(Received for publication, April 28, 1995; and in revised form, June 22, 1995)
From the
In most cases, the transcriptional factor NF-B is a
heterodimer consisting of two subunits, p50 and p65, which are encoded
by two distinct genes of the Rel family. p50 is translated as a
precursor of 105 kDa. The C-terminal domain of the precursor is rapidly
degraded, forming the mature p50 subunit consisted of the N-terminal
region of the molecule. The mechanism of generation of p50 is not
known. It has been suggested that the ubiquitin-proteasome system is
involved in the process; however, the specific enzymes involved and the
mechanism of limited proteolysis, in which half of the molecule is
spared, have been obscure. Palombella and colleagues (Palombella, V.
J., Rando, O. J., Goldberg, A. L., and Maniatis, T.(1994) Cell 78, 773-785) have shown that ubiquitin is required for the
processing in a cell-free system of a truncated, artificially
constructed, 60-kDa precursor. They have also shown that proteasome
inhibitors block the processing both in vitro and in
vivo. In this study, we demonstrate reconstitution of a cell-free
processing system and demonstrate directly that: (a) the
ubiquitin-proteasome system is involved in processing of the intact
p105 precursor, (b) conjugation of ubiquitin to the precursor
is an essential intermediate step in the processing, (c) the
recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of
p53, is also required for the limited processing of the p105 precursor,
and (d) a novel,
320-kDa species of ubiquitin-protein
ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3
,
the ``N-end rule'' ligase.
NF-B and other members of the Rel family of transcriptional
activators have recently gained considerable attention because of their
unique mechanism of activation, and their active role in
cytoplasmic-nuclear signaling in response to a variety of extracellular
stimuli. In most cases, NF-
B is a heterodimeric complex composed
of p50 and p65 subunits. p50 is initially synthesized as an inactive
105-kDa precursor that is processed to the mature protein following
removal of the C-terminal domain of the molecule. A second precursor
molecule, p100, gives rise to the p52 DNA-binding subunit of NF-
B,
probably via a similar mechanism. The heterodimeric complex was
originally identified as an inducible B cell-specific transcription
factor able to bind the
light chain enhancer(1) .
NF-
B is now recognized to be a ubiquitously expressed factor that
is present as an inactive ternary complex in the cytosol of most cells.
In this ternary complex, a third inhibitory protein belonging to the
I
B family is associated with the heterodimer (for recent reviews,
see (2, 3, 4) ). A wide range of stimuli such
as bacterial products, parasites, viruses and viral products,
inflammatory cytokines, T cells, B cells, fibroblast mitogens, protein
synthesis inhibitors, physical and oxidative stresses, and drugs lead
to accelerated processing of the precursor, degradation of the
inhibitor, and consequent translocation of the active heterodimeric
factor from the cytoplasm to the nucleus, where it exerts its
transcriptional activity(2) .
Cellular target genes are
largely involved in the acute-phase response, inflammation, lymphocyte
activation, and cell growth and differentiation. These genes include,
among others, immunoreceptors such as the and
chains of the
T cell receptor, major histocompatibility protein class I and II
molecules, cell adhesion molecules such as ELAM-1 and VCAM-1,
cytokines, hematopoietic growth factors such as granulocyte-macrophage
colony-stimulating factor and interleukin-2, and acute phase proteins
such as angiotensinogen and complement factor B. Interestingly, it also
affects regulation of transcription of transcriptional activators,
including p105, c-Rel, and I
B
(2, 5) . The
physiological role of NF-
B has been recently studied by using mice
that lack the p50 subunit. The disruption leads to multifocal defects
in a whole array of immune responses. These involve, for example,
inability of B lymphocytes to respond to bacterial products, defect in
basal and specific antibody production, and high susceptibility to
certain pathogens(6) . However, it is not clear whether the
absence of other members of the Rel family, p65 or RelB for example,
display a similar phenotype.
The NF-B/Rel proteins are
subjected to multiple regulatory influences. A major component of this
regulation involves control of their intracellular localization, with
the inactive proteins maintained in the cytosol. Regulation is
controlled by two major pathways: (i) control of p105 and p100
processing and (ii) interaction of the p50/p65 heterodimer with a group
of inhibitor molecules, I
Bs. The two pathways may respond to
different stimuli, thus providing a tight control of the response. (i)
The precursor molecules p105 and p100 contain in their N-terminal
region the p50 and the p52 moieties, whereas the C-terminal domain
contains ankyrin repeats, homologous to
I
B(7, 8) . The p50 subunit of NF-
B is
generated by ATP-dependent processing of p105 in vivo and in vitro ((9) ; in vitro, the researchers
used an artificially truncated form of p105 with a molecular mass of 60
kDa, p60). The processing is regulated. It is accelerated, for example,
after treatment of cells with tumor necrosis factor
,
double-stranded RNA, and phorbol esters(10, 11) .
Recent evidence suggests that the ubiquitin-proteasome pathway is
involved in processing of the NF-
B precursor, p105(12) . In vitro, addition of ubiquitin to a Fraction II which does
not contain the protein, stimulated processing of p60 to p50. Addition
of ubiquitin-Arg
, a derivative of ubiquitin that cannot
generate polyubiquitin chains, inhibited processing. However, in these
two experiments the researchers have not demonstrated the formation of
ubiquitin-p60 adducts, the essential intermediates in the process. In a
different set of experiments, it was shown that inhibitors of the 20 S
proteasome complex block processing not only of p60 in vitro,
but also of p105 in intact cells(12) . Inhibition of the
proteasome does not necessarily indicate intermediacy of the ubiquitin
system in the process. Ornithine decarboxylase, for example, is
degraded in a proteasome-dependent, but ubiquitin-independent,
process(13) . (ii) The interaction with the inhibitor, I
B,
masks a nuclear localization signal and thus leads to retention of the
heterodimer in the cytosol(14) . In response to a variety of
extracellular stimuli, I
B is rapidly degraded, thus exposing the
nuclear localization signal and allowing translocation of the p50/p65
dimer to the nucleus. Recently, it has been shown that extracellular
stimuli lead to phosphorylation of the inhibitor, but that this
modification is not sufficient for activation of NF-
B (15, 16, 17, 18) . It was proposed
that phosphorylation signals the protein for degradation, and the
protein is probably degraded while associated as a complex with the
other two components of the ternary complex. Indeed, Brown and
colleagues (19) have shown that phosphorylation of Ser residues
32 or 36 may serve as a proteolysis recognition signal. Furthermore,
inhibitors of the 20 S proteasome stabilize specifically the
phosphorylated form(20) . Recent evidence from our laboratories
indicate that the ubiquitin system is involved in rapid degradation of
the stimulation-induced phosphorylated form of the inhibitor. The
unmodified form is not recognized. (
)
The
ubiquitin-dependent pathway plays an important role in the degradation
of several key short-lived regulatory proteins (reviewed recently in (21) ). Degradation of a protein via the ubiquitin pathway
involves two distinct steps, both requiring ATP: signaling of the
protein by covalent attachment of multiple molecules of ubiquitin, and
degradation of the targeted protein by a multisubunit 26 S proteasome
complex. Conjugation of ubiquitin proceeds via a three-step mechanism.
Initially, ubiquitin is activated to a high energy intermediate by the
ubiquitin-activated enzyme, E1. ()Following
activation, ubiquitin-carrier protein, E2, transfers ubiquitin
from E1 to a ubiquitin-protein ligase, E3, to which
the target protein is bound. E3, therefore, appears to play a
major role in selection of proteins for conjugation and degradation.
The multiply ubiquitinated substrate is specifically recognized by the
26 S proteasome complex. The protein moiety of the adduct is degraded
with the release of free and reutilizable ubiquitin (reviewed in Refs.
22, 23).
Processing of the p105 precursor is the first example in
which the ubiquitin-proteasome pathway is involved in limited
proteolysis and not in complete destruction of its protein substrates.
The finding that E1 (and the whole ubiquitination machinery)
is required for limited processing of antigenic proteins and generation
of antigenic peptides (24) has been challenged
recently(25) . It is not known whether the C-terminal domain is
first cleaved and then degraded, or if processing proceeds via
successive removal of amino acid residues from the C-terminal residue
of p105. Consequently, the nature of the cleavage site, or the
``stop'' signal, is also obscure. Thus, because of the
peculiar mechanisms involved and the central role that NF-B plays
in many basic physiological and pathological processes, dissection of
the mechanism involved in ubiquitin modification and processing of p105
has broad biological implications. Analysis of the enzymatic process
can provide an important insight into the regulation and the
recognition signals involved.
Using a cell-free system, we have been
able to reconstitute processing of the intact p105 precursor into the
mature p50 protein. We demonstrate that the precursor is multiply
ubiquitinated and that formation of the high molecular weight
conjugates is essential for the processing to occur. We also
demonstrate that the process is mediated by a recently described
ubiquitin-carrier protein, E2-F1 (26) that is
involved in the degradation of p53(27) . In addition, a novel,
still unidentified, 320-kDa species of ubiquitin-protein ligase, E3, appears to catalyze the final step in the conjugation
reaction, transfer of ubiquitin to the substrate. The ligase is clearly
distinct from E6-AP, the p53-recognizing ligase, and from E3
, the N-end rule ligase. However, like E6-AP,
chemical modification of -SH groups of the new E3 enzyme
inhibits its activity.
Figure 1: ATP-dependent processing of p105. Labeled p105 was incubated in the presence of C3.F6 cytosolic extract. Incubation was carried out on ice or at 37 °C in the absence or presence of ATP as described under ``Experimental Procedures'' and in the figure, and appearance of p50 was monitored. Ori. and D.F. denote origin of gel and dye front, respectively. Molecular size markers are: phosphorylase b, 97.4 kDa; bovine serum albumin, 69.0 kDa; ovalbumin, 46.0 kDa; carbonic anhydrase, 30.0 kDa; soybean trypsin inhibitor, 21.0 kDa; lysozyme, 14.3 kDa.
Figure 2: Lymphocyte and reticulocyte extracts, but not wheat germ extract, can process p105. Wheat germ-translated p105 was incubated in reticulocyte lysate (lanes 1-3), in C3.F6 cytosolic extract (lane4), and in wheat germ extract (lane5) as described under ``Experimental procedures.'' Lane1, incubation on ice; lane2, incubation in the absence of ATP; lanes3-5, incubation in the presence of ATP. Notes and molecular size markers are as described in the legend to Fig. 1.
Figure 3:
Lymphocyte and reticulocyte extracts, but
not wheat germ extract, can conjugate ubiquitin to p105. Labeled p105
was incubated in the presence of reticulocyte lysate (lanes2 and 3), C3.F6 cytosolic extract (lanes4 and 5), and wheat germ extract (lanes6 and 7) in the absence (lanes2, 4, and 6) or presence (lanes3, 5, and 7) of ATPS. Lane1, substrate incubated in the absence of cell extract,
but in the presence of ATP
S. Conjugates were synthesized as
described under ``Experimental Procedures.'' Conj. denotes ubiquitin conjugates. Notes and molecular size markers are
as described in the legend to Fig. 1.
Figure 4:
Ubiquitin-dependent conjugation of p105.
Conjugation of ubiquitin to p105 was monitored in reaction mixtures
containing C3.F6 cytosolic extract as described under
``Experimental Procedures.'' Ubiquitin and ATPS were
added as indicated. Conj. denotes ubiquitin conjugates. Notes
and molecular size markers are as described in the legend to Fig. 1.
Figure 5:
MeUb inhibits conjugation of p105, and
suppression is alleviated by the addition of excess free ubiquitin.
Conjugation of ubiquitin to p105 was monitored in reaction mixtures
containing C3.F6 cytosolic extract as described under
``Experimental Procedures.'' Reaction mixtures contained the
indicated amounts of ubiquitin and MeUb. ATPS was added or omitted
as indicated. Conj. denotes ubiquitin conjugates. Notes and
molecular size markers are as described in the legend to Fig. 1.
Figure 6: MeUb inhibits processing of p105, and inhibition is alleviated by excess free ubiquitin. Processing of p105 was monitored in reaction mixtures containing C3.F6 cytosolic extract as described under ``Experimental Procedures.'' Reaction mixtures contained the displayed amounts of ubiquitin and MeUb. ATP was added or omitted from reaction mixtures as marked. Reaction mixtures were incubated on ice or at 37 °C as indicated. Notes and molecular size markers are as described in the legend to Fig. 1.
Figure 7: E2-F1 and UbcH5, but not E2-14 kDa are required for conjugation of p105. Conjugation of ubiquitin to p105 was followed essentially as described under ``Experimental Procedures'' in Fraction II derived from rabbit reticulocyte lysate. Panel A, involvement of E2-F1 in p105 conjugation. A-I, the labeled substrate was added to the reaction mixture after synthesis and without any further treatment. A-II, the labeled substrate was first treated with NEM, followed by neutralization with DTT as described under ``Experimental Procedures.'' LanesI-1 and II-1, reaction mixtures incubated without ubiquitin. Lanes I-2 and II-2, reaction mixtures incubated in the presence of 4 µg of ubiquitin. LanesI-3 and II-3, same as lanes I-2 and II-2, but with 0.2 µg of E2-14 kDa. Lanes I-4 and II-4, same as lanes I-2 and II-2, but with 0.2 µg of E2-F1. Panel B, involvement of UbcH5 in p105 conjugation. Substrate was treated with NEM and neutralized with DTT prior to the addition to the reaction mixture (similar to the experiment presented in A-II). Lane1, reaction contained all the components except for Fraction II. Lane2, reaction contained all the components except for ubiquitin. Lane3, as lane2, but in the presence of ubiquitin. Lane4, as lane3, but with 0.4 µg of recombinant UbcH5. Lane5, as lane3, but with 0.2 µg of purified E2-F1. Conj. denotes ubiquitin conjugates. Notes and molecular size markers are as described in the legend to Fig. 1.
Figure 8:
Ubiquitin-p105 ligase is a novel species
of E3 distinct from E6-AP. Ubiquitin conjugates of
p105 (panelA) and p53 (panelB)
were generated essentially as described under ``Experimental
Procedures.'' The p53 image was derived from PhosphorImager
analysis. Reactions mixtures contained: 0.5 µg of E1, 0.15
µg of E2-F1, and a 1.0-µl aliquot from the different
fractions derived from gel filtration chromatography of Fraction IIA. E6 (1.8 ng; 6-fold the molar amount of p53; 27) was added
to the conjugation reactions involving p53. LaneA,
labeled substrate incubated in the presence of E1 and E2 alone. LaneB, as laneA, but with added Fraction IIA (7.5 µg). Numbers denote fraction numbers. Arrows indicate molecular mass
of peak fractions as determined by calibration of the column with a set
of molecular mass standards (not shown). Conj. denotes
ubiquitin conjugates. Notes and molecular size markers are as described
in the legend to Fig. 1.
Figure 9:
Ubiquitin-p105 ligase is a novel species
of E3 distinct from E3. Ubiquitin conjugates of
p105 (panelA) and
I-labeled lysozyme (panelB) were generated essentially as described
under ``Experimental Procedures.'' The experiment described
in panelA was analyzed by PhosphorImager. LaneA1, incubation in the presence of E1 and E2-F1. LaneA2, same as laneA1, but with Fraction IIA (7.5 µg). LaneA3, same as laneA1, but with E3
(0.6 microunit; 1 unit of enzyme catalyzes the
incorporation of ubiquitin into conjugates at a rate of 1 µmol/min; (31) ). LaneA4, reaction mixture contained E1, E2-14 kDa (0.15 µg), and E3
. LaneB1, with E1 and E2-14 kDa. LaneB2, same as laneB1, but with E3
. E1, and E2-F1 were added in the same amounts described in the legend
to Fig. 8. Conj. denote ubiquitin conjugates. Notes and
molecular size markers are as described in the legend to Fig. 1.
To further characterize the novel E3, we investigated the possibility that, like E6-AP(41) , it also contains an active -SH group. As can be seen in Fig. 10, the activity of the enzyme is inhibited by a variety of alkylating agents. It should be noted that since we did not use a purified enzyme, the possibility always exists that the sensitive protein is not the ligase, but a factor that is necessary for its activity. In this case, the fractions that contain the ligase activity represent an overlapping region between the ligase and the putative factor. If this is indeed the case, it is expected that combination of aliquots from the margins of the peak (that represent the individual peaks of the ligase and the factor) should give a synergistic effect in conjugate formation. This is clearly not the case (not shown). Thus, it is likely that the alkylating agents affected the E3 directly.
Figure 10: The ubiquitin-p105 ligase has an active-SH group. Conjugation of ubiquitin to p105 was monitored essentially as described under ``Experimental Procedures.'' All reaction mixtures contained E1, E2-F1, and C3.F6 cytosolic extract as a source for E3. The amounts of the enzymes added are as described under ``Experimental Procedures'' and in the legend to Fig. 8. Lane1, complete reaction mixture containing all three conjugating enzymes, but without ATP. Lane2, same as lane1, but with ATP. Lane3, same as lane2, but the cytosolic extract was treated first with sodium iodoacetate and neutralized with DTT. Lane4, same as lane2, but the extract was treated first with NEM and neutralized with DTT. Lane5, same as lane2, but the extract was treated first with p-hydroxymercuribenzoate and neutralized with DTT. Lane6, same as lane2, but NEM was added after the addition of DTT. In all cases the inhibitor was added at 10 mM and the extract incubated for 10 min at room temperature. DTT was then added to a final concentration of 6 mM. Conj. denotes ubiquitin conjugates. Notes and molecular size markers are as described in the legend to Fig. 1.
Figure 11: Inhibition of the 20 S proteasome suppresses processing of p105. Processing of p105 was carried out in reticulocyte lysate in the presence or absence of 50 µM MG115, a 20 S proteasome inhibitor(12) . Notes and molecular size markers are as described in the legend to Fig. 1.
We have shown that the ubiquitin-proteasome pathway is
involved in processing of intact p105 in vitro. In a previous
study, Palombella and colleagues (12) utilized a truncated form
of p105, p60, to demonstrate the involvement of ubiquitin in
processing. They have not demonstrated, however, formation of
conjugates as essential intermediates in the process. While analysis of
mutant/truncated proteins can be powerful, one should be cautious. The
ubiquitin system may recognize abnormal/truncated/misfolded proteins,
but not their normal counterparts. For example, a truncated form of E1A adenovirus protein (amino acid residues 1-85) is
much more sensitive to degradation then the wild type(1-289)
protein(29, 43) . The case of p53 is even more
illustrative, although the direction appears to be
``opposite.'' A single mutation can render the wild type
protein resistant to conjugation and degradation because of a
significant crucial alteration in the three-dimensional
structure(27, 44, 45) . Processing of p105 to
p50 occurs post-translationally. It requires ATP and occurs in a
time-dependent manner. The process occurs only in mammalian cell
extracts and not in plant. Our analysis revealed that the plant
extract, most probably, does not contain the required ubiquitin-protein
ligase, E3 (see below). Further investigation revealed that
conjugation of ubiquitin precedes processing and is an essential
intermediate step in the process; MeUb inhibits both processes, and the
inhibition is alleviated by the addition of excess free ubiquitin. To
study the conjugating enzymes involved, we reconstituted a cell-free
system and demonstrated that E2-F1, a recently characterized
ubiquitin-carrier protein isolated from crude reticulocyte Fraction
I(26) , is required for the process. We have also demonstrated
that the human homolog of E2-F1, UbcH5(30) , also
catalyzes conjugation of p105. Interestingly, E2-F1 is also
involved in the conjugation and degradation of
glyceraldehyde-3-phosphate dehydrogenase(26) ,
p53(27) , and c-Fos, ()and therefore, does not
appear to be specific. However, another species of ubiquitin-carrier
protein, E2-14 kDa, is not able to promote conjugation.
We have shown that a novel, still unidentified, species of E3
is involved in the process. This ligase, which is not present in wheat
germ, is distinct from E6-AP, the ligase involved in E6-dependent tagging of p53(37) , and from E3
, the ``N-end rule'' ligase. Thus, it appears
that E2-F1 can act in concert with several species of E3 s, and the E3 enzymes are responsible for the
specific recognition of the substrates. The novel E3 appears
to contain an -SH group essential for its activity. As expected,
processing is inhibited by inhibitors of the 20 S proteasome complex.
An important problem involves the mechanism of processing. Unlike
all other substrates of the ubiquitin system, p105 is only partially
degraded. It is not known whether the C-terminal domain is first
cleaved and then degraded, or whether digestion starts in the
C-terminal residue of p105 and stops at certain point. The cleavage
site, or the ``stop'' signal, has not been identified,
although it is clear that it may reside in the region of amino acid
residue 420(46) . Another problem concerns regulation of
processing. It has been suggested that stimulation-induced
phosphorylation of the precursor may accelerate its
processing(47) . Also, it is possible that activation of one or
more specific components of the ubiquitin system occurs following
stimulation. Interestingly, phosphorylation of the inhibitor
IB
leads to its rapid
degradation(18, 19, 20) . This process also
appears to be mediated by the ubiquitin system and to involve E2-F1.
Therefore, it is clear that reconstitution
of the cell-free proteolytic system and identification of the enzymes
involved are essential for further understanding of the mechanisms of
recognition and regulation of the components of the transcriptional
activator complex by the proteolytic system.