105 processing (Fig.
2). However, Orian et al. (47) reported that FII from rabbit
reticulocytes was unable to conjugate Ub to p105 unless supplemented by
E2s present in FI (E2F1 or UBCH5), which could be provided by the WGE
in which the substrate was synthesized. They reported that inactivation
of the E2s in the WGE by treatment with N-ethylmaleimide
prevented p105 ubiquitination in FII and that addition of FI or of E2F1
(or UBCH5) alone could restore this reaction (47). These authors
therefore concluded that E2F1 (or UBCH5) was essential for p105
processing. However, Fig. 5 shows that
under the same conditions, recombinant E2-25K (lane 6) can
stimulate p60 ubiquitination to the same extent as FI or UBCH5C
(lanes 4 and 5), even though this E2 is already
present in FII. Similar stimulatory effects of addition of FI, UBCH5C or E2-25K were observed with p105 as substrate and when the WGE was
not pretreated with N-ethylmaleimide (data not shown).
Therefore, the conclusion that E2F1 or UBCH5 is essential for p105
ubiquitination seems unwarranted, especially because Orian et
al. (47) did not test E2-25K in their experiments. Interestingly,
because the addition of exogenous E2-25K stimulated NF-
B1
ubiquitination in FII, the content of E2-25K appears to be
rate-limiting for p105 ubiquitination in this fraction.

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Fig. 5.
Several E2s can stimulate NF- B1
ubiquitination in FII. The reactions (20 µl) were performed at
37 °C for 20 min in the presence of 2 mM ATP, 12 µg of
GST-Ub, 0.5 µg of E1, p60 (equivalent to 0.67 µl of the initial
translation mixture). Lane 1, buffer; lane 2, FI
(31 µg); lane 3, FII (31 µg); lanes 4, FI + FII; lane 5, FII + UBCH5C (0.55 µg, 2
µM); lane 6, FII + E2-25K (1 µg, 2
µM); lane 7, FII + UBCH5C + E2-25K. For this
experiment, the WGE was treated with 10 mM
N-ethylmaleimide to inactivate E1 and E2s. After incubation
at room temperature for 10 min, 10 mM DTT was added to
inactivate unreacted N-ethylmaleimide. The extracts were
then diluted by addition of 2 volumes of 20 mM Tris (pH
7.5), 100 mM NaCl, 1 mM DTT and filtered
through a Nick Spin column (Amersham Pharmacia Biotech)
equilibrated with the same buffer. The bar indicates the
position of ubiquitinated p60.
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There is an apparent contradiction concerning the effect of addition of
FI to FII between Fig. 2 (no effect) and Fig. 5 (stimulation). However,
direct comparison of the two experiments is difficult, because the
conditions used were quite different. In addition, in Fig. 2, showing a
processing assay, only a limited amount of FI protein was added to
prove that FI did not contain any factor absolutely required for the
processing reaction, whereas in Fig. 5, showing a shorter
ubiquitination assay, similar amounts of protein from FI and FII were
compared.
A New E3, E3
B, Is Required for p105 Ubiquitination--
To
further characterize the E3 (E3
B) responsible for p105
ubiquitination, we purified this activity from crude extracts by a
combination of chromatographic methods. The following steps were
employed: removal of proteasomes by prolonged ultracentrifugation; preparation of FII using DE52 chromatography; and removal of
Ub-conjugating enzymes (E1 and E2s) and other Ub-binding proteins by
Ub-affinity chromatography, hydroxyapatite chromatography, ion-exchange
chromatography, and gel filtration. E3
B activity was assayed by its
ability to promote p105 ubiquitination in the presence of E1,
recombinant E2s, and GST-Ub (Fig. 6).
Several findings are noteworthy. 1) When FII was loaded onto a
Ub-affinity column, the E3 activity was found in the flow-through.
Thus, this activity does not have a strong affinity for free Ub. 2)
When loaded onto the hydroxyapatite column (CHT20, Bio-Rad), the
activity was eluted over a broad range of phosphate concentrations
(Fig. 6A). Although most bound weakly and was eluted between
20 and 30 mM phosphate, some activity was eluted
continuously until the phosphate concentration reached
200
mM. The activities eluted at high and low phosphate
concentrations behaved similarly upon subsequent chromatography on
MonoQ and Sephacryl S100 and therefore seemed to correspond to the same molecule. This unusual behavior upon hydroxyapatite chromatography most
likely reflects some heterogeneity in the enzyme, possibly due to
posttranslational modification or interactions with other proteins. 3)
Upon gel filtration on a Sephacryl S100 column, E3
B was eluted with
an apparent molecular mass of
50 kDa (Fig. 6C), which is
consistent with the 60-kDa value obtained earlier using a Superose 12 column (data not shown). Because its molecular mass is significantly
lower than that of other E3s, E3
B is apparently a new E3. After the
gel filtration step, several protein bands could be detected upon
SDS-PAGE in the fractions containing E3
B activity, but no protein of
about 50 kDa (or potential smaller subunits) was found reproducibly
with a elution profile matching that of the E3kB activity.

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Fig. 6.
Purification of E3 B. A,
hydroxyapatite chromatography. The flow-through of the Ub-affinity
column (42 ml) was loaded onto a hydroxyapatite column (CHT20,
Bio-Rad), and the bound proteins were eluted with a two-slope gradient
of potassium phosphate (20-120 mM and then 120-350
mM) in the presence of 1 mM DTT and 10%
glycerol. Fractions of 5 ml were collected. 11 µl of each fraction
were assayed for their ability to support p105 ubiquitination in a
20-µl reaction containing 5 mM MgCl2, 2 mM ATP, 12 µg of GST-Ub, 0.5 µg of human E1, and a
mixture of three recombinant E2s: bovine E2-25K (1 µg), human UBCH5C
(0.55 µg), and human E2F1 (1 µg). Top panel,
chromatogram; bottom panel, autoradiography of the SDS-gel.
The vertical bar indicates the position of the ubiquitinated
p105; the diamond indicates the position of p105. The
fraction numbers are shown at the top. The horizontal
bars indicate the fractions pooled and used in subsequent steps.
B, anion-exchange chromatography. The pooled fractions from
the hydroxyapatite column were loaded onto a MonoQ 5/5 column (Amersham
Pharmacia Biotech) equilibrated with a buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM DTT, and 10%
glycerol. The bound proteins were eluted with a NaCl gradient (from 0 to 0.5 M). Fractions of 1 ml were collected, and 3 µl of
each were assayed as in A. The horizontal bars
indicate the fractions pooled. C, gel filtration of purified
E3 B on Sephacryl S100. Left panel, E3 B activity in
different column fractions. The pool from the MonoQ column was
concentrated using a centrikon C10 (Amicon); 0.8 ml of the concentrated
pool was loaded onto a Sephacryl S100 HR HiPrep 16/60 column (Amersham
Pharmacia Biotech), equilibrated in buffer containing 20 mM
Tris (pH 7.5), 150 mM NaCl, 1 mM DTT, and 10%
glycerol. Fractions of 4 ml were collected, and 12.5 µl of each
fraction were assayed as in A. The fraction numbers are
shown at the top. Lane labeled Start corresponds
to an assay with 0.5 µl of the material loaded onto the column.
Right panel, molecular mass estimation of E3 B
(dotted lines). The proteins used to standardize the column were bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa). The
void volume was determined using blue dextran ( 2,000 kDa).
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Reconstitution of the Proteolytic Process Generating p50--
A
critical issue in this project was to determine whether the E2s and E3
we purified could actually support the conversion of p105 or p97 to
p50. Initial experiments used the truncated form p97, which lacks the
89 C-terminal amino acids of p105 and is more efficiently processed
in vivo into p50 than p105 (5). In the absence of 26S
proteasomes, p97 was efficiently ubiquitinated in vitro if
pure E1, E2s, and E3
B were present (Fig.
7A), as expected from our
previous results with p105 and p60. The addition of pure 26S
proteasomes to this mixture led to the disappearance of the
ubiquitinated form of p97 and to the concomitant appearance of p50
(Fig. 7A). This new band was identified as p50 because it
migrated similarly to the p50 band formed upon incubation of p97 with
the crude extract (Fig. 7A, lanes labeled S100).
Thus, the purified E3
B can catalyze the ubiquitination reaction, and the 26S proteasome can provide all of the proteolytic activities necessary for p50 formation.

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Fig. 7.
E3 B, E2-25K, and UBCH5C support the
ubiquitin-proteasome-dependent processing of p97. A, the
processing of p97 requires E3 B and proteasomes. The ubiquitination
and processing of p97 were monitored in a 20 µl reaction for 2 h
at 37 °C, in the presence of 2 mM ATP and 0.5 mg/ml Ub,
using 1 µl of the undiluted translation mixture containing p97 in
each assay. The reaction mixtures were supplemented with either 3 or 5 µl of crude extract (S100) or with purified human E1 (0.4 µg), a mixture of three recombinant E2s (bovine E2-25K, human
UBCH5C, and E2F1, 2 µM each) and with the fractions and
volumes indicated at the top. Lane F corresponds
to the FII depleted of ubiquitinating enzymes by Ub-affinity
chromatography and depleted of proteasomes by ultracentrifugation.
E3 B was purified as shown in Fig. 6. As indicated, either buffer
( 26S) or 2 µg of purified HeLa cell 26S proteasome
(+26S) was added. The vertical bar indicates the
position of ubiquitinated p97. B, E2-25K and UBCH5C can
each support proteasome-dependent processing of p97. The
experiment was performed as in A, using 6 µl of the
fraction II depleted of ubiquitinating enzymes and proteasomes as a
source of E3 B. Lane 1, no E2 and no incubation;
lane 2, no E2; lane 3, E2-25K, UBCH5C, and E2F1
(2 µM each); lane 4, E2-25K alone; lane
5, UBCH5C alone; lanes 6, E2F1 alone. The assays were
all performed at 30 °C in the presence of 1 mM
CaCl2, which did not affect the reaction. The
vertical bar indicates the position of ubiquitinated
p97.
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We then tested whether E2-25K and UBCH5C were individually able to
support p50 formation (Fig. 7B), using as a source of E3
B FII depleted of E1 and E2s by Ub-affinity chromatography. In the presence of 26S proteasomes (Fig. 7B, right panel, lane 2),
this mixture allowed a little p50 formation, probably due to some E2s in the WGE in which p97 was synthesized. Despite this background activity, addition of either E2-25K or UBCH5C (lanes 4 and
5) markedly stimulated the formation of p50, in accord with
our earlier findings. We also tested whether E2F1, an E2 present in
fraction I that was reported to support p105 processing (47), could
also function in this reconstituted system. In contrast to the other E2s tested, little or no stimulation of p97 processing was found with
purified recombinant E2F1 (Fig. 7, lane 6), although we
cannot conclude that E2F1 has no activity in this assay because of the high background in this reaction.
Differences in Rates of p97 and p105 Processing--
One
surprising finding was that although these extracts and reconstituted
preparations efficiently converted p97 to p50, they were consistently
less active in forming p50 from the full-length p105 precursor (Fig.
8A). This finding is in accord
with previous observations that truncated forms of p105, including p97,
are more efficiently processed into p50 than the full-length protein in
intact cells (5, 49). We therefore tested whether the more efficient
processing of p97 was due to differences in rates of ubiquitination or
to differences in some other step. Surprisingly, ubiquitination did not
appear to be the rate-limiting step in the processing of p105 into p50.
In several experiments, p97 seemed to be a better substrate for
ubiquitination than p105 (Fig. 8A), but in others (Fig.
8B), p97 and p105 were ubiquitinated to a similar extent. In
these cases, addition of pure 26S proteasomes caused a significant
increase in p50 production above background levels only when p97 was
used as the substrate. Thus, the failure of p105 to be efficiently
processed into p50 does not seem to be due to a deficiency in its rate
of ubiquitination. This observation suggests that the processing of
p105 by the 26S proteasome is inhibited by its C-terminal region after
the precursor has been ubiquitinated.

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Fig. 8.
Comparison of the ubiquitination and
processing of the truncated version p97 and the full-length p105.
A, the processing of p97 is more efficient than the
processing of p105. The ubiquitination and conversion of p97 or p105 to
p50 were monitored at 37 °C in an 80-µl reaction. At the times
indicated, 20 µl were removed and analyzed by SDS-PAGE and
autoradiography. The substrates were incubated in the presence of 2 mM ATP, 0.5 mg/ml Ub, 10 µg/ml human E1, 2 µM of each recombinant E2-25K and UBCH5C, and an
ATP-regenerating system. The source of E3 B was the FII depleted of
ubiquitinating enzymes by Ub-affinity chromatography and of proteasomes
by ultracentrifugation (5 µl (about 20 µg of protein) per 20 µl
of reaction mixture). The translation mixtures containing p97 or p105
were diluted 20-fold. As indicated at the bottom of the
figure, either buffer ( 26S) or 2 µg of purified HeLa
cell 26S proteasome (+26S) was added. B, although
ubiquitination of p105 and p97 was similar, processing was less
efficient for ubiquitinated p105. The ubiquitination and processing of
p97 (1 µl of the undiluted translation mixture) (left) and
p105 (0.5 µl of the undiluted translation mixture) (right)
were monitored as in Fig. 7A. The following enzymes were
added: E2s, 2 µM final concentration of E2-25K and
UBCH5C; E3, 4 µl of purified E3 B; 26S, 2 µg of purified HeLa
cell 26S proteasomes. The bottom two panels show
quantification using a Fujix Bas 1000 (Fuji) of the radioactivity that
accumulated in each lane as p50 or as ubiquitinated proteins
(brackets). In each case, the values obtained in the absence
of E3 B and of proteasomes were subtracted.
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DISCUSSION |
Enzymes Catalyzing p105 Ubiquitination in HeLa Cells--
These
studies demonstrate that E2-25K, as well as the members of the UBCH5
family of E2s, can support the ubiquitination of p105. Although E2-25K
was described and its cDNA was cloned several years ago (39), p105
is the first protein directly shown to be a substrate for E2-25K.
However, Huntingtin, the product of the gene altered in Huntington's
disease, is another likely substrate of E2-25K (50). E2-25K has the
ability to form chains of poly-Ub in the absence of a protein substrate
and of an E3 (51). However, this property does not appear to be
important in p105 ubiquitination, which strictly requires the presence
of an E3.
The UBCH5 family in yeast includes two closely related heat-shock
proteins, UBC4 and UBC5 (52), and in higher species it contains several
isoforms (37, 38, 53). In humans, three members of this family are
known (UBCH5A, UBCH5B, and UBCH5C), which are co-expressed in most
cells and tissues (37, 40, 48). In our study, both recombinant UBCH5B
and recombinant UBCH5C were found to support p105 ubiquitination.
Presumably, therefore, all E2s from the UBCH5 family can function in
this process. In mammalian cells, this family of E2s is involved in the
ubiquitination of a broad range of substrates, including p53 (40, 48)
and the cyclins (26).
In addition to E1 and E2s, ubiquitination of p105 requires a novel E3,
E3
B. Unlike certain E3s, such as E3
(24), E3
B does not bind to
an Ub-affinity column. Also, we could not detect formation of a thiol
ester with radioactive Ub when E3
B was incubated with E1 and E2-25K
or UBCH5B (Fig. 3B). In this respect, E3
B seems to behave
similarly to E3
and to differ from E6AP and other members of the
Hect family of E3s (54), which form thiol ester intermediates while
transferring Ub from E2s to the substrate (25). However, our inability
to detect a Ub-thiol ester with E3
B may simply mean that the amount
(or the stability) of this intermediate is too low for it to be
detected.
A surprising property of E3
B is its small size, 50 kDa, which is
much smaller than that of other E3s. Most of the known E3s have native
molecular masses greater than 100 kDa (23); the closest in size to
E3
B is the Pub1 protein (85 kDa), a yeast protein involved in cdc25
degradation (55). Our several purifications of E3
B, using different
E2s or combinations of E2s for screening, all yielded an enzyme of 50 kDa. Because this small size was found consistently, it is probably not
due to proteoytic cleavage during purification. Thus, E3
B
appears to be a new E3, although definitive proof will require
further characterization.
As noted above, the p105 ubiquitination enzymes characterized here from
HeLa cells apparently differ from those reported to catalyze this
process in reticulocytes (47). Orian et al. (47) reported
that no active E2 is found in FII and that the crucial E2s, either E2F1
or UBCH5A, are in FI. They also described a new E3 of 320 kDa that
catalyzes p105 ubiquitination (47). Their conclusion that the active
E2s are in FI is contradicted by our observation that E2-25K can
support p105 ubiquitination and processing. Moreover, in our
experiments, recombinant E2F1 showed little activity, if any, in p105
ubiquitination and processing, even in crude fractions (see Fig.
7B). However, Orian et al. (47) used an E2F1
isolated from rabbit reticulocyte FI (and not a recombinant protein),
which may be contaminated by other E2s. To avoid this potential
problem, they used the recombinant human UBCH5A, which they assumed to be the human homolog of the rabbit E2F1. However, it is now clear that
E2F1 and UBCH5A are distinct enzymes (38). Because both E2s are small
proteins with neutral pI, present in FI, it seems possible that their
E2F1 preparation was in fact contaminated by UBCH5s, which accounted
for the stimulation of p105 ubiquitination.
It is possible that distinct pathways for p105 ubiquitination function
in different cell types, with some cells using the enzymes described
here and others using the activities found in reticulocytes. However,
it is also possible that the 50-kDa E3
B is actually a component of
the 320-kDa activity found by Orian et al. (47), which was
isolated only by gel filtration of FIIA, a purification step that might
preserve a possible complex between E3
B and other proteins. In fact,
upon gel filtration of FII, we could detect p105 ubiquitination over a
broad range of fractions (data not shown), perhaps indicating the
presence of large complexes containing E3
B that are dissociated
during the extensive purification, which always yielded E3
B.
The physiologic relevance of the ubiquitination enzymes that we
isolated is suggested by their ability to support the generation of
p50. No evidence was presented as to whether the reticulocyte enzymes
can also function in this process. Rigorous conclusions about the
relative importance in vivo of these different enzymes in
p105 ubiquitination will require information on their relative concentrations and kinetic properties or their genetic
inactivation.
Reconstitution and Control of p105 Processing--
The present
data provide the first direct demonstration that the processing of p105
and its truncated form p97 requires their ubiquitination, as proposed
previously (6, 47). In the absence of E3
B, no ubiquitination
occurred, and no p50 was formed. We also show that the 26S proteasome
provides all the proteolytic activities necessary for this process,
because once p97 was ubiquitinated, it was processed only if 26S
proteasomes were added (Figs. 7 and 8).
It remains unclear how the 26S proteasome produces p50 from the
ubiquitinated p105 (p97). Proteolysis in this complex occurs within the
20S particle, the active sites of which are isolated inside its central
chamber (22, 28). Substrate entrance and product exit are restricted by
a small opening (28) into which only unfolded proteins can enter (56).
Within the 20S particle, protein degradation is highly processive and
generally converts polypeptides to small peptides without substrate
release (34). It is therefore likely that the N-terminal part of p105,
p50, is spared because it never reaches the central chamber of the 20S
proteasome. Either degradation of p105 occurs from its C-terminal half
with much of the protein still outside the 20S chamber, and stops
before p50 enters, or an endoproteolytic cleavage first releases p50
from the C-terminal half of p105, which is subsequently degraded. A
recent study presented evidence for such an endoproteolytic cleavage
that is determined by an upstream glycine-rich region present in p50
(57). If such an endoproteolytic activity actually exists, it must be
associated with the 26S proteasome, presumably in the 19S component,
because the 26S proteasome clearly can provide all proteolytic
activities necessary for p50 generation.
An important observation was the unexpected finding that the processing
of the C-terminally truncated forms of p105, in the reconstituted
system, was much more efficient than that of the full-length protein
(Fig. 8), as had been observed previously in cultured cells. (5, 49).
Thus, both in vivo and in vitro, the C-terminal
89 residues of p105 seem to inhibit the conversion of p105 to p50.
Recently, Mackichan et al. (49) reported an enhancement of
p105 processing upon treatment of cells with phorbol esters and
ionomycin. This effect required the phosphorylation of a PEST sequence
within the C-terminal region of p105. Moreover, after deletion of this
region, the truncated forms were more susceptible to constitutive
processing (49). These results confirm the inhibitory effect of the
C-terminal region of p105 and suggest that phosphorylation relieves
this effect. Therefore, it seems likely that in our purified system,
p105 was not efficiently processed because the appropriate kinase was
missing.
It is noteworthy that p105 ubiquitination, although necessary for
further processing, does not per se ensure conversion to p50
(Fig. 8B). Although the ubiquitinated p105 was not
efficiently processed to p50, it disappeared upon addition of the 26S
proteasome (Fig. 8). Therefore, the ubiquitinated p105 may have two
fates: 1) it may be recognized as a typical substrate by the 26S
proteasome and degraded completely, or 2) alternatively, it may be
de-ubiquitinated by the isopeptidase activity of the 26S proteasome
(31, 32). Either process could constitute an important type of
regulation of NF-
B production in vivo. Our findings
indicate that p105 ubiquitination is not rate-limiting for formation of
p50 under these in vitro conditions (Fig. 8B).
Moreover, in vivo, the unprocessed form of p105 accumulates
as the full-length p105 and not as larger, ubiquitinated species (5,
49, 58). These observations suggest that after ubiquitination, p105
molecules that are not been processed into p50 are de-ubiquitinated by
the 26S proteasome rather than totally degraded. A stimulation of p105
processing could thus be achieved by modifying the kinetic partitioning
between the isopeptidase and the "processing" activities of the 26S
proteasome. Indeed, recent studies have also suggested regulatory
functions for the isopeptidase activity of the 26S proteasome (32).
Future experiments involving measurements of the different possible
fates of ubiquitinated p105 in extracts and in vivo should
allow us to test this suggestion.
We are grateful to D. H. Lee and also to S. Lecker, H. C. Huang, C. Sears, M. A. Read, V. J. Palombella, and T. Maniatis for critical reading of the manuscript and
to a number of colleagues for providing reagents.