|
|
|
|||||||
|
|||||||||
(Received for publication, October
5, 1995; and in revised form, January 11, 1996) From the
In unstimulated cells, the transcription factor NF-
The NF- 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 I In unstimulated cells, NF- To
determine the pathway of signal-induced degradation of I
Figure 1:
Effect of proteasome inhibition on
activation and nuclear translocation of NF-
Figure 2:
Effect of proteasome inhibition on
signal-induced degradation of I
Figure 3:
Ubiquitination of I
To independently prove that I
Figure 5:
Ubiquitinated I
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 I
Figure 4:
HA-tagged ubiquitin is covalently bound to
I
A similar situation is apparent when HA-tagged ubiquitin is
cotransfected with a plasmid expressing epitope-tagged I
Figure 6:
Residues Ser-32 and Ser-36 are required
for ubiquitination of I
Peptide aldehyde inhibitors, which block the activity of the
proteasome, inhibit signal induced degradation of I Mutational analysis has indicated that both the amino and carboxyl
termini of I
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7844-7850
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
B
Ubiquitination in Signal-induced Activation of NF-
B in Vivo(*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B is
held in the cytoplasm in an inactive state by the inhibitor protein
IB
. Stimulation of cells results in rapid phosphorylation and
degradation of IB, 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
IB that dissociate from NF-B and are consistent with
ubiquitin conjugation. Removal of the high molecular weight forms of
IB by a recombinant ubiquitin carboxyl-terminal hydrolase and
reactivity of the immunopurified material with a monoclonal antibody
specific for ubiquitin indicated that IB was conjugated to
multiple copies of ubiquitin. Western blot analysis of immunopurified
IB from cells expressing epitope-tagged versions of
IB and ubiquitin revealed the presence of multiple copies of
covalently bound tagged ubiquitin. An S32A/S36A mutant of IB
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 IB, which targets
the protein for rapid degradation by the proteasome and releases
NF-B for translocation to the nucleus.
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) .B (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 IB or MAD3(25, 26) , which like the
carboxyl-terminal region of p105, the proto-oncogene bcl-3 (27) , and the recently described IB
(28) contains multiple ankyrin repeats. How IB 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 IB and I
B,
respectively(29, 24, 30) . Mutational
analysis of IB, IB, 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) . IB 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) . B is held in the cytoplasm by
IB, but signal induction releases NF-B, which
translocates to the nucleus and activates responsive genes. Following
signal induction, IB 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
IB(44, 45, 46, 47, 48, 49, 50) .
Inhibition of protein degradation via the 26 S proteasome results in
accumulation of the hyperphosphorylated form of IB and a
failure to activate NF-B, indicating that IB proteolysis
is a necessary step in NF-B activation(20, 50) .
Degradation of IB is rapidly followed by induction of
IB mRNA in a mechanism that is regulated by interaction of
NF-B with the promoter of the IB
gene(51, 52, 41) . Resynthesized IB
protein appears transiently in the nucleus where it negatively
regulates NF-B dependent transcription(53) .B, 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 IB and a failure to
activate NF-B. Although the phosphorylated forms of IB
remain bound to NF-B, the multiply ubiquitinated forms of
IB were not associated with the transcription factor. It is
likely that prior phosphorylation is required for signal-induced
ubiquitination as an S32A/S36A IB 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.
Materials
Z-LLL-H was synthesized as described
previously (55) , isolated by reverse-phase high performance
liquid chromatography (>95% purity) and the structure confirmed by
NMR spectroscopy. Human recombinant TNF (
)was provided
by the MRC ADP reagent program. Okadoic acid was purchased from Sigma.Plasmids
The plasmid expressing HA-tagged
ubiquitin under control of the cytomegalovirus immediate early promoter (56) was obtained from D. Bohmann. The plasmid expressing
IBctag also under control of the cytomegalovirus immediate
promoter was as described previously(57) . Plasmid DNA was
prepared from saturated cultures of Escherichia coli using
Qiagen columns as described by the manufacturer. A plasmid containing
the gene for a Drosophila ubiquitin carboxyl-terminal
hydrolase fused to the glutathione S-transferase gene (58) was obtained from M. Bownes.Cell Culture and Transfections
HeLa S3 cells were
grown in suspension in minimal essential medium without calcium,
containing 5% calf serum and supplemented with penicillin and
streptomycin (culture medium). COS-7 cells were grown in
Dulbecco's modified Eagle medium containing 5% fetal calf serum
and passaged every 3 days. A total of 60 µg of plasmid DNA was
transfected for 16 h in subconfluent COS-7 cells seeded in 150 cm
flasks using Lipofectamine according to instructions provided by
the manufacturer (Life Technologies, Inc.). The transfection mix
contained 48 µg of HA-ubi made up to a total of 60 µg with
either empty vector (pCDNA1, Invitrogen) or IBctag. 16 h after
transfection, cells were trypsinized and aliquots were seeded in 9-cm
plates and cultured for an additional 24 h. Cells were treated with
Z-LLL-H or TNF and Z-LLL-H or were untreated, and extracts were
prepared as described below. The expression of both wild-type and
S32A/S36A IBctag proteins was stabilized in HeLa cells using
neomycin selection with DNA vectors driven by an immediate early
cytomegalovirus promoter (pCDNAIII, InVitrogen) or an Rous sarcoma
virus promoter (RcRSV, InVitrogen), respectively. Single cell clones
were obtained by limiting dilution of the neomycin resistant cells and
selected on the basis of tagged protein expression.Preparation of Cytoplasmic and Nuclear
Extracts
HeLa cells were concentrated to 5 
10
cells ml prior to treatment with the indicated
concentrations of Z-LLL-H or the Me
SO vehicle for 45 min.
Human recombinant TNF was added to a final concentration of 10 ng
ml, and incubation at 37 °C continued for the
indicated time. Cells were collected by centrifugation, washed once
with ice-cold phosphate-buffered saline (PBS), and cytoplasmic extracts
were prepared by lysis in 20 mM sodium phosphate buffer, pH
7.5, 50 mM sodium fluoride, 5 mM tetrasodium
pyrophosphate, 10 mM
-glycerophosphate, 2 mM EDTA, 0.5% Nonidet P-40, protease inhibitors (1 µM leupeptin, 1 µM pepstatin, 1 mM pefablock,
20 µML-1-tosylamido-2-phenylethyl chloromethyl
ketone, 40 µg ml bestatin), and 10 mM iodoacetamide. After centrifugation to remove nuclei, the
iodoacetamide was quenched by the addition of dithiothreitol to 10
mM. To prepare nuclei, cells were lysed as above, but
iodoacetamide was omitted and nuclei were extracted by resuspension in
the above buffer containing 425 mM NaCl. After 30 min at 4
°C, the lysate was clarified by centrifugation at 14,000 rpm for 15
min at 4 °C in an Eppendorf microcentrifuge.
Gel Electrophoresis DNA Binding Assays
NF-B
DNA binding assays were performed as described previously(53) ,
and contained 8 µg of nuclear protein and a P-labeled
double-stranded DNA representing the
B motif present in the human
immunodeficiency virus type-1 enhancer. Free DNA was resolved from
DNA-protein complexes on a native 6% polyacrylamide gel, and the
positions of the radioactive species were determined by autoradiography
of the dried gel.Western Blot Analysis
Cytoplasmic extracts (40
µg) of protein were resolved in a 10% polyacrylamide gel containing
SDS, transferred to polyvinylidine difluoride membranes (Sigma) by
electroblotting, and processed for Western blotting as described
previousloy(53) . Where indicated, membranes were stripped by
incubation in 50 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM -mercaptoethanol at 70 °C for 15 min. After extensive
washing in PBS containing 0.1% Tween 20, membranes were processed as
described previously(53) . Primary antibodies used to detect
IB were monoclonal antibody MAD3 10B, which recognizes an
epitope located between amino acids 21 and 48 of IB (34) and affinity-purified immunoglobulins obtained from a
rabbit immunized with recombinant IB. p50 was detected with
affinity-purified immunoglobulins obtained from an immune polyclonal
rabbit serum generated by immunization with recombinant p50 (residues
35-381). A monoclonal antibody (4F3) recognizing ubiquitin was
obtained from Dr. L. Guarino(59) . Polyclonal antibodies
specific for ubiquitin were a kind gift from R. J. Mayer. The SV5 Pk
tag (60) monoclonal antibody recognizes the sequence
GKPIPNPLLGLDST and was obtained from Dr. R. E. Randall. Monoclonal
antibody 12CA5, specific for the 9-amino acid HA peptide sequence
YPYDVPDYA from influenza HA was obtained from BabCo. An
affinity-purified rabbit polyclonal anti-actin antibody (A-2066) was
purchased from Sigma.Immunoprecipitation
Cells were collected by
centrifugation and washed once with ice-cold PBS and cytoplasmic
extracts prepared as described above. Immunoglobulins from a preimmune
rabbit serum or from anti-IB or anti-NF-Bp50 rabbit
antisera were covalently cross-linked to Protein A-Sepharose using
dimethyl pimelimidate. 10 µl packed volume of Protein A beads bound
to antibody were incubated with 1 mg of cytoplasmic protein for 2 h at
room temperature, and the beads were washed 4 times with 10 ml of lysis
buffer without iodoacetamide and once with PBS containing 0.1% Tween
20. Immunoprecipitation under denaturing conditions was performed in
RIPA buffer as described previously(39) . Immunoprecipitated
proteins were fractionated in a 10% polyacrylamide gel containing SDS,
transferred to polyvinylidine difluoride, and detected by Western
blotting using ECL as described above.Treatment with Ubiquitin Carboxyl-terminal
Hydrolase
Cytoplasmic extracts were prepared as described, but
iodoacetamide was omitted from the lysis buffer. A Drosophila ubiquitin carboxyl-terminal hydrolase (58) was expressed
in E. coli as a GST fusion protein (GST-UCH) and purified over
glutathione-agarose essentially as described for NF-B
p50(61) . GST alone was purified in an identical fashion.
Cytoplasmic protein (50 µg) was incubated at 37 °C for 30 min
in a buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM dithiothreitol, and 10 µM GST or
GST-UCH. Reaction products were fractionated in an 8% polyacrylamide
gel containing SDS and electroblotted onto polyvinylidine difluoride,
and IB was detected by Western blotting using MAD3 10B
monoclonal antibody and ECL. The membrane was stripped and reprobed
with a rabbit polyclonal anti-ubiquitin antibody (62) that was
detected by ECL. The membrane was again stripped as described above and
probed with an affinity-purified rabbit polyclonal anti-actin antibody
(Sigma, A-2066).
Proteasome Inhibitor Z-LLL-H Blocks TNF
Activation of NF--induced
Activation of NF-BB and
signal-induced degradation of IB can be blocked by inhibiting
the activity of the multicatalytic protease or
proteasome(20, 50) . To define events that lead to
targeting of IB for degradation, the peptide aldehyde Z-LLL-H
was synthesized as a tool to accumulate intermediates in the
degradative process. HeLa suspension cells pretreated with Z-LLL-H were
exposed to TNF, and the cells were fractionated into nucleus and
cytoplasm. NF-B DNA binding activity, determined in a native
polyacrylamide gel, was low in nuclei of unstimulated cells but was
present at an elevated level in cells treated with TNF (Fig. 1a). Characterization by specific competition
with unlabeled DNA, recognition by p50 and p65 antibodies, and
inhibition by recombinant IB indicated that the
TNF-induced DNA-protein complex in HeLa cells was composed of p50,
p65 heterodimers, and p65 homodimers(53) . This
TNF-induced increase was largely, but not completely, abrogated by
pretreatment of the cells with either 10 or 25 µM Z-LLL-H (Fig. 1a). Western blot analysis of nuclear extracts
with an antibody recognizing p50 revealed a low level of nuclear p50 in
untreated cells and high levels of nuclear p50 in TNF-induced
cells. Preincubation of the cells with Z-LLL-H prior to TNF
induction blocked the nuclear accumulation of p50 (Fig. 1b). Z-LLL-H inhibition of nuclear translocation
was not specific to TNF but was also observed with other inducers such
as interleukin-1 (data not shown).
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.
Slowly Migrating Forms of I
Western blot analysis, with
monoclonal or affinity-purified polyclonal antibodies recognizing
IB Accumulate in the
Presence of TNF and Z-LLL-HB(34) , reveals that TNF induces rapid
degradation of IB, but this process is inhibited in cells
previously treated with Z-LLL-H (Fig. 2, short exposure). As
expected(20, 50) , phosphorylated IB
accumulates in the presence of the inhibitor (Fig. 2, short
exposure). Further exposure of the Western blot reveals that in the
presence of Z-LLL-H, TNF induction results in accumulation of a
ladder of more slowly migrating forms of IB, with a major
form that is 8 kD larger than IB (Fig. 2, long
exposure). Also detected are more rapidly migrating forms of
IB that are likely to be intermediates in the degradation of
IB. With Z-LLL-H alone, small amounts of more slowly
migrating forms of IB are also detected (Fig. 2, long
exposure), suggesting that even in the resting state, IB is
being turned over by the same mechanism that operates after TNF
induction. The appearance and molecular weight of the more slowly
migrating forms of IB that are detected in the presence of
the proteasomal inhibitor are highly suggestive of multiple additions
of ubiquitin. This phenomenon was also observed in monocytic cells
(U937) and with other inducers of NF-B (interleukin-1) when
cells were preincubated with Z-LLL-H (data not shown).
B. HeLa cells were pretreated
with Z-LLL-H and exposed to TNF as indicated. Cytoplasmic extracts
(40 µg) were fractionated by SDS-PAGE, and IB protein was
analyzed by ECL Western blotting using monoclonal antibody MAD3 10B (34) . The positions of prestained molecular weight markers
(Sigma), IB and the phosphorylated form of the protein (P) are indicated. More slowly migrating and faster migrating
forms of IB are indicated by the brackets. A short exposure
is displayed to demonstrate Z-LLL-H inhibition of TNF-induced
IB degradation, while a long exposure is shown to display the
IB species that accumulate in the presence of
Z-LLL-H.
Ubiquitination of I
To
demonstrate that the more slowly migrating forms of IB in VivoB are a
result of multiple additions of ubiquitin, extracts from cells treated
with TNF and Z-LLL-H were incubated with either a recombinant
ubiquitin carboxyl-terminal hydrolase (58) (GST-UCH) or GST.
Western blot analysis indicates that the more slowly migrating forms of
IB present in the 20-min sample are lost after treatment with
GST-UCH but remain after treatment with GST alone (Fig. 3a). In contrast phosphorylated IB and
more rapidly migrating IB forms are unchanged after
incubation with either GST-UCH or GST (Fig. 3a). Thus
the more slowly migrating forms of IB represent the addition
of one or more copies of ubiquitin, whereas faster migrating
IB species are not linked to ubiquitin and may be
intermediates in IB degradation. To determine the fate of the
bulk of cellular ubiquitin conjugates, the blot in Fig. 3a was
reprobed with an antibody recognizing protein-ubiquitin conjugates.
Cells incubated with Z-LLL-H accumulate high molecular weight
conjugates, some of which are removed by the GST-UCH, but not by GST
alone (Fig. 3b). An unidentified 47-kDa species is
detected after incubation with GST but not GST-UCH (Fig. 3b), presumably as a result of ubiquitin removal.
Thus the Drosophila UCH has a limited specificity for
protein-ubiquitin conjugates. Reprobing the blot with an anti-actin
antibody revealed an equivalent signal in each lane (Fig. 3c), confirming the absence of nonspecific
proteolysis.
B 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
IB-specific monoclonal antibody MAD3 10B. IB, 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-IB
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.
B is linked to
ubiquitin, cells were treated with TNF and Z-LLL-H, and extracts
were immunoprecipitated with antibodies to IB, 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 IB is
degraded, but ubiquitinated adducts on the remaining protein are
detected in immunoprecipitates with IB 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 IB are
detected in immunoprecipitates with antibodies specific to
IB. Again antibodies to p50 and p65 only precipitate amounts
of ubiquitinated IB that are comparable with that obtained
with the preimmune serum, even although they can precipitate bound
IB 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
IB-ubiquitin conjugates dissociate from NF-B.
B dissociates
from NF-B whereas phosphorylated IB 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% MeSO vehicle alone
for 45 min. Cytoplasmic extracts were prepared and immunoprecipitated
with either rabbit preimmune serum (PI), rabbit
anti-IB serum (a-IkB), or rabbit anti-p50 serum (a-p50). Immunoprecipitates were fractionated by SDS-PAGE and
analyzed by Western blotting with anti-IB monoclonal antibody
MAD3 10B. Molecular mass markers and the position of IB and
its phosphorylated derivative are indicated. IB-ubiquitin
conjugates are indicated by the upper bracket, while
IB 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 IB detected by Western
blotting.
B (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 IB or preimmune serum, and analyzed by Western
blotting using first the monoclonal antibody recognizing the HA tag and
reprobed with an IB-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
IB even in the absence of TNF and Z-LLL-H (Fig. 4b, upper left panel), again indicating
that IB is being constantly turned over. Multiply
ubiquitinated IB 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
IB antibody as only a single epitope is present. Although
treatment of the cells with TNF results in a proportion of the
IB being degraded (Fig. 4b, upper right
panel), this does not translate into a reduction in the amount of
multiply HA-tagged, ubiquitinated IB detected (Fig. 4b, upper left panel). While the rate of
IB degradation is increased in the presence of TNF, the
rate of ubiquitination of IB is also increased, with the
consequence that the steady-state level of ubiquitinated IB
is unaltered. Endogenous IB is thus covalently linked to
HA-tagged ubiquitin as part of a rapidly turning over pool of modified
IB that is stabilized by blocking degradation with Z-LLL-H.
B in vivo. a, structure of the HA-tagged
polyubiquitin gene (HA-ubi, (56) ) with the amino-terminal tag
shaded. The IBctag 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
IB (IP Ab IB, 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 IB-specific monoclonal
antibody MAD3 10B (Blot Ab IB, right
panels).
B
(IBctag). Only a small proportion of the highly expressed
IBctag is degraded (Fig. 4b, upper right
panel), indicating that the IB modification and
degradation machinery has a limited capacity. Thus, only a small amount
of immunopurified IB 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 IB (Fig. 4b, upper right panel). Preimmune serum
did not immunoprecipitate material reactive with the HA-specific
antibody (Fig. 4b, lower left panel). Thus
IB is covalently linked to HA-tagged ubiquitin when
signal-induced degradation is blocked by proteasomal inhibitors.I
To confirm that ubiquitinated IB, Ubiquitinated in Vivo, Is Not Associated
with NF- BB was
not associated with NF-B, cell extracts were immunoprecipitated
with antibodies specific for either IB, p50, or preimmune
serum and bound IB detected by Western blotting. In the
presence of Z-LLL-H, TNF induction results in accumulation of
ubiquitinated forms of the protein that are immunoprecipitated with
antibodies to IB (Fig. 5a). While antibodies
to p50 immunoprecipitate IB, they fail to precipitate
ubiquitinated IB (Fig. 5a), indicating that
ubiquitination of IB releases NF-B. In contrast,
phosphorylated IB remains associated with NF-B and is
precipitated by antibodies to p50 (Fig. 5b). Although
the TNF-induced degradation of IB was incomplete, the
presence of phosphorylated IB indicated that signal induction
had taken place.Residues Ser-32 and Ser-36 Are Required for
Ubiquitination of I
Residues S32 and S36 are
required for the signal-induced phosphorylation and degradation of
IB in VivoB(36, 42, 43) . To determine if
phosphorylation of these residues was required for ubiquitination of
IB, a cell line expressing an S32A/S36A IBctag was
selected. Both wild-type IBctag and S32A/S36A IBctag were
expressed at levels that were comparable with that of the endogenous
IB. Western blot analysis with the tag-specific monoclonal
antibody revealed that the wild-type IBctag was rapidly degraded
in response to TNF or TNF plus okadaic acid, whereas okadaic
acid alone did not induce degradation (Fig. 6b). In the
presence of TNF, okadaic acid and the proteasome inhibitor Z-LLL-H
degradation was blocked (Fig. 6b), and ubiquitinated
forms of the protein were detected (Fig. 6a). In
contrast, the S32A/S36A mutant was neither degraded nor ubiquitinated (Fig. 6, a and b). Western blot analysis with
an antibody, which detects both endogenous and tagged IB,
revealed that in cells expressing S32A/S36A IBctag, the endogenous
IB was degraded. Thus it is likely that phosphorylation of
Ser-32 and Ser-36 is required for ubiquitination and degradation of
IB in vivo.
B in vivo. HeLa cells stably
expressing either wild-type IBctag or S32A/S36A IBctag 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% MeSO 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-IB monoclonal
antibody MAD 10B (c). The positions of molecular mass markers,
IBctag, IB phosphorylated derivatives, and ubiquitin
adducts are indicated.
B and
activation of NF-B(20, 50) . Although it was
demonstrated that ubiquitination was involved in processing of p105 to
p50(20) , ubiquitination of IB was not reported.
However a recent report (63) has shown that IB is
ubiquitinated, and in vitro studies demonstrated that this
process required residues Ser-32 and Ser-36. Furthermore it was shown
that ubiquitinated IB was a substrate for the 26 S proteasome in vitro. Here we demonstrate that signal induction with
TNF results in phosphorylation-dependent ubiquitination of
IB and release of NF-B. This is in contrast with the
situation observed in vitro where ubiquitinated IB
remains bound to NF-B(63) . Thus it is unlikely that
ubiquitination per se is responsible for the dissociation of
IB from NF-B, but one possibility is that a protein with
chaperonin activity could release ubiquitinated IB from
NF-B in vivo. Ubiquitinated IB must represent a
short lived intermediate in the degradative pathway, as ubiquitinated
IB is only readily detected when proteasomal degradation is
inhibited. The results support a model for signal-induced activation of
NF-B in which IB is first phosphorylated on residues
Ser-32 and Ser-36 (36, 42, 43) by an as yet
unidentified kinase. Phosphorylated IB remains bound to
NF-B but is recognized by proteins involved in ubiquitin addition.
Enzymes responsible for ligation of ubiquitin to IB 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
IB 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 IB is degraded by
the multicatalytic protease or proteasome(66) . Although
ubiquitinated IB is detected when proteasomal degradation is
blocked, only a small proportion of the IB 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
IB-ubiquitin conjugates do not constitute a large proportion
of the total IB pool, the observation that Z-LLL-H only
partially blocks translocation of NF-B to the nucleus (Fig. 1) but efficiently blocks degradation of IB (Fig. 2) is thus explained by release of NF-B from
ubiquitinated IB ( Fig. 3and Fig. 5). B 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 IB, 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.
), tumor
necrosis factor ; HA, hemagglutinin; HA-ubi, HA-tagged ubiquitin;
PBS, phosphate-buffered saline; PAGE, polyacrylamide gel
electrophoresis.)
We thank R. J. Mayer for anti-ubiquitin antibodies, L.
Guarino for the 4F3 monoclonal antibody, R. E. Randall for the SV5 Pk
tag monoclonal antibody, M. Bownes for the strain expressing ubiquitin
carboxyl-terminal hydrolase, D. Bohmann for the HA-tagged ubiquitin
plasmid, and B. Blyth for photography. Comments on the manuscript by A.
Webster and J.L. Virelizier were greatly appreciated.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Hu, R. Zhou, J. Liu, A.-Y. Gong, A. N. Eischeid, J. W. Dittman, and X.-M. Chen MicroRNA-98 and let-7 Confer Cholangiocyte Expression of Cytokine-Inducible Src Homology 2-Containing Protein in Response to Microbial Challenge J. Immunol., August 1, 2009; 183(3): 1617 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hedhli, P. Lizano, C. Hong, L. F. Fritzky, S. K. Dhar, H. Liu, Y. Tian, S. Gao, K. Madura, S. F. Vatner, et al. Proteasome inhibition decreases cardiac remodeling after initiation of pressure overload Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1385 - H1393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Gutierrez, G. W. O'Keeffe, N. Gavalda, D. Gallagher, and A. M. Davies Nuclear Factor {kappa}B Signaling Either Stimulates or Inhibits Neurite Growth Depending on the Phosphorylation Status of p65/RelA J. Neurosci., August 13, 2008; 28(33): 8246 - 8256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sartore-Bianchi, F. Gasparri, A. Galvani, L. Nici, J. W. Darnowski, D. Barbone, D. A. Fennell, G. Gaudino, C. Porta, and L. Mutti Bortezomib Inhibits Nuclear Factor-{kappa}B Dependent Survival and Has Potent In vivo Activity in Mesothelioma Clin. Cancer Res., October 1, 2007; 13(19): 5942 - 5951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Gallagher, H. Gutierrez, N. Gavalda, G. O'Keeffe, R. Hay, and A. M. Davies Nuclear Factor-{kappa}B Activation via Tyrosine Phosphorylation of Inhibitor {kappa}B-{alpha} Is Crucial for Ciliary Neurotrophic Factor-Promoted Neurite Growth from Developing Neurons J. Neurosci., September 5, 2007; 27(36): 9664 - 9669. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yang, X. Liao, M. K. Agarwal, L. Barnes, P. E. Auron, and G. R. Stark Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NF{kappa}B Genes & Dev., June 1, 2007; 21(11): 1396 - 1408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Garcia, J. Gil, I. Ventoso, S. Guerra, E. Domingo, C. Rivas, and M. Esteban Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 1032 - 1060. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Cardoso, V. Durbecq, J.-F. Laes, B. Badran, L. Lagneaux, F. Bex, C. Desmedt, K. Willard-Gallo, J. S. Ross, A. Burny, et al. Bortezomib (PS-341, Velcade) increases the efficacy of trastuzumab (Herceptin) in HER-2-positive breast cancer cells in a synergistic manner Mol. Cancer Ther., December 1, 2006; 5(12): 3042 - 3051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sors, F. Jean-Louis, C. Pellet, L. Laroche, L. Dubertret, G. Courtois, H. Bachelez, and L. Michel Down-regulating constitutive activation of the NF-{kappa}B canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis Blood, March 15, 2006; 107(6): 2354 - 2363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Stark and M. G. Dunlop Nucleolar Sequestration of RelA (p65) Regulates NF-{kappa}B-Driven Transcription and Apoptosis Mol. Cell. Biol., July 15, 2005; 25(14): 5985 - 6004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Gutierrez, V. A. Hale, X. Dolcet, and A. Davies NF-{kappa}B signalling regulates the growth of neural processes in the developing PNS and CNS Development, April 1, 2005; 132(7): 1713 - 1726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Preciado, E. Caicedo, R. Jhanjee, R. Silver, G. Harris, S. K. Juhn, D. I. Choo, and F. Ondrey Pseudomonas aeruginosa Lipopolysaccharide Induction of Keratinocyte Proliferation, NF-{kappa}B, and Cyclin D1 Is Inhibited by Indomethacin J. Immunol., March 1, 2005; 174(5): 2964 - 2973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Misaghi, P. J. Galardy, W. J. N. Meester, H. Ovaa, H. L. Ploegh, and R. Gaudet Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate J. Biol. Chem., January 14, 2005; 280(2): 1512 - 1520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. E. Ackerman IV, B. H. Rovin, and D. A. Kniss Epidermal Growth Factor and Interleukin-1{beta} Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells Biol Reprod, December 1, 2004; 71(6): 2079 - 2086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Minekawa, T. Takeda, M. Sakata, M. Hayashi, A. Isobe, T. Yamamoto, K. Tasaka, and Y. Murata Human breast milk suppresses the transcriptional regulation of IL-1{beta}-induced NF-{kappa}B signaling in human intestinal cells Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1404 - C1411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Rahman, K. N. Anwar, Mohd. Minhajuddin, K. M. Bijli, K. Javaid, A. L. True, and A. B. Malik cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-{kappa}B activation and ICAM-1 expression in endothelial cells Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L1017 - L1024. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gil, M. A. Garcia, P. Gomez-Puertas, S. Guerra, J. Rullas, H. Nakano, J. Alcami, and M. Esteban TRAF Family Proteins Link PKR with NF-{kappa}B Activation Mol. Cell. Biol., May 15, 2004; 24(10): 4502 - 4512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Mistry, K. Deacon, S. Mistry, J. Blank, and R. Patel NF-{kappa}B Promotes Survival during Mitotic Cell Cycle Arrest J. Biol. Chem., January 9, 2004; 279(2): 1482 - 1490. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. P. Atkinson, H. J. Coope, M. Rowe, and S. C. Ley Latent Membrane Protein 1 of Epstein-Barr Virus Stimulates Processing of NF-{kappa}B2 p100 to p52 J. Biol. Chem., December 19, 2003; 278(51): 51134 - 51142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakurai, S. Suzuki, N. Kawasaki, H. Nakano, T. Okazaki, A. Chino, T. Doi, and I. Saiki Tumor Necrosis Factor-{alpha}-induced IKK Phosphorylation of NF-{kappa}B p65 on Serine 536 Is Mediated through the TRAF2, TRAF5, and TAK1 Signaling Pathway J. Biol. Chem., September 19, 2003; 278(38): 36916 - 36923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Parcellier, E. Schmitt, S. Gurbuxani, D. Seigneurin-Berny, A. Pance, A. Chantome, S. Plenchette, S. Khochbin, E. Solary, and C. Garrido HSP27 Is a Ubiquitin-Binding Protein Involved in I-{kappa}B{alpha} Proteasomal Degradation Mol. Cell. Biol., August 15, 2003; 23(16): 5790 - 5802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Mendoza, L.-n. Shen, C. Botting, A. Lewis, J. Chen, B. Ink, and R. T. Hay NEDP1, a Highly Conserved Cysteine Protease That deNEDDylates Cullins J. Biol. Chem., July 3, 2003; 278(28): 25637 - 25643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zou, B. Attuwaybi, and B. C. Kone Effects of NF-kappa B inhibition on mesenteric ischemia-reperfusion injury Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G713 - G721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Burke, M. A. Pattoli, K. R. Gregor, P. J. Brassil, J. F. MacMaster, K. W. McIntyre, X. Yang, V. S. Iotzova, W. Clarke, J. Strnad, et al. BMS-345541 Is a Highly Selective Inhibitor of Ikappa B Kinase That Binds at an Allosteric Site of the Enzyme and Blocks NF-kappa B-dependent Transcription in Mice J. Biol. Chem., January 10, 2003; 278(3): 1450 - 1456. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Tapalaga, G. Tiegs, and S. Angermuller NF{kappa}B and Caspase-3 Activity in Apoptotic Hepatocytes of Galactosamine-sensitized Mice Treated with TNF{alpha} J. Histochem. Cytochem., December 1, 2002; 50(12): 1599 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Kabouridis, M. Hasan, J. Newson, D. W. Gilroy, and T. Lawrence Inhibition of NF-{kappa}B Activity by a Membrane-Transducing Mutant of I{kappa}B{alpha} J. Immunol., September 1, 2002; 169(5): 2587 - 2593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Adams Development of the Proteasome Inhibitor PS-341 Oncologist, February 1, 2002; 7(1): 9 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yang, G.-H. Fan, B. E. Wadzinski, H. Sakurai, and A. Richmond Protein Phosphatase 2A Interacts with and Directly Dephosphorylates RelA J. Biol. Chem., December 14, 2001; 276(51): 47828 - 47833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Jiang, P. Brecher, and R. A. Cohen Persistent Activation of Nuclear Factor-{kappa}B by Interleukin-1{beta} and Subsequent Inducible NO Synthase Expression Requires Extracellular Signal-Regulated Kinase Arterioscler. Thromb. Vasc. Biol., December 1, 2001; 21(12): 1915 - 1920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Rahman, K. N. Anwar, S. Uddin, N. Xu, R. D. Ye, L. C. Platanias, and A. B. Malik Protein Kinase C-{delta} Regulates Thrombin-Induced ICAM-1 Gene Expression in Endothelial Cells via Activation of p38 Mitogen-Activated Protein Kinase Mol. Cell. Biol., August 15, 2001; 21(16): 5554 - 5565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Hay, G. D. Kemp, C. Dargemont, and R. T. Hay Interaction between hnRNPA1 and I{kappa}B{alpha} Is Required for Maximal Activation of NF-{kappa}B-Dependent Transcription Mol. Cell. Biol., May 15, 2001; 21(10): 3482 - 3490. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. S. Neish, A. T. Gewirtz, H. Zeng, A. N. Young, M. E. Hobert, V. Karmali, A. S. Rao, and J. L. Madara Prokaryotic Regulation of Epithelial Responses by Inhibition of Ikappa B-alpha Ubiquitination Science, September 1, 2000; 289(5484): 1560 - 1563. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. Holzl, B. Kapelari, J. Kellermann, E. Seemuller, M. Sumegi, A. Udvardy, O. Medalia, J. Sperling, S. A. Muller, A. Engel, et al. The Regulatory Complex of Drosophila melanogaster 26S Proteasomes: Subunit Composition and Localization of a Deubiquitylating Enzyme J. Cell Biol., July 11, 2000; 150(1): 119 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Renard, Y. Percherancier, M. Kroll, D. Thomas, J.-L. Virelizier, F. Arenzana-Seisdedos, and F. Bachelerie Inducible NF-kappa B Activation Is Permitted by Simultaneous Degradation of Nuclear Ikappa Balpha J. Biol. Chem., May 12, 2000; 275(20): 15193 - 15199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. A. Atencio, M. Ramachandra, P. Shabram, and G. W. Demers Calpain Inhibitor 1 Activates p53-dependent Apoptosis in Tumor Cell Lines Cell Growth Differ., May 1, 2000; 11(5): 247 - 253. [Abstract] [Full Text]
|
|
|
|
|
|
M. T. Sommerfeld, R. Schweigreiter, Y.-A. Barde, and E. Hoppe Down-regulation of the Neurotrophin Receptor TrkB following Ligand Binding. EVIDENCE FOR AN INVOLVEMENT OF THE PROTEASOME AND DIFFERENTIAL REGULATION OF TrkA AND TrkB J. Biol. Chem., March 17, 2000; 275(12): 8982 - 8990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Breitschopf, J. Haendeler, P. Malchow, A. M. Zeiher, and S. Dimmeler Posttranslational Modification of Bcl-2 Facilitates Its Proteasome-Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway Mol. Cell. Biol., March 1, 2000; 20(5): 1886 - 1896. [Abstract] [Full Text]
|
|
|
|
|
|
G. Middleton, M. Hamanoue, Y. Enokido, S. Wyatt, D. Pennica, E. Jaffray, R. T. Hay, and A. M. Davies Cytokine-induced Nuclear Factor Kappa B Activation Promotes the Survival of Developing Neurons J. Cell Biol., January 24, 2000; 148(1): 325 - 332. [Abstract] [Full Text]
|
|
|
|
|
|
G. Middleton, M. Hamanoue, Y. Enokido, S. Wyatt, D. Pennica, E. Jaffray, R. T. Hay, and A. M. Davies Cytokine-induced Nuclear Factor Kappa B Activation Promotes the Survival of Developing Neurons J. Cell Biol., January 24, 2000; 148(2): 325 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Burke, M. K. Wood, R.-P. Ryseck, S. Walther, and C. A. Meyers Peptides Corresponding to the N and C Termini of Ikappa B-alpha , -beta , and -epsilon as Probes of the Two Catalytic Subunits of Ikappa B Kinase, IKK-1 and IKK-2 J. Biol. Chem., December 17, 1999; 274(51): 36146 - 36152. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Didcock, D. F. Young, S. Goodbourn, and R. E. Randall The V Protein of Simian Virus 5 Inhibits Interferon Signalling by Targeting STAT1 for Proteasome-Mediated Degradation J. Virol., December 1, 1999; 73(12): 9928 - 9933. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Giuliano, M. Lauricella, G. Calvaruso, M. Carabillo, S. Emanuele, R. Vento, and G. Tesoriere The Apoptotic Effects and Synergistic Interaction of Sodium Butyrate and MG132 in Human Retinoblastoma Y79 Cells Cancer Res., November 1, 1999; 59(21): 5586 - 5595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakurai, H. Chiba, H. Miyoshi, T. Sugita, and W. Toriumi Ikappa B Kinases Phosphorylate NF-kappa B p65 Subunit on Serine 536 in the Transactivation Domain J. Biol. Chem., October 22, 1999; 274(43): 30353 - 30356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. W. Harhaj and S.-C. Sun Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50 Mol. Cell. Biol., October 1, 1999; 19(10): 7088 - 7095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gil, J. Alcami, and M. Esteban Induction of Apoptosis by Double-Stranded-RNA-Dependent Protein Kinase (PKR) Involves the alpha Subunit of Eukaryotic Translation Initiation Factor 2 and NF-kappa B Mol. Cell. Biol., July 1, 1999; 19(7): 4653 - 4663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakurai, H. Miyoshi, W. Toriumi, and T. Sugita Functional Interactions of Transforming Growth Factor beta -activated Kinase 1 with Ikappa B Kinases to Stimulate NF-kappa B Activation J. Biol. Chem., April 9, 1999; 274(15): 10641 - 10648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Rodriguez, J. Thompson, R. T. Hay, and C. Dargemont Nuclear Retention of Ikappa Balpha Protects It from Signal-induced Degradation and Inhibits Nuclear Factor kappa B Transcriptional Activation J. Biol. Chem., March 26, 1999; 274(13): 9108 - 9115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Mercurio, B. W. Murray, A. Shevchenko, B. L. Bennett, D. B. Young, J. W. Li, G. Pascual, A. Motiwala, H. Zhu, M. Mann, et al. Ikappa B Kinase (IKK)-Associated Protein 1, a Common Component of the Heterogeneous IKK Complex Mol. Cell. Biol., February 1, 1999; 19(2): 1526 - 1538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Giri, R. T. Mehta, R. G. Kansal, and B. B. Aggarwal Mycobacterium avium-intracellulare complex Activates Nuclear Transcription Factor-{kappa}B in Different Cell Types Through Reactive Oxygen Intermediates J. Immunol., November 1, 1998; 161(9): 4834 - 4841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Jeremias, C. Kupatt, B. Baumann, I. Herr, T. Wirth, and K.M. Debatin Inhibition of Nuclear Factor kappa B Activation Attenuates Apoptosis Resistance in Lymphoid Cells Blood, June 15, 1998; 91(12): 4624 - 4631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Burke, K. R. Miller, M. K. Wood, and C. A. Meyers The Multisubunit Ikappa B Kinase Complex Shows Random Sequential Kinetics and Is Activated by the C-terminal Domain of Ikappa Balpha J. Biol. Chem., May 15, 1998; 273(20): 12041 - 12046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Schwartz, V. Marechal, B. Friguet, F. Arenzana-Seisdedos, and J.-M. Heard Antiviral Activity of the Proteasome on Incoming Human Immunodeficiency Virus Type 1 J. Virol., May 1, 1998; 72(5): 3845 - 3850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Hicke, B. Zanolari, and H. Riezman Cytoplasmic Tail Phosphorylation of the alpha -Factor Receptor Is Required for Its Ubiquitination and Internalization J. Cell Biol., April 20, 1998; 141(2): 349 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bergmann, L. Hart, M. Lindsay, P. J. Barnes, and R. Newton Ikappa Balpha Degradation and Nuclear Factor-kappa B DNA Binding Are Insufficient for Interleukin-1beta and Tumor Necrosis Factor-alpha -induced kappa B-dependent Transcription. REQUIREMENT FOR AN ADDITIONAL ACTIVATION PATHWAY J. Biol. Chem., March 20, 1998; 273(12): 6607 - 6610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Luque and C. Gélinas Distinct Domains of Ikappa Balpha Regulate c-Rel in the Cytoplasm and in the Nucleus Mol. Cell. Biol., March 1, 1998; 18(3): 1213 - 1224. [Abstract] [Full Text]
|
|
|
|
|
|
R.-M. Dai, E. Chen, D. L. Longo, C. M. Gorbea, and C.-C. H. Li Involvement of Valosin-containing Protein, an ATPase Co-purified with Ikappa Balpha and 26 S Proteasome, in Ubiquitin-Proteasome-mediated Degradation of Ikappa Balpha J. Biol. Chem., February 6, 1998; 273(6): 3562 - 3573. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Sears, J. Olesen, D. Rubin, D. Finley, and T. Maniatis NF-kappa B p105 Processing via the Ubiquitin-Proteasome Pathway J. Biol. Chem., January 16, 1998; 273(3): 1409 - 1419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Mercurio, H. Zhu, B. W. Murray, A. Shevchenko, B. L. Bennett, J. w. Li, D. B. Young, M. Barbosa, M. Mann, A. Manning, et al. IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science, October 31, 1997; 278(5339): 860 - 866. [Abstract] [Full Text]
|
|
|
|
|
|
M. Velasco, M. J. M. Diaz-Guerra, P. Martin-Sanz, A. Alvarez, and L. Bosca Rapid Up-regulation of Ikappa Bbeta and Abrogation of NF-kappa B Activity in Peritoneal Macrophages Stimulated with Lipopolysaccharide J. Biol. Chem., September 12, 1997; 272(37): 23025 - 23030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-T. Yamin and D. K. Miller The Interleukin-1 Receptor-associated Kinase Is Degraded by Proteasomes following Its Phosphorylation J. Biol. Chem., August 22, 1997; 272(34): 21540 - 21547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tashiro, M. P. Pando, Y. Kanegae, P. M. Wamsley, S. Inoue, and I. M. Verma Direct involvement of the ubiquitin-conjugating enzyme Ubc9/Hus5 in the degradation of Ikappa Balpha PNAS, July 22, 1997; 94(15): 7862 - 7867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. van Delft, R. Govers, G. J. Strous, A. J. Verkleij, and P. M. P. v. B. en Henegouwen Epidermal Growth Factor Induces Ubiquitination of Eps15 J. Biol. Chem., May 30, 1997; 272(22): 14013 - 14016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Rayanade, K. Patel, M. Ndubuisi, S. Sharma, S. Omura, J. D. Etlinger, R. Pine, and P. B. Sehgal Proteasome- and p53-dependent Masking of Signal Transducer and Activator of Transcription (STAT) Factors J. Biol. Chem., February 21, 1997; 272(8): 4659 - 4662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Trawick, S.-L. Wang, D. Bell, and R. A. Davis Transcriptional Induction of Cholesterol 7alpha -Hydroxylase by Dexamethasone in L35 Hepatoma Cells Requires Sulfhydryl Reducing Agents J. Biol. Chem., January 31, 1997; 272(5): 3099 - 3102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xu and R. A.F. Clark A Three-dimensional Collagen Lattice Induces Protein Kinase C-zeta Activity: Role in alpha 2 Integrin and Collagenase mRNA Expression J. Cell Biol., January 27, 1997; 136(2): 473 - 483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F Bachelerie, M. Rodriguez, C Dargemont, D Rousset, D Thomas, J. Virelizier, and F Arenzana-Seisdedos Nuclear export signal of IkappaBalpha interferes with the Rev-dependent posttranscriptional regulation of human immunodeficiency virus type I J. Cell Sci., January 11, 1997; 110(22): 2883 - 2893. [Abstract] [PDF]
|
|
|
|
|
|
A. Benoliel, B Kahn-Peries, J Imbert, and P Verrando Insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor J. Cell Sci., January 9, 1997; 110(17): 2089 - 2097. [Abstract] [PDF]
|
|
|
|
|
|
Y. Imai, M. Soda, and R. Takahashi Parkin Suppresses Unfolded Protein Stress-induced Cell Death through Its E3 Ubiquitin-protein Ligase Activity J. Biol. Chem., November 10, 2000; 275(46): 35661 - 35664. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |