From the Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8594
Received for publication, September 12, 2000, and in revised form, October 20, 2000
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ABSTRACT |
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IKK The NF- Both IKK The mitogen-activated protein kinase kinase family members NIK (36, 37)
and MEKK1 (27, 31, 32, 35) can stimulate IKK activity via
phosphorylation of serine residues in their activation loop. Mutation
of serine residues to alanine in the activation loop at positions 176 and 180 in IKK In addition to IKK Mutagenesis of the IKK Recent murine gene disruption studies (48-50) and genetic analysis of
families lacking IKK In the current study, we utilized a biochemical approach to address the
function of IKK DNA Constructs--
The murine IKK Transfection and Cellular Fractionation--
COS cells grown in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum were
transfected using Fugene-6 (Roche Molecular Biochemicals) as described
by the manufacturer. For a typical cell fractionation experiment, cells
cultured overnight in 100-mm plates were transfected with 0.6 µg of
each DNA construct. Cells from five transfected plates were pooled, and
S100 extracts were prepared as detailed (53) except that the extracts
were directly loaded onto a Superdex 200 column without dialysis. The
S100 extract containing a total of 2 mg of protein was chromatographed
through a Superdex-200 column (Amersham Pharmacia Biotech) in buffer D (20 mM HEPES (pH 7.9), 0.1 M KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 20% glycerol, and 0.2 mM EDTA) (54), and
fractions of 1 ml each were collected.
Western Blotting and Immunoprecipitation/Kinase
Assays--
Western blotting was done with 30 µg of protein obtained
from each of the column fractions as previously described (53). The
antibodies used in this analysis are specified in the figure legends.
For kinase assays, 50 µl of each column fraction was incubated
overnight at 4 °C with 1-2 µg of the indicated antibodies in 150 µl of PD buffer (40 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1% Nonidet P-40. 6 mM EDTA, 6 mM EGTA, 10 mM In Vivo Phosphorylation Assay--
COS cells (1.3 × 105) were transfected with either 0.5 µg of IKK IKK
In an attempt to assay the effects of IKK
In contrast to these results, coexpression of IKK Assembly of the IKK Complex by IKK
Similar experiments were performed to determine whether IKK
Next we addressed the specificity of IKK IKK
The chromatographic distribution of the kinase defective IKK The Amino-terminal Domain of IKK IKK
To confirm the specificity of IKK Domains in IKK
Finally, we addressed whether the ability of IKK IKK The results presented here using transient expression assays and column
chromatography indicate that the expression of IKK We also determined whether IKK The role of IKK We were able to demonstrate that IKK A model to address IKK/NEMO is a protein that is critical for the
assembly of the high molecular weight I
B kinase (IKK) complex. To
investigate the role of IKK
/NEMO in the assembly of the IKK complex,
we conducted a series of experiments in which the chromatographic
distribution of extracts prepared from cells transiently expressing
epitope-tagged IKK
/NEMO and the IKKs were examined. When expressed
alone following transfection, IKK
and IKK
were present in low
molecular weight complexes migrating between 200 and 400 kDa. However,
when coexpressed with IKK
/NEMO, both IKK
and IKK
migrated at
~600 kDa which was similar to the previously described IKK complex
that is activated by cytokines such as tumor necrosis factor-
. When
either IKK
or IKK
was expressed alone with IKK
/NEMO, IKK
but not IKK
migrated in the higher molecular weight IKK complex.
Constitutively active or inactive forms of IKK
were both
incorporated into the high molecular weight IKK complex in the presence
of IKK
/NEMO. The amino-terminal region of IKK
/NEMO, which
interacts directly with IKK
, was required for formation of the high
molecular weight IKK complex and for stimulation of IKK
kinase
activity. These results suggest that recruitment of the IKKs into a
high molecular complex by IKK
/NEMO is a crucial step involved in IKK function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B proteins are a family of transcription factors that
regulate the expression of a variety of cellular genes involved in the
control of the immune and the inflammatory response (1-4). NF-
B is
sequestered in the cytoplasm of most cells where it is bound to a
family of inhibitory proteins known as I
B (2, 5, 6). A variety of
agents including the cytokines interleukin-1 and
TNF
,1 endotoxin,
double-stranded RNA, and the viral transactivator Tax activate the
NF-
B pathway (4, 7-11). These agents stimulate upstream kinases
that result in the activation of two related I
B kinases, IKK
and
IKK
(9, 12-16). IKK phosphorylation of amino-terminal serine
residues in both I
B
and I
B
results in their ubiquitination
via interaction with
-TrCP and subsequent degradation by the
proteasome (10, 17-26). Following I
B degradation, the NF-
B
proteins translocate from the cytoplasm to the nucleus where they
activate the expression of specific cellular genes (8).
and IKK
are components of a high molecular weight
complex migrating between 600 and 900 kDa that phosphorylates the I
B
proteins (12, 14, 23, 27-30). These kinases have 52% amino acid
identity and a similar domain structure that includes amino-terminal
kinase, leucine zipper, and helix-loop-helix motifs (9, 12-16). IKK
and IKK
can both homodimerize and heterodimerize, and this process
is critical for their kinase activity. Although these kinases have a
number of similarities, IKK
has at least a 20-fold higher level of
kinase activity for I
B than does IKK
(12, 29, 31-35).
and positions 177 and 181 in IKK
inactivates IKK
kinase activity, whereas replacement of these serine residues with
glutamates results in the generation of constitutively active kinases
(12, 29, 38, 39). Whether phosphorylation of IKK
by either NIK or
MEKK1 is the critical event that leads to stimulation of IKK
kinase
activity or whether other mechanisms such as IKK
phosphorylation of
IKK
(39, 58) or IKK
autophosphorylation (38) regulate this
process remains to be determined.
and IKK
, there are additional components of
the IKK complex. A protein known as IKK
/NEMO has also been shown to
be a critical component of the IKK complex (28-30). This 48-kDa
glutamine-rich protein contains a leucine zipper domain and two
coiled-coil motifs but has no known enzymatic activity. IKK
/NEMO was
first identified in a genetic complementation assay as a cellular
factor that was able to restore NF-
B activation to cells that did
not respond to a variety of activators of this pathway (28).
IKK
/NEMO was also isolated independently as a component of the high
molecular weight IKK complex (29, 30) and as a factor, designated
FIP-3, that binds to the adenovirus E3 protein and inhibits the
cytolytic effects of TNF
(40). Cells lacking IKK
/NEMO are unable
to form the high molecular weight IKK complex or respond to cytokines
that activate this pathway (28-30, 41, 42).
/NEMO indicates that several distinct domains
are critical for its function (29, 30, 43). These include the
amino-terminal 100 amino acids that mediate the direct interactions of
IKK
/NEMO with IKK
, the carboxyl terminus which likely functions
in the recruitment of upstream kinases to the IKK complex, and a
coiled-coil domain that mediates oligomerization of IKK
/NEMO.
Although IKK
preferentially binds to IKK
/NEMO as compared with
IKK
(28, 30), IKK
has also been found to bind to IKK
/NEMO
using extracts prepared from IKK
knock-out cells (44). The kinase
RIP which is recruited to the p55 TNF receptor following TNF
treatment binds to IKK
/NEMO (45). The recruitment of IKK
/NEMO by
RIP leads to the subsequent association of IKK
and IKK
. Another
protein, A20 which functions to inhibit NF-
B activation, also binds
to IKK
/NEMO and may serve to down-regulate TNF
signaling pathway
(45). Finally, the human T-cell lymphotrophic virus, type I, Tax
protein can bind to IKK
/NEMO to facilitate the activation of the
IKKs (43, 46, 47). These results indicate that IKK
/NEMO may serve to
link various activators of the NF-
B pathway to the IKK complex.
/NEMO (51) demonstrate its essential role in
regulating the anti-apoptotic and inflammatory properties of the
NF-
B pathway. Mutations in the
IKK
/NEMO gene on the X chromosome are
the cause of incontinentia pigmenti, an X-linked dominant genetic
disorder of the skin that is lethal in males (51). Gene disruption
studies of the IKK
/NEMO gene demonstrate that
although male mice die in utero, heterozygous female mice develop granulocytic infiltration and both hyperproliferation and
increased apoptosis of keratinocytes similar to that seen in
incontinentia pigmenti (48, 50). Homozygous deletion of IKK
/NEMO
leads to embryonic lethality due to massive hepatic apoptosis (48, 49).
Mouse embryo fibroblasts isolated from these mice exhibit
extreme defects in stimulating the NF-
B pathway in response to a
variety of well characterized activators of this pathway (48-50).
However, the mechanism by which IKK
/NEMO activates the NF-
B
pathway remains to be determined.
/NEMO in the recruitment of IKK
and IKK
into
the IKK complex. We demonstrated that the amino terminus of IKK
/NEMO
that interacts with IKK
is crucial for formation of the high
molecular weight IKK complex. Moreover, we found that IKK
/NEMO
stimulates the ability of IKK
but not IKK
to phosphorylate I
B
. These results further establish that IKK
/NEMO association with the IKK complex is critical for stimulation of IKK kinase activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/NEMO coding sequence (28)
(GenBankTM accession number AF069542-1) was obtained
by PCR using a mouse spleen cDNA library. Two oligonucleotide
primers complementary to the 5'- and 3'-coding regions of mouse
IKK
/NEMO were used in PCR assays. The PCR product was cloned into
the expression vector pCMV5/Myc1 fusing the Myc tag to the 5' of
IKK
/NEMO sequence. An amino-terminal deletion that contains amino
acid residues 101-412 of IKK
/NEMO was constructed by PCR followed
by cloning of the product into pCMV5/Myc1. The carboxyl-terminal
deletions of IKK
/NEMO containing either amino acids 1-270 or 1-312
were constructed utilizing PCR to generate these fragments followed by
cloning into pCMV5/Myc1. The leucine zipper mutations were constructed by substitution of leucine residues at positions 315, 322, and 329 with
methionine in the IKK
/NEMO gene cloned into
pCMV5/Myc1. Wild-type and mutant IKK
were cloned into plasmid pCMVFl
such that the FLAG tag was fused to the 5' end of IKK
(12,
35). IKK
was cloned into plasmid pRcBactHA and the HA tag was fused to the 5' end of IKK
(12, 35). The cDNA clone containing Myc-tagged CREB was previously described (52).
-glycerophosphate, 10 mM NaF, 300 µM
Na3VO4, and protease inhibitors (Roche
Molecular Biochemicals)) (12). Immune complexes were precipitated with protein A-agarose (Bio-Rad) for 1-3 h at 4 °C and analyzed by in vitro kinase assays in the presence of 5 µg of
bacterially expressed GST fusion protein consisting of I
B
(aa
1-54) or with serine residues 32 and 36 changed to alanine (53). After
incubation at 30 °C for 30 min, the reactions were mixed with
protein sample buffer (50 mM Tris (pH 8.0), 2% SDS, 0.1%
bromphenol blue, 10% glycerol, and
-mecaptoethanol), heated at
95 °C for 3 min, and loaded on a 12% SDS gel. The phosphoprotein
products were visualized by autoradiography.
alone
or in the presence of 0.5 µg of wild-type or mutant IKK
/NEMO
mutants. After 24 h post-transfection, cells were grown overnight
in 2 ml of Dulbecco's modified Eagle's medium lacking phosphate (Life
Technologies, Inc.) followed by incubating 3 h in 1 ml of
Dulbecco's modified Eagle's medium in the presence of 50 µCi of
[32P]orthophosphate (PerkinElmer Life Sciences). Cells
were then collected in 300 µl of PD buffer. The
32P-labeled IKK
protein was immunoprecipitated from 200 µl of cell extract using 4 µg of anti-FLAG monoclonal antibody
(Sigma, M2) or 4 µg of anti-HA monoclonal antibody (Roche Molecular
Biochemicals, 12CA5). The immunoprecipitates were washed with PD
buffer, and the phosphoproteins were resolved on a 10%
SDS-polyacrylamide gel and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/NEMO Recruits the IKKs into a High Molecular Weight
Complex--
It has been shown previously that cytokines such as
TNF
stimulate IKK activity present in a high molecular complex
migrating between 600 and 900 kDa on gel filtration columns (12, 14, 23, 29, 30). Examination of the chromatographic distribution of the
endogenous IKK
, IKK
, and IKK
/NEMO proteins isolated from
cytoplasmic fractions of COS cells indicated that these proteins were
present in a similarly sized high molecular weight complex in both
untreated and TNF
-treated cells (Fig.
1). In extracts prepared from
TNF
-treated cells, as compared with untreated cells, there was
increased kinase activity for the I
B
substrate in immunoprecipitates isolated using either IKK
or IKK
antibody. There was no phosphorylation of an I
B
mutant in which serine residues 32 and 36 were changed to alanine (data not shown). The level
of these proteins was not increased by TNF
treatment (Fig. 1).
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Fig. 1.
Chromatographic distribution of IKK in
untreated and TNF -treated cells. COS
cells were either untreated (untr) or treated with TNF
(20 ng/ml) for 10 min. Cytoplasmic (S100) extracts were prepared and
subjected to chromatography on a Superdex-200 column (Amersham
Pharmacia Biotech). The fractions derived from the indicated cells
(left of figures) were analyzed by Western blotting using
antibodies (Ab) directed against either IKK
(Santa Cruz
Biotechnology, sc-7606), IKK
(Santa Cruz Biotechnology, sc-7607), or
IKK
(Santa Cruz Biotechnology, sc-8330). In vitro kinase
activity was analyzed utilizing a GST/I
B
(aa 1-54) substrate
following immunoprecipitation of the column fractions with either
IKK
(sc-7606) or IKK
(sc-7607) polyclonal antibodies. Molecular
weight markers and column fraction numbers are indicated at the
top and bottom of the figure, respectively. The
positions of the IKKs and GST/I
B
substrate are indicated at the
right of these figures.
/NEMO on the assembly of
the IKK complex, COS cells were transfected with expression vectors
encoding epitope-tagged IKK
and IKK
in the presence or absence of
epitope-tagged IKK
/NEMO. Cytoplasmic extracts were subjected to gel
filtration, and the column fractions were analyzed by Western blot
analysis and by IKK kinase activity assay (Fig. 2). In extracts prepared from cells
transfected with IKK
and IKK
in the absence of IKK
/NEMO, the
majority of the epitope-tagged IKK
and IKK
proteins was detected
in fractions corresponding to a molecular mass of 200-400 kDa
(Fig. 2A). The majority of the respective IKK kinase
activity for the I
B
substrate was also detected in these same
column fractions (Fig. 2A). The overlapping, yet slightly
different chromatographic positions between IKK
and IKK
may
reflect heterodimeric and homodimeric pools of these kinases.
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Fig. 2.
Effect of IKK /NEMO
on the chromatographic distribution of cotransfected
IKK
and IKK
. COS
cells were transfected with CMV expression vectors containing cDNAs
encoding IKK
and IKK
(A), IKK
, IKK
, and
IKK
/NEMO (B), or IKK
/NEMO only (C).
Fractions isolated from a Superdex-200 column were subjected to either
Western blotting or immunoprecipitation and in vitro kinase
assays utilizing the GST/I
B
(aa 1-54) substrate. Monoclonal
antibodies (Ab) used against the epitope-tagged proteins
included anti-HA (Roche Molecular Biochemicals, 12CA5) for IKK
,
anti-FLAG (Sigma, M2) for IKK
, and anti-Myc (9E10) for IKK
/NEMO.
Molecular weight markers and fraction numbers are indicated at the
top and bottom of the figures, respectively. The
position of the IKKs and GST/I
B
are indicated at the
right of the figure.
and IKK
with
IKK
/NEMO resulted in the migration of these epitope-tagged IKK
and IKK
proteins to a position corresponding to a molecular mass of
~600 kDa (Fig. 2B). The kinase activity of the IKK
proteins correlated with the presence of these proteins in the 600-kDa fractions. The epitope-tagged IKK
/NEMO protein was detected only in
extracts prepared from cells that were transfected with the IKK
/NEMO
cDNA and were mainly present in the 400-500-kDa fractions, regardless of whether it was coexpressed with the IKKs (Fig.
2B) or was expressed alone (Fig. 2C). There was
no IKK activity using an I
B
mutant in which serine residues 32 and 36 were changed to alanine (data not shown). These results suggest
that IKK
/NEMO has the ability to recruit IKK
and IKK
into a
large protein complex, although the majority of IKK
/NEMO does not
necessarily comigrate with this complex.
/NEMO Is Mediated through
Interaction with IKK
--
To address the mechanism by which
IKK
/NEMO leads to changes in the chromatographic mobility of the
IKKs, we performed experiments in which either epitope-tagged IKK
or
IKK
alone was expressed in the presence or absence of epitope-tagged
IKK
/NEMO. In extracts prepared from COS cells transfected with
IKK
in the absence of IKK
/NEMO, the majority of the
epitope-tagged IKK
protein was detected migrating at ~400 kDa
(Fig. 3A). The majority of the IKK
kinase activity was also detected in these fractions (Fig. 3A). When IKK
was coexpressed with IKK
/NEMO, the
chromatographic position of both the IKK
protein and kinase activity
was virtually unchanged when compared with that seen when the IKK
was expressed alone (Fig. 3A). The epitope-tagged
IKK
/NEMO protein was detected only in the cells that were
transfected with this cDNA, and its chromatographic position was at
~400-500 kDa (Fig. 3A).
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Fig. 3.
Effect of IKK /NEMO
on the chromatographic distribution of transfected
IKK
or IKK
. COS
cells were transfected with IKK
alone (top three panels)
or IKK
and IKK
/NEMO (bottom three panels)
(A), IKK
alone (top three panels) or IKK
and IKK
/NEMO (bottom three panels) (B), or
IKK
and CREB (C). Western blotting and in
vitro kinase assays performed on fractions isolated from the
Superdex-200 column were performed using the same antibodies
(Ab) as described in Fig. 2. C, the Myc-tagged
CREB was detected by anti-Myc antibody. Molecular weight markers and
fraction numbers are indicated at the top and the
bottom of the figure, respectively. The positions of the
IKKs and GST/I
B
are indicated at the right of the
figure.
/NEMO
altered the chromatographic distribution of IKK
. In the absence of
IKK
/NEMO, the majority of the epitope-tagged IKK
protein and
kinase activity was found in fractions migrating at 200-300 kDa. A
small fraction of IKK
kinase activity was also seen migrating in
higher molecular fractions likely due to its binding to endogenous
IKK
/NEMO (Fig. 3B). In the presence of transfected
IKK
/NEMO, the majority of the epitope-tagged IKK
protein and
kinase activity was found in fractions migrating at ~600 kDa (Fig.
3B). The epitope-tagged IKK
/NEMO was detected mainly in
fractions migrating at 500 kDa in cells coexpressing IKK
and
IKK
/NEMO (Fig. 3B). These results suggest that
IKK
/NEMO alters the chromatographic behavior of IKK
but not
IKK
.
/NEMO to alter the
chromatographic mobility of IKK
. An unrelated epitope-tagged protein, the cAMP-responsive element-binding protein (CREB) which has
no known role in regulating the IKK complex, was coexpressed with
IKK
, and their chromatographic mobility was determined. Both the
epitope-tagged IKK
protein and IKK
kinase activity were detected
in column fractions migrating at ~200 kDa, indicating that CREB
unlike IKK
/NEMO is unable to alter IKK
mobility (Fig. 3C). Taken together, these results suggest that IKK
/NEMO
is a factor directly involved in changing the chromatographic position of the IKKs. Furthermore, IKK
but not IKK
, serves as a primary target for IKK
/NEMO in the process of formation of the high
molecular weight IKK complex.
Recruitment by IKK
/NEMO Is Independent of Its Kinase
Activity--
To demonstrate whether IKK
/NEMO requires an active
IKK
kinase to result in its incorporation into the high molecular
weight IKK complex, epitope-tagged IKK
mutants were transfected into COS cells in either the presence or the absence of IKK
/NEMO. Either
the constitutively active IKK
mutant, IKK
(SS/EE), which contains
substitutions of serine residues 177 and 181 with glutamic acid
residues or a noninducible IKK
kinase mutant, IKK
(SS/AA), in
which these same serine residues in the activation loop were substituted with alanine were assayed (12, 29). Similar to the results
seen with the wild-type IKK
protein when expressed alone, the
epitope-tagged IKK
(SS/EE) protein was detected in fractions
corresponding to ~200 kDa (Fig.
4A). When the IKK
(SS/EE) protein was coexpressed with the epitope-tagged IKK
/NEMO, the IKK
(SS/EE) protein and kinase activity was detected in the higher molecular weight IKK complex (Fig. 4A).
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Fig. 4.
Effect of IKK /NEMO
on the chromatographic distribution of IKK
mutants. COS cells were transfected with IKK
(SS/EE)
either alone (top three panels) or IKK
(SS/EE) and
IKK
/NEMO (bottom three panels) (A) or
IKK
(SS/AA) alone (top three panels) or IKK
(SS/AA) and
IKK
/NEMO (bottom three panels) (B). Western
blotting and in vitro kinase assays were performed on the
column fractions using the same antibodies (Ab) as described
in Fig. 2. Molecular weight markers and column fraction numbers are
indicated at the top and bottom of the figure,
respectively. The positions of IKK
and the GST/I
B
substrate
are indicated at the right of the figure.
(SS/AA)
protein was then assayed. In the absence of IKK
/NEMO, the majority
of epitope-tagged IKK(SS/AA) protein was present in lower molecular
weight fractions migrating between 200 and 400 kDa (Fig.
4B). In the presence of the epitope-tagged IKK
/NEMO, the
IKK
(SS/AA) protein was detected in the high molecular weight IKK
complex (Fig. 4B). No detectable kinase activity was
detected in any of the fractions containing the kinase-defective
IKK
(SS/AA) protein regardless of whether or not IKK
/NEMO was
coexpressed (Fig. 4B). The chromatographic position of
IKK
/NEMO was unchanged in cells expressing IKK
(SS/EE) and
IKK
(SS/AA) (Fig. 4, A and B). These results
suggest that incorporation of IKK
into high molecular weight IKK
complex by IKK
/NEMO is not dependent on the kinase activity of
IKK
.
/NEMO Is Necessary for Assembly
of the IKK Complex--
Studies were next performed to determine which
domains in IKK
/NEMO are responsible for recruiting IKK
into the
high molecular weight complex. Epitope-tagged IKK
/NEMO deletion
mutants lacking either the carboxyl-terminal 100 amino acid residues
(aa 1-312) or the amino-terminal 100 residues (aa 101-412) were
transfected into COS cells along with both IKK
and IKK
. Similar
to the results obtained with wild-type IKK
/NEMO shown in Fig.
2A, the mutant lacking the carboxyl-terminal portion of
IKK
/NEMO was able to recruit the majority of the epitope-tagged
IKK
protein and kinase activity into the high molecular weight IKK
complex (Fig. 5A). However,
this IKK
/NEMO mutant consistently failed to shift efficiently the
majority of the epitope-tagged IKK
into the higher molecular complex
(Fig. 5A). The epitope-tagged IKK
/NEMO mutant lacking the
amino-terminal portion of this protein was unable to shift either
IKK
or IKK
into the high molecular weight IKK complex (Fig.
5B). Finally, mutations that changed leucine residues 315, 322, and 329 in the leucine zipper motif of IKK
/NEMO to methionine did not have major effects on the ability of IKK
/NEMO to shift the
chromatographic distribution of IKK
(Fig. 5C). These
results suggest that the amino-terminal portion of IKK
/NEMO is
critical for the chromatographic shift of the IKKs.
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Fig. 5.
Effect of IKK /NEMO
mutants on the chromatographic distribution of IKK
and IKK
. COS cells were transfected
with either IKK
, IKK
, and a carboxyl-terminal deletion mutant of
IKK
/NEMO (aa 1-312) (A), IKK
, IKK
, and an
amino-terminal deletion mutant of IKK
/NEMO (aa 101-412)
(B), or IKK
and a IKK
/NEMO mutant that contains
substitutions of leucine residues with methionine at 315, 322, and 329 positions in the leucine zipper motif (C). Molecular weight
markers and column fraction numbers are indicated at the top
and bottom of the figure, respectively. The position of the
IKKs and the GST/I
B
substrate are indicated at the
right of the figure. Ab, antibody.
/NEMO Is Critical for Stimulating IKK
Kinase
Activity--
To demonstrate whether IKK
/NEMO is involved in
activation of the IKKs, COS cells were transfected with either
epitope-tagged IKK
or IKK
cDNAs in the presence of increasing
amounts of IKK
/NEMO. IKK kinase activity was then assayed following
immunoprecipitation of either the epitope-tagged IKK
or IKK
proteins. In the absence of the transfected epitope-tagged IKK
/NEMO,
there was little IKK
activity detected (Fig.
6A, upper panel, lane 2).
However, IKK
kinase activity was dramatically increased by
transfecting increasing amounts of IKK
/NEMO (Fig. 6A, lanes
3-6). Immunoprecipitation of the epitope-tagged IKK
/NEMO from
these extracts followed by in vitro kinase assays of the
associated IKK
activity gave similar results (data not shown). In
contrast, IKK
/NEMO resulted in very little stimulation of IKK
activity (Fig. 6B). This result was consistently seen
regardless of the amount of IKK
that was transfected (data not
shown).
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Fig. 6.
IKK /NEMO is critical
for stimulation of IKK
kinase activity.
A, COS cells (2 × 105) were transfected
with either a CMV expression vector alone (lane 1), 0.05 µg of IKK
alone (lane 2), 0.05 µg of IKK
and
increasing amounts of the CMV vector containing IKK
/NEMO of 0.005 µg (lane 3), 0.05 µg (lane 4), 0.25 µg
(lane 5), or 0.5 µg (lane 6). The total DNA
amounts were adjusted to the same level with the vector DNA for the
different transfections. In vitro IKK
kinase activity was
assayed using the FLAG monoclonal antibody (Sigma, M2), and Western
blotting was performed with the FLAG antibody to assay the expression
of IKK
and the Myc monoclonal antibody (Roche Molecular
Biochemicals, 9E10) to assay IKK
/NEMO. B, COS cells were
transfected with an IKK
expression vector (0.5 µg) rather than an
IKK
vector as in A. The HA monoclonal antibody (Roche
Molecular Biochemicals, 12CA5) was used to detect IKK
. C,
COS cells were transfected with vector alone (lane 1),
IKK
(0.05 µg), or the mutants indicated in the presence or absence
of IKK
/NEMO (lanes 2-9). In vitro kinase
activity and protein expression were determined. Ab,
antibody.
/NEMO induction of IKK
kinase
activity, epitope-tagged IKK
mutants in either the activation loop,
IKK
(SS/AA) and IKK
(SS/EE), or the kinase-defective mutant IKK
(K/M) were expressed in either the presence or the absence of
IKK
/NEMO. Whereas IKK
/NEMO stimulated wild-type IKK
activity, it failed to stimulate the kinase activity of any of the IKK
mutants
(Fig. 6C). These results would be consistent with a
potential role of IKK
/NEMO in stimulating the phosphorylation of
serine residues in the IKK
activation loop with resultant increases in kinase activity.
/NEMO That Regulate the Kinase Activity and
Phosphorylation of IKK
--
Next we determined which domains in
IKK
/NEMO were important for stimulation of the IKK
kinase
activity. COS cells were transfected with epitope-tagged IKK
alone
or with either the wild-type or mutated IKK
/NEMO cDNAs.
Mutations in either the carboxyl terminus, the amino terminus, or the
leucine zipper of IKK
/NEMO reduced its ability to stimulate IKK
kinase activity (Fig. 7A, upper panel). The amounts of the epitope-tagged IKK
and IKK
/NEMO
proteins were comparable with these transfections (Fig. 7A,
bottom two panels).
View larger version (32K):
[in a new window]
Fig. 7.
Domains in IKK /NEMO
that regulate IKK
kinase activity and
phosphorylation. A, COS cells were transfected with
vector alone (lane 1), IKK
(0.05 µg) alone (lane
2), or IKK
(0.05 µg) with either 0.1 µg of the wild-type or
mutant IKK
/NEMO cDNAs as indicated (lanes 3-7).
In vitro kinase assays were performed to determine IKK
kinase activity (top panel), and Western blotting was
performed to detect the expression of IKK
and IKK
/NEMO
(bottom two panels). PhosphorImager analysis of seven sets
of transfections were quantitated as follows: lane 1, 3.04;
lane 2, 24.74; lane 3, 100; lane 4, 36.01; lane 5, 38.79; lane 6, 23.43; and
lane 7, 69.94. B, COS cells were transfected for
36 h with vector alone (lane 1), 0.5 µg of IKK
alone (lane 2), or 0.5 µg of IKK
and the indicated
IKK
/NEMO mutants (0.5 µg) (lanes 3-7). The cells were
either incubated with [32P]orthophosphate for 3 h at
37 °C or harvested for Western analysis (bottom two
panels). The radiolabeled proteins were immunoprecipitated using
anti-FLAG monoclonal antibody (Sigma, M2) and subjected to
electrophoresis and autoradiography (top panel).
/NEMO to stimulate
IKK
kinase activity correlated with the phosphorylation of IKK
.
Epitope-tagged wild-type or mutant IKK
/NEMO cDNAs were transfected into COS cells in the presence of IKK
. Cells were then
incubated with [32P]orthophosphate, and the
epitope-tagged IKK
proteins were immunoprecipitated. Analysis of
these samples following SDS-polyacrylamide gel electrophoresis and
autoradiography demonstrated that wild-type IKK
/NEMO and both the
carboxyl-terminal deletion and leucine-zipper mutants stimulated IKK
phosphorylation. However, the amino-terminal deletion of IKK
/NEMO
prevented IKK
phosphorylation (Fig. 7B). The increased phosphorylation of IKK
in the presence of IKK
/NEMO appears to require an intact IKK
activation loop as phosphorylation of the mutants IKK
(SS/EE) and IKK
(SS/AA) was not induced by IKK
/NEMO (data not shown). Taken together, these results suggest that
IKK
/NEMO is involved in activation of IKK
kinase activity
possibly through stimulation of IKK
phosphorylation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/NEMO is an essential factor required for NF-
B activation
(28, 41, 42, 48-50). The ability of IKK
/NEMO to stimulate the
NF-
B pathway is likely due to its effects on the assembly of the IKK
complex (28-30) and the recruitment of upstream kinases that increase
IKK activity (45). However, the mechanisms involved in the assembly of
the IKK complex by IKK
/NEMO has not been elucidated.
/NEMO leads to the
recruitment of the IKKs into a high molecular weight complex. Moreover,
IKK
/NEMO can stimulate IKK
phosphorylation of the I
B
protein. The ability to assay IKK
/NEMO recruitment of IKK
into
the high molecular weight IKK complex allowed us to identify domains in
these proteins that are involved in this process. We found that the
amino terminus of IKK
/NEMO is crucial for recruitment of IKKs into
the high molecular weight IKK complex. These results are consistent
with previous data indicating that the amino terminus of IKK
/NEMO is
critical for its association with IKK
(29, 30, 43). Neither the
carboxyl terminus nor the leucine zipper domains of IKK
/NEMO were
required for recruitment of IKK
into the IKK complex. However, both
domains are important for the ability of IKK
/NEMO to stimulate
maximally IKK
kinase activity. This suggests that the carboxyl
terminus and possibly the leucine zipper of IKK
/NEMO are involved in
additional functions such as the recruitment of upstream kinases
including RIP into the IKK complex (45). These results are consistent
with a role for different domains in IKK
/NEMO in regulating distinct
functions that lead to activation of the NF-
B pathway.
kinase activity was necessary for its
recruitment into the high molecular weight IKK complex. We found that
both constitutively active and kinase-defective IKK
proteins were
recruited into the IKK complex by IKK
/NEMO. These results suggest
that activation of IKK
kinase activity is not a requirement to
facilitate IKK
association with the IKK complex. In the absence of
IKK
/NEMO, both IKK
and IKK
migrate in lower molecular weight
complexes. Although IKK
/NEMO does not appear to efficiently recruit
IKK
alone into the high molecular weight IKK complex, it is able to
recruit IKK
through its interactions with IKK
. A
carboxyl-terminal deletion mutant of IKK
/NEMO consistently exhibited
defects in recruiting IKK
into the high molecular weight complex.
Previous data suggest that IKK
can associate with IKK
/NEMO in
IKK
-deficient fibroblasts (44) and in cotransfection assays performed in 293 cells (46). These results raise the possibility that
the carboxyl terminus and potentially other domains of IKK
/NEMO can
facilitate IKK
binding via either a direct association or by
indirect association with upstream kinases that interact with IKK
/NEMO. Thus, IKK
/NEMO leads to the recruitment of both IKK
and IKK
into the IKK complex.
/NEMO in regulating IKK kinase activity and
stimulating the NF-
B pathway has previously been studied using transient expression assays (29, 30, 42, 43, 55). Antisense IKK
reduces IKK activation in response to upstream activators (30, 43);
whereas the expression of carboxyl-terminal IKK
/NEMO mutants
functions as dominant negative inhibitors of IKK
activity (29, 30).
These results are consistent with a positive role of IKK
/NEMO on
activating the NF-
B pathway. However, overexpression of wild-type
IKK
/NEMO in transient expression assays can in some cases reduce IKK
activation likely by a squelching mechanism (40).
/NEMO markedly stimulates IKK
kinase activity. This stimulation is dependent on intact serine
residues in the IKK
activation loop. The amino terminus of
IKK
/NEMO is crucial for this process although the carboxyl terminus
and leucine zipper region of IKK
/NEMO are also involved. In
contrast, IKK
/NEMO does not significantly stimulate IKK
kinase activity. The ability of IKK
/NEMO to stimulate IKK
activity is
likely mediated in part by the ability of IKK
/NEMO to increase phosphorylation of serine residues in the IKK
activation loop. The
amino terminus of IKK
/NEMO is important for both increasing IKK
phosphorylation and stimulating IKK
kinase activity suggesting that
IKK
/NEMO association with IKK
is critical for both of these events. Whether this process is due to IKK
/NEMO-mediated stimulation of IKK
autophosphorylation or the ability of IKK
/NEMO to recruit other kinases remains to be determined. Thus, IKK
/NEMO appears to be
important for regulating both IKK
phosphorylation and kinase activity.
/NEMO function would suggest that its
interaction with IKK
/IKK
is critical for formation of the high molecular weight IKK complex that is responsive to various activators of the NF-
B pathway (28-30). The amino terminus of IKK
/NEMO is critical for IKK
association with the high molecular weight complex, whereas the carboxyl terminus of IKK
/NEMO likely mediates
interactions with upstream kinases such as RIP (45, 57) or
MEKK12 or potentially
regulates IKK
activation of IKK
kinase activity (39, 58). Thus,
IKK
/NEMO likely serves as a unique scaffold protein to facilitate
the assembly and activity of the IKK complex. Further studies on the
regulation of IKK
/NEMO will be important in defining its ability to
regulate NF-
B and potentially other signal transduction pathways.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sharon Johnson and Alex Herrera for
preparation of the manuscript and the figures, respectively. We also
thank Dean Ballard for providing us with IKK/NEMO deletion mutants.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA74128 and a grant from the Welch Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of
Hematology-Oncology, Dept. of Medicine, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX
75390-8594. Tel.: 214-648-4996; Fax: 214-648-4152; E-mail:
gaynor@utsw.swmed.edu.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M008353200
2 X.-H. Li and R. B. Gaynor, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor-
;
IKK, I
B kinase;
PCR, polymerase chain reaction;
GST, glutathione S-transferase;
CMV, cytomegalovirus;
HA, hemagglutinin;
aa, amino acid;
CREB, cAMP-responsive element-binding
protein.
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