From the Department of Biochemistry and the Rappaport
Family Institute for Research in the Medical Sciences, Bruce Rappaport
Faculty of Medicine, Haifa 31096, Israel, the ¶ Department of
Pathology and the Kaplan Comprehensive Cancer Center, New York
University Medical Center, New York, New York 10016, and the
Department of Immunology and Cell Biology, Graduate School of
Medicine, Kyoto University, Sankyo-ku, Kyoto 606-8501, Japan
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ABSTRACT |
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The last step in the activation of the
transcription factor NF- Generation and activation of the transcription factor NF- The ubiquitin system targets a wide array of short-lived regulatory
proteins such as transcriptional activators, tumor suppressors and
growth modulators, cell cycle regulators, and signal transduction pathway components. Consequently, it is involved in the regulation of
many basic cellular processes. Among these are cell cycle and division,
differentiation, and development; response to stress and extracellular
stimuli; modulation of cell-surface receptors; DNA repair; regulation
of the immune and inflammatory responses; biogenesis of organelles; and
apoptosis. Degradation of a protein by the ubiquitin system involves
two distinct and successive steps: (i) covalent attachment of multiple
ubiquitin molecules to the target protein and (ii) degradation of the
tagged substrate by the 26 S proteasome. Conjugation proceeds via a
three-step mechanism involving three enzymes. Initially, ubiquitin is
activated by the ubiquitin-activating enzyme (E1). One of several E2
enzymes (ubiquitin carrier proteins or ubiquitin-conjugating enzymes
(designated UBCs)) transfers ubiquitin from E1 to the substrate, either
directly or via a member of the ubiquitin-protein ligase family of
enzymes (E3) to which the substrate protein is specifically bound. The first ubiquitin moiety typically binds via its C-terminal Gly and
generates an isopeptide with an A few E2 enzymes, such as E2-C, which is involved in the targeting
cyclins (18), appear to be E3- and substrate group-specific. Other E2
enzymes are involved in transfer of ubiquitin to several E3 enzymes and
in the targeting of different groups of substrates. Three homologous
human enzymes (UBCH5a, UBCH5b, and UBCH5c) have been described (19)
that are closely related to Saccharomyces cerevisiae Ubc4
and Ubc5, which are involved in targeting many cellular proteins,
particularly under stress. They are also homologous to Drosophila
melanogaster UBCD1 and to Arabidopsis thaliana
UBC8-12. UBCH5b and UBCH5c are ~98% identical, whereas UBCH5a is
~90% similar to the b and c species. Members of the family have been
shown to be involved in cell-free conjugation of several proteins
(e.g. p53 (20) and p105 (21)) and to interact with certain
E3 enzymes such as members of the HECT domain family of ligases (22).
The cellular substrates of these enzymes have, however, remained
obscure. S. cerevisiae cdc34/ubc3, which is
involved in G1 Materials
High pressure liquid chromatography-purified synthetic peptides
were purchased from SynPep (Dublin, CA). cDNAs coding for wild-type
and S32A,S36A I Methods
Preparation and Fractionation of Crude Cell
Lysates E1 and E3 Enzymes--
E1 was purified from human erythrocytes
as described (29). Crude Fraction II or Fraction IIA was used as a
source of E3 (30).
E2 Enzymes--
E2-14K was purified from rabbit reticulocytes as
described (31). Recombinant E2-C was as described (18). cDNA coding
for UBCH7 was as described (32). Following induction and cell
disruption, the bacterial cell extract was resolved on DEAE-cellulose,
and the enzyme that was contained in Fraction I was further purified via gel filtration chromatography on a Superdex 75 HiLoad column (16 × 600 mm; Amersham Pharmacia Biotech). Recombinant E2-8A (33) was expressed in bacteria, and the protein was partially purified as
described above for UBCH7. cDNAs coding for UBCH5a (20) and UBCH5b
and UBCH5c (19) were obtained from Dr. Allan Weissman (National
Institutes of Health). cDNAs coding for human CDC34/UBC3 (24),
E2-25K (34), and E2-20K (35) were cloned by reverse transcription-polymerase chain reaction from 293 cells. Active-site, dominant-negative mutants of the different E2 enzymes (C85A UBCH5a, C85A UBCH5b, C85A UBCH5c, C93A CDC34/UBC3, C92A E2-25K, and C87A E2-20K) were generated by a two-step polymerase chain reaction as
described (36). For bacterial expression, the cDNAs coding for
wild-type and mutant UBCH5a, UBCH5b, and UBCH5c, for CDC34/UBC3, and
for E2-25K and E2-20K were subcloned into the pT7-7 vector (37)
following their modification by polymerase chain reaction to contain an
N-terminal His6 tag. For transient expression in mammalian
cells, the cDNAs were subcloned into the pCAGGS vector (38),
whereas for tetracycline-inducible expression, they were subcloned into
the pUHG10-3 vector (39). Sequences of all the constructs were verified
using the ABI 310 or ABI 377 autosequencers (Perkin-Elmer).
Identification of the transfected cDNAs was carried out by reverse
transcription-polymerase chain reaction of cellular RNA following
induction by doxycycline and primers from noncoding regions in the
vectors used for transfection. Amplified cDNAs were diagnosed by
specific restriction analysis. For purification of the His-tagged E2
enzymes, BL21(DE3) cells, transformed with the appropriate expression
constructs, were cultured in 2× YT medium, and
isopropyl- Conjugation of I Degradation of I Degradation of MyoD--
Degradation of bacterially expressed
MyoD was followed in crude HeLa cell Fraction II by Western blot
analysis as described (31).
Degradation of the General Population of Short-lived
Proteins--
HeLa Tet-on cells stably transfected with C85A UBCH5b
and C85A UBCH5c were labeled with [35S]methionine (100 µCi/ml) for 5 min (pulse). Following removal of the labeling amino
acid, the cells were incubated for the indicated time periods in the
presence of excess unlabeled methionine (chase), and degradation was
monitored by measuring release of trichloroacetic acid-soluble
radioactivity into the medium as described (43).
Determination of Ubiquitin Conjugates in Cells--
Cell
extracts derived from uninduced or doxycycline-induced HeLa cells
stably transfected with Cys-to-Ala mutant UBCH5b and UBCH5c or from 293 cells transiently transfected with Cys-to-Ala mutant CDC34/UBC3 were
resolved via SDS-PAGE (10%). Resolved proteins were blotted onto
nitrocellulose paper and probed with anti-ubiquitin antibody as
described (31).
Protein Concentration--
Protein concentration was determined
according to Bradford (44) using bovine serum albumin as a standard.
Iodination of Ubiquitin--
Ubiquitin was iodinated, using the
chloramine-T method, as described (29).
UBCH5b, UBCH5c, and CDC34/UBC3 Are the Ubiquitin Carrier Proteins
Involved in Conjugation of NF-
Recent evidence suggests that human
To dissect further the mechanism of action of the E2 enzymes and to
enable the development of tools with which it will be possible to
analyze the function of these enzymes in vivo, we tested the
effect of catalytic site mutant species of the different E2 enzymes on
the conjugation of the inhibitor in vitro. As shown in Fig.
2A, a mutant species of UBCH5c
in which the ubiquitin-binding residue Cys85 was
substituted with Ala, strongly inhibit the wild-type enzyme-catalyzed reaction. C85A mutant UBCH5b had a similar effect (Fig. 2B).
Interestingly, C85A mutant UBCH5a also inhibited the reaction, although
to a lesser extent (Fig. 2B). It is possible that this
enzyme, which is 90% homologous to UBCH5b and UBCH5c, binds to the
I
Considering the relative small number of E2 enzymes and the numerous
cellular proteins targeted by the ubiquitin system, it appears that
each E2 enzyme may be involved in the targeting of many cellular
substrates, most probably via its interaction with several E3 enzymes.
Indeed, as shown in Fig. 3, UBCH5a,
UBCH5b, and UBCH5c as well as UBCH7, E2-8A, and E2-14K have each many endogenous cellular targets, whereas E2-25K, E2-20K, and E2-C appear to
be relatively more specific. Because of the inhibitory activity of
Fraction II on the activity of CDC34/UBC3 in the cell-free system (see
above), it is impossible to assess the abundance of its substrates in
this system. Despite this apparently large number of substrates, the
pI UBCH5b, UBCH5c, and CDC34/UBC3 Are the Ubiquitin Carrier Proteins
Involved in Signal-induced Degradation of I
To test for the specificity of the inhibitory effect of the mutant E2
enzymes on the degradation of I We have shown that UBCH5b, UBCH5c, and CDC34/UBC3 are the
ubiquitin carrier proteins involved in the conjugation of
signal-induced phosphorylated and NF- Why does the degradation of a single protein employ different species
of E2 enzymes? As for UBCH5b and UBCH5c, because the two enzymes are
almost identical (19), they are probably functionally redundant, act
via the same E3 enzyme, and can substitute for each other. This
assumption is supported by the findings that mutant UBCH5b can inhibit
UBCH5c conjugation in a cell-free system (Fig. 2B) and that
there is no difference between the effect of the two enzymes in
vivo (Fig. 6): expression of either one or two of the UBCH5
species displays a similar inhibitory effect on I The reason for the inhibitory effect of Fractions II and IIA on the
activity of CDC34/UBC3 is not known. It is possible that the enzyme
binds to an E3 in Fraction II and is not available for ubiquitin
transfer to the substrate-bound E3. Initial experiments in which we
increased the concentration of CDC34/UBC3 in the reaction mixture
failed to corroborate this hypothesis (Fig. 1C,
lane 4), although did not exclude it altogether.
Another possibility is that the conjugates generated by CDC34/UBC3 are
different from those generated by the UBCH5 enzymes and are more
sensitive to the activity of ubiquitin C-terminal hydrolases
(isopeptidases). These hydrolases must be insensitive to ubiquitin
aldehyde, which is included in all reactions.
Interestingly, C85A UBCH5a also inhibits the conjugation of the
inhibitor in vitro (Fig. 2B) and its subsequent
degradation in vivo (Fig. 6D), although the
inhibition is weaker than that exerted by its b and c counterparts.
Wild-type UBCH5a also catalyzes pI The nonspecific conjugation catalyzed by UBCH5a, UBCH7, and E2-8A in
the cell-free system (Fig. 1, A and B) also
deserves some attention. It is possible that these E2 enzymes are
involved in the conjugation and subsequent degradation of the free
dissociated inhibitor. This process is clearly signal-independent and
must be slower then the signal-induced proteolysis of the inhibitor. It
is clear that this process does not require phosphorylation of the
inhibitor, as the S32A,S36A mutant species of the inhibitor is also
conjugated, and the process is not inhibited by the I In a recent study, it has been reported that UBC9/HUS5 is the E2
involved n IB is signal-induced, ubiquitin- and
proteasome-mediated degradation of the inhibitor I
B
. Although
most of the components involved in the activation and degradation
pathways have been identified, the ubiquitin carrier proteins (E2) have
remained elusive. Here we show that the two highly homologous members
of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian
homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the
process. The conjugation reaction they catalyze in vitro is
specific, as they do not recognize the S32A,S36A mutant species of
I
B
that cannot be phosphorylated and conjugated following an
extracellular signal. Furthermore, the reaction is specifically
inhibited by a doubly phosphorylated peptide that spans the ubiquitin
ligase recognition domain of the inhibitor. Cys-to-Ala mutant species
of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor
-induced degradation of the inhibitor in vivo. Not
surprisingly, they have a similar effect in a cell-free system as well.
Although it is clear that the E2 enzymes are not entirely specific to
I
B
, they are also not involved in the conjugation and degradation
of the bulk of cellular proteins, thus exhibiting some degree of
specificity that is mediated probably via their association with a
defined subset of ubiquitin-protein ligases. The mechanisms that
underlie the involvement of two different E2 species in I
B
conjugation are not clear at present. It is possible that different
conjugating machineries operate under different physiological
conditions or in different cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
involve two successive ubiquitin- and proteasome-mediated proteolytic steps: (i) processing of the precursor protein p105 to the active subunit p50 and (ii) signal-induced degradation of the inhibitor I
B
. Degradation of I
B
is triggered by a broad array of
stimuli, such as binding of cytokines or viral products to their
appropriate receptors. The receptors then recruit adaptor proteins such
as TRADD and TRAF. Consequently, activated NF-
B-inducing kinase (1)
is released and sequestered into a large complex that contains, among
other proteins, I
B kinases
and
(see, for example, Ref. 2),
NEMO (NF-
B essential modulator)
(3) or I
B kinase
(4), and I
B kinase complex-associated
protein (5). Complex formation leads, most probably, to activation of
the I
B kinases that phosphorylate NF-
B-complexed I
B
on
serines 32 and 36. This modification leads to its targeting and rapid
degradation by the ubiquitin-proteasome pathway (6-10). Most of the
upstream components of the signaling pathway have been identified.
Also, recent studies have identified an SCF complex that contains
Skp1p, Cullin1, and the F-box protein
-TrCP as the
ubiquitin-I
B
ligase complex (see for example Refs. 11-13).
However, all these studies have not identified the ubiquitin carrier
protein(s) involved in targeting of phosphorylated I
B
(pI
B
),1 and the
identity of the enzyme(s) has remained elusive.
-NH2 group of a Lys
residue of the protein substrate. In successive reactions, a
polyubiquitin chain is synthesized by transfer of additional activated
ubiquitin moieties to Lys48 of the previously conjugated
molecule. The structure of the ubiquitin system appears to be
hierarchical: a single E1 carries out activation of ubiquitin required
for all modifications. It can transfer ubiquitin to several species of
E2 enzymes. Each E2 acts in concert with either one or several E3
enzymes. Following conjugation, the protein moiety of the adduct is
recognized, most probably via its polyubiquitin chain, and degraded by
the 26 S proteasome complex. Free and reutilizable ubiquitin is
released via the activity of isopeptidases (for recent reviews and a
monograph on the ubiquitin system, see, for example, Refs. 14-17).
S transition, encodes a 295-amino acid
E2 (23). The human homolog of this enzyme has been cloned (24). Recent
evidence indicates that CDC34/UBC3 acts in concert with different SCF
ubiquitin ligase complexes that target, among other substrates,
phosphorylated G1 regulatory proteins (reviewed recently in
Refs. 16, 17, and 25-27). It is also involved, along with SCF
complexes, in other processes such as down-regulation of methionine
biosynthesis gene products (28).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
proteins were as described (9-11). Antibodies
to I
B
, p65, and MyoD were from Santa Cruz Biotechnology, whereas
an antibody to human CDC34/UBC3 was from Transduction Laboratories. The
wheat germ-based coupled transcription-translation kit and TNF-
were
from Promega. Ni2+-nitrilotriacetic acid-agarose and
anti-RGS-His antibody were from QIAGEN Inc. Materials for SDS-PAGE were
from Bio-Rad. Hexokinase and okadaic acid were from Roche Molecular
Biochemicals. L-[35S]Methionine and
Na125I were obtained from NEN Life Science Products.
Ubiquitin, ATP, phosphocreatine kinase, phosphocreatine,
2-deoxyglucose, bestatin, doxycycline, Tris buffer, and
isopropyl-
-D-thiogalactopyranoside were from Sigma.
HEPES was from Calbiochem. DEAE-cellulose (DE52) was from Whatman.
Tissue culture sera and media were purchased from Biological Industries
(Kibbutz Bet Haemek, Israel) or from Sigma. Restriction and modifying
enzymes were from New England Biolabs Inc. Immobilized protein A was
from Amersham Pharmacia Biotech. Reagents for ECL were from Pierce.
Centricons and Centripreps (10-kDa molecular mass cutoff) for rapid
concentration and dialysis by centrifugation were from Amicon.
Nitrocellulose paper was from Schleicher & Schüll.
Oligonucleotides were synthesized by the local facility in the
Department of Immunology and Cell Biology at the University of Kyoto
and by Biotechnology General (Rehovot, Israel). All other reagents used
were of high analytical grade.
HeLa cells grown in suspension in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum were washed
twice in a buffer containing 20 mM HEPES (pH 7.4), 1 mM dithiothreitol, and 150 mM NaCl. Following resuspension (10 ml of 108 cells/ml) in a similar buffer
without NaCl, the cells were exposed, for two cycles of 15 min each, to
1500 p.s.i. of N2 in a high pressure cell (Parr
Instrument Co., Moline, IL). The disrupted cells were centrifuged
successively for 30 min at 1500, 12,000, and 50,000 × g. The supernatant from the last centrifugation step was
collected and either used as a crude extract or further fractionated over DEAE-cellulose onto unadsorbed material (Fraction I) and a high
salt eluate (Fraction II) as described (29). Fraction II was further
fractionated by (NH4)2SO4 into
Fraction IIA (0-38%) and Fraction IIB (42-80%) as described
(30).
-D-thiogalactopyranoside (400 µM) was added when A600 nm attained 0.6. The
induced proteins were purified over Ni2+-nitrilotriacetic
acid-agarose according to the manufacturer's instructions. For
transient expression of the various E2 enzymes, 293 cells were
transfected with the various E2 constructs using the DEAE-dextran
method (40). Transfection efficiency was always >75% as determined by
parallel transfection with a gene encoding green fluorescent protein.
Experiments were carried out 40-48 h following transfection. Stable
transformants of HeLa Tet-on cells (CLONTECH) for
inducible expression of mutant E2 enzymes were established by calcium
phosphate transfection as described (41). Protein expression was
induced by the addition of 1 µg/ml doxycycline for 48 h.
Induction of the proteins was monitored by Western blot analysis using
anti-His tag antibody.
B
and Cellular Proteins in
Vitro--
[35S]Methionine-labeled I
B
complexed to
HeLa p50/p65 was generated as described (9, 10). Briefly, cDNAs
coding for wild-type or S32A,S36A mutant I
B
were
translated/transcribed in vitro in wheat germ extract in the
presence of [35S]methionine. For phosphorylation and
incorporation into endogenous cellular NF-
B complex, the labeled
protein was incubated in HeLa cell extract in the presence of okadaic
acid. Following incubation, anti-p65 antibody was added, and the immune
complex was immobilized on protein A-Sepharose beads. The washed
immobilized complex was used as a substrate in the different
conjugation assays. Conjugation of I
B
to ubiquitin was monitored
essentially as described (9, 10, 30, 31). Briefly, the reaction mixture
contained (in final volume of 25 µl) 4 µl of packed and washed
protein A-Sepharose beads containing the labeled I
B
protein
(~20,000 cpm), 40 mM Tris-HCl (pH 7.6), 5 mM
MgCl2, 2 mM dithiothreitol, 5 µg of
ubiquitin, and 0.5 µg of the isopeptidase inhibitor ubiquitin
aldehyde (42). Crude HeLa cell extract (60 µg), Fraction II (45 µg
of protein), Fraction I (15 µg; as a source of E2), and Fraction IIA
(25 µg; as a source of E3); E1 (0.75 µg); E2-14K (0.75 µg); E2-C
(0.5 µg); E2-8A (0.75 µg); UBCH7 (0.75 µg); and E2-25K (1.0 µg)
were added as indicated. UBCH5a, UBCH5b, and UBCH5c were added at 0.75 µg or as indicated, whereas CDC34/UBC3 was added at 1.25 µg or as
indicated. The different Cys-to-Ala mutant species of UBCH5a, UBCH5b,
and UBCH5c and of CDC34/UBC3 were added as indicated. E2-20K was added
at 0.8 µg or as indicated. The biological activity of the different
E2 enzymes was monitored using formation of E2-S~ubiquitin thiol
ester in the presence of E1 as described (29). The I
B
phosphopeptide and S32A,S36A peptide were added at 40 µM.
In mixtures containing the peptides, bestatin was added at 20 µg/ml
and was incubated for 15 min at room temperature with all the
components of the reaction except for the peptides and the labeled
substrate. Following addition of the peptides, the reaction mixture was
further incubated for 5 min at 30 °C prior to the addition of the
labeled substrate. All mixtures containing the peptides contained also 2 µM okadaic acid. When complete HeLa cell extract (25 µg) was used as a source of endogenous substrates, endogenous E1 and
E2 and E3 enzymes were inactivated by N-ethylmaleimide (10 mM; 10 min at room temperature) followed by neutralization
with dithiothreitol (6 mM; 1 min at room temperature). E1,
the different E2 enzymes, Fraction IIA (as a source of E3 enzymes), and
125I-labeled ubiquitin (0.1 µg, ~100,000 cpm) were
added as described above and in the figure legends. The complete
reaction mixtures were incubated for 30 min at 37 °C in the presence
of ATP (0.5 mM ATP and ATP-regenerating system) (30, 31).
Reactions were terminated by the addition of 12.5 µl of 3-fold
concentrated sample buffer and, following boiling, were resolved via
SDS-PAGE (10%). Gels were dried, and
[35S]methionine-labeled proteins were visualized using a
PhosphorImager (Fuji, Japan). 125I-Labeled proteins were
visualized following exposure to Kodak XAR-5 film.
B
in Vivo--
The fate of I
B
was
monitored in cells that were stably or transiently transfected with the
different species of E2 enzymes. Following incubation in the presence
of TNF-
(10 ng/ml), cells were harvested at the indicated time
points, lysed in sample buffer, and resolved via SDS-PAGE (10%). The
resolved proteins were blotted onto nitrocellulose paper, and the
inhibitor was visualized by Western blot analysis using a specific
antibody, a secondary horseradish peroxidase-conjugated antibody, and
ECL reaction as described (31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-complexed and Phosphorylated
I
B
in Vitro--
To identify the ubiquitin carrier proteins
involved in conjugation of pI
B
in the context of the
heterotrimeric pI
B
·p50·p65 complex, a cell-free system was
reconstituted. The system contained E1, Fraction IIA (as a source of
E3), and different species of purified E2 enzymes. As shown in Fig.
1A, only UBCH5b and UBCH5c conjugated the inhibitor in a specific manner. The adducts are of high
molecular mass, but more important, they are specific to the
phosphorylated species of the inhibitor: they are not generated when
the S32A,S36A mutant species of I
B
is used as a substrate. UBCH5a, UBCH7, and E2-8A also conjugated the inhibitor; however, the
conjugates are mostly of the low molecular mass type and do not appear
to be specific: S32A,S36A I
B
is also targeted. E2-20K, E2-25K,
E2-C, and E2-14K did not conjugate the inhibitor at all. To further
establish the specificity of these two E2 enzymes, we tested the effect
of a specific phosphopeptide that spans the phosphorylation domain of
I
B
. This peptide blocks specifically the conjugation reaction by
interfering with the recognition by E3 (10). As shown in Fig.
1B, the phosphopeptide inhibited specifically the UBCH5b-
and UBCH5c-mediated conjugation, but not UBCH5a-, UBCH7-, and
E2-8A-mediated conjugation. The S32A,S36A peptide had no effect on the
specific conjugation. As noted, the conjugates generated by UBCH5a,
UBCH7, and E2-8A are of low molecular mass. UBCH5b and UBCH5c also
catalyzed formation of such conjugates (see Fig. 1, A and
B). However, they were also generated when S32A,S36A mutant
I
B
was used as a substrate, and the process was not sensitive to
the phosphopeptide. Thus, they do not appear to be specific to the
complexed and signal-induced phosphorylated inhibitor and/or do not
lead to its degradation (for the possible significance of these
conjugates, see "Discussion").
View larger version (35K):
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Fig. 1.
Specificity of different species of E2
enzymes in conjugating I B
in vitro. A, effect of different
species of E2 enzymes on the conjugation of wild-type and S32A,S36A
mutant I
B
proteins. Conjugation reactions were carried out as
described under "Experimental Procedures" and contained labeled,
NF-
B-complexed wild-type and phosphorylated (pWT) or
S32A,S36A (S/A) mutant I
B
proteins, E1, HeLa cell
Fraction IIA (as a source of E3 enzymes), and the indicated species of
E2 enzymes. E2 enzymes were added as described under "Experimental
Procedures." Following incubation, reactions were resolved via
SDS-PAGE, and proteins were visualized by a PhosphorImager.
B, effect of the I
B
phosphopeptide and S32A,S36A
peptide on the conjugation of I
B
by different E2 enzymes.
Conjugation reactions were carried out, and proteins were visualized as
described for A and under "Experimental Procedures."
E2 and Peptide denote the different species of
the E2 enzymes and peptides added to the mixtures, respectively.
PP denotes the I
B
phosphopeptide, whereas
NR denotes the S32A,S36A I
B
peptide. Conj.
denote conjugates. I
B
and pI
B
denote unmodified and phosphorylated I
B
, respectively.
C, effect of Fraction I, CDC34/UBC3, and UBCH5c on the
conjugation of pI
B
. Conjugation reactions were carried out
essentially as described under "Experimental Procedures" and contained labeled wild-type
pI
B
, ubiquitin, and, when indicated, HeLa cell Fraction I
(FrI; 15 µg), Fraction II (FrII; 45 µg), extract
(Ext.; 60 µg), CDC34/UBC3 (0.75 µg), and UBCH5c (0.75 µg). D, distribution of CDC34/UBC3 between Fractions I and
II. HeLa cell extract (60 µg), Fraction I (15 µg; derived from
~60 µg of HeLa cell extract), and Fraction II (15 and 45 µg; 45 µg was derived from ~60 µg of crude HeLa cell extract) were
resolved via SDS-PAGE (12.5%). Following transfer to nitrocellulose
paper, CDC34/UBC3 was detected via Western blot analysis as described
under "Experimental Procedures." E, effect of the
I
B
phosphopeptide, the S32A,S36A peptide, and Fraction II on
conjugation of pI
B
by CDC34/UBC3. Conjugation reactions were
carried out essentially as described under "Experimental
Procedures" and for A-C. All E2 enzymes employed were
added at a concentration that catalyzes the transfer of ~2 pmol of
125I-labeled ubiquitin to the E2 enzyme under the
conditions employed (29, 30).
-TrCP is the receptor subunit of
the I
B
ligase (11-13).
-TrCP is an F-box-containing WD
protein that, unlike the initial finding report (11), does not act
alone. It associates with Skp1p and Cullin1 to generate an SCF complex
that serves as the I
B
E3 (12, 13). As mentioned above, these
complexes are involved also in cell cycle control via their ability to
target certain phosphorylated cell cycle regulators. They act in
concert with CDC34/UBC3 that serves as the E2 enzyme. Therefore, it was
important to study the role of this E2 in pI
B
conjugation. Our
initial results indicated that CDC34/UBC3 is not the E2 enzyme involved
in targeting the inhibitor. As shown in Fig. 1C, the enzyme
did not conjugate the inhibitor (compare lanes 4 and
5). Furthermore, the enzyme was clearly confined to Fraction
II (Fig. 1D), and one would expect that if it plays a role
in the conjugation of the inhibitor, ubiquitin-supplemented Fraction II
alone would be sufficient to support conjugation. However, this was not
the case (Fig. 1C, lane 2), and the
process required the addition of either Fraction I as a source of a
different E2 (Fig. 1C, lane 3) or an
E2 enzyme such as UBCH5c that is contained in Fraction I
(lane 5) (all members of the UBCH5 family are
contained in Fraction I (19, 20, 30, 32)). The confinement of
CDC34/UBC3 to Fraction II implies that potential CDC34/UBC3-mediated
conjugation of pI
B
should not require the addition of Fraction I
or of an E2 contained in this fraction. Other studies have yielded
similar results and have shown that pI
B
conjugation requires an
E2 enzyme contained in Fraction I. Alkalay et al. (9) and
Winston et al. (12) used crude Fraction I as a source of E2
in the reconstituted cell-free conjugation reaction. They could not
demonstrate any conjugating activity in Fraction II. Similarly, Yaron
et al. (11) and Spencer et al. (13) used, for the
reconstitution assays, UBCH5c and UBCH5, respectively, (the species of
the UBCH5 enzyme is not indicated), which are contained in Fraction I
(both groups, however, have not shown the specificity of the enzyme,
the role of other members of the family or other E2 enzymes in the
process, and the involvement of the enzyme in I
B
proteolysis
in vivo; see below). Taken together, these findings strongly
suggested that CDC34/UBC3 is not involved in I
B
conjugation.
However, more careful reconstitution assays have shown a specific role for CDC34/UBC3 in conjugation of the inhibitor in vitro
(Fig. 1E) and in vivo (see below). As shown
clearly in Fig. 1E (lanes 2 and
7), the phosphorylated pI
B
·p50·p65 substrate
recruited into the complex the E3 enzyme, and conjugation of the
inhibitor required only E1 and an E2 (similar results as for the
presence of endogenous E3 in the signal-induced phosphorylated
substrate complex have been reported also by Yaron et al.
(11)). In the absence of added exogenous E3, CDC34/UBC3 supported
conjugation of the inhibitor (Fig. 1E, lane
2). Conjugation was specific, as it was inhibited by the
I
B
phosphopeptide (Fig. 1E, lane 3), but not by the S32A,S36A mutant peptide (lane
4). Also, the S32A,S36A mutant species of I
B
was not
conjugated by CDC34/UBC3 (Fig. 1E, lane
6). Interestingly, however, the addition of crude Fraction
II as a source of E3 strongly inhibited the CDC34/UBC3-catalyzed conjugation, although additional endogenous CDC34/UBC3 was contained in
this fraction (Fig. 1, C, lanes 2 and
4; and E, lane 5). Similar results were obtained using Fraction IIA as a source of E3 (data not
shown). In striking contrast, the addition of Fraction II did not
inhibit the UBCH5c-catalyzed reaction. On the contrary, this reaction
was further stimulated by Fraction II, a stimulation that was probably
due to the addition of exogenous E3 complex (Fig. 1E,
compare lanes 7 and 8; compare also
the amount of conjugates in Fig. 1, C, lane 5;
and E, lane 7). The inhibition of
CDC34/UBC3-mediated conjugation by Fractions II and IIA is probably the
reason for the conclusion in other studies (9, 11-13) that the
I
B
E2 is contained in Fraction I. Although the reason for the
inhibitory effect is not clear (see "Discussion"), it appears that
CDC34/UBC3 is also involved in signal-induced targeting of the
inhibitor in vivo (see below), thus suggesting a
physiological role for the enzyme in the turnover of the inhibitor in
the intact cell (see "Discussion").
B
E3, but cannot catalyze the ubiquitin transfer reaction (see
also below). Not surprisingly, E2-20K, which cannot catalyze
conjugation, did not inhibit it as well (Fig. 2B).
Similarly, wild-type E2-25K and the Cys-to-Ala mutant species of E2-20K
and E2-25K did not have any effect (data not shown). Thus, it appears
that these enzymes do not interact with any component of the
I
B
-conjugating machinery (see also below). Not surprisingly, C93A
CDC34/UBC3 had an inhibitory effect similar to that of the mutant
species of UBCH5b and UBCH5c (Fig. 2C).
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Fig. 2.
Effect of the catalytic site Cys-to-Ala
mutant species of E2 enzymes on the conjugation of
pI B
.
A, effect of C85A (C/A) UBCH5c on wild-type
(wt) UBCH5c-mediated conjugation of pI
B. Conjugation
reactions were carried out in the presence of E1 and the indicated
species and amounts of E2 enzymes, Fraction IIA (as a source of E3),
and ubiquitin as described under "Experimental Procedures."
Proteins were visualized following exposure to a PhosphorImager screen.
B, effect of C85A UBCH5a, C85A UBCH5b, and E2-20K on
wild-type UBCH5c-mediated conjugation of pI
B
. Conjugation
reactions were carried out as described for A. C,
effect of C93A mutant CDC34/UBC3 on wild-type CDC34/UBC3-mediated
conjugation of pI
B
. Conjugation reactions were carried out as
described for A and B, except that reaction
mixtures did not contain Fraction IIA. Conj.,
conjugates.
B
conjugation reaction is relatively specific, and pI
B
is
conjugated only by UBCH5b, UBCH5c, and CDC34/UBC3. This is due, most
probably, to the specific interaction of these E2 enzymes with the
specific SCF E3 complex that recognizes only the post-translationally
modified and complexed inhibitor. To further demonstrate the limited,
although not entirely specific, scope of these enzymes and to
corroborate the notion that they are not involved in targeting the bulk
of cellular proteins in vitro, we have shown that their
corresponding mutant species do not inhibit conjugation of labeled
ubiquitin to the general population of cellular proteins (Fig.
4A). In addition, they do not
inhibit ubiquitin-mediated degradation of MyoD (Fig. 4B), a
bona fide substrate of the ubiquitin system (31). For this
substrate, it has been reported that it is targeted by E2-14K (31), but interestingly, also by CDC34/UBC3 (45).
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Fig. 3.
Conjugation of endogenous HeLa proteins by
different species of E2 enzymes. Conjugation of
125I-ubiquitin to endogenous HeLa cell proteins was monitored as
described under "Experimental Procedures." Reaction mixtures
contained 125I-labeled ubiquitin
(125I-Ub), E1, the indicated species of E2 enzymes,
HeLa cell Fraction IIA (as a source of E3 enzymes),
N-ethylmaleimide-treated HeLa cell extract as a source of
endogenous substrates, ubiquitin, and ATP. Proteins were visualized by
autoradiography. Conj., conjugates.
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Fig. 4.
Specificity of the conjugating activity of
UBCH5b and UBCH5c. A, lack of effect of C85A
(C/A) UBCH5a, C85A UBCH5b, and C85A UBCH5c on the
conjugation of ubiquitin to endogenous HeLa cell proteins. The C85A
species of UBCH5c, UBCH5b, and UBCH5a were added at the indicated
concentrations to a reaction mixture containing
125I-labeled ubiquitin (125I-Ub), crude
HeLa cell extract, and ATP. Reactions were carried out as described
under "Experimental Procedures," and conjugates (Conj.)
were visualized by autoradiography. B, lack of effect of the
C85A mutant species of UBCH5a, UBCH5b, and UBCH5c on the degradation of
MyoD. Degradation of MyoD was carried out in ubiquitin-supplemented
crude HeLa cell Fraction II and ATP as described under "Experimental
Procedures." Mixtures also contained the indicated amounts the of
C85A mutant species of UBCH5a, UBCH5b, and UBCH5c.
B
in Vivo--
To
study the role of UBCH5b, UBCH5c, and CDC34/UBC3 in signal-induced
degradation of I
B
in vivo, we tested the effect of catalytic site mutant species of the enzymes on TNF-
-induced degradation of I
B
in cells. As shown in Fig.
5, transient expression of mutant UBCH5b
and UBCH5c (panel B) as well as of CDC34/UBC3 (panel C) significantly inhibit the degradation
of the inhibitor. Expression of any one of the two UBCH5 mutant enzymes
alone had a similar effect monitored with both of them (data not shown; see below, however). Not surprisingly, mutant E2-20K and E2-25K did not
have any effect in the transiently transfected cells (Fig. 5D). A similar effect was observed also in stably
transfected cells (Fig. 6). UBCH5b and
UBCH5c, expressed either singly or together, strongly inhibited the
degradation of the inhibitor (Fig. 6, A-C). Like its effect
in vitro (see Fig. 2B), UBCH5a also inhibited the
degradation of I
B
, although the effect was weaker compared with
that of UBCH5b and UBCH5c (Fig. 6D).
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Fig. 5.
Effect of transiently expressed catalytic
site mutant species of UBCH5b and UBCH5c, CDC34/UBC3, E2-20K, and
E2-25K on TNF- -induced degradation of
I
B
in 293 cells.
Empty pCAGGS vector (A) or vectors containing C85A UBCH5b
and UBCH5c (B), C93A CDC34/UBC3 (C), and C87A
E2-20K or C92A E2-25K (D) were transiently transfected into
293 cells. The effect of expression of the different E2 enzymes on
TNF-
-induced degradation of I
B
was monitored 48 h
following transfection as described under "Experimental Procedures"
using Western blot analysis and ECL.
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Fig. 6.
Effect of mutant species of UBCH5a, UBCH5b,
and UBCH5c on TNF- -induced degradation of
I
B
in Tet-on HeLa
cells. C85A UBCH5b (A), C85A UBCH5c (B),
C85A UBCH5b and C85A UBCH5c (C), and C85A UBCH5a
(D) were stably transfected into Tet-on HeLa cells as
described under "Experimental Procedures." Stability of endogenous
I
B
was monitored via Western blot analysis and ECL in uninduced
and doxycycline-induced cells following the addition of TNF-
.
Numbers indicate time (minutes) following addition of the
cytokine. E demonstrates the effect of doxycycline on
expression of UBCH5c. Here, UBCH5c was detected using an anti-His
antibody as described under "Experimental Procedures."
B
in vivo, we monitored the effect of expression of these enzymes on the degradation of the
general bulk of short-lived proteins that are known to be targeted by
the ubiquitin system (46). As shown in Fig.
7A, expression of mutant
UBCH5b and UBCH5c did not affect the stability of the general
population of short-lived proteins. Furthermore, it did not decrease
the steady-state level of ubiquitin conjugates in the cell (Fig. 7,
panel B1). Similarly, transient expression of
C93A CDC34/UBC3 did not decrease the steady-state level of cellular
ubiquitin conjugates either (Fig. 7, panel B2).
Although it is clear that UBCH5b and UBCH5c, but also CDC34/UBC3, are
not entirely specific to I
B
, it is obvious that they are also not involved in the degradation of the bulk of short-lived cellular proteins, but rather in targeting of a more limited set of unstable proteins.
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Fig. 7.
Effect of mutant UBCH5b and UBCH5c on the
degradation of short-lived proteins (A) and effect of
mutant UBCH5b, UBCH5c, and CDC34/UBC3 on the steady-state level of
ubiquitin conjugates in cells (B).
Doxycycline-induced ( ) and uninduced (
) HeLa Tet-on cells
carrying stably transfected C85A mutant UBCH5b and UBCH5c were labeled
with [35S]methionine. Degradation of short-lived proteins
was monitored as described under "Experimental Procedures"
(A). In a parallel experiment (B), uninduced
(U) and induced (I) HeLa cells stably expressing
either C85A UBCH5b or C85A UBCH5c (B1) or transiently
transfected 293 cells expressing empty vector (control
(Cont.)) or C93A mutant CDC34/UBC3 (B2) were
lysed in sample buffer. Proteins were resolved via SDS-PAGE (10%), and
following blotting onto nitrocellulose paper, endogenous ubiquitin
conjugates (Conj.) were detected by Western blot analysis
using anti-ubiquitin serum and ECL as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-complexed I
B
. In
vitro, conjugation is specific to the post-translationally
modified protein: the S32A,S36A mutant is not conjugated, and the
reaction is inhibited specifically by a doubly phosphorylated peptide
that spans the I
B
recognition domain. Catalytic site mutant
species of the UBCH5 enzymes and of CDC34/UBC3 inhibit conjugation of
I
B
in vitro and degradation of the protein in
vivo. They probably act via binding to the E3 enzyme and render it
inaccessible to association with wild-type endogenous E2. It should be
noted that the in vivo results were obtained using HeLa and
293 cells. One cannot exclude the possibility that different E2 enzymes
may be involved in the process in other cell types.
B
degradation.
As for the involvement of CDC34/UBC3 in the process, it is possible
that this enzyme acts via an E3 that is different from the one with
which the UBCH5 enzymes catalyze their reaction. Alternatively, the two
enzymes can catalyze conjugation of pI
B
via the same E3, but
carry it out in different cell types or in the same cell, but under
different pathophysiological conditions. As noted, the activity of
CDC34/UBC3 appears to be different from that of its UBCH5 counterparts
as it is strongly inhibited by Fraction II or IIA (Fig. 1,
C, lanes 2 and 4; and
E, lane 5; for possible mechanism of
this inhibition, see below). In contrast, the activity of UBCH5 is
further stimulated following the addition of Fraction II (Fig. 1,
E, compare lanes 7 and 8).
Experimentally, the distinct E3 enzyme hypothesis can be tested, at
least initially, using cross-inhibition by catalytic site, mutant
species of one class of E2 along with the wild-type species of the
other class. If distinct E3 enzymes are involved, C93A CDC34/UBC3 will
not be able to inhibit wild-type UBCH5b- and UBCH5c-mediated
conjugation, and similarly, C85A UBCH5b and C85A UBCH5c will not be
able to inhibit the CDC34/UBC3-catalyzed reaction. However, because the reaction requires Fraction II, which may provide one or more E3 enzymes, and because Fraction II is inhibitory for CDC34/UBC3 activity,
experimental examination of this theory may be difficult at present.
Also, experiments with inhibitory inactive mutant species are not
conclusive at times. The inhibitory effects of UBCH5a demonstrated in
this study illustrate the limit of such probes as faithful experimental
tools. It appears therefore that elucidation of the underlying
mechanisms will have to await reconstitution of the system from the
different purified components. Of note is that a requirement for two
distinct E2 enzymes has been demonstrated previously for different
substrates. We have shown that lysozyme can be targeted by E2-14K and
E2-F1 (UBCH7) and that each acts with a distinct E3 enzyme that
recognizes a different structural motif in the protein (47). Chen
et al. (48) have shown that the yeast transcriptional
activator Mat
2 is also targeted by two distinct E2 enzymes, Ubc6 and
Ubc7; however, the mechanistic basis for their differential activity
has not been resolved.
B
conjugation, although the
reaction appears to be nonspecific (Fig. 1, A and
B). UBCH5a is ~90% similar to UBCH5b and UBCH5c, which
are 98-99% identical to each other (19). It is possible that UBCH5a
recognizes and binds to the I
B
E3, although with a lower affinity
than UBCH5b and UBCH5c, but cannot catalyze ubiquitin transfer to the inhibitor.
B
phosphopeptide. The protein is probably recognized by a constitutive, non-regulated motif. It is possible that during incubation, a small
part of the complexed inhibitor is dissociated and targeted by
different E2 enzymes. Alternatively, the complexed inhibitor may be
recognized simultaneously via fast-reacting regulated and specific and
slow-reacting non-regulated signals. Of note is that all the
nonspecific conjugates are of relatively low molecular mass and that
even UBCH5b and UBCH5c catalyze generation of such low molecular mass,
nonspecific adducts (Fig. 1, A and B). Only the
high molecular mass adducts appear to be regulated. Thus, it appears
that two conjugation events that serve different purposes occur in
parallel. The physiological significance of these processes is
still obscure. Another explanation for the involvement of multiple E2
enzymes in the nonspecific conjugation of I
B
is that the cell-free system does not faithfully reproduce the cellular events, and
these E2 enzymes are inactive toward I
B
in the intact cell.
B
degradation (49). However, this enzyme cannot
conjugate ubiquitin and is involved in modification of substrate
proteins with the ubiquitin-like protein SUMO-1. Thus, if it acts at
all on I
B
degradation, it has an indirect effect. The recent
report that modification by SUMO-1 renders I
B
stable and protects
it from ubiquitin-mediated degradation (50) makes the involvement of
UBC9 in the process, even in an indirect manner, highly unlikely.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Allan Weissman for the cDNA clones of UBCH5a, UBCH5b, and UBCH5c; Simon S. Wing (McGill University) for the clone of E2-8A; Martin Scheffner (Deutsches Krebsforschungszentrum) for the clone of UBCH7; Avram Hershko (Technion) for purified E2-C; Guojun Bu (Washington University School of Medicine) for the cDNA encoding green fluorescent protein; and Avraham Yaron for thoughtful advice during the initial phase of the work.
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FOOTNOTES |
---|
* This work was supported by grants from the Israel Science Foundation founded by the Israeli Academy of Sciences and Humanities-Centers of Excellence Program, the German-Israeli Foundation for Scientific Research and Development, the United States-Israel Binational Science Foundation, the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum and the Israeli Ministry of Science, the European Community (a TMR grant), Signal Pharmaceuticals, Inc., the Foundation for Promotion of Research at the Technion, and the Vice President of the Technion for Research (to A. C.); the Ministries of Education and Science and of Health and Welfare of Japan (to K. I.); and the Israel Cancer Research Fund (to H. G.). DNA sequences were partially determined using an ABI 310 autosequencer purchased with the support of a special grant for procurement of equipment from the Israel Science Foundation.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.
§ These two authors contributed equally to this work.
** These these senior corresponding authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Biochemistry, Faculty of Medicine, Technion-Israel Institute of
Technology, Efron St., Bat Galim, P. O. Box 9649, Haifa 31096, Israel.
Tel.: 972-4-829-5365/5379/5356; Fax: 972-4-851-3922 and 972-4-855-2296; E-mail: mdaaron{at}tx.technion.ac.il.
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ABBREVIATIONS |
---|
The abbreviations used are:
pIB
, phosphorylated I
B
;
E1, ubiquitin-activating enzyme;
E2, ubiquitin
carrier protein;
E3, ubiquitin-protein ligase;
TNF-
, tumor necrosis
factor
;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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