(Received for publication, October 5, 1995; and in revised form, January 11, 1996)
From the
In unstimulated cells, the transcription factor NF-B is
held in the cytoplasm in an inactive state by the inhibitor protein
I
B
. Stimulation of cells results in rapid phosphorylation and
degradation of I
B
, thus releasing NF-
B, which
translocates to the nucleus and activates transcription of responsive
genes. Here we demonstrate that in cells where proteasomal degradation
is inhibited, signal induction by tumor necrosis factor
results
in the rapid accumulation of higher molecular weight forms of
I
B
that dissociate from NF-
B and are consistent with
ubiquitin conjugation. Removal of the high molecular weight forms of
I
B
by a recombinant ubiquitin carboxyl-terminal hydrolase and
reactivity of the immunopurified material with a monoclonal antibody
specific for ubiquitin indicated that I
B
was conjugated to
multiple copies of ubiquitin. Western blot analysis of immunopurified
I
B
from cells expressing epitope-tagged versions of
I
B
and ubiquitin revealed the presence of multiple copies of
covalently bound tagged ubiquitin. An S32A/S36A mutant of I
B
that is neither phosphorylated nor degraded in response to signal
induction fails to undergo inducible ubiquitination in vivo.
Thus signal-induced activation of NF-
B involves
phosphorylation-dependent ubiquitination of I
B
, which targets
the protein for rapid degradation by the proteasome and releases
NF-
B for translocation to the nucleus.
The NF-B/rel family of transcription factors are involved
in the activation of a wide variety of genes, including the HIV-1
provirus, that respond to immune and inflammatory signals (for review,
see (1) and (2) ). In humans, the family of proteins
consists of p50 (3, 4) ,
p52(5, 6, 7) , p65(8, 9) ,
c-Rel(10) , and RelB(11) . While almost all
combinations of homo- and heterodimer can exist, the typical form of
NF-
B that is activated in response to extracellular signals is a
heterodimer of p50 and p65. Disruption of genes coding for the p50 or
RelB components of NF-
B complexes results in transgenic animals
with defects in immune and inflammatory
responses(12, 13) . NF-
B proteins share a highly
conserved region known as the rel homology domain, which is
responsible for DNA binding, dimerization, and nuclear localization.
DNA is recognized by NF-
B in an unusual way involving base and
backbone contacts with the DNA over one complete helical
turn(14, 15) . Structural analysis of p50 homodimers
bound to DNA reveals that the protein recognizes DNA by an extended
network of loops that arise from noncontiguous regions of the
protein(16, 17) . Although p50 does not possess a
transcriptional activation domain, its p65 partner does have an acidic
activation domain that accounts for the transcriptional activity of the
NF-
B heterodimer(18, 19) .
p50 represents the
amino-terminal region of a p105 precursor from which it is processed,
by a pathway thought to involve ubiquitination of the
protein(20) . The carboxyl-terminal region of p105 contains
multiple repeats of a 30-35-amino acid sequence present in the
erythrocyte protein ankyrin(21) . In lymphoid cells the
carboxyl-terminal region of p105 has been identified as an independent
entity known as IB
(22, 23) that
preferentially inhibits the DNA binding activity of p50 homodimers. In
p105, the carboxyl-terminal region is thought to function as a cis-acting inhibitor of DNA binding activity (24) .
NF-
B activity is regulated by its association with the inhibitor
protein I
B
or MAD3(25, 26) , which like the
carboxyl-terminal region of p105, the proto-oncogene bcl-3 (27) , and the recently described I
B
(28) contains multiple ankyrin repeats. How I
B proteins
inhibit both nuclear translocation and DNA binding of NF-
B
proteins has not been established, but the nuclear localization signals
of p50 and p65 are occluded by bound I
B
and I
B
,
respectively(29, 24, 30) . Mutational
analysis of I
B
, I
B
, and pp40 has demonstrated that
both the ankyrin repeats and carboxyl-terminal acidic domains are
required for interaction with the corresponding NF-
B
proteins(31, 32, 33) . I
B
displays
a tripartite organization with a central domain containing five ankyrin
repeats, an unstructured amino-terminal extension, and a small highly
acidic carboxyl-terminal domain connected to the core of the protein by
a protease-sensitive linker that is occluded by bound p65(34) .
In unstimulated cells, NF-B is held in the cytoplasm by
I
B
, but signal induction releases NF-
B, which
translocates to the nucleus and activates responsive genes. Following
signal induction, I
B
is rapidly phosphorylated and
degraded(35, 36, 37, 38, 39, 40, 41) .
Mutational analysis has indicated that residues Ser-32 and Ser-36 are
the likely sites of inducible
phosphorylation(36, 42, 43) , which targets
the protein for degradation but does not disrupt complexes of NF-
B
and
I
B
(44, 45, 46, 47, 48, 49, 50) .
Inhibition of protein degradation via the 26 S proteasome results in
accumulation of the hyperphosphorylated form of I
B
and a
failure to activate NF-
B, indicating that I
B
proteolysis
is a necessary step in NF-
B activation(20, 50) .
Degradation of I
B
is rapidly followed by induction of
I
B
mRNA in a mechanism that is regulated by interaction of
NF-
B with the promoter of the I
B
gene(51, 52, 41) . Resynthesized I
B
protein appears transiently in the nucleus where it negatively
regulates NF-
B dependent transcription(53) .
To
determine the pathway of signal-induced degradation of IB
, we
have made use of a peptide aldehyde inhibitor that blocks the
proteolytic activity of the proteasome(54) . In the presence of
this inhibitor, signal induction results in the accumulation of
phosphorylated and ubiquitinated forms of I
B
and a failure to
activate NF-
B. Although the phosphorylated forms of I
B
remain bound to NF-
B, the multiply ubiquitinated forms of
I
B
were not associated with the transcription factor. It is
likely that prior phosphorylation is required for signal-induced
ubiquitination as an S32A/S36A I
B
mutant is neither
phosphorylated, degraded, nor ubiquitinated. Our observations emphasize
the importance of signal-induced protein degradation in activation of
the NF-
B transcription factor and demonstrate a crucial role for
the ubiquitin-proteasome pathway in this process.
Figure 1:
Effect of proteasome inhibition on
activation and nuclear translocation of NF-B. a, HeLa
cells were pretreated with the proteasome inhibitor Z-LLL-H and exposed
to TNF
as indicated. NF-
B DNA binding activity in nuclear
extracts was determined in a gel electrophoresis DNA binding assay with
the positions of DNA-protein complexes (B) and free DNA (F) indicated. b, as in a, but nuclear and
cytoplasmic extracts were fractionated in an 8% polyacrylamide gel
containing SDS and transferred to polyvinylidine difluoride, and the
p50 subunit of NF-
B and its p105 precursor protein were detected
by ECL Western blotting using an affinity-purified polyclonal antibody
raised against the p50 protein.
Figure 2:
Effect of proteasome inhibition on
signal-induced degradation of IB
. HeLa cells were pretreated
with Z-LLL-H and exposed to TNF
as indicated. Cytoplasmic extracts
(40 µg) were fractionated by SDS-PAGE, and I
B
protein was
analyzed by ECL Western blotting using monoclonal antibody MAD3 10B (34) . The positions of prestained molecular weight markers
(Sigma), I
B
and the phosphorylated form of the protein (P) are indicated. More slowly migrating and faster migrating
forms of I
B
are indicated by the brackets. A short exposure
is displayed to demonstrate Z-LLL-H inhibition of TNF
-induced
I
B
degradation, while a long exposure is shown to display the
I
B
species that accumulate in the presence of
Z-LLL-H.
Figure 3:
Ubiquitination of IB
in
vivo. a-c, HeLa S3 cells either treated with 10
µM Z-LLL-H for 45 min or untreated were exposed to 10 ng
ml
TNF
for the indicated time. Cytoplasmic
extracts were incubated with either 10 µM purified GST or
10 µM of a GST-ubiquitin carboxyl-terminal hydrolase
fusion protein (GST-UCH) at 37 °C for 30 min. a, reaction
products were analyzed by Western blotting with the
I
B
-specific monoclonal antibody MAD3 10B. I
B
, its
phosphorylated derivative, and anomalously migrating forms are
indicated as described in the legend to Fig. 2. b, the
blot displayed in a was stripped and reprobed with an
anti-ubiquitin antibody(62) . c, the same blot was
again stripped and reprobed with an anti-actin antibody (Sigma). d, cytoplasmic extracts were prepared and immunoprecipitated with
either rabbit preimmune serum (PI), rabbit anti-I
B
serum, rabbit anti-p50 serum, or rabbit anti-p65 serum cross-linked to
protein A-Sepharose. Immunoprecipitates were fractionated by SDS-PAGE
and analyzed by ECL Western blotting with anti-ubiquitin monoclonal
antibody 4F3. The positions of molecular mass markers are
shown.
To independently prove that IB
is linked to
ubiquitin, cells were treated with TNF
and Z-LLL-H, and extracts
were immunoprecipitated with antibodies to I
B
, NF-
B p50,
NF-
B p65, or nonimmune serum prior to detection of bound ubiquitin
by Western blotting with a monoclonal antibody directed against
ubiquitin(59) . In TNF
-treated cells, most I
B
is
degraded, but ubiquitinated adducts on the remaining protein are
detected in immunoprecipitates with I
B
antibodies but not
with antibodies to p50, p65, or nonimmune serum (Fig. 3d). In the presence of TNF
and Z-LLL-H, a
considerable increase in ubiquitinated forms of I
B
are
detected in immunoprecipitates with antibodies specific to
I
B
. Again antibodies to p50 and p65 only precipitate amounts
of ubiquitinated I
B
that are comparable with that obtained
with the preimmune serum, even although they can precipitate bound
I
B
that is not linked to ubiquitin (see Fig. 5). The
failure of NF-
B antibodies to immunoprecipitate material
recognized by the ubiquitin antibody (Fig. 3d), suggests that
I
B
-ubiquitin conjugates dissociate from NF-
B.
Figure 5:
Ubiquitinated IB
dissociates
from NF-
B whereas phosphorylated I
B
remains bound to
NF-
B. a, HeLa S3 cells were treated with either 10
µM Z-LLL-H for 45 min (ZLLLH),
10 ng of ml
TNF
for 15 min (TNF
), 10
µM Z-LLL-H for 45 min followed by a further 15 min in the
presence of 10 ng ml
TNF
(TNF
+ ZLLLH) or 0.1% Me
SO vehicle alone
for 45 min. Cytoplasmic extracts were prepared and immunoprecipitated
with either rabbit preimmune serum (PI), rabbit
anti-I
B
serum (a-IkB), or rabbit anti-p50 serum (a-p50). Immunoprecipitates were fractionated by SDS-PAGE and
analyzed by Western blotting with anti-I
B
monoclonal antibody
MAD3 10B. Molecular mass markers and the position of I
B
and
its phosphorylated derivative are indicated. I
B
-ubiquitin
conjugates are indicated by the upper bracket, while
I
B
degradation products are indicated by the lower
bracket. b, HeLa cells were pretreated with Z-LLL-H and
exposed to TNF
. Cytoplasmic extracts were immunoprecipitated with
anti-p50 antibodies and bound I
B
detected by Western
blotting.
To
independently confirm these results, an HA-tagged version of human
ubiquitin (56) was introduced into cells by transfection (Fig. 5a). Cells were transfected with either empty
vector DNA, the plasmid expressing HA-tagged ubiquitin, or
cotransfected with plasmids expressing epitope-tagged IB
(57) and HA-tagged ubiquitin. Transfected cells were
trypsinized, seeded into three separate dishes and after 16 h treated
with either TNF
, TNF
plus Z-LLL-H or untreated. Cytoplasmic
extracts were denatured in SDS, immunoprecipitated with polyclonal
antibodies to I
B
or preimmune serum, and analyzed by Western
blotting using first the monoclonal antibody recognizing the HA tag and
reprobed with an I
B
-specific monoclonal antibody. HA
immunoreactive material is not detected in cells transfected with the
empty vector (Fig. 4b, upper left panel) but
in cells transfected with the plasmid expressing HA tagged ubiquitin,
the expressed protein appears to be associated with endogenous
I
B
even in the absence of TNF
and Z-LLL-H (Fig. 4b, upper left panel), again indicating
that I
B
is being constantly turned over. Multiply
ubiquitinated I
B
species are relatively rare, but they can be
detected by the HA antibody as a large number of epitopes are present.
In contrast, these species are difficult to detect with the
I
B
antibody as only a single epitope is present. Although
treatment of the cells with TNF
results in a proportion of the
I
B
being degraded (Fig. 4b, upper right
panel), this does not translate into a reduction in the amount of
multiply HA-tagged, ubiquitinated I
B
detected (Fig. 4b, upper left panel). While the rate of
I
B
degradation is increased in the presence of TNF
, the
rate of ubiquitination of I
B
is also increased, with the
consequence that the steady-state level of ubiquitinated I
B
is unaltered. Endogenous I
B
is thus covalently linked to
HA-tagged ubiquitin as part of a rapidly turning over pool of modified
I
B
that is stabilized by blocking degradation with Z-LLL-H.
Figure 4:
HA-tagged ubiquitin is covalently bound to
IB
in vivo. a, structure of the HA-tagged
polyubiquitin gene (HA-ubi, (56) ) with the amino-terminal tag
shaded. The I
Bctag gene (57) is displayed with the ankyrin
repeats (filled boxes), a region involved in interaction with p65
(shaded box), the acidic region (open box) and the carboxyl-terminal
tag (hatched box). Both genes used for transfections are under the
control of the cytomegalovirus immediate early promoter. b,
COS7 cells in 150-cm
flasks were transfected with the
indicated plasmids, and after 16 h the cells were trypsinized and split
into three, and growth continued in 9-cm plates. After a further 24 h
of growth, cells were either untreated, treated with 10 ng/ml TNF
for 15 min, or pretreated for 45 min with 10 µM Z-LLL-H
and then incubated with 10 ng/ml TNF
for a further 15 min. At the
end of the incubation period, cytoplasmic extracts were prepared,
denatured in the presence of SDS, and immunoprecipitated with
matrix-bound immunoglobulins from either rabbit preimmune serum (IP
PI, lower panels) or a polyclonal serum specific for
I
B
(IP Ab I
B
, upper panels).
Immunoprecipitates were fractionated by SDS-PAGE and analyzed by
Western blotting with the HA tag-specific monoclonal antibody 12CA5
(Blot Ab HA, left panels). After ECL development, the blots
were stripped and reprobed with the I
B
-specific monoclonal
antibody MAD3 10B (Blot Ab I
B
, right
panels).
A similar situation is apparent when HA-tagged ubiquitin is
cotransfected with a plasmid expressing epitope-tagged IB
(I
Bctag). Only a small proportion of the highly expressed
I
Bctag is degraded (Fig. 4b, upper right
panel), indicating that the I
B
modification and
degradation machinery has a limited capacity. Thus, only a small amount
of immunopurified I
B
is associated with HA-tagged ubiquitin
in the absence or presence of TNF
(Fig. 4b, upper right panel), but when TNF
and Z-LLL-H are present,
a substantial quantity of HA-tagged ubiquitin is linked to I
B
(Fig. 4b, upper right panel). Preimmune serum
did not immunoprecipitate material reactive with the HA-specific
antibody (Fig. 4b, lower left panel). Thus
I
B
is covalently linked to HA-tagged ubiquitin when
signal-induced degradation is blocked by proteasomal inhibitors.
Figure 6:
Residues Ser-32 and Ser-36 are required
for ubiquitination of IB
in vivo. HeLa cells stably
expressing either wild-type I
Bctag or S32A/S36A I
Bctag were
treated with either 10 ng/ml TNF
for 20 min (TNF), 0.5
µM okadaic acid for 20 min (OKA), 10 ng/ml
TNF
plus 0.5 µM okadaic acid for 20 min (TNF
+ OKA), 10 µM Z-LLL-H for 45 min followed by a
further 20 min in the presence of 10 ng/ml TNF
plus 0.5 µM okadaic acid (TNF + OKA + ZLLLH) or 0.1% Me
SO vehicle alone
for 65 min(-). Cytoplasmic extracts were fractionated by SDS-PAGE
and analyzed by Western blotting with either SV5 Pk tag monoclonal
antibody (a, b) or anti-I
B
monoclonal
antibody MAD 10B (c). The positions of molecular mass markers,
I
Bctag, I
B
phosphorylated derivatives, and ubiquitin
adducts are indicated.
Peptide aldehyde inhibitors, which block the activity of the
proteasome, inhibit signal induced degradation of IB
and
activation of NF-
B(20, 50) . Although it was
demonstrated that ubiquitination was involved in processing of p105 to
p50(20) , ubiquitination of I
B
was not reported.
However a recent report (63) has shown that I
B
is
ubiquitinated, and in vitro studies demonstrated that this
process required residues Ser-32 and Ser-36. Furthermore it was shown
that ubiquitinated I
B
was a substrate for the 26 S proteasome in vitro. Here we demonstrate that signal induction with
TNF
results in phosphorylation-dependent ubiquitination of
I
B
and release of NF-
B. This is in contrast with the
situation observed in vitro where ubiquitinated I
B
remains bound to NF-
B(63) . Thus it is unlikely that
ubiquitination per se is responsible for the dissociation of
I
B
from NF-
B, but one possibility is that a protein with
chaperonin activity could release ubiquitinated I
B
from
NF-
B in vivo. Ubiquitinated I
B
must represent a
short lived intermediate in the degradative pathway, as ubiquitinated
I
B
is only readily detected when proteasomal degradation is
inhibited. The results support a model for signal-induced activation of
NF-
B in which I
B
is first phosphorylated on residues
Ser-32 and Ser-36 (36, 42, 43) by an as yet
unidentified kinase. Phosphorylated I
B
remains bound to
NF-
B but is recognized by proteins involved in ubiquitin addition.
Enzymes responsible for ligation of ubiquitin to I
B
have not
been identified, but it is likely that this process is mediated by a
specific E1-E2-E3 type thioester cascade of the type involved in the
E6-induced ubiquitination of p53(64, 65) . Although
I
B
contains nine lysines, five of which are located in the
amino-terminal region, it is clear that residues Lys-21 and Lys-22 are
the primary sites of phosphorylation dependent ubiquitination. (
)Given the specificity of the peptide aldehyde inhibitors (54) and the recent in vitro experiments(63) ,
it is then likely that the ubiquitinated I
B
is degraded by
the multicatalytic protease or proteasome(66) . Although
ubiquitinated I
B
is detected when proteasomal degradation is
blocked, only a small proportion of the I
B
accumulates in the
ubiquitinated form. This is probably due to the activity of ubiquitin
carboxyl-terminal hydrolases, which remove ubiquitin from
ubiquitin-protein conjugates and process the primary products of
polyubiquitin gene mRNA translation(67) . Although
I
B
-ubiquitin conjugates do not constitute a large proportion
of the total I
B
pool, the observation that Z-LLL-H only
partially blocks translocation of NF-
B to the nucleus (Fig. 1) but efficiently blocks degradation of I
B
(Fig. 2) is thus explained by release of NF-
B from
ubiquitinated I
B
( Fig. 3and Fig. 5).
Mutational analysis has indicated that both the amino and carboxyl
termini of IB
are required for signal-induced
degradation(36, 42, 43, 57) . The
requirement for the amino terminus can be explained by the location of
sites for signal-induced phosphorylation and ubiquitination, while the
carboxyl terminus contains PEST sequences, which are thought to
destabilize proteins. A role for ubiquitin conjugating enzymes in the
degradation of S and M phase cyclins has been established in
vivo(68) , and in vitro studies have demonstrated
that like I
B
, ubiquitination of the G
cyclin Cln
2p is preceded by phosphorylation of the protein(69) . Our
observations emphasize the importance of signal-induced protein
degradation in activation of NF-
B and demonstrate a crucial role
for the ubiquitin-proteasome pathway (66) in this process.