From the Departments of Microbiology and Molecular Genetics and Medicine, Program in Virology, Harvard Medical School, Boston, Massachusetts, 02115
Received for publication, February 23, 2001
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ABSTRACT |
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Homozygosity for the aly point
mutation in NF- NF- NIK has an essential role in lymphoid organ development (12). The
aly mutation results in a single amino acid change of glycine to arginine at mNIK codon 855 and can be rescued by transgenic expression of wild type NIK (12). Alymphoplasia
(aly/aly) mice not only lack lymph nodes and
Peyer's patches but also have abnormal spleen and thymus development,
low serum Ig levels, and impaired B cell proliferation in response to
LPS or CD40L (12, 13). LT Epstein-Barr virus (EBV) latent infection of human B lymphocytes causes
B lymphocyte proliferation through expression of nuclear proteins and
an integral membrane protein, LMP1, which mimics constitutively
activated TNFRs (16). LMP1 has a short, arginine-rich, N-terminal
cytoplasmic domain that is important for anchoring the first
transmembrane domain, six hydrophobic transmembrane domains that
mediate LMP1 aggregation in lipid rafts, and a 200-amino acid
C-terminal cytoplasmic domain that has two sites that mediate EBV-induced B cell proliferation and NF- Expression Vectors--
Wild type hNIK624-947
was amplified by PCR of hNIK cDNA with oligonucleotides NF3 (5'
GGATCCCCTCTCACAGCCCAGGCCATC 3') and NR1 (5'
GAATTCTTAGGGCCTGTTCTCCAGCTGGC 3') that included BamHI and
EcoRI sites and cloned into pGEX-2TK (Amersham Pharmacia
Biotech) for bacterial expression. The aly
hNIK624-947 GST mutant was made by site-directed
mutagenesis of the hNIK codon 860 with primers NF8 (5'
AGCTATTTCAATCGGGTGAAAGTCCAAATA CAG 3') and NR6 (5'
CTGTATTTGGACTTTCACCCGATTGAAATAGCTTGG 3') followed by PCR with the
BamHI and EcoRI primers and cloned into pGEX-2TK.
The BamHI/EcoRI fragments were also used to make
wt and aly DN-NIK for mammalian expression by subcloning
into pCDNA3 (Invitrogen). Plasmids encoding TRAF1, TRAF2, TRAF3,
IKK Cell Lines--
293 and 293T cells were cultured as previously
described (7). MEFs from aly/aly and wild type mice were
obtained from Dr. T. Honjo. Primary MEFs were not amenable to
transfection. Therefore, cells were infected with a human papilloma
virus 16 E6/E7 retrovirus containing a neomycin resistance cassette
(obtained from Dr. P. Howley), and transformed cell lines were
selected for G418 resistance and growth advantage. Cells were
subsequently grown in Dulbecco's modified Eagle's medium with 20%
fetal calf serum and antibiotics.
Transfections and Reporter Gene Assays--
Transfections and
reporter assays (7) were done with 350 ng per well of the
3xNF- [35S]Met in Vitro Translations and GST
Pulldowns--
In vitro transcription and translation reactions (IVTs)
were done with (18) 1 µg of expression plasmid for TRAF1, 2, 3, or 6 diluted into 8 µl of distilled H2O and 40 µl of a TnT
quick-coupled rabbit reticulocyte lysate in vitro
transcription and translation system (Promega) in the presence of 2 µl of [35S]Met (10 µCi/µl). After incubation at
30 °C for 1 h and preclearing with GST-bound
glutathione-Sepharose (Amersham Pharmacia Biotech) for 1 h at
4 °C, IVTs were split into fractions for incubation with ~5 µg
of wt or aly NIK-GST fusion proteins or GST alone. The
GST/IVT mix was rotated at 4 °C for 1 h, beads were washed 3-5
times with GST lysis buffer, and boiled in 30 µl of SDS-PAGE loading
buffer. The samples were analyzed by SDS-PAGE and phosphorimaging (Molecular Dynamics).
Immune Precipitations--
For transfections, 293T cells were
seeded in 6-well plates at 5 × 105 cells/well, and
24 h later 3 wells were transfected with 1 µg of
pCDNA3-F-TRAF2 or pRK5-myc-IKK Effects of wt or aly DN-NIK on EBV LMP1-mediated Activation of
NF- Effects of wt or aly DN-NIK on LT LMP1, LMP1/LT aly NIK Interacts with TRAFs in Vitro and in Vivo--
The
aly mutation at codon 860 falls within both the TRAF binding
domain (aa 624-947) and the IKK binding domain (aa 735-947) of hNIK
(1, 25) (Fig. 5A). The
aly/aly phenotype is presumed to be because of the failure
of aly NIK to interact with TRAFs (25). To more precisely
determine the biochemical basis for the aly effect on NIK
interactions, we compared the in vitro and in
vivo binding of wt and aly NIK to TRAFs.
hTRAFs 1, 2, and 3 and mTRAF6 were transcribed and translated with
radiolabeled [35S]methionine, and their ability to bind
in vitro to equal amounts of wt or aly hNIK aa
624-947 fused to GST (NIK624-947-GST) was assessed. The
aly mutation did not affect TRAFs binding to NIK624-947-GST in vitro (Fig. 5B).
However, TRAFs differed in their binding to
NIK624-947-GST; TRAF1 and -2 bound to NIK624-947-GST at the 10 to 15% level, whereas less than 3% of TRAF3 or -6 bound to NIK624-947-GST (Fig.
5B).
Because TRAF2 is an important mediator of TNFR1-induced NF- Differential Association of wt and aly NIK with IKK
In similar experiments, the binding of FLAG-tagged IKK
To evaluate the association of Myc-IKK The data presented here indicate that LMP1, LMP1/LT The simplest explanation of the inability of the aly DN-NIK
to block LMP1 or LMP1/LT During the preparation of this manuscript, two publications appeared
that are relevant to these experiments. In one, NIK is found to
associate with the p100 precursor to the NF-B-inducing kinase (NIK) results in alymphoplasia in
mice, a phenotype similar to that of homozygosity for deletion of the
lymphotoxin
receptor (LT
R). We now find that NF-
B activation
by Epstein-Barr virus latent membrane protein 1 (LMP1) or by an
LMP1 transmembrane domain chimera with the LT
R signaling
domain in human embryonic kidney 293 cells is selectively
inhibited by a wild type dominant negative NIK comprised of amino acids
624-947 (DN-NIK) and not by aly DN-NIK. In contrast,
LMP1/CD40 is inhibited by both wild type (wt) and aly
DN-NIK. LMP1, an LMP1 transmembrane domain chimera with the LT
R
signaling domain, and LMP1/CD40 activate NF-
B in wt or
aly murine embryo fibroblasts. Although wt and
aly NIK do not differ in their in vitro binding
to tumor necrosis factor receptor-associated factor 1, 2, 3, or 6 or in
their in vivo association with tumor necrosis factor
receptor-associated factor 2 and differ marginally in their very poor
binding to I
B kinase
(IKK
), only wt NIK is able to bind to
IKK
. These data are compatible with a model in which activation of
NF-
B by LMP1 and LT
R is mediated by an interaction of NIK or a
NIK-like kinase with IKK
that is abrogated by the aly
mutation. On the other hand, CD40 mediates NF-
B activation through a
kinase that interacts with a different component of the IKK complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-inducing kinase,
NIK,1 is a TRAF2-interacting
mitogen-activated protein kinase kinase kinase that potently activates NF-
B (1-3). NIK can activate the I
B kinase (IKK) complex
by phosphorylating serine 176 in the activation loop of IKK
and may directly phosphorylate IKK
(4-6). NIK activation of IKK results in phosphorylation of I
B
serines 32 and 36, I
B
ubiquitination and degradation, and NF-
B translocation to the
nucleus. Overexpression of a catalytically inactive mutant of NIK (NIK
K429A/K430A) has a dominant negative effect on NF-
B
activation through most known stimuli including LMP1, TNFR1, TNFR2,
RANK, hTollR, CD3/CD28, interleukin-1R, human T-cell lymphotropic
virus-1 Tax, and LPS (1, 7-11).
R
/
and
aly/aly mice have similar developmental and
immunological defects, and NIK has been implicated in LT
R-mediated
activation of NF-
B (14, 15). Indeed, LT
R up-regulation of VCAM-1
is abnormal in aly/aly murine embryo fibroblasts
(15). Also, CD40L-induced phosphorylation of I
B
is abnormal in B
lymphocytes from aly/aly mice, although
phosphorylation of I
B
in dendritic cells is normal (13).
B activation (for review see
Ref. 17). One site binds TRAF 3, 1, 2, and 5, whereas the second site
binds TNFR-associated death domain protein (18, 19). NF-
B
activation from either site is inhibited by overexpression of
K429A/K430A kinase-negative DN-NIK (7). Thus, previous data are
consistent with NIK having a significant role in LMP1 activation of
NF-
B. The experiments reported here further investigate the role of
NIK and of the aly mutation in NF-
B activation by LMP1, LT
R, and CD40.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IKK
, LMP1, CD40, and LMP1/CD40 have been described (7, 18,
20). The pCR-F-mTRAF6 plasmid was obtained from Dr. J. Inoue. The
LMP1/LT
R construct was made by cloning the cytoplasmic domain from
LT
R into the previously described LMP1 pCDNA3 construct.
B-luc reporter plasmid and 350 ng per well of
pGK-
-galactosidase as a transfection control. Measurements of
luciferase and
-galactosidase activities were done with an Optocomp
I luminometer (MGM Systems).
per well and 2, 3, or 4 µg/well of wt or aly DN-NIK or pCDNA3 for each
immune precipitation. Cells were lysed in Nonidet P-40 lysis buffer
(1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, 20 mM Tris, pH 7.5) containing phenylmethylsulfonyl fluoride
(1 mM) and aprotinin (22 µg/ml) for 30 min on ice.
Following lysis, cells were centrifuged at high speed for 10 min,
precleared with protein G-Sepharose (Amersham Pharmacia Biotech) for
1 h at 4 °C, normalized for total protein concentration, and 2 µg of TRAF2 (C-20; Santa Cruz Biotechnology) antibody or 30 µl of M2 beads (Amersham Pharmacia Biotech) were added. After rotation for
1 h at 4 °C for C-20, protein G-Sepharose was added to the samples for an additional 1 h. Immune precipitates were washed 3-5 times with Nonidet P-40 lysis buffer. Samples were denatured in
SDS-PAGE loading buffer and subjected to SDS-PAGE followed by
immunoblotting (IB). IB antibodies were H-248 (NIK; Santa Cruz Biotechnology), M-110 (IKK
; Santa Cruz Biotechnology), 9E10 (Myc; see Ref. 21), and C-20 (TRAF2; Santa Cruz Biotechnology).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B--
LMP1 expression in 293T cells strongly activated NF-
B
as measured by a co-transfected reporter with three NF-
B sites from the major histocompatibility complex I promoter upstream of
luciferase and a pGK-
-galactosidase control plasmid (see Fig.
1 and Refs. 7, 22, and 23). LMP1-mediated
NF-
B activation was inhibited in a dose-dependent manner
by co-expression of the wt DN-NIK fragment (aa 624-947) (Fig.
1A). In contrast to the effect of wt DN-NIK, co-expression
of the DN-NIK containing the aly point mutation G860R did
not inhibit LMP1 activation of NF-
B (Fig. 1B). wt and aly DN-NIK were expressed at equivalent levels,
-galactosidase levels did not vary more than 2-fold, and LMP1
expression was not affected by either wt or aly DN-NIK (Fig.
1C), excluding an artifactual basis for the differential
effect. Thus, LMP1 activation of NF-
B is resistant to aly
but not to wt DN-NIK as had been previously noted for LT
R (12).
Similar results were obtained in multiple experiments in 293 and 293T
cells.
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Fig. 1.
Inhibition of LMP1-mediated
NF- B activation by wt DN-NIK but not
aly DN-NIK. A and B, 293T
cells were cotransfected with 50 ng of LMP1 expression plasmid
pCDNA3-LMP1, 350 ng of the NF-
B reporter construct 3x
B-luc,
and 350 ng of pGK-
-galactosidase. Also, increasing amounts (0.1, 1, and 2 µg) of either wt or aly DN-NIK were transfected. Raw
luciferase values were divided by
-galactosidase values to control
for transfection efficiency. The effects of DN-NIK on LMP1-mediated
NF-
B activation are expressed as a percentage of the activation with
LMP1 transfected alone. Values represent the average from three
independent experiments. C, lysates from transfected cells
were subjected to SDS-PAGE and IB using either LMP1 or NIK antibody.
For LMP1 IB, lanes 1-4 represent cells transfected with
LMP1 plus 0, 0.1, 1, or 2 µg of wt DN-NIK. No change in expression of
LMP1 was detected in the presence of aly DN-NIK (not shown).
For NIK, IB of lysates from cells transfected with LMP1 and 0, 0.1, or
2 µg of wt DN-NIK (lanes 1-3) and 0.1 or 2 µg of
aly DN-NIK (lanes 4-5) are shown.
R and CD40 Activation of
NF-
B--
To evaluate whether the differential effect of the wt and
aly DN-NIK is specific for LMP1 and LT
R signaling as
opposed to other TNFRs, the effect of wt and aly DN-NIK on
NF-
B activation by LMP1, LT
R, and CD40 was assayed in 293T and
293 cells. The LMP1 transmembrane domains were used to provide
constitutive, ligand-independent receptor aggregation. Isogenic
expression constructs were made in which the LMP1 C-terminal
cytoplasmic domain was replaced with the LT
R or CD40 C-terminal
cytoplasmic domains. LMP1 and the LMP1/LT
R chimera activated
NF-
B, and the activation was inhibited by wt but not by
aly DN-NIK (see Fig. 1 and Fig. 2, A and
B). wt and aly DN-NIK did not affect LMP1/LT
R
expression, and
-galactosidase levels did not vary more than 2-fold
(Fig. 2C and data not shown).
These results are consistent with the previous observation that NF-
B
activation following LT
R overexpression is inhibited by wt but not
by aly DN-NIK (12). In contrast to the effects of wt but not
aly DN-NIK on LMP1 or LMP1/LT
R activation of NF-
B,
NF-
B activation mediated by an LMP1/CD40 cytoplasmic domain chimera
or by CD40 overexpression was inhibited by both wt and aly
DN-NIK (Fig. 3, A and
B and data not shown). CD40 and LMP1/CD40 expression were
not affected by either wt or aly DN-NIK (Fig. 3C
and data not shown). These data support a model in which LMP1 and
LT
R activation of NF-
B involves a downstream molecular interaction that can be inhibited by wt hNIK aa 624-947 but not by the
corresponding fragment with the glycine to arginine mutation at codon
860.
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Fig. 2.
Inhibition of
LMP1/LT R-mediated
NF-
B activation by wt DN-NIK but not
aly DN-NIK. A and B,
experiments were performed as in Fig. 1, except that 5 µg of
LMP1/LT
R was used instead of LMP1. Results are averages from three
independent experiments and are represented as a percent of the NF-
B
activation observed with LMP1/LT
R alone. C, IB for
FLAG-tagged LMP1/LT
R using M2/M5 anti-FLAG antibody (Sigma).
Lanes 1-3 represent cells transfected with LMP1/LT
R
alone or with 0.1 or 2 µg of wt DN-NIK. Expression of LMP1/LT
R was
identical with aly DN-NIK (not shown). Equivalent amounts of
wt as compared with aly DN-NIK were detected by anti-NIK IB
(not shown).
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Fig. 3.
CD40-mediated NF- B
activation is inhibited by both wt and aly
DN-NIK. A and B, this experiment was
identical to that shown in Fig. 1, except that 1 µg of CD40 was
transfected instead of LMP1. The data shown are from one representative
experiment of two with CD40 and three with LMP1/CD40 (not shown).
C, IB of CD40 from cells transfected with CD40 alone
(lane 1) or with CD40 plus 0.1 or 2 µg of wt DN-NIK
(lanes 2 and 3). Identical expression of CD40 was
seen with CD40 plus aly DN-NIK (not shown).
R, and LMP1/CD40 Activation of NF-
B in wt and
aly/aly MEFs--
To evaluate the direct effect of the aly
mutation on LMP1, LT
R, and CD40 activation of NF-
B, NF-
B
activation by isogenic LMP1, LMP1/LT
R, and LMP1/CD40 was assessed in
wt and aly/aly MEFs using the co-transfected
NF-
B-dependent luciferase reporter and control
pGK-
-galactosidase expression plasmids. Before initiating this
series of experiments, the MEFs were first transformed with a human
papilloma virus 16 E6 and E7-expressing retrovirus so as to enhance
their growth and transfection efficiency (24). The surprising result
was that LMP1, LMP1/LT
R, and LMP1/CD40 activated NF-
B similarly
in wt and aly/aly MEFs indicating that NIK is not
essential for NF-
B activation by these receptors in fibroblasts
(Fig. 4). Thus, the insensitivity of LMP1
and LMP1/LT
R to aly DN-NIK inhibition of NF-
B
activation is not because of a specific and exclusive dependence of
these receptors on wt NIK for NF-
B activation.
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Fig. 4.
LMP1, LMP1/LT R, and
LMP1/CD40 activate NF-
B at an equivalent level
in wt and aly MEFs. LMP1 (100 ng), LMP1/LT
R (5 µg), or LMP1/CD40 (100 ng) were transfected into either wt (
) or
aly/aly
(
)
MEFs, along with 3x
B-luc and pGK-
-galactosidase. Luciferase
assays were performed as in Fig. 1. NF-
B activation relative to
control plasmid is shown for one representative of three independent
experiments.
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Fig. 5.
wt and aly NIK bind to TRAFs
in an equivalent manner. A, a schematic diagram of NIK
is shown. N-terminal regions were recently defined to play a role in
regulating NIK activity by preventing the interaction between the C
terminus of NIK and IKK (39). The ATP binding pocket within the
conserved MAP kinase domain is ablated by the K429A/K430A
mutation (1). Thr 559 is the critical residue in the activation loop of
the kinase domain that is phosphorylated by Tpl2 and is required
for kinase activation (3). The region from aa 624-947 has been defined
as the necessary and sufficient TRAF binding domain (1),2
whereas aa 735-947 within this domain is necessary and sufficient for
IKK
binding (3). The alymphoplasia phenotype is caused by a single
point mutation (Gly to Arg) occurring at amino acid 855 in mNIK (12)
and is in a highly conserved region in hNIK at amino acid 860. BR, basic region; PRR, proline-rich region. B,
[35S]methionine-labeled, in vitro-translated
TRAFs 1, 2, and 3 or mTRAF6 were incubated with wt
NIK624-947-GST, aly
NIK624-947-GST, or GST alone. The 10% input lane
indicates 10% of the total IVT reaction lysate used for each binding
assay. Samples were resolved by SDS-PAGE, and binding was analyzed by
phosphorimaging. C, 293T cells were co-transfected with
F-TRAF2 and either wt or aly DN-NIK. F-TRAF2·NIK
complexes were immunoprecipitated with anti-FLAG resin (M2; Sigma)
followed by IB with anti-NIK antibody. 2% of whole cell lysates were
probed with anti-TRAF2 and anti-NIK antibody.
B
activation (26), TRAF2 associations with wt and aly NIK were evaluated in 293 cells that were co-transfected with a FLAG-tagged TRAF2 expression construct and either wt or aly DN-NIK (aa
624-947). F-TRAF2 was immune-precipitated using M2-conjugated
Sepharose beads, and the complexes were blotted for the presence of
NIK. F-TRAF2 consistently brought down equivalent amounts of wt or aly DN-NIK (Fig. 5C). These data indicate that
the aly/aly phenotype is unlikely to be because of the
previously postulated inability of TRAFs to engage and associate with
aly NIK.
and
IKK
--
hNIK aa 735-947 is not only part of the domain that binds
to TRAFs but is also a sufficient domain for interaction with IKK
(3, 27). hNIK aa 735-947 efficiently competes with wt NIK for binding
to IKK
and is a dominant negative inhibitor of TNF-mediated NF-
B
activation (3). Surprisingly, only wt but not aly
NIK624-947-GST was able to pull down Myc-IKK
from
lysates of 293T cells in which Myc-IKK
was expressed. About 10% of
the Myc-tagged IKK
bound to wt NIK624-947-GST, whereas
binding to similar amounts of aly
NIK624-947-GST was below the limits of detection (Fig.
6A).
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Fig. 6.
wt and aly NIK bind to
IKK ; however aly NIK does not
bind to IKK
. A, wt
NIK624-947-GST, aly
NIK624-947-GST, or GST were used to precipitate either
Myc-IKK
or F-IKK
from transfected 293T cells. GST-bound IKKs were
subjected to SDS-PAGE followed by IB for the appropriate tag. IB with
IKK
antibody (M110; Santa Cruz Biotechnology) resulted in an
identical result (data not shown). B, 293T cells
co-transfected with the indicated amounts of wt or aly
DN-NIK, along with a constant amount of Myc-IKK
expression vector,
and were immunoprecipitated with anti-myc antibody followed by IB with
anti-NIK antibody. Lysates contained equivalent amounts of IKK
and
DN-NIK proteins (not shown).
expressed in
293T cells to wt or aly NIK624-947-GST was
assessed. As reported for full-length NIK (27), FLAG-tagged IKK
bound very weakly to wt NIK624-947-GST, at a level of
about 1% of input IKK
(Fig. 6A). Binding of F-IKK
to
aly NIK624-947-GST was about half the level of
binding to wt and about twice as strong as to GST alone (Fig.
6A).
with wt or aly
DN-NIK in vivo, 293T cells were co-transfected with
Myc-IKK
and wt or aly DN-NIK expression vectors, lysed in
non-ionic detergent, and immune-precipitated with anti-Myc antibody
followed by immune blotting for NIK. The efficiency of the Myc-IKK
IP was about 10% (data not shown). wt DN-NIK was readily detected in
the immune precipitate, whereas aly DN-NIK was not
detectable (Fig. 6B). Thus, the aly mutation
results in virtually complete loss of
interaction or association of DN-NIK with IKK
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R, and
LMP1/CD40 activate NF-
B similarly in wild type and
aly/aly murine embryo fibroblasts. Therefore, NIK either
does not have a direct role in NF-
B activation from any of these
receptor cytoplasmic domains in MEFs, or another kinase substitutes for
NIK in the transfected aly/aly fibroblasts and is unable to
substitute under more physiologic conditions in vivo.
Although this latter possibility seems a priori unlikely,
similar substitution effects have been noted among MAP kinases in
yeast, for example (28-33). Further, the similarity between the
aly/aly and LT
R
/
phenotypes is most
consistent with a physiologically significant role for the
aly mutation in LT
R signaling. Moreover, the inability of
the aly DN-NIK to specifically block LMP1 and LMP1/LT
R
activation of NF-
B in 293 cells supports the notion that these
cytoplasmic domains signal through a pathway that is specifically
blocked by wt and not by aly DN-NIK. Indeed, the biochemical
studies further support the hypothesis that the effect is at the level
of NIK per se, in indicating that aly hNIK is
most abnormal in loss of interaction with IKK
, is not abnormal in
interaction with TRAF1, 2, 3, or 6, and is only minimally evident in
diminished very weak interaction with IKK
.
R activation of NF-
B is that
aly NIK is unable to bind to an essential mediator of that
pathway. The aly mutation is within the NIK TRAF binding
domain, and the failure of aly NIK to block some TNFRs has
been attributed to a putative affect of the aly mutation on
TRAF binding. Our data indicate that the aly mutation does
not affect NIK binding to or association with TRAF1, 2, 3, or 6, making
it less likely that the aly effect is at the level of TRAF
interaction with NIK. Instead, we find aly NIK to be highly
deficient in binding to IKK
and that both wt and aly
mutant NIK bind poorly to IKK
. Thus, the ability of wt NIK and the
inability of aly NIK to block NF-
B activation from LMP1
or LMP1/LT
R are most consistent with a key role for a NIK-like
kinase and IKK
in LMP1 and LT
R activation of NF-
B. Further,
the blockade of CD40 and LMP1/CD40 activation of NF-
B by both wt and
aly DN-NIK are most compatible with the possibility that
CD40 signaling through the IKK complex is mediated by a protein that
can be blocked by either the wt or aly NIK C terminus.
B subunit p52 and induce
its phosphorylation and proteolytic processing (34). aly
NIK, however, is unable to associate with p100 or induce p100
phosphorylation and processing (34). Consistent with these
observations, p52 is not detected in aly/aly cells, despite
the presence of p100 (34, 35). However, although p52
/
mice have major defects in germinal center formation and splenic architecture similar to aly/aly and LT
R
/
mice, serum Ig levels and proliferation in response to LPS and CD40L
appear relatively normal in p52
/
mice but are abnormal
in aly/aly mice (36, 37). Therefore, the deficiency in p100
processing is likely to account for only part of the
aly/aly phenotype. In a second very recent paper, B cells from IKK
/
mice are found to be quite similar
to aly/aly B cells in their response to LPS and CD40L (38).
This report is consistent with our finding that aly NIK is
unable to interact with IKK
. Moreover, serum Ig levels are depressed
in IKK
/
bone marrow chimeras and in
aly/aly mice but not in p52
/
mice (38).
Thus, the inability of aly NIK to process p100 into p52 and
the inability of aly NIK to interact with IKK
may both contribute to the aly/aly phenotype.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Shinkura, T. Honjo, J. Inoue, E. Hung, B. Sylla, P. Howley, E. Cooper, and D. Goeddel for reagents. Also, we are grateful for helpful comments and discussion from the Kieff laboratory.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA47006.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.
Present address: Biomedical Sciences Research Center "Al
Fleming", Institute of Immunology, 14-16 Al Fleming Str.,
Vari, 16672, Athens, Greece.
§ To whom correspondence should be addressed. Tel.: 617-525-4250; Fax: 617-525-4251; E-mail: ekieff@rics.bwh.harvard.edu.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.C100103200
2 M. L. and E. K., unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
NIK, NF-B-inducing kinase;
h, human;
LPS, lipopolysaccharide;
IKK, I
B kinase;
TNFR(s), tumor necrosis factor receptor (s);
TRAF, TNFR-associated factor;
DN-NIK, dominant negative hNIK (aa 624-947);
GST, glutathione S-transferase;
LMP1, Epstein-Barr virus
latent membrane protein 1;
LT
R, lymphotoxin
receptor;
IB, immunoblotting;
MEF(s), mouse embryo fibroblast(s);
F, FLAG-tagged;
IVT(s), in vitro translation(s);
EBV, Epstein-Barr virus;
PCR, polymerase chain reaction;
wt, wild type;
luc, luciferase;
PAGE, polyacrylamide gel electrophoresis;
aa, amino acid;
IP, immunoprecipitation;
MAP, mitogen-activated protein.
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