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
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ABSTRACT |
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Various members of the tumor necrosis factor
(TNF) receptor superfamily interact directly with signaling molecules
of the TNF receptor-associated factor (TRAF) family to activate nuclear factor B (NF-
B) and the c-Jun N-terminal kinase (JNK) pathway. The receptor activator of NF-
B (RANK), a recently described TNF receptor family member, and its ligand, RANKL, promote survival of
dendritic cells and differentiation of osteoclasts. RANK contains 383 amino acids in its intracellular domain (residues 234-616), which
contain three putative TRAF-binding domains (termed I, II, and III). In
this study, we set out to identify the region of RANK needed for
interaction with TRAF molecules and for stimulation of NF-
B and JNK
activity. We constructed epitope-tagged RANK (F-RANK616) and
three C-terminal truncations, F-RANK330, F-RANK427, and F-RANK530,
lacking 85, 188, and 285 amino acids, respectively. From this deletion
analysis, we determined that TRAF2, TRAF5, and TRAF6 interact with RANK
at its C-terminal 85-amino acid tail; the binding affinity appeared to
be in the order of TRAF2 > TRAF5 > TRAF6. Furthermore,
overexpression of RANK stimulated JNK and NF-
B activation. When the
C-terminal tail, which is necessary for TRAF binding, was deleted, the
truncated RANK receptor was still capable of stimulating JNK activity
but not NF-
B, suggesting that interaction with TRAFs is necessary
for NF-
B activation but not necessary for activation of the JNK
pathway.
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INTRODUCTION |
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To date, over 20 members of the tumor necrosis factor
(TNF)1 ligand and receptor
superfamilies have been identified. Most of these receptors activate
signaling cascades involving the activation of nuclear factor B
(NF-
B), protein kinases (MAPK/JNK/p38), and apoptosis through
engagement of various adaptor proteins (1-3). Activation of apoptosis
is typically transmitted through death domain-containing receptors (4).
Additionally, many TNFR family members activate NF-
B and JNK
pathways via interaction with various TRAF family members (1, 3,
5-12). The TRAF family consists of six distinct proteins, each
containing a ring and zinc finger motif in their N termini and
C-terminal domains that appear to be responsible for self-association
and protein interaction. TRAF1, TRAF2, and TRAF3 bind to distinct
motifs within CD40, CD30, ATAR/HVEM, and p80 TNFR (6-8, 13). The
PXQX(T/S) motif is characteristic for binding
TRAF1, TRAF2, and TRAF5 (6, 7, 14). Moreover, TRAF6 interacts with CD40
via a 16-amino acid region (residues 230-245) (7). Of the TRAF
molecules, only TRAF2, TRAF5, and TRAF6 have been demonstrated to
mediate signaling of NF-
B and JNK (3, 5, 10, 11).
RANK (for receptor activator of NF-B), a recently described novel
TNFR family member, bears high similarity in its extracellular domain
to CD40 (15). It consists of a 616-amino acid transmembrane receptor,
of which 383 amino acids reside in the intracellular domain. The
intracellular domain does not show any homology to any of the known
TNFR family members. RANK mRNA is ubiquitously expressed in human
tissues, but cell surface RANK is expressed only on dendritic cells,
the CD4+ T cell line MP-1, and foreskin fibroblasts (15). CD40L greatly
enhances expression of RANK on mature dendritic cells (15), suggesting
a potential role for RANK in dendritic cell function.
The human and mouse ligands for RANK (RANKL) share 85% identity (15).
This ligand consists of 317 residues and is a type II transmembrane
protein, whose expression is restricted to primary T cells, T cell
lines, and lymphoid tissue (15). Furthermore, RANKL was cloned
independently by three groups as an osteoclast differentiation factor
(16), as an apoptosis-regulatory gene (TRANCE, for
TNF-related
activation-induced
cytokine) (17), and as a ligand for the soluble
TNFR family member osteoprotegerin (18). Overexpression of RANK and
RANKL has been demonstrated to activate NF-B (15). RANKL was also
shown to stimulate JNK activity in mouse thymocytes and T cell
hybridomas, but not B cells (17), and was partially inhibited in
thymocytes from dominant negative TRAF2 transgenic mice (19).
Additionally, RANKL appears to enhance T cell growth and dendritic cell
survival by up-regulation of Bcl-XL (15, 17).
To date, there is no report to indicate the region of the RANK receptor
necessary for activation of JNK and NF-B. Thus, we constructed
various C-terminal truncations of RANK and transiently expressed them
in human cultured cell lines to characterize their ability to interact
with various TRAF family members and to activate JNK and NF-
B. From
this deletion analysis, we have identified specific regions of RANK
that interact with TRAF2, TRAF5, and TRAF6 and that stimulate JNK
and NF-
B activation.
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EXPERIMENTAL PROCEDURES |
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Reagents, Cell Lines, and Antibodies-- HeLa, an epithelial carcinoma cell line, and 293, a human embryonic kidney 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. Affinity-purified rabbit anti-TRAF2 (SC-876, C-20) and anti-JNK1 (SC-474, C-17) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit IgG-conjugated horseradish peroxidase was obtained from Bio-Rad. Anti-FLAG (monoclonal antibody M2) and anti-FLAG (M2)-conjugated agarose were obtained from Eastman Kodak Co. (New Haven, CT). Goat anti-mouse IgG-conjugated horseradish peroxidase was obtained from Transduction Laboratories (Lexington, KY). Protein A/G-Sepharose was obtained from Pierce.
Expression Plasmids--
The complete cDNA for RANK
(pSPORT3.0-TR8) was identified through a homology search of an
expressed sequence tag cDNA data base (Human Genome Sciences, Inc.,
Rockville, MD) obtained from a primary dendritic cell cDNA library
for proteins containing the cysteine-rich repeat characteristic of TNFR
family members. This cDNA is identical to RANK (15). To
generate FLAG-tagged RANK616, the 5'-primer
CTAAGAAAGCTTTGTACCAGTGAGAAGCAT and the 3'-primer
GACGTAGTCGACTCAAGCCTTGGCCCCGCC were used in a PCR reaction with
pSPORT3.0-TR8 to generate a PCR product that would encode residues
33-616 (lacking the signal sequence), which was cloned into the
HindIII/SalI site of the expression vector
pCMVFLAG1 (Eastman Kodak Co.). RANK deletion mutants were generated by
PCR using the above 5' primer and the 3' primers (for RANK330:
TCCTACGTCGACTCAGCTGACCAATGAGAGAGCATCCT; RANK427:
AACGGCGTCGACTCAACTGTCCACCTCTTTTTGCAA; and RANK530:
CGCTGAGTCGACTCAGGAGTTACTTGTTTCCAGTCAC) and cloned into the
HindIII/SalI site of pCMVFLAG1. All
plasmids were verified by automated DNA sequencing. Human TRAF2
cDNA (pcDNA3HisTRAF2) was a generous gift from Dr. T. Kamitani
(University of Texas Health Science Center, Houston, TX). The complete
cDNA for TRAF2 was cloned by PCR using primers containing
BamHI (5') and SalI (3') sites and
pcDNA3HisTRAF2 as a template. The TRAF2 PCR product was digested
with BamHI/SalI and cloned into pRKmyc, resulting in pRKmycTRAF2. The plasmid encoding cDNA for TRAF6 (pSR-TRAF6) was a generous gift from Dr. S. Reddy (M. D. Anderson Cancer Center, Houston, TX). The cDNA for TRAF6 was digested from pSR
-TRAF6 with KpnI/EcoRI and cloned into pBS(KS
) to give
rise to pBS-TRAF6.
In Vitro Translation of 35S-Labeled TRAFs-- Expression vectors encoding for TRAF2 (pRKmycTRAF2), TRAF5 (pcDNA3mycTRAF5), and TRAF6 (pBS-TRAF6) were in vitro transcribed and translated with 35S-Met (Amersham Pharmacia Biotech) using the TNT system as described by the manufacturer (Promega, Madison, WI).
Transient Transfections--
HeLa (1.5 × 106
cells/100-mm dish) and 293 (2 × 106 cells/100-mm
dish) cells were plated and transfected the next day with 7.5-10 µg
of expression vectors by using LipofectAMINE (Life Technologies, Inc.)
as described by the manufacturer; transfection was allowed to proceed
for an additional 24 h. Alternatively, 293 cells (0.6 × 106 cells/well, 6-well plate) were plated and transfected
the next day by the calcium phosphate method as described by the
manufacturer (Life Technologies, Inc.). Cells were harvested 36-40 h
after transfection; half were analyzed for expression of epitope-tagged receptors and JNK activity, and the other half for NF-B. Lysates were prepared in lysis buffer (20 mM Tris, pH 8, 250 mM NaCl, 1 mM dithiothreitol, 2 mM
EDTA, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mg/ml benzamidine, and 2 mM sodium vanadate). After a
30-min incubation on ice, the samples were cleared by centrifugation
for 10 min. Protein was estimated using a Bio-Rad protein determination
kit.
Western Blotting-- Whole cell lysates (15 µg) or proteins from immunoprecipitations were separated by 8.5% SDS-PAGE and electroblotted onto nitrocellulose membranes (Bio-Rad). Western blot analysis was performed using the indicated antibodies, and membranes were developed using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech).
Immunoprecipitations and JNK Kinase Assays-- From transiently transfected cells, lysates were prepared and immunoprecipitated using anti-FLAG-conjugated agarose or anti-JNK1 and protein A/G-Sepharose for 1 h. Where indicated, 35S-labeled proteins were added to the lysate prior to immunoprecipitation. Beads were collected by centrifugation and washed four times in lysis buffer and then two times in kinase buffer (20 mM Tris, pH 8, 50 mM NaCl, and 1 mM dithiothreitol). For coimmunoprecipitation, proteins were eluted in SDS-sample buffer, boiled, and subjected to SDS-PAGE. JNK activity was analyzed using exogenous GST-Jun-(1-79) as a substrate as described previously (20). JNK activity and 35S-labeled TRAF binding were quantitated using a PhosphorImager and Imagequant software (Molecular Dynamics, Sunnyvale, CA).
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts were prepared from transfected cells essentially as described
(20). Equivalent amounts of nuclear protein were used in an EMSA
reaction with 32P-labeled NF-B oligonucleotide from the
human immunodeficiency virus-long terminal repeat as described (20).
NF-
B activation was quantitated using a PhosphorImager and
Imagequant software.
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RESULTS AND DISCUSSION |
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A human cDNA (TR8, for TNF receptor-like 8) encoding a TNFR-related protein was identified through a homology search of an expressed sequence tag cDNA library. The full-length cDNA encodes a protein of 616 amino acid residues. The extracellular domain (residues 1-208) contains a signal sequence and the conserved cysteine-rich repeats characteristic of the TNFR family (21). The intracellular domain (residues 234-616) is the largest of all the TNFR family members to date and contains no homology to other members of this family. This cDNA was found to be identical to a previously reported TNFR family member known as RANK (15).
Construction and Expression of Epitope-tagged RANK-- To facilitate detection and immunoprecipitation of RANK in cultured cells, we constructed a FLAG epitope-tagged version of RANK in the plasmid pCMVFLAG1. The mature polypeptide encodes residues 33-616 (F-RANK616) with a FLAG epitope tag at its N terminus (Fig. 1A). To identify which region of the cytoplasmic domain is needed for signaling, we constructed three C-terminal deletions, designated F-RANK530, -427, and -330 (Fig. 1A) and lacking 85, 188, and 285 amino acids, respectively.
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TRAF2, TRAF5, and TRAF6 Interact with the C Terminus of RANK-- Because most TNFR family members utilize TRAFs as signaling components and RANK contains putative TRAF-binding domains, we examined the ability of RANK to interact with various TRAFs. We transiently transfected HeLa and 293 cells with vectors directing expression of F-RANK616 and F-RANK deletion mutants. After 24-36 h, cell lysates were prepared, and epitope-tagged receptors were immunoprecipitated with anti-FLAG-conjugated agarose. Coprecipitation of endogenous TRAF2 was detected by Western blotting with anti-TRAF2 polyclonal antibodies. When expressed in HeLa (Fig. 3A, top) and 293 cells (Fig. 3A, bottom), only F-RANK616 and none of the F-RANK deletion mutants precipitated endogenous TRAF2. Membranes were also probed with anti-FLAG to ensure the precipitation of epitope-tagged receptors (data not shown).
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RANK Deletion Mutants Lacking TRAF Binding Domains (II and III) Activate JNK-- TRAF2, TRAF5, and TRAF6 are involved in JNK activation (3) by various members of the TNFR family and the interleukin-1 receptor (5) (i.e. TRAF6). We tested whether RANK and the various C-terminal deletion mutants were capable of activating JNK. When overexpressed in cultured cell lines, most TNFR family members activate signal transduction pathways in the absence of ligand (2). Thus, we transiently transfected 293 cells with increasing amounts of F-RANK expression vectors. Cell lysates were prepared 36 h after transfection and analyzed for receptor expression by Western blotting with anti-FLAG antibodies (Fig. 4A). Furthermore, the cell lysates were assayed for JNK activation by immune complex kinase assays using GST-Jun-(1-79) as a substrate. Transient overexpression of F-RANK616 in 293 cells activated JNK (Fig. 4B). Furthermore, F-RANK530 and -427 deletion mutants, which lack 85 and 188 residues from the C terminus, respectively, could still activate JNK (Fig. 4B). However, C-terminal truncation of 285 residues (which leaves approximately 98 amino acids intact) could not activate JNK (Fig. 4B). From at least three independent transfection experiments, we found that F-RANK616, -530, and -427 could increase JNK activity between 4- and 10-fold, whereas F-RANK330 increased activity by no more than 1.5-fold relative to vector-transfected cells. These data suggest that F-RANK530 and F-RANK427 may stimulate JNK activation without binding directly to TRAFs. Because F-RANK330 had no significant effect on JNK activation, we tentatively localized a JNK activation domain between residues 330 and 427 within the cytoplasmic domain of RANK.
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The C Terminus of RANK Is Necessary for NF-B
Activation--
According to gel mobility shift assays, overexpression
of RANK in 293 cells activates NF-
B (15). To explore whether RANK deletion mutants activate NF-
B, we transiently transfected 293 cells
with F-RANK616 and the F-RANK deletion mutants. Western blotting with
anti-FLAG antibodies indicated expression of the epitope-tagged
receptors (Fig. 5A). Analysis
of NF-
B by a gel mobility shift assay indicated that only F-RANK616
activated NF-
B (Fig. 5B). None of the F-RANK deletions
were capable of activating NF-
B in three independent transient
transfection experiments, even though from the same transfections
F-RANK530 and -427 could activate JNK.
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FOOTNOTES |
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* This research 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, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3503/6459; Fax: 713-794-1613; E-mail: aggarwal{at}audumla.mdacc.tmc.edu.
The abbreviations used are:
TNF, tumor necrosis
factor; TNFR, TNF receptor; NF-B, nuclear factor
B; RANK, receptor activator of NF-
B; TRAF, TNF receptor-associated factor; TRANCE, tumor necrosis factor-related activation-induced cytokine; JNK, c-Jun N-terminal kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; GST, glutathione S-transferaseEMSA, electrophoretic mobility shift assay(s).
2 B. G. Darnay and B. B. Aggarwal, unpublished observations.
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REFERENCES |
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