Activation of NF-
B by RANK Requires Tumor Necrosis Factor
Receptor-associated Factor (TRAF) 6 and NF-
B-inducing Kinase
IDENTIFICATION OF A NOVEL TRAF6 INTERACTION MOTIF*
Bryant G.
Darnay
,
Jian
Ni§,
Paul A.
Moore§, and
Bharat B.
Aggarwal
¶
From the
Cytokine Research Laboratory,
Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and § Human
Genome Sciences, Inc., Rockville, Maryland 20850
 |
ABSTRACT |
Various members of the tumor necrosis factor
(TNF) receptor superfamily activate nuclear factor
B (NF-
B) and
the c-Jun N-terminal kinase (JNK) pathways through their interaction
with TNF receptor-associated factors (TRAFs) and NF-
B-inducing
kinase (NIK). We have previously shown that the cytoplasmic domain of
receptor activator of NF-
B (RANK) interacts with TRAF2, TRAF5, and
TRAF6 and that its overexpression activates NF-
B and JNK pathways.
Through a detailed mutational analysis of the cytoplasmic domain of
RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF
binding motifs located in the C terminus at positions 565-568 and
606-611, respectively. In contrast, TRAF6 interacts with a novel motif
located between residues 340 and 358 of RANK. Furthermore, transfection
experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340-358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-
B activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-
B activation. However,
RANK-mediated JNK activation required a distal portion (427-603) of
RANK containing the TRAF2-binding domain. Thus, our results indicate
that RANK interacts with various TRAFs through distinct motifs and
activates NF-
B via a novel TRAF6 interaction motif, which then
activates NIK, thus leading to NF-
B activation, whereas RANK most
likely activates JNK through a TRAF2-interacting region in RANK.
 |
INTRODUCTION |
RANK1 (for
receptor activator of
NF-
B), a new member of the tumor necrosis
factor (TNF) receptor superfamily, is a 616-amino acid receptor that
includes a 383-amino acid intracellular domain with no significant
homology to other members of this family (1). Although RANK is
ubiquitously expressed in human tissues, its cell surface expression is
limited to dendritic cells, the CD4+ T cell line MP-1, and foreskin
fibroblasts (1, 2). Human RANK ligand (RANKL/TRANCE/OPGL/ODF), a type
II transmembrane protein with an approximate molecular mass of 45 kDa,
is expressed primarily on primary T cells, T cell lines, and lymphoid
tissue (1, 3-5). Like other ligands of the TNF superfamily, RANKL has
been demonstrated to activate nuclear factor
B (NF-
B) (1) and
c-Jun-terminal kinase (JNK) (3). Furthermore, stimulation of dendritic
cells with RANKL up-regulates the expression of the
anti-apoptotic protein Bcl-XL, suggesting a potential
role for RANK/RANKL in dendritic cell survival (2). Moreover, RANKL has
been demonstrated to play an essential role in osteoclast
differentiation and activation (4, 5).
Many of the TNF receptor superfamily members interact with a family of
adaptor proteins referred to as TNF receptor-associated factors
(TRAFs), which are characterized by a ring and zinc finger motif in
their N termini and C-terminal domains that appear to be responsible
for self- and non-self associations (6). Of the six known TRAF family
members, only TRAF2, TRAF5, and TRAF6 activate NF-
B and JNK (7), and
only TRAF2 has been demonstrated to activate p38 kinase (8, 9). TRAF1,
TRAF2, and TRAF5 interact with a characteristic TRAF binding motif,
PXQXT, in the cytoplasmic domain of several
members of the TNF receptor family (10-16). TRAF6 interacts with the
cytoplasmic domain of RANK (16) and with CD40 via a distinct 16-amino
acid region (residues 230-245) (11). Furthermore, TRAF6 interacts with
interleukin-1 receptor-associated kinase 1 and 2 (IRAK1 and IRAK2)
(17-19).
Besides TRAFs, the activation of NF-
B is also mediated through a
recently identified novel member of the mitogen-activated protein
kinase kinase kinase family termed NF-
B-inducing kinase (NIK) (20).
NIK was originally identified as a TRAF2-interacting protein (20) and
subsequently was found to interact with all TRAF molecules, except
TRAF4 (7). When overexpressed in cultured cells, NIK, but not a
kinase-inactive mutant (NIK-KM), activates NF-
B (7, 15, 20) and JNK
(15, 21). Furthermore, overexpression of NIK-KM inhibits NF-
B
activation by TNF, interleukin-1, CD27, human T-cell leukemia virus
type 1 TAX, and Epstein-Barr virus-transforming protein latent
infection membrane protein 1 (7, 15, 20, 22-24). Consequently, the
activation of NF-
B by NIK is mediated through its interaction with
the I
B
kinase (IKK
and IKK
) complex (25-28), which results
in the phosphorylation and degradation of I
B
.
Previous studies from our laboratory showed that the cytoplasmic domain
of RANK interacts with TRAF2, TRAF5, and TRAF6 and that its
overexpression activates NF-
B and JNK pathways (16). However, it is
not known whether these TRAFs bind to the same region of RANK or which
TRAF or TRAFs are necessary for activation of NF-
B and JNK.
Similarly, it is not known whether NIK is involved in RANK-induced
NF-
B activation. In addressing the role of various TRAFs and NIK in
NF-
B and JNK activation mediated by RANK, we now demonstrate
that RANK activates NF-
B by interacting with TRAF6 via a novel TRAF6
interaction motif and TRAF6 potentially activates NIK, leading to
NF-
B activation, whereas RANK activates JNK through a
TRAF2-interacting region in RANK.
 |
EXPERIMENTAL PROCEDURES |
Reagents, Cell Lines, and Antibodies--
Human embryonic kidney
293 cell line were obtained from the American Type Culture Collection
(Rockville, MD) and cultured in minimal essential medium supplemented
with 10% fetal bovine serum and antibiotics. Monoclonal antibody to
Myc (SC40) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA);
anti-HA from Boehringer Mannheim (Indianapolis, IN); goat anti-rabbit
IgG-conjugated horseradish peroxidase from Bio-Rad (Hercules, CA);
anti-FLAG (monoclonal antibody M2) from Eastman Kodak Co. (New Haven,
CT); goat anti-mouse IgG-conjugated horseradish peroxidase from
Transduction Laboratories (Lexington, KY); protein A/G-Sepharose beads
from Pierce (Rockford, IL); and the alkaline phosphatase fluorescent
substrate 4-methylumbelliferyl phosphate (M3168) from Sigma.
Expression Plasmids--
Expression plasmids encoding mouse
FLAG-tagged TRAF5 and TRAF6 (15) were generously provided by H. Nakano
(Juntendo University, Tokyo, Japan). Expression plasmids encoding
FLAG-tagged NIK and NIK (KK429-430AA) (2) were kindly provided by D. Wallach (Weizmann Institute of Science, Rehovot, Israel). An expression
plasmid encoding pSR
-HA-JNK1
was kindly provided by F.-X. Claret
(University of Texas M. D. Anderson Cancer Center, Houston, TX).
Expression plasmids encoding Myc-tagged TRAF2 and FLAG-tagged
C-terminal truncated versions of RANK (RANK616, -530, -427, and -330)
were prepared as described previously (16). To generate deletion mutants of the cytoplasmic domain of RANK as glutathione
S-transferase (GST) fusion proteins, specific 5' and 3'
primers containing EcoRI and SalI sites,
respectively, were used in PCR reactions with pSPORT3.0-TR8 (16). The
resulting PCR products were digested with
EcoRI/SalI and cloned in-frame with GST in
pGEX-KG (29). To generate cytoplasmic deletion mutants of FLAG-tagged
RANK, a parental plasmid containing the extracellular and transmembrane domains of RANK (residues 33-240) was first amplified by specific 5'
and 3' primers containing HindIII and EcoRI,
respectively, and cloned in-frame with the FLAG epitope in pCMVFLAG1,
resulting in pF-RANK241, which contains an in-frame stop codon 4 residues downstream of residue 240. All the RANK deletion mutants were prepared from the same PCR products utilized for subcloning into pGEX-KG and then subcloned into pF-RANK241 at the
EcoRI/SalI site. Due to the subcloning at the
EcoRI site, all FLAG-tagged RANK deletion mutants contained
homologous amino acid substitutions (i.e.
Ala241-Leu242 to
Gly241-Ile242). These substitutions, however,
did not effect the ability of RANK to activate signaling cascades (see
"Results"). The sequence of all plasmids were verified by automated
DNA sequencing. Expression and purification of GST-Jun(1-79) and the
GST-RANK deletion mutant fusion proteins were essentially as described
(30).
Transient Transfections and Western Blotting--
Human
embryonic 293 cells (0.6 × 106 cells/well on 6-well
plates) were plated and transfected as described (16). The total amount
of plasmid DNA was kept constant by addition of the control plasmid
pCMVFLAG1. Cells and the conditioned supernatants were harvested 24-36
h after transfection. Lysates were prepared as described (16). For
Western blot analysis whole cell lysates (15-30 µg) or proteins from
GST affinity precipitations were separated by 8.5% SDS-PAGE,
electroblotted onto nitrocellulose membranes (Bio-Rad), and incubated
with the indicated antibodies. The membranes were then developed using
the enhanced chemiluminescence (ECL) system (Amersham).
GST-RANK Fusion Protein Affinity Binding Assays--
Equivalent
amounts of each GST-RANK fusion protein attached to 20 µl of
glutathione-agarose beads were mixed with lysates (50 µg) from 293 cells programmed to express the epitope-tagged TRAF protein in binding
buffer (20 mM Tris, pH 8, 150 mM NaCl, 1 mM dithiothreitol, 2 mM EDTA, and 0.1% Nonidet
P-40) and allowed to rotate for 1 h at 4 °C. The beads were
collected by centrifugation, washed three times in binding buffer, and
then washed once in low-salt buffer (20 mM Tris, pH 8, 50 mM NaCl, and 1 mM dithiothreitol). Bound
proteins were eluted with addition of SDS sample buffer and boiled. The
eluted proteins were subjected to 7.5% SDS-PAGE and Western blot
analysis was performed with either anti-Myc (TRAF2) or anti-FLAG (TRAF5
and TRAF6) as indicated in the figure legend.
JNK Kinase Assays--
From transiently transfected 293 cells,
lysates were prepared. Approximately 120 µg was then used for
immunoprecipitation with anti-HA and protein A/G-Sepharose beads for
1 h. Beads were collected by centrifugation, washed three times in
lysis buffer, and then washed two times in low-salt buffer. JNK
activity was analyzed using exogenous GST-Jun(1-79) as a substrate as
described previously (16). JNK activity was quantitated using a
PhosphorImager and Imagequant Software (Molecular Dynamics, Sunnyvale,
CA). To verify equal transfection, lysates from the transiently
transfected cells were subjected to Western blotting with anti-HA.
NF-
B SEAP Reporter Assays--
To construct a synthetic
NF-
B-containing promoter element, a PCR-based strategy was used. The
upstream primer
(5'-GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG-3') contained four tandem copies of the NF-
B-binding site
(GGGGACTTTCCC) and 18 base pairs of a sequence complementary to the 5'
end of the SV40 early promoter sequence, and was flanked with an
XhoI site. The downstream primer
(5'-GCGGCAAGCTTTTTGCAAAGCCTAGGC-3') was complementary to the 3' end of
the SV40 promoter and was flanked with a HindIII site. PCR
was performed using the SV40 promoter template. The resulting PCR
fragment was digested with XhoI/HindIII and
subcloned into a likewise digested SEAP2-promoter plasmid to replace
the SV40 minimal promoter element.
Human embryonic 293 cells were transiently transfected with
pNF-
B-SEAP2 (0.5 µg) and the expression plasmids as indicated in
the figure legends. After transfection (24-36 h), the conditioned medium was removed and assayed for SEAP activity essentially as described by the manufacturer (CLONTECH, Palo Alto,
CA). In brief, medium (25 µl) was mixed with 30 µl of 5 times
buffer (500 mM Tris, pH 9, and 0.5% bovine serum albumin)
in a total volume of 100 µl in a 96-well plate and incubated at
65 °C for 30 min. The plate was chilled on ice for 2 min. Then 50 µl of 1 mM 4-methylumbelliferyl phosphate was added to
each well and incubated at 37 °C for 2 h. The activity of SEAP
was assayed on a 96-well fluorescent plate reader (Fluoroscan II, Lab
Systems, Needham, Heights, MA) with excitation set at 360 nm and
emission at 460 nm. The average (±S.D.) number of relative fluorescent
light units for each transfection was then determined and reported as
fold activation with respect to control vector-transfected cells. The
NF-
B SEAP reporter used in these assays was shown to be activated by
TNF and by overexpression of
TNFR2.2 Similar to the case
in previously published reports (7, 9, 20), the specificity was
established of this reporter system by the fact that TNF-induced
NF-
B SEAP activity was inhibited by overexpression of either an
I
B
mutant lacking Ser32/36, a kinase-inactive NIK, or
a dominant negative TRAF2 mutant.2
 |
RESULTS |
In previous studies, we found that the intracellular domain
(residues 234-616) of RANK contains three putative TRAF binding motifs
of the sequence PXQXT: two located at the C
terminus and one localized in the middle of the cytoplasmic domain
(16). We also previously reported that TRAF2, TRAF5, and TRAF6 interact with RANK (16) and that overexpression of RANK in 293 cells activates
the NF-
B and JNK pathways (1, 16). Therefore, in the present study,
we sought to identify more specifically which regions of RANK are
responsible for the activation of NF-
B and JNK and, furthermore, to
define which TRAF molecules are responsible for these signaling pathways.
Different Regions of RANK Are Responsible for Binding TRAF2, TRAF5,
and TRAF6--
As we previously reported (16), RANK contains three
putative TRAF binding motifs (Fig.
1A) and RANK interacts with
TRAF2, TRAF5, and TRAF6. To identify which region of the cytoplasmic domain of RANK is necessary for binding TRAF2, TRAF5, and TRAF6, we
constructed a series of deletion mutants of the cytoplasmic domain of
RANK encompassing the various putative TRAF-binding domains (Fig.
1A). Each of these deletion mutants were fused in-frame with
GST and purified by glutathione-agarose affinity chromatography. We
examined the ability of each GST-RANK fusion protein to precipitate epitope-tagged TRAF2, TRAF5, and TRAF6 upon their overexpression in 293 cells (Fig. 1B). We observed strong interaction of TRAF2 and
TRAF5 with GST-RANK fusion proteins containing residues 529-616. However, while TRAF2 was still capable of binding to GST-RANK fusion
proteins lacking the last 13 amino acids, TRAF5 was not (Fig.
1B, top and middle). These data
suggest that TRAF-binding domain III is responsible for TRAF5
interaction and that both TRAF II and TRAF III binding motifs are
required for high-affinity binding of TRAF2, but the TRAF III binding
motif is not essential for RANK's interaction with TRAF2. Unlike TRAF2
and TRAF5, TRAF6 did not interact with TRAF-binding domains II and III
(Fig. 1B, bottom). This is consistent with a data
indicating that TRAF6 does not bind to the PXQXT
motif (11). Surprisingly, TRAF6 did bind to RANK between residues 326 and 427 (Fig. 1B, bottom). Conversely, GST-RANK
deletion mutants that did not contain residues 326-427 did not bind
TRAF6 (Fig. 1, A and B). Inspection of the amino acid sequence between residues 326 and 427 revealed a putative TRAF6
binding motif (see below). Hence, the cytoplasmic domain of RANK
appears to interact with TRAF2, TRAF5, and TRAF6 molecules using three
distinct motifs.

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Fig. 1.
TRAF2, TRAF5, and TRAF6 associate with
different regions of RANK. A, diagrammatic
representation of GST-RANK fusion proteins. All GST-RANK deletion
mutants were fused in-frame with GST in the expression vector pGEX-KG
as described under "Experimental Procedures." The Roman numerals
I, II, and III represent putative TRAF-binding
domains within the cytoplasmic domain of RANK (RANK-CD). B,
distinct regions of RANK interact with TRAF2, TRAF5, and TRAF6.
Approximately equal amounts of GST and each GST-RANK deletion mutant (5 µg) were mixed with lysates (50 µg) from 293 cells that were
transiently transfected with Myc-tagged TRAF2 (top),
FLAG-tagged TRAF5 (middle), or FLAG-tagged TRAF6
(bottom), and binding was performed as described under
"Experimental Procedures." Beads were collected and washed, bound
proteins were eluted with SDS sample buffer. Samples were subjected to
7.5% SDS-PAGE, and co-precipitating Myc-TRAF2, FLAG-TRAF5, and
FLAG-TRAF6 were detected by Western blotting with the indicated
monoclonal antibody.
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A Minimal Region of RANK (Residues 326-427) Activates
NF-
B--
How interactions of different TRAFs with RANK affect
RANK's ability to activate NF-
B and JNK is not known. To examine
this, we constructed FLAG-tagged RANK deletion mutants (identical to those deletion mutants used to construct GST-RANK) in pCMVFLAG1 (Fig.
2A). Their expression was
determined by transient transfection in 293 cells and Western blotting
with anti-FLAG (Fig. 2B). As expected, all of the
FLAG-tagged RANK deletion mutants were expressed similarly in 293 cells.

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Fig. 2.
Schematic diagram and expression of
FLAG-tagged RANK and its deletion mutants. A,
diagrammatic representation of RANK and its deletion mutants. All RANK
deletion mutants were fused with a FLAG epitope tag at the N terminus
using the signal sequence in the expression vector pCMVFLAG1 as
described under "Experimental Procedures." RANK-616, -530, -427, and -330 were previously described (16). The Roman numerals I,
II, and III represent putative TRAF-binding domains
within the cytoplasmic domain of RANK. ED, extracellular
domain; TM, transmembrane domain; CD, cytoplasmic
domain. B, expression of FLAG-tagged RANK and its deletions.
Human embryonic 293 cells on 6-well plates were transiently transfected
with the indicated RANK expression vectors (2.5 µg) using a total of
3 µg of plasmid DNA. After 24 h, cell lysates were prepared and
subjected to SDS-PAGE and Western blotting with an anti-FLAG monoclonal
antibody as described under "Experimental Procedures."
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Next, we examined the ability of each RANK deletion mutant to activate
a NF-
B-dependent SEAP reporter construct. Transient overexpression of RANK616 in 293 cells activated
NF-
B-dependent reporter activity (Fig.
3A), which could be inhibited
by co-transfection of an I
B
mutant lacking its N-terminal
phosphorylation sites (data not shown). Deletion of the C-terminal
region up to residue 427 (RANK427) had no effect on
NF-
B-dependent reporter activity, but further deletion
to residue 330 (RANK330) failed to activate NF-
B (Fig.
3A). Furthermore, when only the C-terminal region was fused
to the transmembrane domain of RANK, NF-
B-dependent activity was either very weak (RANK429-616) or failed to respond (RANK529-616) (Fig. 3A), although each of the deletion
mutants RANK429-616 and RANK529-616 interacted strongly with TRAF2
and TRAF5 (Fig. 2). Truncation of the TRAF5-binding region (RANK603 and
RANK326-603) did not appear to affect NF-
B-dependent
reporter activity. Together, these data indicate that residues 326-427 are responsible for activation of NF-
B. This was further confirmed by transfection of a deletion mutant, containing only residues 326-427
fused to the transmembrane region of RANK, which activated NF-
B-dependent reporter activity similar to that of
RANK616 (Fig. 3A). The observations that some RANK deletion
mutants (i.e. R530) activate NF-
B stronger than the
full-length suggest that other factors may regulate RANK signaling such
as cell surface receptor expression, other receptor-associated factors,
and receptor processing. Nevertheless, taken together, these data
suggest that the interaction of TRAF2 and TRAF5 with RANK is not
required for RANK-induced NF-
B, but that the interaction of TRAF6
with RANK is necessary and sufficient for mediating NF-
B activation
by RANK.

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Fig. 3.
A minimal region of RANK (residues 326-427)
activates NF- B-dependent SEAP
reporter activity via NIK. A, localization of a minimal
region of RANK for NF- B activation. Human 293 cells were transiently
transfected with 3 µg of total plasmid DNA in duplicate with
pNF- B-SEAP (0.5 µg) and the indicated RANK expression plasmids
(2.5 µg) as described under "Experimental Procedures." At 36 h post-transfection, the conditioned medium was assayed for SEAP
activity as described under "Experimental Procedures." Lysates were
prepared and Western blotting with anti-FLAG was performed to verify
receptor expression. The results are representative of at least six
independent transfection experiments with similar results.
B, inhibition of RANK-induced NF- B activation by NIK-KM.
Human 293 cells transiently transfected with 5 µg of total plasmid
DNA were transfected in duplicate with pNF- B-SEAP (0.5 µg),
pFLAG-NIK or pFLAG-NIK (KM) (1.5 µg), and the indicated RANK
expression plasmids (1 µg) as described under "Experimental
Procedures." At 36 h post-transfection, the conditioned medium
and cells were processed as described in A. The data are
representative of at least three independent transfection experiments
that produced similar results.
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A Kinase-inactive NIK Inhibits NF-
B-dependent SEAP
Activity Induced by RANK and RANK326-427--
When transiently
overexpressed in cultured cell lines, NIK, but not a kinase-inactive
mutant (NIK-KM), activates NF-
B (7, 15, 20) (Fig. 3B), while NIK-KM
inhibits TNF-induced NF-
B dependent reporter activity (7, 20) (data
not shown). We thus examined the effect of NIK-KM on RANK616- and
RANK326-427-induced NF-
B reporter activity. Co-transfection of
NIK-KM with RANK616 or RANK326-427 inhibited
NF-
B-dependent reporter activity (Fig. 3B).
Collectively, these data indicate that RANK activates NF-
B via
residues 326-427, which interacts with TRAF6. Since TRAF6 has been
demonstrated to interact with NIK (7), RANK most likely utilizes the
TRAF6-NIK pathway for activation of NF-
B.
TRAF2-binding Domain of RANK Is Required for JNK
Activation--
Transient overexpression of RANK in 293 cells (16) or
treatment of T cells with RANKL (3) has been demonstrated to activate the JNK pathway. We therefore examined the ability of each RANK deletion mutant to activate co-transfected HA-JNK1. RANK616 activated JNK strongly, RANK427 and RANK530 activated JNK marginally, while RANK330 failed to activate JNK (Fig.
4A). Similar to our results in
the NF-
B-dependent reporter assay, the C-terminal region
of RANK (residues 529-616) failed to activate JNK. Moreover,
truncation of the TRAF5-binding domain, residues 604-616 (RANKE603),
had no effect on JNK activation, which suggests that RANK's
interaction with TRAF5 is not required for JNK activation. The
inability of these deletion mutants to activate JNK was not due to a
lack of expression of transfected HA-JNK (Fig. 4B).
Furthermore, unlike NF-
B activation by RANK, truncation of the
TRAF2-binding domain (i.e. RANK326-427 and RANK326-530)
reduced JNK activation by 3-fold when compared with RANKE616 (Fig.
4A). These data suggest that, unlike the TRAF6-binding
domain of RANK, which is required for NF-
B activation, the
TRAF2-binding domain is required but not sufficient for activation of
JNK.

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Fig. 4.
The region in RANK that activates JNK
overlaps the region in RANK necessary for induction of
NF- B. Panels A and
B, immune complex kinase assays of HA-JNK1 induced by
RANK. Human 293 cells transiently transfected with 5 µg of total
plasmid DNA were transfected with pNF- B-SEAP (0.5 µg),
pSR -HA-JNK1 (0.5 µg), and the indicated RANK expression
plasmids (1.5 µg) as described under "Experimental Procedures."
At 36 h post-transfection, the conditioned medium and cells were
collected and JNK kinase assays (A) and Western blotting
with anti-HA (B) were performed as described under
"Experimental Procedures." The data are representative of at least
three independent transfection experiments that produced similar
results.
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Identification of a Novel TRAF6 Binding Motif in RANK--
Of all
the members of the TNF receptor superfamily, only CD40 (11) and RANK
(16) bind directly to TRAF6. Interestingly, the interleukin-1 receptor
interacts indirectly with TRAF6 via its association with IRAK1 and
IRAK2, which bind the adaptor protein MyD88 (17-19). Unlike TRAF1,
TRAF2, and TRAF5, which interact with receptors through a common
PXQXT motif (10-16), no known binding motif has
been described for TRAF6. However, deletion analysis of the cytoplasmic
domain of CD40 has implicated a region between residues 230 and 245 of
CD40 that interacts with TRAF6 (11). Inspection of this sequence in
CD40, residues 326-427 of RANK, and the C terminus of IRAK1 and IRAK2
has revealed a putative TRAF6 binding motif (Fig.
5A). Alignment of these
protein sequences suggests that TRAF6 interacts with a consensus
sequence having the characteristic pattern, basic
QXPXE acidic (Fig. 5A).

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Fig. 5.
Identification of a novel TRAF6 binding
motif. A, alignment of the amino acid sequences of four
proteins that interact with TRAF6. The region within the amino acid
sequence of CD40 (residues 230-245) that specifically interacts with
TRAF6 (11) was used as the template for alignment of RANK, IRAK1, and
IRAK2. Homologous residues are outlined in bold and
shaded. A consensus motif is shown on the bottom.
Bc, basic residue; Ac, acidic residue. B,
diagrammatic representation of additional RANK deletion mutants. All
RANK deletion mutants were constructed as described in the legends to
Figs. 1A and 2A. C, a minimal region
of RANK interacts with TRAF6. Approximately equal amounts of GST and
each GST-RANK deletion mutant (5 µg) were mixed with lysates (50 µg) from 293 cells that were transiently transfected with FLAG-tagged
TRAF6, and binding was performed as described under "Experimental
Procedures." D, localization of the minimal region of RANK
for NF- B activation. Human 293 cells were transiently transfected
with 2.5 µg of total plasmid DNA in duplicate with pNF- B-SEAP (0.5 µg) and the indicated RANK expression plasmids (0.1 µg) as
described under "Experimental Procedures." At 24 h
post-transfection, the conditioned medium was assayed for SEAP activity
as described under "Experimental Procedures." The results are
representative of at least three independent transfection experiments
with similar results.
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To further confirm that this putative TRAF6 interaction motif does
indeed bind TRAF6, we constructed various deletion mutants between
residues 326 and 427 (Fig. 5B). As stated above, each of the
deletion mutants was constructed as a GST fusion protein and as a
FLAG-tagged receptor fused to the transmembrane and extracellular region of RANK. First we examined the ability of these GST-RANK fusion
proteins to precipitate epitope-tagged TRAF6 upon its overexpression in
293 cells. In contrast to RANK330 which did not bind to TRAF6 (Fig. 1B), RANK358 interacted strongly with TRAF6
implicating residues between 330 and 358 for binding TRAF6 (Fig.
5C). Furthermore, we observed strong interaction of TRAF6
with all of the deletion mutants shown in Fig. 5B, except
for RANK358-427, which failed to bind TRAF6 (Fig. 5C).
Moreover, a GST-RANK fusion protein containing only residues 340-358
interacted with TRAF6 (Fig. 5C). Overall, these data support
our observation that TRAF6 interacts with RANK via a novel motif
located between residues 340 and 347.
Next, we examined whether this putative TRAF6 interaction motif is
sufficient to activate a NF-
B-dependent SEAP reporter construct. Transient overexpression of RANK616, RANK326-427, and RANK358 activated NF-
B-dependent reporter activity,
while RANK330 and RANK358-427 failed to activate NF-
B (Figs.
3A and 5D). These data suggest that residues
located between 330 and 358 are critical for NF-
B activation by
RANK, which was further confirmed by a RANK deletion mutant containing
only residues 326-358 (Fig. 5D). Furthermore, a RANK
deletion mutant containing only 19 residues (340-358) linked to the
transmembrane and extracellular domain of RANK was sufficient to
activate NF-
B-dependent reporter activity, which was not
further increased by addition of residues 358-427 (i.e.
RANK340-427) (Fig. 5D). The ability of these RANK deletion mutants to activate NF-
B is consistent with their ability to interact with TRAF6, supporting the notion that RANK activates NF-
B
via TRAF6. Thus, as with the PXQXT TRAF binding
motif, this putative TRAF6 binding motif (Fig. 5A) may prove
useful in identifying other proteins and receptors that could
potentially interact with TRAF6.
 |
DISCUSSION |
Most members of the TNF receptor superfamily activate NF-
B and
JNK via their association with TRAF molecules. With the exception of
TRAF4, which has no known function, various members of this receptor
family, most notably CD30, CD40, CD27, LT
receptor, TNFR2, and RANK,
interact directly with more than one TRAF family member (10, 12, 15,
16, 31-33). Although it is not fully understood how each of these TRAF
molecules participates in signaling by these receptor family members,
it appears that only TRAF2, TRAF5, and TRAF6 are functionally competent
to activate downstream signaling pathways (7).
We have demonstrated that TRAF2, TRAF5, and TRAF6 interact with RANK
via three distinct motifs in the cytoplasmic domain of RANK.
Furthermore, our data suggest that NF-
B activation by RANK is
dependent upon its interaction with TRAF6, while JNK activation by RANK
is dependent in some way on TRAF2. This is consistent with the
observation in mouse thymocytes that RANKL could activate JNK and that
this activation could be inhibited by transgenic expression of a
dominant negative TRAF2 (2). The significance of TRAF5 interaction with
RANK is not clear. Moreover, on the basis of previous deletion studies
with CD40 (11) and our deletion studies of RANK reported here, we have
identified a novel TRAF6 binding motif (Fig. 5A) that is
also present in two other TRAF6-interacting proteins, IRAK1 and IRAK2
(17-19). Whether the TRAF6-binding region in RANK is required for
other biological signaling events in addition to NF-
B activation is
unclear; however, it is likely that more than one signal could emanate
from different regions of RANK and could cooperate to induce biological responses.
While our report was under review, a similar study was published
indicating the interaction of various TRAF molecules with the mouse
homolog of RANK (34). Consistent with our data, that study demonstrated
that the C terminus of mouse RANK interacts with TRAF2 and TRAF5, but
TRAF6 appears to interact near the middle and N terminus of the
cytoplasmic domain of RANK. Furthermore, their data implicated the N
terminus and middle regions of RANK for activation of NF-
B, although
the C terminus of RANK could activate NF-
B, albeit weakly. In
addition, these authors co-transfected dominant negative mutants of
TRAF2, TRAF5, and TRAF6 with RANK to examine whether these TRAFs could
block RANK-induced NF-
B activation. Similar to our
observations,2 these authors were unable to completely
inhibit NF-
B activation by coexpression of dominant negative
versions of these TRAF molecules (34). Moreover, besides activation of
NF-
B, transient expression of RANK also activated c-Jun- and
Elk-1-dependent transcriptional activity (34). These data
would suggest that like CD30 (35), RANK may activate TRAF-independent
signaling pathways or interact with unknown TRAF-like molecules to
activate NF-
B and possibly other transcription factors.
Overall, we have demonstrated the ability of RANK to interact with
various TRAF molecules through distinct motifs in the cytoplasmic domain of RANK. That more than one TRAF molecule interacts with RANK
may suggest a cell-type- and TRAF-dependent signaling
cascade initiated by RANK. To date, RANK activates at least three
transcription factors, i.e. NF-
B, c-Jun, and Elk-1, which
may be linked to the expression of anti-apoptotic genes in dendritic
cells and the expression of genes involved in differentiation of
osteoclasts. The identification of these genes may lead to the
understanding of the function of RANKL/RANK in dendritic cells and osteoclasts.
 |
ACKNOWLEDGEMENTS |
We thank Drs. H. Nakano, D. Wallach, and
F.-X. Claret for providing expression plasmids for TRAF5 and TRAF6, NIK
and NIK-KM, and JNK, respectively. We also thank J. Reddy and K. Malhotra for technical assistance.
 |
Note Added in Proof |
While our paper was under review, a
similar study was reported by Galibert et al. (Galibert, L.,
Tometsko, M. E., Anderson, D. M., Cosman, D., and Dougall, W. C. (1998)
J. Biol. Chem. 273, 34120-34127) that demonstrated
TRAF1, 2, 3, 5, and 6 interaction with RANK. Similar to our results,
these authors observed interaction of TRAF1, 2, 3, and 5 with the C
terminus of RANK whereas TRAF6 interacted with a membrane proximal
region between residues 340 and 421 of RANK. Additionally, like our
results, these authors demonstrated that deletion of residues 340-421
inhibited RANK-induced NF-
B activation, whereas JNK activation was
only partially inhibited.
 |
FOOTNOTES |
*
This work was supported by the Clayton Foundation for
Research.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: Cytokine Research
Laboratory, Dept. of Molecular Oncology, Box 143, The University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3503 or 792-6459; Fax: 713-794-1613; E-mail: aggarwal{at}audumla.mdacc.tmc.edu.
2
B. G. Darnay and B. B. Aggarwal,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
RANK, receptor
activator of NF-
B;
TNF, tumor necrosis factor;
NF-
B, nuclear
factor
B;
RANKL, RANK ligand;
TRAF, TNF receptor-associated factor;
TRANCE, TNF-related activation-induced cytokine;
JNK, c-Jun N-terminal
kinase;
PAGE, polyacrylamide gel electrophoresis;
IRAK1, interleukin-1
receptor-associated kinase;
GST, glutathione S-transferase;
PCR, polymerase chain reaction;
NIK, NF-
B-inducing kinase.
 |
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