From the Division of Cellular Immunology, the
National Institute for Medical Research, Mill Hill,
London NW7 1AA, United Kingdom and
Abbott Bioresearch
Center, Worcester, Massachusetts 01605-4314
Received for publication, February 26, 2001, and in revised form, April 3, 2001
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ABSTRACT |
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The p105 precursor protein of NF- NF- In unstimulated cells, NF- The best characterized member of the I The constitutive proteolytic generation of NF- Unprocessed p105 functions as an I It has been shown recently that the mammalian IKK complex
immunoprecipitated from transfected cells will phosphorylate
recombinant p105 in vitro in a region of the C-terminal PEST
domain that appears to be important for TNF In this study, the role of the IKK complex in controlling p105
proteolysis was investigated genetically using knockout cell lines
lacking IKK component subunits. These experiments demonstrated that a
functional IKK complex is essential for TNF cDNA Constructs and Antibodies--
p105 constructs were
subcloned into the pcDNA3 expression vector (Invitrogen) for
transient mammalian cell expression experiments and into the pMX-1
expression vector (Ingenius) for generation of stably transfected HeLa
cell lines. Addition of an N-terminal HA epitope tag to the cDNA
encoding human p105 (28) and generation of p105 point mutants were done
using the polymerase chain reaction (PCR). PCR was also used to
generate a cDNA encoding a GST-p105-(758-967) fusion protein and
its S927A derivative, subcloned into the pGEX-6P vector (Amersham
Pharmacia Biotech). For baculovirus expression, IKK1 and IKK2
constructs were tagged at the N terminus with a His6 tag by
PCR and subcloned into the pFastBac vector (Life Technologies, Inc.).
DNA templates for PCR of IKK1 (clone 1321982) and IKK2 (clone 3126643)
were obtained from Incyte Genomics. All constructs were verified by DNA
sequencing. FLAG-IKK2, subcloned in the pCMV vector, has been described
previously (30).
The following anti-peptide antisera were raised in rabbits to synthetic
peptides coupled to keyhole limpet hemocyanin (Pierce): human
p105 amino acids 952-967 (anti-hp105-C); murine p105 amino
acids 955-971 (anti-mp105-C); human IKK1 amino acids
13-28 (anti-IKK1); and human IKK2 amino acids 572-588 (anti-IKK2). To
generate the anti-phospho-Ser927 antibody, a peptide was
synthesized corresponding to residues 922-935 of human p105 in which
serine 927 is phosphorylated. After high pressure liquid chromatography
purification, this phosphopeptide was coupled to keyhole limpet
hemocyanin and injected into rabbits. Anti-I Cell Lines--
All cells were cultured in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
calf serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (50 units/ml) and maintained in a rapid
growth phase for experiments. Mouse embyronic fibroblasts (MEFs)
lacking component subunits of the IKK complex were generously provided
by the originating laboratories (see Fig. 1 legend).
To stably transfect HeLa cells (Ohio subline from ECACC) with HA-p105,
7 × 105 cells were plated in a 90-mm dish (Life
Technologies, Inc.) and, after 18 h in culture, transfected using
LipofectAMINE (Life Technologies, Inc.). Transfected cells were
cultured for a further 48 h and then selected for neomycin
resistance with 1 mg/ml G418 (Life Technologies, Inc.). After 4 weeks,
clones were picked manually and then expanded. Expression of HA-p105
was determined by Western blotting.
Pulse-Chase Metabolic Labeling--
All pulse-chase metabolic
labeling experiments were performed at least twice with similar
results. For MEFs, 3 × 105 cells were plated per
60-mm dish. After 18 h, cells were washed with phosphate-buffered
saline and cultured in methionine/cysteine-free minimal essential
Eagle's medium (Sigma) for 1 h. Cells were pulse-labeled with
2.65 MBq of [35S]methionine/[35S]cysteine
(Pro-Mix, Amersham Pharmacia Biotech) for 30-45 min and chased for the
indicated times in complete medium (Dulbecco's modified Eagle's
medium plus 2% fetal calf serum) alone (0) or complete medium
supplemented with TNF
NIH-3T3 cells were transiently transfected using LipofectAMINE (Life
Technologies, Inc.). Pulse-chase metabolic labeling and immunoprecipitation were carried out as described previously (22). Labeled bands were quantified by laser densitometry (Calibrated Imaging
Densitometer, Bio-Rad). To analyze HA-p105 proteolysis in stably
transfected HeLa clones, cells were plated at 6 × 105
per well of a 6-well plate (Life Technologies, Inc.). After 18 h
of culture, pulse-chase metabolic labeling was carried out as for MEFs.
HA-p105 was isolated by immunoprecipitation with 12CA5 anti-HA mAb
after lysis with buffer A supplemented with 0.1% SDS and 0.5% deoxycholate.
Analysis of p105 Phosphorylation--
To analyze in
vivo phosphorylation of p105 on serine 927, 5 × 106 HeLa cells were plated per 100-mm dish. After 18 h
in culture, cells were pretreated with 20 µM MG132
proteasome inhibitor for 30 min (Biomol Research Labs) and then
stimulated for the indicated times with IL-1
For in vitro phosphorylation experiments with endogenous IKK
complex, control or TNF
His6-IKK1 and His6-IKK2 were expressed in
Sf9 insect cells by baculovirus infection. Proteins were
isolated from 1 liter of cell pellet extracted with 50 ml of lysis
buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10%
glycerol, and 1.0% Triton X-100) plus protease inhibitor mix (Roche
Molecular Biochemicals). Centrifuged lysate was applied to a 5-ml
chelating Sepharose HiTrap column (Amersham Pharmacia Biotech) that was
pretreated with NiCl2 and equilibrated in 50 mM
HEPES, pH 7.5, 300 mM NaCl. The column was then washed with
50 mM imidazole and bound protein eluted with 250 mM imidazole. The protein was buffer exchanged into 50 mM HEPES, pH 7.5, 10% glycerol, 5 mM
dithiothreitol using a 5-ml Desalt HiTrap column (Amersham Pharmacia
Biotech) and stored at Analysis of p105 Degradation in Fibroblast Cells Lacking Component
Subunits of the IKK Complex--
A genetic approach was taken to
investigate the role of the IKK complex in p105 degradation induced by
physiological stimulation to avoid potential problems associated with
the use of dominant negative kinase mutants (see the Introduction). To
do this, a series of pulse-chase metabolic labeling experiments were
carried out in embryonic fibroblast (MEF) cells isolated from the
indicated knockout mice to determine p105 turnover after cytokine stimulation.
In cells lacking either IKK1 (32) or IKK2 (33), both TNF The PEST Domain of p105 Contains a Sequence Highly Related to the
IKK Target Sequence of I
In addition, a discrepancy was noted at residue 927, which is a serine
in three of the published human p105 sequences
(GenBankTM accession numbers: 227315, AAF35232, and
XP003398), and also in the corresponding residue in mouse, rat, and
chicken p105. However, in three other published human p105 sequences
there is a threonine at residue 927 (GenBankTM
accession numbers: P19838, AAA36361, and AAA94041). To confirm directly
the sequence in this region of human p105, clones encoding p105
cDNA were obtained from two different laboratories and sequenced
over the DNA region encoding amino acids 918-935. Both sequences
predicted a serine at amino acid 927, although one of these had been
reported previously to encode a threonine at this position (28). This
difference is accounted for by a single base pair error in the original
published DNA sequence. DNA was also extracted from primary human
lymphocytes from four donors and sequenced. Again these sequences all
predicted a serine at amino acid 927. Together these analyses clearly
indicated that residue 927 in human p105 is a serine rather than a threonine.
Human, mouse, rat, and chicken p105, therefore, all contain a
sequence (DnSGVETn+5) that is closely related to the I Identification of Serine Residues Involved in
IKK2-mediated p105 Proteolysis--
A series of serine or threonine to
alanine mutants were generated in the region of the human p105 PEST
domain containing the two potential overlapping IKK target sites (Fig.
2C). Overexpressed IKK2 triggers proteolysis of
co-transfected p105 (24). Accordingly, to investigate whether any of
the p105 mutations affected IKK2-promoted p105 proteolysis, 3T3
fibroblasts were co-transfected with plasmids encoding HA
epitope-tagged p105 together with IKK2 or empty vector. HA-p105
turnover was then assessed by immunoprecipitating HA-p105 from lysates
of pulse-chase metabolically labeled cells and resolving isolated
protein by SDS-PAGE. Overexpressed IKK2 dramatically decreased the
half-life of pulse-labeled HA-p105 compared with empty vector
control (Fig. 3, A and
B). The S921A, S923A, and T931A mutants of HA-p105 were all
degraded similar to wild type HA-p105 in response to IKK2 co-expression
(Fig. 3B). HA-p105(S932A) was also degraded when
co-expressed with IKK2 but consistently with delayed kinetics compared
with wild type HA-p105 (Fig. 3B). However, IKK2 had little
detectable effect on the proteolysis of HA-p105(S927A) (Fig. 3,
A and B). Thus serine 927 plays a crucial role in
controlling the proteolysis of p105 triggered by IKK2 overexpression.
TNF TNF
To test this reagent, 3T3 cells were transfected with plasmids encoding
wild type HA-p105 or point mutants thereof plus and minus an IKK2
plasmid and cultured for 24 h. Cells were incubated with MG132
inhibitor for the last 4 h of culture to block proteasome-mediated proteolysis (6). HA-p105 immunoprecipitated from cells co-expressing IKK2 was clearly recognized in Western blots as two distinct bands by
the anti-phospho-Ser927 antibody (Fig.
5A, upper panel). The slower
mobility band was probably caused by IKK2-induced phosphorylation of
HA-p105 on multiple amino acids. Similar results were obtained with the
S921A, S923A, T931A, and S932A mutants. However, neither of the bands were detected in cells co-transfected with HA-p105(S927A), although HA-p105(S927A) protein was clearly present in the immunoprecipitates as
revealed by immunoblotting with anti-hp105-C antibody (Fig.
5A, lower panel). In addition, the IKK2-induced bands detected in cells co-transfected with HA-p105 were completely lost when blots were incubated with anti-phospho-Ser927
antibody plus Ser927 phosphopeptide but not with the
unphosphorylated peptide (data not shown). Together these data
confirmed the specificity of the anti-phospho-Ser927
antibody and demonstrated that IKK2 overexpression induces
phosphorylation of co-transfected HA-p105 on serine 927.
To investigate whether pro-inflammatory cytokines induce
phosphorylation of endogenous p105 on serine 927, HeLa cells were preincubated with MG132 for 30 min to block the proteasome and then
stimulated for the indicated times with either TNF-
The data in this section confirm that p105 serine 927 is phosphorylated
under stimulatory conditions that promote p105 proteolysis and strongly
suggest that the inhibitory effects of the S927A mutation on
signal-induced p105 proteolysis are due to removal of a regulatory
phosphorylation site.
The IKK Complex Phosphorylates a Recombinant p105 Fusion Protein on
Serine 927 in Vitro--
Previous research has established that the
IKK-1/2 complex preferentially phosphorylates serine residues over
threonine. A mutant I
To investigate directly whether the IKK complex could phosphorylate
p105 serine 927, a GST fusion protein was purified from Escherichia coli transformed with a plasmid encoding the C
terminus of p105 (residues 758-967; GST-p105-(758-967)) and a
corresponding mutant in which the residue equivalent to p105 serine 927 was mutated to alanine (GST-p105-(758-967)(S927A)). HeLa cells were stimulated with TNF
IKK-1 and IKK-2 were also individually produced using a baculovirus
expression system and isolated by affinity purification to over 90%
purity (data not shown). These proteins each displayed constitutive
kinase activity in the absence of NEMO, as noted previously (12).
In vitro kinase assays demonstrated that both His6-IKK-1 and His6-IKK-2 phosphorylated the
GST-p105-(758-967) fusion protein on Ser927 (Fig.
6C). No signal was detected with the control
GST-p105-(758-967)(S927A) fusion protein confirming the specificity of
the anti-phospho-Ser927 antibody. Thus, both IKK-1 and
IKK-2 can directly phosphorylate serine 927 of p105, consistent with
ability of TNF The combined genetic and biochemical data in this study
demonstrate that the mammalian IKK complex directly regulates the signal-induced degradation of NF- Activation of the IKK complex by TNF A previous study from this laboratory demonstrated that the C terminus
of NF- An earlier study showed that the IKK complex could phosphorylate
p105 in vitro (21). Analysis of internal deletion mutants revealed that the major in vitro phosphorylation sites
resided between residues 920 and 936 which contained three serine
residues according to original sequences of p105 published by this and other laboratories. A triple mutant in which these serine residues were
mutated to alanine (S921A,S923A,S932A) was no longer phosphorylated by
IKK in vitro and was not degraded in response to TNF Heissmeyer et al. (29) demonstrate a critical role for
serine 927 in IKK2-induced p105 proteolysis and a minor role for serine
923 in transiently transfected 293 cells. By analysis of serine/threonine to alanine mutants of p105, it is also shown that
purified baculovirus IKK2 phosphorylated a p105 fusion protein on both
of these residues in vitro (29). The effects of mutating serine 927 to alanine on p105 proteolysis are consistent with the
present study, which also extends these observations by directly demonstrating in vivo phosphorylation of this residue (Fig.
5) and its critical importance in cytokine-induced p105 degradation (Fig. 3A). A regulatory role for serine 923 in p105
proteolysis was not observed in the present study, as HA-p105(S923A) is
degraded similarly to wild type HA-p105 when co-expressed with IKK2 in 3T3 cells (Fig. 3B). This discrepancy may relate to use of
different cell lines for transient transfections. It remains to be
determined, however, whether p105 serine 923 is actually phosphorylated
in vivo when co-expressed with IKK2 or after cytokine stimulation.
Mutation of serine 932 to alanine consistently delayed, but did not
completely block, HA-p105 proteolysis triggered by IKK2 overexpression
in 3T3 cells (Fig. 3B), suggesting that this residue may
also play a regulatory role in p105 degradation. Serine 932 is not
phosphorylated by IKK2 in vitro (29), and its mutation to
alanine does not affect IKK2-mediated phosphorylation of serine 927 in vivo (Fig. 5A). Thus it is possible that a
serine 932 kinase, distinct from the IKK complex, is also involved in
the regulation of p105 proteolysis. A regulatory role for this residue
may be cell type-specific, however, as Heissmeyer et al.
(29) did not detect any inhibitory effect of mutation of this residue
on IKK2-induced p105 degradation in 293 cells.
The Asp-Ser927-Gly-Val-Glu-Thr931 motif
in the PEST domain of p105 is closely related to the N-terminal IKK
target sequence of I In preliminary experiments, it has been shown that serine 927 is
essential for signal-induced ubiquitination of p105 (data not
shown). It is likely that this reflects a requirement for serine 927 phosphorylation to recruit an ubiquitin-protein isopeptide ligase to p105, thereby facilitating its subsequent ubiquitination and
degradation by the proteasome. Recent published data have indicated
that The Drosophila genome contains three NF- Thus it appears that distinct kinases regulate the degradation of
different IB1 acts as an
NF-
B inhibitory protein, retaining associated Rel subunits in the
cytoplasm of unstimulated cells. Tumor necrosis factor
(TNF
) and
interleukin-1
(IL-1
) stimulate p105 degradation, releasing
associated Rel subunits to translocate into the nucleus. By using
knockout embryonic fibroblasts, it was first established that the I
B
kinase (IKK) complex is essential for these pro-inflammatory cytokines
to trigger efficiently p105 degradation. The p105 PEST domain contains
a motif (Asp-Ser927-Gly-Val-Glu-Thr), related to the
IKK target sequence in I
B
, which is conserved between human,
mouse, rat, and chicken p105. Analysis of a panel of human p105 mutants
in which serine/threonine residues within and adjacent to this motif
were individually changed to alanine established that only serine 927 is essential for p105 proteolysis triggered by IKK2 overexpression.
This residue is also required for TNF
and IL-1
to stimulate p105
degradation. By using a specific anti-phosphopeptide antibody, it was
confirmed that IKK2 overexpression induces serine 927 phosphorylation
of co-transfected p105 and that endogenous p105 is also rapidly
phosphorylated on this residue after TNF
or IL-1
stimulation.
In vitro kinase assays with purified proteins demonstrated
that both IKK1 and IKK2 can directly phosphorylate p105 on serine 927. Together these experiments indicate that the IKK complex regulates the
signal-induced proteolysis of NF-
B1 p105 by direct phosphorylation
of serine 927 in its PEST domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B transcription factors play a critical role in regulating
the expression of genes that function in inflammation, cell proliferation, apoptosis, and development (1, 2). NF-
B is composed
of homodimeric and heterodimeric complexes of NF-
B/Rel family
polypeptides, in which each has a conserved N-terminal Rel homology
domain (RHD),1 containing
DNA-binding domains and dimerization domains (3). Five mammalian
members of the Rel family have been identified as follows: NF-
B1
(p50), NF-
B2 (p52), c-Rel, Rel-A (p65), and RelB. The first two of
these are produced as inactive precursor molecules of 105 (p105) and
100 (p100) kDa, respectively (4, 5), which are proteolytically
processed by the 26 S proteasome to produce the smaller,
transcriptionally active forms (6, 7).
B is sequestered in the cytoplasm in an
inactive form bound to inhibitory proteins, termed I
Bs. In response
to agonist stimulation with ligands such as the pro-inflammatory cytokines tumor necrosis factor (TNF)
and interleukin-1 (IL-1), I
Bs are proteolytically degraded, releasing associated NF-
B dimers to translocate into the nucleus and induce gene expression (1,
2). I
Bs consist of a family of structurally related proteins, which
includes I
B
, I
B
, and I
B
together with the precursor
forms of NF-
B1 (p105) and NF-
B2 (p100) (3). These all contain
multiple ankyrin repeats that interact with the nuclear localizing
signals of RHDs to prevent nuclear translocation of associated Rel subunits.
B family is I
B
, which
binds p50/Rel-A and p50/c-Rel heterodimers (3). In response to
stimulation with an NF-
B agonist, I
B
is rapidly phosphorylated in a conserved D(P)SG
XS(P) motif at its N terminus
(8-11). The regulatory serines of I
B
are phosphorylated by a
700-kDa complex, the I
B kinase (IKK) complex, which contains three
polypeptides as follows: the serine kinases IKK1 (IKK
) and IKK2
(IKK
) and a structural subunit NEMO (IKK
) (2). IKK
and IKK
form a heterodimer that directly phosphorylates I
B
(12) and is
activated by both TNF
and IL-1. NEMO plays an essential role in
coupling the IKK complex to upstream signals (13). The phosphorylated sequence is recognized by the SCF
TRCP
ubiquitin ligase complex which then promotes rapid ubiquitination on
two adjacent lysine residues and subsequent degradation of I
B
by
the 26 S proteasome to release associated NF-
B dimers (14, 15).
B1 p50 from its
precursor p105 involves ubiquitination and is mediated by the 26 S
proteasome (6). This is an unusual example of limited proteolysis by
the proteasome resulting from the presence of a glycine-rich region in
the C-terminal half of the p50 moiety of p105 that appears to act as a
physical barrier to proteasome entry (16, 17). Processing to p50 is
inefficient, and the majority of p105 is simply slowly degraded.
Therefore, p105 levels may be regulated by two proteolytic pathways,
limited (processing to p50) and complete (degradation). An alternative
mechanism has also been suggested in which p105 is processed
co-translationally by the proteasome to produce p50 (18). The relative
importance of post-translational versus co-translational
processing of p105 is presently unclear and may be cell
type-specific.
B through the association of its
C-terminal ankyrin repeats with p50, c-Rel, or Rel-A, which are thereby
retained in the cytoplasm (4, 5). Following stimulation with TNF
,
and other NF-
B agonists, p105 is phosphorylated and then proteolyzed
more rapidly by the proteasome (5, 19-21). This predominantly results
in accelerated p105 degradation, rather than increased processing to
p50 (21, 22). Freed Rel subunits can then translocate into the nucleus
to activate gene transcription. A physiological role for p105 in the
correct regulation of NF-
B has been suggested by the generation of
mice lacking its C-terminal (I
B-like) half, while still expressing
p50 product (23). Such mice display a chronic inflammatory phenotype
that correlates with increased nuclear NF-
B1 p50 homodimers and
involves altered function of T cells, B cells, and macrophages.
-induced degradation in
HeLa cells (21). Overexpressed IKK2 also promotes the proteolysis of
co-transfected p105 in COS cells (24). Together these data are
consistent with the hypothesis that the IKK complex directly
phosphorylates the PEST domain of p105 to promote its proteolysis after
TNF
stimulation. However, such in vitro and
overexpression experiments are prone to artifact, as exemplified by
recent genetic data showing that NIK is not actually required for
NF-
B activation by pro-inflammatory cytokines (25) contrary to
previous transfection data in cell lines (26). Indeed, the spacing of
the putative IKK target serine residues (underlined) in the p105 PEST
domain
(920DSDSVCDSGVETS)
identified in vitro (21) differs considerably from the
regulatory phosphorylation sites on I
B
(see above). Thus it is
possible that immunoprecipitated IKK complex does not directly
phosphorylate p105 in vitro, but this is mediated by an
associated downstream kinase.
and IL-1
to trigger
efficiently p105 degradation. Analysis of a panel of point mutants
demonstrated that serine 927 in the PEST domain of p105, which is part
of a motif related to the IKK phosphorylation site on I
B
, is
directly phosphorylated by the IKK complex to regulate its
signal-induced degradation. Significantly, this residue does not
correspond to any of the three serine residues previously proposed to
be important for TNF
-induced degradation of p105 (21). A simple
explanation for this apparent discrepancy is the incorrect designation
of this residue as a threonine when the cDNA for p105 was initially
cloned and sequenced (27, 28). This was confirmed during the
preparation of this manuscript by Heissmeyer et al. (29),
who also concluded that phosphorylation of serine 927 by the IKK
complex regulates signal-induced p105 degradation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
antibody was
kindly provided by Ron Hay (University of St. Andrews, Scotland, UK)
and anti-GST mAb by Steve Dillworth (Royal Postgraduate Medical
School, London, UK). 12CA5 mAb was used for immunoprecipitation of
HA-p105, whereas a high affinity anti-HA mAb (Roche Molecular
Biochemicals) was used for its detection in Western blots. Anti-NEMO
(IKK
) and anti-phospho-Ser32 I
B
antibodies were
purchased from Santa Cruz Biotechnology.
(20 ng/ml; Amersham Pharmacia Biotech) or
IL-1
(4 ng/ml; R & D Systems). Cells were lysed in buffer A (31) and
endogenous p105 immunoprecipitated as described previously (22) using a
1:1 mixture of anti-hp105-C and anti-mp105-C
(10 µl of antiserum total per immunoprecipitate).
(4 ng/ml), TNF
(20 ng/ml), or control medium. Endogenous p105 was then immunoprecipitated
using anti-hp105-C antiserum, following extraction in
buffer A, and Western-blotted with anti-phospho-Ser927
antiserum. Bovine serum albumin (Sigma) was used as a blocking agent
for blots.
-stimulated HeLa cells (7 × 105) were lysed in Buffer A and immunoprecipitated using
anti-NEMO antibody. Immunoprecipitates were washed four times in Buffer A and once in kinase buffer (25 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol,
0.1 mM sodium vanadate, 10 mM
MgCl2). Immunoprecipitates were then incubated at room
temperature in 50 µl of kinase buffer plus 40 µM ATP
and 5 µg of purified GST-p105-(758-967) or
GST-p105-(758-967)-(S928A) fusion protein. The reaction was stopped by
addition of 50 µl of 2× Laemmli sample buffer and proteins Western
blotted with anti-phospho-Ser927 antiserum.
80 °C. Protein was >90% pure as judged by
Coomassie Blue staining of SDS-PAGE gels (data not shown). For in
vitro kinase assays with baculovirus IKK-1/2, 25 ng of recombinant
IKK protein was used per reaction which were carried out as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IL-1
increased the proteolysis of p105 to a similar degree to their wild
type counterparts (Fig. 1, A
and B). Control experiments also demonstrated that neither
IKK1 nor IKK2 was absolutely essential for TNF
and IL-1
to
trigger degradation of I
B
, although the extent of I
B
degradation was markedly decreased in cells lacking IKK2 (Fig. 1,
A and B). In contrast, cytokine-induced
degradation of p105 was significantly reduced, and I
B
was
completely blocked, in MEF cells lacking NEMO expression ((34) Fig.
1C). Control experiments confirmed that both TNF
and
IL-1
stimulation of NEMO-negative cells promoted phosphorylation of
p38 mitogen-activated protein kinase, indicating that the TNF
and
IL-1 signaling pathways were still intact in these cells (data not
shown). Since NEMO is essential for IKK activation by pro-inflammatory
cytokines (13, 34, 35), these data demonstrated that a functional IKK
complex is required for both TNF
and IL-1
to trigger efficiently
p105 proteolysis. IKK1 and IKK2, however, are redundant in this
signaling pathway.
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Fig. 1.
The IKK complex is essential for
TNF and IL-1
to
trigger efficiently p105 proteolysis. Mouse embryonic fibroblasts
lacking IKK1 (32) (A), IKK2 (33) (B), or NEMO
(34) (C) and their matched +/+ controls were metabolically
pulse-labeled with
[35S]methionine/[35S]cysteine (45 min) and
then chased for times indicated in complete medium plus TNF
,
IL-1
, or with no addition (lanes C).
Anti-p105C immunoprecipitates (anti-p105C Ip) were resolved
by 10% SDS-PAGE and revealed by fluorography (upper
panels). In parallel experiments, knockout and control fibroblasts
were stimulated for the indicated times with TNF
, IL-1
, or
control medium, and lysates were Western-blotted for I
B
and the
relevant IKK subunits (lower panels).
B
--
The experiments with IKK knockout
cell lines suggested that p105 might be a direct target of IKK.
Interestingly, a data base search revealed a conserved motif in p105
that is similar to the IKK complex phosphorylation site in I
B
.
Fig. 2A compares an alignment
of residues 918-935 from the PEST domain of human p105 with p105
sequences from other species. This region contains two overlapping
sequences related to the IKK target sequence in I
that are
identical in the p105 sequences from human, mouse, and rat. In chicken
p105, however, only the second of these overlapping motifs are
identical, and the first differed at two positions from the human p105
sequence (Fig. 2A). Interestingly, the regulatory IKK
phosphorylation site at the N terminus of I
B
is identical between
human, mouse, rat, and chicken sequences (36).
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Fig. 2.
Identification of a putative IKK
phosphorylation site in the PEST domain of p105. A,
alignment of a conserved sequence in the PEST domains of human, mouse,
rat, and chicken p105 with the IKK target sequence at the N terminus of
I B
and I
B
. B, schematic representation of
mammalian NF-
B1 p105. The relative positions of the RHD, nuclear
localizing signal (NLS), glycine-rich region
(GRR), ankyrin repeats (ANK); death domain
(DD (59)) and PEST domain are shown. C,
serine/threonine to alanine point mutants of HA-p105. WT,
wild type.
B
regulatory sequence
(DnSGLDSn+5) and contains conservative
substitutions at residues n + 3 and n + 4 but in
which the second serine at n + 5 is replaced with a
threonine (Fig. 2, A and B).
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Fig. 3.
Serine 927 is essential for HA-p105
proteolysis triggered by IKK2 overexpression. A and
B, 3T3 cells were transfected with 0.5 µg each of
expression vectors encoding IKK2 or no insert (EV) and the
indicated HA-p105 constructs. After 24 h, cells were metabolically
pulse-labeled with
[35S]methionine/[35S]cysteine (30 min) and
then chased for the times indicated. Anti-HA immunoprecipitates
(Anti-HA Ip) were resolved by 7.5% SDS-PAGE and revealed by
fluorography. A, the position of the HA-p105 band is
indicated for wild type (p105 WT) plus EV or IKK2 and for
HA-p105(S927A) plus IKK2. The stability of HA-p105(S927A) when
co-expressed with control EV was very similar to HA-p105 WT (data not
shown). B, amounts of immunoprecipitated HA-p105 from the
pulse-chase experiments in A and other similar experiments
were quantified by laser densitometry for the indicated HA-p105
constructs.
and IL-1 Cannot Trigger Proteolysis of an S927A Mutant of
p105--
It was important to demonstrate that serine 927 is important
for proteolysis of p105 induced by physiological stimulation with
cytokines, in addition to its essential role in proteolysis triggered
by IKK2 overexpression. To this end, HeLa cells were transfected with
plasmids encoding HA-p105 or HA-p105(S927A) and stable clones isolated
after G418 selection. A pulse-chase experiment was carried out with a
representative clone expressing levels of HA-p105(S927A) similar to
that of a control clone expressing HA-p105 (data not shown). Both
TNF
and IL-1
increased the rate of turnover of HA-p105 (Fig.
4A). In contrast, neither
cytokine affected the turnover of HA-p105(S927A), although both induced a p105 mobility shift in SDS-PAGE which was probably due to
phosphorylation on amino acids other than serine 927 (20, 22). The
turnover of stably transfected HA-p105 in unstimulated cells was
consistently found to be much greater than endogenous p105 in parental
HeLa cells (data not shown), as found previously by Heissmeyer et
al. (21). However, this basal turnover was also blocked in the
HA-p105(S927A) mutant (Fig. 4A). Control experiments
demonstrated that I
B
was degraded following stimulation with
either TNF
or IL-1
in the HA-p105(S927A) clone, similar to the
HA-p105 wild type clone (Fig. 4B). The TNF
and IL-1
signaling pathways controlling the IKK complex were, therefore, intact
in the HA-p105(S927A) clone. Similar results were obtained with an
independently derived HA-p105(S927A) clone (data not shown). Thus
introduction of an S927A mutation into p105 completely blocks its
proteolysis induced by two different pro-inflammatory cytokines,
confirming the importance of serine 927 in regulating signal-induced
p105 degradation.
View larger version (35K):
[in a new window]
Fig. 4.
Serine 927 is essential for
TNF or IL-1
to
trigger proteolysis of p105. A, clones of HeLa cells
stably transfected with HA-p105 or HA-p105(S927A) were metabolically
pulse-labeled with
[35S]methionine/[35S]cysteine (30 min) and
then chased for times indicated in complete medium (control)
or complete medium supplemented with TNF
or IL-1
. Anti-HA
immunoprecipitates were resolved by 7.5% SDS-PAGE and revealed by
fluorography. The positions of HA-p105 and background bands are
indicated. Western blotting of lysates demonstrated that HA-p105 and
HA-p105(S927A) were expressed at similar levels in the two cell lines
(data not shown). B, the stably transfected HeLa clones used
in A were stimulated for the indicated times with TNF
,
IL-1
, or left unstimulated. Lysates were Western-blotted for
I
B
levels.
and IL-1 Induce Rapid Phosphorylation of p105 on Serine
927--
The previous experiments indicated an important role for
serine 927 of p105 in its signal-induced proteolysis. However, it was
important to obtain direct evidence that serine 927 was phosphorylated in vivo, as it was possible that the S927A mutation might
mediate its inhibitory effects on p105 proteolysis via a structural
alteration, rather than by preventing serine 927 phosphorylation. To
investigate this possibility, an antibody was raised against a
synthetic peptide corresponding to residues 921-933 of human p105 in
which serine 927 was phosphorylated (Fig. 2, A and
B).
View larger version (43K):
[in a new window]
Fig. 5.
p105 is rapidly phosphorylated on serine 927 after stimulation of HeLa cells with TNF or
IL-1
. A, 3T3 cells were transiently
transfected with expression vectors encoding wild type (WT)
HA-p105 or the indicated mutants and IKK2 (+) or no insert (
) and
treated with MG132 for the last 4 h of an 18-h culture. HA-p105
was immunoprecipitated (Ip), and isolated protein was
Western-blotted with anti-phospho-Ser927 antibody
(upper panel). The blot was then stripped and reprobed with
anti-hp105C (middle panel). In the lower
panel, cell lysates were Western-blotted for transfected FL-IKK2.
B, HeLa cells were preincubated for 30 min with MG132 and
then stimulated for the indicated times with TNF
, IL-1
, or
control medium. Immunoprecipitates of endogenous p105 were then
Western-blotted and probed with the indicated antibodies. Lysates were
re-immunoprecipitated with anti-I
B
antiserum and Western blots
sequentially probed with an antibody specific for phosphoserine 32 of
I
B
(upper panel) and then with anti-I
B
antibody
(lower panel).
or IL-1
(Fig.
5B). p105 was isolated from cell lysates by
immunoprecipitation and then immunoblotted with the
anti-phospho-Ser927 antibody. TNF
stimulation induced
rapid serine 927 phosphorylation that reached a maximum at 15 min and
then gradually declined. Stimulation with IL-1
also induced
phosphorylation of p105 on Ser927 but with delayed kinetics
(peaking at 30-60 min) and reduced amplitude compared with TNF
.
Immunoblotting with anti-hp105C antibody confirmed that
equal amounts of p105 were present in all of the immunoprecipitates.
Lysates were re-immunoprecipitated with an anti-I
B
antibody, and
isolated protein was immunoblotted with an antibody specific for
phospho-Ser32 of I
B
. The kinetics of I
B
Ser32 phosphorylation (Fig. 5B, lower panels)
were very similar to those of p105 phosphorylation on
Ser927, and the TNF
signal was greater than that
detected with IL-1
, as also found with p105 Ser927 phosphorylation.
B
in which the regulatory phosphorylation
sites (Ser32/Ser36) are mutated to threonine is
phosphorylated and degraded at significantly reduced levels in
stimulated cells compared with the wild type protein (11, 30, 37). The
experiments in the previous sections indicated that IKK2 overexpression
triggered degradation of co-transfected p105 as a result of inducing
phosphorylation of p105 on serine 927 (Figs. 3 and 5A). To
initially investigate whether the IKK complex might directly
phosphorylate p105 on serine 927 to regulate its degradation, a mutant
was generated in which residue 927 was altered to threonine. Similar to
HA-p105(S927A), HA-p105(S927T) was not degraded when co-expressed with
IKK2 (Fig. 6A). In contrast, IKK2 co-expression induced the rapid degradation of HA-p105 wild type,
as expected. These data indicate that the kinase that phosphorylates p105 at residue 927 is serine-specific, consistent with the hypothesis that it might correspond to the IKK complex.
View larger version (29K):
[in a new window]
Fig. 6.
The IKK complex directly phosphorylates
serine 927 of p105 in vitro. A, 3T3
cells were transfected with expression vectors (0.5 µg each) encoding
IKK2 or no insert (EV) and either HA-p105, HA-p105(S927A),
or HA-p105(S927T). After 24 h, cells were metabolically
pulse-labeled with
[35S]methionine/[35S]cysteine (30 min) and
then chased for the times indicated. Anti-HA immunoprecipitates were
resolved by 7.5% SDS-PAGE and revealed by fluorography. p105 was
quantified by laser densitometry. B, HeLa cells were
stimulated for 15 min with TNF or left unstimulated. The endogenous
IKK complex was isolated from cell lysates by immunoprecipitation with
an anti-NEMO antibody. Control immunoprecipitates were carried out with
non-immune rabbit serum (NRS). Immunoprecipitates were
tested for their ability to phosphorylate in vitro
GST-p105-(758-967) or GST-p105-(758-967)(S927A) fusion proteins (as
indicated). Phosphorylation was determined by Western blotting kinase
reaction mixtures with anti-phospho-Ser927 antibody
(upper panel). Blots were then stripped and reprobed
sequentially with anti-NEMO antibody (middle panel) and
anti-GST mAb (lower panel). C, in
vitro kinase assays were carried out with baculovirus-produced
purified His6-IKK1 or His6-IKK2 (25 ng), using
as substrates either GST-p105-(758-967) or GST-p105-(758-967)(S927A)
fusion protein. Phosphorylation was assessed by Western blotting kinase
reactions with anti-phospho-Ser927 antibody (upper
panel). Equal loading of the fusion proteins was confirmed by
reprobing blots with anti-GST mAb (lower panel).
for 15 min, or left unstimulated, and the endogenous IKK complex was isolated by immunoprecipitation using an
anti-NEMO antibody. An in vitro kinase assay was then
carried out using purified GST-p105-(758-967) protein as a substrate, and serine 927 phosphorylation was assessed by Western blotting using
the anti-phospho-Ser927 antibody. Immunopurified IKK
complex phosphorylated the GST fusion protein on serine 927, and this
was dramatically increased after TNF
stimulation (Fig.
6B), which activates the IKK complex (15). As expected, no
signal was detected when GST-p105-(758-967)(S927A) was used as a
substrate for anti-NEMO immunoprecipitates from TNF
-stimulated cells
(lane 5). In addition, control immunoprecipitates with
non-immune serum (lanes 1 and 2) did not isolate
any p105 Ser927 kinase activity either from control or
TNF
-stimulated cells. These data indicate that the endogenous IKK
complex isolated from HeLa cells directly phosphorylates serine 927 of p105.
and IL-1
to induce p105 degradation in IKK1
/
and IKK2
/
MEFs (see Fig. 1). The data in this section, therefore,
show that p105 serine 927 is a direct target of the IKK complex.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B1 p105 (12). These data strengthen the notion that the IKK complex plays a central role in the regulation of NF-
B activation by inducing degradation of the major I
B
proteins (I
B
, I
B
, and p105) in mammals (15, 38).
and IL-1 involves distinct
upstream signaling intermediates (15, 38-41). However, both of these
cytokines induce the IKK complex to phosphorylate serine 927 of
NF-
B1 p105 to trigger its degradation. Similar to I
B
(1, 15),
therefore, the signal-induced degradation of p105 by different stimuli
involves a common phosphorylation-based mechanism, and the IKK complex
integrates the signals from these two different signaling pathways to
trigger p105 degradation. The signal-induced proteolysis of p105,
therefore, is controlled at the level of IKK complex activation and
perhaps also recruitment of the IKK complex to p105 (29).
Interestingly, basal turnover of p105 is also blocked by mutation of
serine 927 (Fig. 4A) and, therefore, may also be regulated
by the IKK complex.
B1 p105 is stably associated at high stoichiometry with the
mitogen-activated protein 3-kinase, TPL-2/Cot (22). Overexpression of
TPL-2/Cot induces phosphorylation and degradation of co-expressed p105
in 3T3 cells. TPL-2/Cot, however, cannot directly phosphorylate p105 on
serine 927 in vitro but does induce serine 927 phosphorylation when co-expressed with HA-p105 in 3T3 cells.2 Since TPL-2/Cot
overexpression has been shown to activate the IKK complex (42), it is
likely that TPL-2/Cot regulates serine 927 phosphorylation and
subsequent degradation of p105 via the IKK complex. This possibility is
currently being investigated.
stimulation when expressed in HeLa cells as a C-terminal p105 fragment.
Surprisingly, in the present study, it was found that individual
mutation of each of these residues to alanine did not block
IKK2-triggered proteolysis of HA-p105 in co-transfected 3T3 cells,
whereas an S927A p105 mutation completely prevented HA-p105 proteolysis
promoted by IKK2 co-expression. A serine-to-threonine mutation at
residue 927 was also found to block IKK2-mediated proteolysis of
HA-p105 (Fig. 6A), consistent with known preference of the
IKK complex for serine residues over threonine (11, 30, 37, 43). The most likely explanation for the discrepancy between the results of the
present study and those of Heissmeyer et al. (21) is that a 927 serine to threonine mutation was inadvertently introduced into the triple mutant of p105 during PCR, which was presumably performed on the assumption that residue 927 was a threonine. This
hypothesis was confirmed in a more recent paper (29) from the same
laboratory published while this article was in preparation.
B
(Asp-Ser32-Gly-Leu-Asp-Ser36) (8, 9, 11, 30,
37), except for the substitution of a serine for threonine at the
residue 931 of p105, which corresponds to serine 36 of I
B
.
Previous experiments demonstrated that mutation of serine 36 to
threonine only slightly reduces I
B
degradation in response to
TNF
stimulation (11). However, mutation of serine 32 of I
B
to
threonine has a more dramatic inhibitory effect. In vitro
experiments have also established that the IKK complex can
phosphorylate serine 32 in an I
fusion protein containing an
Ser36 to Thr mutation (12). Together, these data are
consistent with the findings in the present study showing a crucial
role for serine 927 of the Asp-Ser927-Gly-Val-Glu-Thr motif
in the regulation of p105 proteolysis by IKK2 overexpression and its
direct phosphorylation by the IKK complex in vitro. Western
blot analyses indicate that p105 is only partially degraded after
TNF
stimulation of HeLa cells, whereas degradation of I
B
is
complete (data not shown). The presence of a threonine, rather than a
serine, at residue 931 of p105 may contribute to the inefficient
proteolysis of p105 triggered by cytokine stimulation.
TrCP, a component subunit of an SCF-type
ubiquitin-protein isopeptide ligase, mediates the ubiquitination
of phosphorylated p105 (24), similar to its established role in
I
B
degradation (44-46). Deletion of p105 residues 917-933
prevents both the interaction of
TrCP with phospho-p105 and the
ubiquitination of phospho-p105 in vitro (24). Furthermore, a
quadruple mutant of p105, encoding serine to alanine mutations at
residues 921, 923, 927, and 933, is no longer ubiquitinated when
co-transfected with IKK2 and
TrCP (29). It will be important in
future studies to determine whether serine 927 phosphorylation is
required for
TrCP-mediated p105 ubiquitination and in particular
whether phospho-Ser927 is the binding site for
TrCP on p105.
B/Rel family
members: Dorsal, Dorsal-related immunity factor, and Relish (47). Dorsal-related immunity factor and Dorsal are retained in the cytoplasm
of unchallenged flies by binding to the I
B
homolog Cactus (48).
Cactus is degraded in response to an immune stimulus via a Toll
signaling pathway, releasing associated Dorsal-related immunity factor
and Dorsal to activate the expression of anti-fungal peptide genes
(49-51). Relish contains an N-terminal RHD and an I
B-like
ankyrin-repeat in its C terminus similar to mammalian p105 (52). During
anti-bacterial immune responses, Relish is cleaved by DREDD caspase to
release an N-terminal fragment that translocates into the nucleus to
promote expression of anti-bacterial peptides (53).
Drosophila IKK2 (DmIKK
) and NEMO (DmIKK
/Kenny) are
essential for this signaling pathway but not for signal-mediated degradation of Cactus during anti-fungal responses (54-56). Since Cactus degradation is triggered by phosphorylation at its N-terminal regulatory domain followed by ubiquitination similar to I
B
(46), a separate IKK complex may be involved in regulating Cactus proteolysis (54).
Bs in Drosophila. This contrasts with the
situation in mammals where the IKK complex is now known to regulate
directly the degradation of three I
B proteins: I
B
, I
B
(12), and NF-
B1 p105 (this paper). I
B
may also be directly
phosphorylated by the IKK complex (57, 58). However, mammalian NF-
B2
p100 is not phosphorylated by the IKK complex in vitro (21),
and recent data have indicated that signal-induced proteolysis of this
protein is regulated by a distinct kinase (60).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Shizuo Akira, Ron Hay, Alain Israel, Michael Karin, and Tak Mak for reagents used in this study. We are also grateful to Lee Johnston (National Institute for Medical Research) for critical reading of the manuscript; Glenn Cowley (Abbott) for sequencing p105 cDNAs from human peripheral blood mononuclear cells; Richard Marais (Institute of Cancer Research, London, UK) for advice on generating anti-phosphopeptide antisera; Pete Fletcher (National Institute for Medical Research) for peptide synthesis; and the Photo-Graphics department (National Institute for Medical Research) for help with the figures.
![]() |
FOOTNOTES |
---|
* This work was supported by the Medical Research Council, UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Current address: Abbott Bioresearch Center, 100 Research Dr., Worcester, MA 01605-4314.
** To whom correspondence should be addressed: Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Tel.: 44-20-8913-8589; Fax: 44-20-8906-4477; E-mail: sley@nimr.mrc.ac.uk.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101754200
2 A. Salmerón, J. Janzen, H. Allen, and Steven C. Ley, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RHD, Rel homology
domain;
IL, interleukin;
IKK, IB kinase;
MEF, mouse embryonic
fibroblast;
TNF, tumor necrosis factor;
PCR, polymerase chain reaction;
HA, hemagglutinin;
mAb, monoclonal antibody;
GST, glutathione
S-transferase;
PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve] |
2. | Karin, M., and Delhase, M. (2000) Semin. Immunol. 12, 85-98[CrossRef][Medline] [Order article via Infotrieve] |
3. | Siebenlist, U., Franzoso, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, 405-455[CrossRef] |
4. | Rice, N. R., MacKichan, M. L., and Israel, A. (1992) Cell 71, 243-253[Medline] [Order article via Infotrieve] |
5. | Mercurio, F., DiDonato, J. A., Rosette, C., and Karin, M. (1993) Genes Dev. 7, 705-718[Abstract] |
6. | Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785[Medline] [Order article via Infotrieve] |
7. | Betts, J. C., and Nabel, G. J. (1996) Mol. Cell. Biol. 16, 6363-6371[Abstract] |
8. | Brockman, J. A., Scherer, D. C., McKinsey, T. A., Hall, S. M., Qi, X., Lee, W. Y., and Ballard, D. W. (1995) Mol. Cell. Biol. 15, 2809-2818[Abstract] |
9. | Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267, 1485-1488[Medline] [Order article via Infotrieve] |
10. | Traenckner, E. B.-M., Pahl, H. L., Henkel, T., Schmidt, K. N., Wilk, S., and Baeuerle, P. A. (1995) EMBO J. 14, 2876-2883[Abstract] |
11. | DiDonato, J., Mercurio, F., Rosette, C., Wu-Li, J., Suyang, H., Ghosh, S., and Karin, M. (1996) Mol. Cell. Biol. 16, 1295-1304[Abstract] |
12. |
Zandi, E.,
Chen, Y.,
and Karin, M.
(1998)
Science
281,
1360-1363 |
13. | Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S. T., Weil, R., Agou, F., Kirk, H. E., Kay, R. J., and Israel, A. (1998) Cell 93, 1231-1240[Medline] [Order article via Infotrieve] |
14. |
Maniatis, T.
(1999)
Genes Dev.
13,
505-510 |
15. | Karin, M., and Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18, 621-663[CrossRef][Medline] [Order article via Infotrieve] |
16. | Lin, L., and Ghosh, S. (1996) Mol. Cell. Biol. 16, 2248-2254[Abstract] |
17. |
Orian, A.,
Schwartz, A. L.,
Israel, A.,
Whiteside, S.,
Kahana, C.,
and Ciechanover, A.
(1999)
Mol. Cell. Biol.
19,
3664-3673 |
18. | Lin, L., DeMartino, G. N., and Greene, W. C. (1998) Cell 92, 819-828[Medline] [Order article via Infotrieve] |
19. | Mellits, K. H., Hay, R. T., and Goodbourn, S. (1993) Nucleic Acids Res. 21, 5059-5066[Abstract] |
20. |
MacKichan, M. L.,
Logeat, F.,
and Israel, A.
(1996)
J. Biol. Chem.
271,
6084-6091 |
21. |
Heissmeyer, V.,
Krappmann, D.,
Wulczyn, F. G.,
and Scheidereit, C.
(1999)
EMBO J.
18,
4766-4788 |
22. | Belich, M. P., Salmeron, A., Johnston, L. H., and Ley, S. C. (1999) Nature 397, 363-368[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Ishikawa, H.,
Claudio, E.,
Dambach, D.,
Raventos-Suarez, C.,
Ryan, C.,
and Bravo, R.
(1998)
J. Exp. Med.
187,
985-996 |
24. |
Orian, A.,
Gonen, H.,
Bercovich, B.,
Fajerman, I.,
Eytan, E.,
Israel, A.,
Mercurio, F.,
Iwai, K.,
Schwartz, A. L.,
and Ciechanover, A.
(2000)
EMBO J.
19,
2580-2591 |
25. | Shinkura, R., Kitada, K., Matsuda, F., Tashiro, K., Ikuta, K., Suzuki, M., Kogishi, K., Serikawa, T., and Honjo, T. (1999) Nat. Genet. 22, 74-77[CrossRef][Medline] [Order article via Infotrieve] |
26. | Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544[CrossRef][Medline] [Order article via Infotrieve] |
27. | Ghosh, S., Gifford, A. M., Riviere, L. R., Tempst, P., Nolan, G. P., and Baltimore, D. (1990) Cell 62, 1019-1029[Medline] [Order article via Infotrieve] |
28. | Kieran, M., Blank, V., Logeat, F., Vandekerckhove, J., Lottspeich, F., Le Bail, O., Urban, M. B., Kourilsky, P., Baeuerle, P. A., and Israel, A. (1990) Cell 62, 1007-1018[Medline] [Order article via Infotrieve] |
29. |
Heissmeyer, V.,
Krappmann, D.,
Hatada, E. N.,
and Scheidereit, C.
(2001)
Mol. Cell. Biol.
21,
1024-1035 |
30. |
Mercurio, F.,
Zhu, H.,
Murray, B. W.,
Shevchenko, A.,
Bennett, B. L.,
Li, J. W.,
Young, D. B.,
Barbosa, M.,
Mann, M.,
Manning, A.,
and Rao, A.
(1997)
Science
278,
860-866 |
31. | Salmeron, A., Ahmad, T. B., Carlile, G. W., Pappin, D., Narsimhan, R. P., and Ley, S. C. (1996) EMBO J. 15, 817-826[Abstract] |
32. |
Takeda, K.,
Takeuchi, O.,
Tsujimura, T.,
Itami, S.,
Adachi, O.,
Kawai, T.,
Sanjo, H.,
Yoshikawa, K.,
Terada, N.,
and Akira, S.
(1999)
Science
284,
313-316 |
33. |
Li, Q.,
Antwerp, D. V.,
Mercurio, F.,
Lee, K.-F.,
and Verma, I. M.
(1999)
Science
284,
321-325 |
34. |
Rudolph, D.,
Yeh, W.-C.,
Wakeham, A.,
Rudolph, B.,
Nallainathan, D.,
Potter, J.,
Elia, A. J.,
and Mak, T. W.
(2000)
Genes Dev.
14,
854-862 |
35. | Makris, C., Godfrey, V. L., Krahn-Senftleben, G., Takahashi, T., Roberts, J. L., Schwarz, T., Feng, L., Johnson, R. S., and Karin, M. (2000) Mol. Cell 5, 969-979[Medline] [Order article via Infotrieve] |
36. | Whiteside, S. T., Ernst, M. K., Lebail, O., Laurent-Winter, C., and Rice, N. (1995) Mol. Cell. Biol. 15, 5339-5345[Abstract] |
37. | DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve] |
38. | Israel, A. (2000) Trends Cell Biol. 10, 129-133[CrossRef][Medline] [Order article via Infotrieve] |
39. | Kopp, E. B., and Medzhitov, R. (1999) Curr. Opin. Immunol. 11, 13-18[CrossRef][Medline] [Order article via Infotrieve] |
40. | Aderem, A., and Ulevitch, R. J. (2000) Nature 406, 782-787[CrossRef][Medline] [Order article via Infotrieve] |
41. | O'Neill, L. A. J., and Dinarello, C. A. (2000) Immunol. Today 21, 206-209[CrossRef][Medline] [Order article via Infotrieve] |
42. | Lin, X., Cunningham, E. T., Mu, Y., Geleziunas, R., and Greene, W. C. (1999) Immunity 10, 271-280[Medline] [Order article via Infotrieve] |
43. |
D'Adamio, L.,
Clayton, L. K.,
Awad, K. M.,
and Reinherz, E. L.
(1992)
J. Immunol.
149,
3550-3553 |
44. |
Winston, J. T.,
Strack, P.,
Beer-Romero, P.,
Chu, C. Y.,
Elledge, S. J.,
and Harper, J. W.
(199)
Genes Dev.
13,
270-283 |
45. | Yaron, A., Hatzubai, A., Davis, M., Lavon, I., Amit, S., Manning, A. M., Anderson, J. S., Mann, M., Mercurio, F., and Ben-Neriah, Y. (1998) Nature 396, 590-594[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Spencer, E.,
Jiang, J.,
and Chen, Z. J.
(1999)
Genes Dev.
13,
284-294 |
47. |
Hoffmann, J. A.,
Kafatos, F. C.,
Janeway, C. A.,
and Ezekowitz, R. A.
(1999)
Science
284,
1313-1318 |
48. | Belvin, M. P., and Anderson, K. V. (1996) Annu. Rev. Cell Dev. Biol. 12, 393-416[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Manfruelli, P.,
Reichhart, J. M.,
Steward, R.,
Hoffmann, J. A.,
and Lemaitre, B.
(1999)
EMBO J.
18,
3380-3391 |
50. |
Meng, X.,
Khanuja, B. S.,
and Ip, Y. T.
(1999)
Genes Dev.
13,
792-797 |
51. | Rutschmann, S., Jung, A. C., Hetru, C., Reichhart, J. M., Hoffmann, J. A., and Ferrandon, D. (2000) Immunity 12, 569-580[Medline] [Order article via Infotrieve] |
52. |
Dushay, M. S.,
Asling, B.,
and Hultmark, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10343-10347 |
53. |
Stoven, S.,
Ando, I.,
Kadalayil, L.,
Engstrom, Y.,
and Hultmark, D.
(2000)
EMBO Rep.
1,
347-352 |
54. |
Silverman, N.,
Zhou, R.,
Stoven, S.,
Pandey, N.,
Hultmark, D.,
and Maniatis, T.
(2000)
Genes Dev.
14,
2461-2471 |
55. | Rutschmann, S., Jung, A. C., Zhou, R., Silverman, N., Hoffmann, J. A., and Ferrandon, D. (2000) Nat. Immunol. 1, 342-347[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Lu, Y.,
Wu, L. P.,
and Anderson, K. V.
(2001)
Genes Dev.
15,
104-110 |
57. |
Nakano, H.,
Shindo, M.,
Sakon, S.,
Nishinaka, S.,
Mihara, M.,
Yagita, H.,
and Okumura, K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3537-3542 |
58. |
Shirane, M.,
Hatakeyama, S.,
Hattori, K.,
Nakayama, K.,
and Nakayame, K.
(1999)
J. Biol. Chem.
274,
28169-28174 |
59. | Wallach, D., Boldin, M., and Varfolomeev, E. (1995) Trends Biochem. Sci. 20, 342-344[CrossRef][Medline] [Order article via Infotrieve] |
60. | Xiao, G., Harhaj, E. W., and Sun, S.-C. (2001) Mol. Cell. 7, 401-409[Medline] [Order article via Infotrieve] |