CD27, a Member of the Tumor Necrosis Factor Receptor Superfamily,
Activates NF-
B and Stress-activated Protein Kinase/c-Jun
N-terminal Kinase via TRAF2, TRAF5, and NF-
B-inducing
Kinase*
Hisaya
Akiba
,
Hiroyasu
Nakano
§,
Shigeyuki
Nishinaka¶,
Masahisa
Shindo,
Tetsuji
Kobata
,
Machiko
Atsuta
,
Chikao
Morimoto
,
Carl F.
Ware**,
Nikolai L.
Malinin
,
David
Wallach
,
Hideo
Yagita
, and
Ko
Okumura
From the Department of Immunology, Juntendo University School of
Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, the ¶ Exploratory
Group, Sumitomo Pharmaceuticals Research Center, 4-2-1 Takatsukasa,
Takarazuka 665, the
Department of Clinical Immunology,
Institute of Medical Science, the University of Tokyo,
4-6-1 Shirokanedai, Minatoku, Tokyo 108, Japan, the ** Division
of Molecular Immunology, La Jolla Institute for Allergy and
Immunology, La Jolla, California 92121, the

Department of Membrane Research and
Biophysics, The Weizmann Institute of Science, 76100 Rehovot, Israel,
and the
Core Research for Evolutional Science and
Technology of Japan Science and Technology Corporation, 2-3 Surugadai,
Kanda, Chiyoda-ku, Tokyo 101, Japan
 |
ABSTRACT |
CD27 is a member of the tumor necrosis factor
(TNF) receptor superfamily and is expressed on T, B, and NK cells. The
signal via CD27 plays pivotal roles in T-T and T-B cell interactions. Here we demonstrate that overexpression of CD27 activates NF-
B and
stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK).
Deletion analysis of the cytoplasmic domain of CD27 revealed that the
C-terminal PIQEDYR motif was indispensable for both NF-
B and
SAPK/JNK activation and was also required for the interaction with TNF
receptor-associated factor (TRAF) 2 and TRAF5, both of which have been
implicated in NF-
B activation by members of the TNF-R superfamily.
Co-transfection of a dominant negative TRAF2 or TRAF5 blocked NF-
B
and SAPK/JNK activation induced by CD27. Recently, a TRAF2-interacting
kinase has been identified, termed NF-
B-inducing kinase (NIK). A
kinase-inactive mutant NIK blocked CD27-, TRAF2-, and TRAF5-mediated
NF-
B and SAPK/JNK activation. These results indicate that TRAF2 and
TRAF5 are involved in NF-
B and SAPK/JNK activation by CD27, and NIK
is a common downstream kinase of TRAF2 and TRAF5 for NF-
B and
SAPK/JNK activation.
 |
INTRODUCTION |
CD27 is a member of the tumor necrosis factor receptor
(TNF-R)1 superfamily and is
expressed on T, B, and NK cells as a disulfide-linked homodimer (1).
CD27 ligand (CD70) belongs to the TNF superfamily and is expressed on
the surface of activated T and B cells. Cross-linking of CD27 along
with a suboptimal dose of phytohemagglutinin, phorbol 12-myristate
13-acetate, anti-CD2, or anti-CD3 antibodies resulted in vigorous
proliferation of T cells, indicating that CD27 transmits a
co-stimulatory signal in T cells (2). On the other hand, ligation of
CD27 on B cells enhanced IgG production (3). These studies implicated
the important roles of CD27/CD70 interaction in immunoregulation
through T-T and T-B cell interactions. A recent report also
demonstrated a critical role of CD27/CD70 interaction in T cell
development (4). Although biological function of CD27/CD70 interaction
has been extensively investigated, the mechanism by which CD27
transmits the signal has been largely unknown, except for a previous
study, which demonstrated involvement of the protein tyrosine kinase
cascade (2).
TNF receptor-associated factors (TRAFs) have emerged as signal
transducers for some members of the TNF-R superfamily (5-10). All
TRAFs, except for TRAF1, are composed of N-terminal zinc RING finger,
multiple zinc fingers, coiled-coil, and C-terminal receptor binding
(TRAF) domains (5, 6, 9-11). Whereas an N-terminal RING finger domain
of TRAF2, TRAF5, and TRAF6 is responsible for NF-
B activation, the
TRAF and coiled-coil domains are required for homo- and
heterodimerization and receptor association (5, 6, 9, 10, 12). With the
exception of TRAF4, all other TRAFs have been shown to interact
directly with the non-death domain receptors, CD30, CD40, TNFR80,
lymphotoxin-
R (LT-
R), and interleukin-1R (9, 10, 12-16). TRAF2
has been shown to interact indirectly with death domain receptors,
TNFR60 and death receptor 3, via the adapter molecule TRADD (17-19).
TRAF2, TRAF3, and TRAF5 also interact with latent infection membrane
protein 1, the transforming protein of Epstein-Barr virus (11). TRAF2 also participates in the activation of stress-activated kinase (SAPK)/c-Jun N-terminal kinase (JNK) induced by TNF (20-22). TRAF3 and
TRAF5 are involved in CD23 up-regulation by CD40 (6, 16). TRAF3 has
been also shown to be involved in apoptosis by LT-
R (23). The
mechanism by which TRAFs activate NF-
B remains to be solved.
Recently, a serine/threonine kinase was identified that interacts with
TRAF2 and activates NF-
B, named NF-
B-inducing kinase (NIK) (24).
However, the functional role of NIK in TRAF5- or TRAF6-mediated NF-
B
activation is uncertain.
CD27 is functionally similar to other co-stimulatory receptors OX40,
4-1BB, and the herpesvirus entry mediator (HVEM) (25). HVEM was
recently shown to interact with TRAF2 and TRAF5 (26, 27). Here we
demonstrate that CD27 activates both NF-
B and SAPK/JNK, which are
mediated by TRAF2 and TRAF5. We also demonstrate that a kinase-inactive
mutant of NIK inhibited TRAF2-, TRAF5-, and CD27-mediated NF-
B and
SAPK/JNK activation. These results indicated a crucial role of TRAF2
and TRAF5 in CD27 signaling and that NIK is a common downstream kinase
of TRAF2 and TRAF5 for NF-
B and SAPK/JNK activation.
 |
MATERIALS AND METHODS |
Reagents and Cell Line--
Biotin-conjugated monoclonal
antibody (mAb) to Flag (M2) (Kodak), anti-hemagglutinin A (HA) (12CA5)
(Boehringer Mannheim), horseradish peroxidase-conjugated rabbit
anti-mouse IgG (Zymed), rabbit antibodies to NF-
B subunits, p50 and
p65 (Santa Cruz Biotechnology) were obtained from the indicated
commercial sources. The anti-human CD27 mAb (1A4) has been described
previously (28). The human embryonic kidney (HEK) 293 cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum.
Expression Vectors--
Flag-tagged expression vectors for TRAF2
(Dr. T. Watanabe, Institute of Medical Science, University of Tokyo),
TRAF3 (Dr. G. Mosialos, Harvard Medical School), TRAF5, and TRAF6 were
constructed by inserting each cDNA into pCR-Flag vector, which has
two copies of Flag tag sequence downstream of cytomegalovirus promoter
in pCR-3 (Invitrogen). Deletion mutants of
TRAF2-(272-501) and
TRAF5-(233-558) were amplified by polymerase chain reaction (PCR)
using primers corresponding to the numbered amino acids. The PCR
products were ligated directly into pCR-Flag vector. The C-terminal
deletion mutants of human CD27 (
5,
8,
10, and
16) were made
by PCR, and the appropriate PCR fragments were inserted into pCR-3
vector. pCR-CD30 (15) (Dr. T. Watanabe, Institute of Medical Science, University of Tokyo), pCDM8-CD40 (29) (Dr. H. Kikutani, Research Institute for Microbial Disease, University of Osaka),
pcDNA-LT-
R (9), pcDNA-NIK, pCR-Flag-NIK,
pcDNA-NIK-KM(KK429-430AA), and pCR-Flag-NIK-KM(KK429-430AA) (24),
and pSR
-HA-SAPK (30) (Dr. E. Nishida, Kyoto University) were
described previously. Recombinant GST-c-Jun-(1-79) (Dr. E. Nishida,
Kyoto University) was expressed and purified as described previously
(31).
Electrophoretic Mobility Shift Assay (EMSA)--
HEK293 cells
(4 × 106) were plated in 100-mm dishes. The following
day the cells were transfected with 5 µg of pCR-CD27 or an empty
vector using LipofectAMINE reagent (Life Technologies, Inc.) according
to a manufacturer's instruction. After 36 h, nuclear extracts
were prepared from transfected cells, and EMSA was performed as
described previously (9).
NF-
B-dependent Reporter Assays--
HEK293 cells
(1 × 106) were plated in 35-mm dishes. The following
day the cells were transfected using LipofectAMINE. Transfections included 50 ng of
-actin-
-galactosidase (Dr. K. Yokota, NIH, Japan),
-actin promoter-driven
-galactosidase expression plasmid to normalize for transfection efficiency, together with 100 ng of
reporter plasmid and various amounts of each expression vector. Total
DNA (1 µg) was kept constant by supplementation with pCR-3. A
reporter plasmid, 3x-
B-tk-luc, has three repeats of the NF-
B site
upstream of a minimal thymidine kinase promoter and a luciferase reporter gene in pGL-2 vector (Promega) (32) (Dr. M. Kashiwada, NIH,
Japan). After 24 h, the cells were harvested in phosphate-buffered saline and lysed in luciferase lysis buffer, LC-
(Piccagene). The
lysates were assayed for luciferase and
-galactosidase activities using a luminometer (Berthold).
Co-immunoprecipitation and Western Blotting--
HEK293 cells
(1.5 × 106) were plated in 60-mm dishes and
transfected with various expression vectors using LipofectAMINE. After 24-36 h, the cells were washed in ice-cold phosphate-buffered saline
and lysed for 30 min on ice in 1 ml of a lysis buffer containing 1%
Nonidet P-40, 50 mM HEPES (pH 7.3), 250 mM
NaCl, 2 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin,
1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride.
Cellular debris was removed by centrifugation, and the supernatant was
precleared with protein A-conjugated beads (Bio-Rad) for 1 to 2 h.
Cleared lysates were incubated with anti-CD27 mAb (1A4) for 1 h at
4 °C. After the addition of protein A beads, the lysates were
rotated at 4 °C for 1 h. The beads were washed three times with
the lysis buffer, and bound proteins were eluted with 1% SDS sample
buffer, subjected to 10% SDS-polyacrylamide gel electrophoresis, and
then blotted onto polyvinylidene difluoride membrane (Millipore).
Expression of transfected constructs was verified by immunoblotting of
aliquots of cell lysates. Flag-tagged TRAFs were detected using
biotin-conjugated anti-Flag mAb followed by incubation with
avidin-biotin complex (Vectastain) and enhanced chemiluminescence (ECL)
Western Blotting Detection System (Amersham Pharmacia Biotech)
according to the instructions of the manufacturer. Expression of
full-length and deletion mutants of CD27 was detected using anti-CD27
mAb followed by horseradish peroxidase-conjugated rabbit anti-mouse Ig
and ECL.
SAPK/JNK Assay--
Twenty-four hours after the transfection
with the indicated expression vectors together with pSR
-HA-SAPK,
HEK293 cells were lysed in 1 ml of a lysis buffer containing 1% Triton
X-100, 20 mM HEPES (pH 7.3), 150 mM NaCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 0.1 mM sodium orthovanadate. Lysates were then clarified by
centrifugation at 15,000 rpm for 5 min. The supernatants were
immunoprecipitated with anti-HA mAb (12CA5) for 1 h at 4 °C.
After the addition of protein A-conjugated beads, the lysates were
incubated for an additional 1 h. The immunoprecipitates were
washed three times with the lysis buffer and twice with a kinase buffer
containing 20 mM HEPES (pH 7.3), 20 mM
MgCl2, 20 mM MnCl2, 1 mM EDTA, 1 mM NaF, 0.1 mM sodium
orthovanadate, 1 mM dithiothreitol and subjected to
in vitro kinase assay. The immunoprecipitates were incubated with 1 µg of GST-c-Jun-(1-79) and [
-32P]ATP (10 µCi) in the kinase buffer for 20 min at 30 °C. The reaction was
stopped by the addition of Laemmli's sample buffer. Phosphorylated proteins were subjected to 12% SDS-polyacrylamide gel electrophoresis, visualized by autoradiography, and quantified by using an image analyzer (Fujix, BAS2000). In all cases, expression of the transfected proteins was verified by immunoblotting of aliquots of the cell lysates.
 |
RESULTS |
Overexpression of CD27 Activates NF-
B--
Transient
transfection of HEK293 cells with full-length CD27 activated a
significant amount of NF-
B as measured by DNA binding activity (Fig.
1). Supershift assay with antibodies
against each component of the NF-
B complex demonstrated that this
complex was mainly composed of p50/p65 subunits.

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Fig. 1.
NF- B activation induced by CD27.
HEK293 cells (4 × 106) were transfected with 5 µg
of pCR control (lane 1) or full-length CD27 (lane
2). Nuclear extracts were prepared 24 h after transfection,
and 5 µg of the nuclear extracts were incubated with a radiolabeled
double-stranded B oligonucleotide. Unlabeled competitor
oligonucleotide containing either B oligonucleotide (WT)
(lane 4) or random oligonucleotide (MT)
(lane 3) was added at a 50-fold molar excess. For supershift
assays, reaction mixtures were incubated with 1 µl of either
preimmune serum (lane 5), anti-p50 (lane 6), or
anti-p65 (lane 7) serum. B and F
indicate the position of the bound and free fraction,
respectively.
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|
To delineate the functional domain of CD27 for NF-
B activation, we
constructed a series of C-terminal deletion mutants (Fig. 2A) and performed a reporter
gene assay after transfection into HEK293 cells. As expected from the
result of EMSA, full-length human CD27 increased
NF-
B-dependent luciferase expression approximately 6-7-fold over the level seen in vector-transfected cells (Fig. 2B), whereas C-terminal deletion mutants CD27
5 and
CD27
8 exhibited only slight decrease in activation of NF-
B
compared with the full-length CD27. By contrast, CD27
16 failed to
activate NF-
B (Fig. 2B), suggesting that amino acids
225-232 are required for NF-
B activation. This region contains the
sequence PIQED that is similar to the TRAF binding sequence, with a
consensus sequence PXQX(T or S) found in several
other receptors, including CD30, CD40, or latent infection membrane
protein 1 (13, 15, 33). To determine if this region of CD27 was
sufficient for supporting TRAF binding, HEK293 cells were transiently
co-transfected with Flag-TRAFs and full-length or mutant CD27
cDNAs. Flag-TRAF2 and Flag-TRAF5 were efficiently co-precipitated
with CD27 and CD27
8 but not with CD27
10 or CD27
16 (Fig.
3A), consistent with their ability to activate NF-
B. That CD27
10 failed to interact with TRAFs indicated that PIQED sequence in CD27 is not sufficient to
support the interaction in a cellular context. Flag-TRAF3 only weakly
interacted with CD27 and Flag-TRAF6 did not bind at all. Immunoblot
analysis of the total cell lysates demonstrated that the expression
level of each TRAF (Fig. 3A) and CD27 mutant (Fig. 3B) was equivalent. These results indicated that CD27
interacts with TRAF2 and TRAF5, and the PIQEDYR motif in the
cytoplasmic region is responsible for TRAF binding and NF-
B
activation.

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Fig. 2.
Delineation of the region required for
NF- B activation in the cytoplasmic domain of CD27. A,
computer alignment of the C-terminal region of murine and human CD27.
The alignment was performed with GCG software. Conserved residues are
indicated with asterisks. The putative core sequence of TRAF
binding is boxed. The C-terminal ends of CD27 mutants ( 5,
8, 10, and 16) are indicated by the arrows.
B, PIQEDYR motif is required for NF- B activation. HEK293
cells were transiently co-transfected with 3x- B-tk-luc reporter gene
plasmid with 1 µg of the indicated expression vectors for full-length
or deletion mutants of CD27. After 24 h the cells were collected,
and luciferase activity was determined for each sample, and the values
were normalized to the expression of -galactosidase. The level of
induction in luciferase activity is indicated as compared with cells
transfected with the control vector. The data represent one of three
independent experiments with similar results. Values are shown as the
mean ± S.D. of triplicate samples.
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Fig. 3.
Binding of CD27 to TRAF2, TRAF3, or TRAF5.
A, HEK293 cells were co-transfected with full-length or deletion
mutants of CD27 and Flag-TRAF2, Flag-TRAF3, Flag-TRAF5, or Flag-TRAF6.
After 24 h, cell lysates were prepared and immunoprecipitated with
anti-CD27 mAb. Co-immunoprecipitated TRAFs were detected by
immunoblotting with anti-Flag mAb. Expression of Flag-TRAFs
(A) and deletion mutants of CD27 (B) was verified
by immunoblotting of the total lysates. The positions of molecular mass
standards are indicated at the left.
|
|
A Dominant Negative TRAF2 or TRAF5 Block NF-
B Activation
Induced by CD27--
To determine whether TRAF2 and TRAF5 are involved
in NF-
B activation by CD27, we tested whether N-terminal truncated
mutants of TRAF2 and TRAF5 would act as dominant negative mutants based on previous results that showed that the zinc binding domain of TRAF2
and TRAF5 is required for NF-
B activation (9, 12). Co-transfection
of CD27 and truncated TRAF2(
TRAF2:272-499) or TRAF5(
TRAF5:233-558) blocked NF-
B activation as revealed by the
reporter assay (Fig. 4A),
suggesting that these two TRAFs are required for NF-
B activation by
CD27.

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Fig. 4.
Involvement of TRAFs and NIK in CD27-mediated
NF- B activation. A, N-terminal deletion mutants of TRAF2
or TRAF5 blocked NF- B activation by CD27. 293 cells were
co-transfected with 100 ng of 3x- B-tk-luc reporter gene plasmid, 50 ng of -actin- -galactosidase, and the indicated amounts of
expression plasmids. B, NIK-KM blocks NF- B activation by
TRAF2, TRAF5, TRAF6, CD27, CD30, CD40, and LT- R. HEK293 cells were
co-transfected with 100 ng of 3x- B-tk-luc reporter gene plasmid, 50 ng of -actin- -galactosidase, and 0.5 µg of indicated expression
vectors. Data are shown as the mean ± S.D. of triplicate samples
and represent one of three independent experiments with similar
results.
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|
Involvement of NIK in TRAF5- and CD27-mediated NF-
B
Activation--
NIK has been identified as a TRAF2-interacting kinase
(24) and, thus, a likely candidate for mediating NF-
B activation by
CD27. Co-transfection of a kinase-inactive mutant NIK (NIK-KM) with
TRAF2, TRAF5, or CD27 blocked NF-
B activation (Fig. 4B). In addition, we also observed a dominant negative effect of NIK-KM on
TRAF6-, CD30-, CD40-, and LT-
R-mediated NF-
B activation (Fig. 4B). These results indicated that NIK is a common downstream
kinase of TRAF2, TRAF5, and TRAF6 for NF-
B activation in HEK293
cells.
CD27 Activates SAPK/JNK Pathway via TRAF2 and TRAF5--
It has
been shown that ligation of TNFR60 activated SAPK/JNK pathway, and this
activation is dependent on TRAF2 (20-22). Together the data suggests
that TRAF2 interacts with CD27 and thus CD27 may also activate SAPK/JNK
pathway. To test this, HEK293 cells were co-transfected with HA-tagged
SAPK and the C-terminal deletion mutants of CD27. The extent of
SAPK/JNK activity was determined by immunoprecipitation of SAPK/JNK
followed by in vitro kinase assay using GST-c-Jun-(1-79) as
a substrate. Full-length CD27, CD27
5, and CD27
8, but not
CD27
16, induced a 2.3- to 5.4-fold increase in SAPK/JNK activity
(Fig. 5A). Overexpression of
TRAF2 or TRAF5, but not TRAF3, also increased SAPK/JNK activity (Fig. 5B). These results indicated that CD27 activates SAPK/JNK
pathway, and this activation is probably mediated by TRAF2 and TRAF5.
This hypothesis was confirmed by the demonstration that N-terminal deletion mutants of TRAF2 or TRAF5 partially blocked SAPK/JNK activation by CD27 (Fig. 5C), indicating that TRAF2 and
TRAF5 are signaling molecules for SAPK/JNK activation as well as for NF-
B activation by CD27.

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Fig. 5.
CD27 activates SAPK/JNK via TRAF2 and TRAF5
SAPK/JNK activation by CD27 (A), TRAF2 and TRAF5
(B). HEK293 cells were co-transfected with 0.5 µg of
HA-SAPK and 1.5 µg of CD27, deletion mutants of CD27, Flag-TRAF2,
Flag-TRAF3, or Flag-TRAF5. HA-SAPK was immunoprecipitated, and kinase
activity was assayed as described under "Materials and Methods."
Phosphorylation of GST-c-Jun-(1-79) was quantified by an image
analyzer, and the fold increase in the kinase activity is indicated
below the autoradiograms. The expression levels of CD27 and its
deletion mutants, Flag-TRAF2, Flag-TRAF3, and Flag-TRAF5 were monitored
by immunoblotting with anti-CD27 mAb (A) or anti-Flag mAb
(B). The positions of Flag-TRAFs are indicated at the
right. C, inhibition of CD27-mediated SAPK/JNK
activation by N-terminal deletion mutants of TRAF2 ( TRAF2) or TRAF5
( TRAF5). HEK293 cells were co-transfected with 0.5 µg HA-SAPK and
1 µg of CD27 along with or without 0.5 µg of Flag- TRAF2 or
Flag- TRAF5. HA-SAPK was immunoprecipitated, and in vitro
phosphorylation of GST-c-Jun-(1-79) was performed as in A
and B. The expression levels of CD27, Flag- TRAF2, and
Flag- TRAF5 were determined by immunoblotting with anti-CD27 or
anti-Flag mAb. The positions of Flag- TRAF2 and Flag- TRAF5 are
indicated at the right.
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|
NIK Activates SAPK/JNK and Is Involved in CD27-, TRAF2-, and
TRAF5-mediated SAPK/JNK Activation--
Considering that NIK is
structurally related to mitogen-activated protein kinase kinase kinase,
we tested whether NIK activates SAPK/JNK. Overexpression of NIK, but
not a kinase-inactive mutant NIK (NIK-KM), substantially increased
SAPK/JNK activity to 6.9-fold compared with the vector-transfected
cells (Fig. 6A). We next examined the effect of NIK-KM on SAPK/JNK activation by CD27, TRAF2,
and TRAF5. As shown in Fig. 6, B and C,
co-transfection of NIK-KM partially inhibited CD27-, TRAF2-, and
TRAF5-mediated SAPK/JNK activation, suggesting that NIK is a common
downstream kinase for SAPK/JNK activation as well as for NF-
B
activation, induced by these molecules.

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Fig. 6.
Involvement of NIK in SAPK/JNK activation by
CD27, TRAF2, and TRAF5. A, activation of SAPK/JNK by NIK.
HEK293 cells were co-transfected with 0.5 µg of HA-SAPK and 1.5 µg
of Flag-NIK or Flag-NIK-KM. HA-SAPK was immunoprecipitated, and
in vitro phosphorylation of GST-c-Jun-(1-79) was performed
as in Fig. 5. The expression levels of Flag-NIK and Flag-NIK-KM were
determined by immunoblotting with anti-Flag mAb. B,
inhibition of CD27-mediated SAPK/JNK activation by NIK-KM. HEK293 cells
were co-transfected with 0.5 µg of HA-SAPK and 1 µg of CD27 along
with or without 0.5 µg of Flag-NIK-KM. HA-SAPK was
immunoprecipitated, and in vitro phosphorylation of
GST-c-Jun-(1-79) was performed as in Fig. 5. The expression levels of
CD27 and Flag-NIK-KM were determined by immunoblotting with anti-CD27
or anti-Flag mAb. C, inhibition of TRAF2- and TRAF5-mediated
SAPK/JNK activation by NIK-KM. HEK293 cells were co-transfected with
0.3 µg of HA-SAPK and 1 µg of Flag-TRAF2 or Flag-TRAF5 along with
or without 1 µg of Flag-NIK-KM. HA-SAPK was immunoprecipitated, and
in vitro phosphorylation of GST-c-Jun-(1-79) was performed
as in Fig. 5. The expression levels of Flag-TRAF2, Flag-TRAF5, and
Flag-NIK-KM were determined by immunoblotting with anti-Flag mAb. The
positions of Flag-tagged proteins are indicated at the
right.
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 |
DISCUSSION |
Accumulating data have indicated important roles of TRAFs in
signaling through certain members of the TNF-R superfamily. Here, we
demonstrated that CD27 associates with TRAF2 and TRAF5, and these TRAFs
are implicated in NF-
B and SAPK/JNK activation by CD27. Our results
suggest that other members of the TNF-R superfamily, such as 4-1BB and
OX40, may also interact with TRAFs and activate NF-
B and SAPK/JNK
pathways. We also demonstrated that a kinase-inactive mutant NIK
blocked TRAF5- and TRAF6-mediated NF-
B activation, indicating that
NIK is a common downstream kinase of TRAF2, TRAF5, and TRAF6 for
NF-
B activation. Previous studies (20, 34) implied that
mitogen-activated protein kinase/ERK kinase kinase-1 may be responsible
for TNF- and TRAF-mediated SAPK/JNK activation. Here we demonstrated
that NIK also activates SAPK/JNK pathway and a kinase-inactive mutant
NIK partially inhibited SAPK/JNK activation by TRAF2, TRAF5, and CD27.
The dominant negative effect of NIK-KM on SAPK/JNK activation by CD27
and TRAFs appeared to be weaker than that on NF-
B activation. These
results suggest that other mitogen-activated protein kinase kinase
kinases, such as mitogen-activated protein kinase/ERK kinase kinase-1,
could compensate the dominant negative effect of NIK-KM on SAPK/JNK activation. The mechanism by which NIK activates SAPK/JNK pathway is
currently under investigation. A previous study showed that CD27
cross-linking induced tyrosine phosphorylation of several signal
transducing molecules including ZAP70, and the treatment with a Src
family kinase inhibitor, herbimycin, completely blocked co-stimulatory
signal through CD27 (2). Collectively, these data suggest that CD27
activates two distinct signaling pathways, one is protein tyrosine
kinase-dependent and another is TRAF-dependent pathway, the latter of which activates NF-
B and SAPK/JNK.
To date, it has been shown that CD27, CD30, CD40, LT-
R, and HVEM
interact with both TRAF2 and TRAF5 (9, 15, 16, 26, 27). A common
consequence of signaling through these receptors is the activation of
NF-
B that can now be explained by their use of a similar set of TRAF
proteins. However, these receptors display unique functions in
vivo at several levels. For example, CD40 is involved in class
switching and proliferation in B cells (35), and CD27 enhances IgG
production in B cells (3). CD30 is involved in some types of negative
selection in the thymus (36), and LT-
R is implicated in lymph node
development (37, 38). The unique functions in vivo may
result in part from the different tissue expression patterns, in
combination with the distinct expression patterns of their cognate
ligands, as well as for each TRAF. Indeed, follicular dendritic cells,
which play an important role in germinal center formation, exhibit high
expression of LT-
R and TRAF5 (39) but little or no detectable TRAF2.
The absence of LT-
R on lymphocytes distinguishes this receptor from lymphocyte-specific CD27. Clearly, TRAF2 and TRAF5 have some redundant functions in these tissue culture models; however, other interacting molecules may distinguish the roles of TRAF2 and TRAF5 in
vivo. For example, TRAF5 does not interact with the TRAF2-binding
proteins, TRADD2 or IAPs
(40). On the other hand, TRAF5 interacts with TRAF3, but TRAF2 does
not.2 These results suggested that TRAF2 and TRAF5 could
exert unique functions through interaction with distinct sets of
downstream signaling molecules. The recent generation of
TRAF2-deficient mice revealed that SAPK/JNK activation, but not NF-
B
activation, by TNF is abrogated in these mice (41). Our preliminary
results showed that a truncated TRAF5 partially inhibited NF-
B
activation by TNF, suggesting that TRAF2 and TRAF5 may act redundantly
in NF-
B activation by TNF. In contrast, SAPK/JNK activation may be
more efficiently mediated by TRAF2 than TRAF5. Consistent with this
notion, a higher potential of TRAF2 to activate SAPK/JNK (Fig.
5B) and truncated TRAF2 to inhibit CD27-mediated SAPK/JNK activation were noted (Fig. 5C). At this moment, it remains
to be determined whether TRAF2 and TRAF5 are functionally redundant in
signals via CD27 and other members of the TNF-R family in TRAF2- and
TRAF5-deficient mice.
 |
ACKNOWLEDGEMENTS |
We thank Masaki Kashiwada, Kyoko Yokota,
Hitoshi Kikutani, Toshiki Watanabe, Eisuke Nishida, George Mosilaos,
and Elliott Kieff for reagents. We also thank Sachiko Sakon for
technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Education, Science, and Culture and the Ministry of Health, Japan. and
the Uehara Memorial 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: Dept. of Immunology,
Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo
113, Japan. Tel.: 81-3-5802-1045; Fax: 81-3-3813-0421; E-mail: hnakano{at}med.juntendo.ac.jp.
1
The abbreviations used are: TNF, tumor necrosis
factor; TNF-R, TNF receptor; TRAF, TNF receptor-associated factor;
LT-
R, lymphotoxin-
receptor; HEK, human embryonic kidney; HVEM,
herpesvirus entry mediator; SAPK, stress-activated protein kinase; JNK,
c-Jun N-terminal kinase; NIK, NF-
B-inducing kinase; EMSA,
electrophoretic mobility shift assay; mAb, monoclonal antibody; HA,
hemagglutinin; PCR, polymerase chain reaction.
2
H. Akiba and H. Nakano, unpublished data.
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