TNF Receptor Family Member BCMA (B Cell Maturation)
Associates with TNF Receptor-Associated Factor (TRAF) 1,
TRAF2, and TRAF3 and Activates NF-?B, Elk-1, c-Jun
N-Terminal Kinase, and p38 Mitogen-Activated Protein
Anastassia Hatzoglou,2* Je ´ro ˆme Roussel,2†Marie-Franc ¸oise Bourgeade,2†Edith Rogier,†
Christine Madry,†Junichiro Inoue,‡Odile Devergne,†and Andreas Tsapis3†
BCMA (B cell maturation) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B
lymphocytes. Previously, we reported in a human malignant myeloma cell line that BCMA is not primarily present on the cell
surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. We now show that in transiently or stably
transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. We also show that overex-
pression of BCMA in 293 cells activates NF-?B, Elk-1, the c-Jun N-terminal kinase, and the p38 mitogen-activated protein kinase.
Coimmunoprecipitation experiments performed in transfected cells showed that BCMA associates with TNFR-associated factor
(TRAF) 1, TRAF2, and TRAF3 adaptor proteins. Analysis of deletion mutants of the intracytoplasmic tail of BCMA showed that
the 25-aa protein segment, from position 119 to 143, conserved between mouse and human BCMA, is essential for its association
with the TRAFs and the activation of NF-?B, Elk-1, and c-Jun N-terminal kinase. BCMA belongs structurally to the TNFR family.
Its unique TNFR motif corresponds to a variant motif present in the fourth repeat of the TNFRI molecule. This study confirms
that BCMA is a functional member of the TNFR superfamily. Furthermore, as BCMA is lacking a “death domain” and its
overexpression activates NF-?B and c-Jun N-terminal kinase, we can reasonably hypothesize that upon binding of its correspond-
ing ligand BCMA transduces signals for cell survival and proliferation. The Journal of Immunology, 2000, 165: 1322–1330.
through cell-to-cell contact, and those that are secreted proteins act
on distant target cells. The TNFR are a heterologous family, of
which 18 members are known. They mediate the action of TNF-
related cytokines leading to cell death or cell proliferation and
differentiation (1, 2). Most members of the TNFR family are type
I transmembrane proteins with an extracellular ligand-binding do-
main, a single membrane-spanning region, and a cytoplasmic re-
gion that activates cell functions (3). The common characteristic of
all TNFR family members is the repetition of a six-cysteine motif
in the extracellular N-terminal part of the molecule. In contrast to
the extracellular parts of the receptors, the sequences of the cyto-
plasmic tails are generally dissimilar, and none possess sequences
he TNF-related cytokines are a large family of pleiotropic
mediators of host defense and immune system regulators.
Those that are integral membrane proteins act locally
suggestive of catalytic activity. However, several motifs in the
C-terminal part of TNFR have been shown to bind protein factors
transducing the signal initiated by ligand binding and receptor tri-
merization. One of these motifs, the “death domain,” is present in
TNFRI, Fas, DR3, DR4, and DR5 and is responsible for the ca-
pacity of these receptors to induce apoptosis (4, 5). A second group
of motifs binds signal transducers, TNFR-associated factors
(TRAFs).4TRAFs interact directly with several TNFRs, like
TNFRII, CD40, CD30, and lymphotoxin ? receptor (6–9) and
with the EBV oncogene LMP1 (10). TRAF2, TRAF5, and TRAF6
mediate the activation of the transcriptional factor NF-?B (11–13)
and activate the c-Jun N-terminal protein kinase (JNK) (14).
TRAF6 also mediates the activation of extracellular signal-regu-
lated kinase (ERK) (15).
Recently, we identified of a novel TNFR (16, 17) through the
molecular analysis of a t(4;16) translocation (16, 18), characteristic
of a malignant human T cell lymphoma. The gene product is se-
lectively expressed in mature B lymphocytes (19) and was there-
fore named BCMA for B cell maturation protein. The BCMA gene
codes for a nonglycosylated integral membrane type I protein. The
N-terminal part of both mouse and human proteins contains a con-
served six-cysteine motif (17). A sensitive method of sequence
analysis, hydrophobic cluster analysis (20), indicated that this con-
served motif is similar to the six-cysteine repeat motif found in the
*Laboratory of Experimental Endocrinology, Faculty of Medicine, University of
Crete, Heraklion, Greece;†Institut National de la Sante ´ et de la Recherche Me ´dicale,
Unite ´ 131, Institut Paris-Sud sur les Cytokines, Clamart, France; and‡Department of
Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Received for publication January 10, 2000. Accepted for publication May 18, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a grant from the Comite ´ Departmental des Hauts
de Seine de la Ligue Nationale contre le Cancer (to A.T.) and by a grant from the
Association de Recherche contre le Cancer (Grant 9907 to A.T.).
2A.H., J.R., and M.-F.B. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Andreas Tsapis, Institut Na-
tional de la Sante ´ et de la Recherche Me ´dicale Unite ´ 131, Institut Paris-Sud sur les
Cytokines, 32, rue des Carnets, 92140 Clamart, France. E-mail address:
4Abbreviations used in this paper: TRAF, TNFR-associated factor; JNK, c-Jun
N-terminal kinase; ERK, extracellular signal-related kinase; BCMA, B cell matura-
tion; HA, hemagglutinin; h, human; m, mouse; ATF, activating transcription factor;
GFP, green fluorescence protein; MEK, MAPK/ERK kinase; MEKK, MEK kinase;
PI3-K, phosphatidylinositol 3-kinase.
Copyright © 2000 by The American Association of Immunologists0022-1767/00/$02.00
extracellular part of TNFRs. There are two notable differences be-
tween the BCMA protein and other members of the TNFR family.
The first is that BCMA contains only one six-cysteine-rich motif,
whereas the members of the TNFR family contain more than one
copy. The second is that the six-cysteine motif of BCMA is not the
canonical motif of TNFRs but corresponds to a variant motif
present in the fourth repeat of the TNFRI molecule. The full name
for BCMA in the new TNF nomenclature scheme is TNFRSF17.
The human BCMA gene is the first TNFR gene that has been
implicated in chromosome translocation.
We report a study of the cellular localization of BCMA in tran-
siently and stably transfected cells. We show that overexpression
of BCMA activates the NF-?B, Elk-1, p38, and JNK. We also
studied the association of the six known TRAFs with BCMA and
defined the region of the BCMA protein responsible for this
Materials and Methods
Abs and reagents
The rabbit polyclonal anti-TRAF1 (H-132), anti-TRAF2 (C-20), anti-
TRAF3 (H-122), anti-TRAF5 (H-257), anti-JNK1 (sc-474), and goat poly-
clonal anti-TRAF4 (N-16) and anti-goat HRP-conjugated Abs were pur-
chased from Santa Cruz Biotechnology (Santa Cruz, CA). M2 anti-FLAG
mAb, M2 mAb bound to agarose beads, and FLAG peptide and protease
inhibitor mixture were purchased from Sigma-Aldrich (St. Louis, MO).
Anti-F mAb was a generous gift from M. C. Rio (Institut National de la
Sante ´ et de la Recherche Me ´dicale, Unite ´ 184, Strasbourg, France). 12CA5
anti-hemagglutinin (anti-HA) mAb was purchased from Roche Diagnostics
(Somerville, NJ). PE-, FITC-, and HRP-conjugated goat anti-mouse IgG
polyclonal Abs and HRP-conjugated donkey anti-rabbit IgG polyclonal
Abs were purchased from Immunotech (Marseille, France). Rabbit poly-
clonal anti-phosphatidylinositol 3 kinase (PI3-K) p85 Abs were obtained
from Upstate Biotechnology (Lake Placid, NY). RPMI 1640, DMEM,
FCS, additional reagents for cell culture, optimem, and lipofectamine were
purchased from Life Technologies (Grand Island, NY).
The following primers were used in this study: BCMA5?ATG (5?-
AAGCTTATGTTGCAGATGGCTGGGCA-3?), BCMA3?TAA (5?-GGAT
CCTTACCTAGCAGAAATTGATTTC-3?), 37 (5?-CCCAAGCTTATGGCT
GGGCAGTGCTCC-3?), 43 (5?-CGCGGATCCTTATGGTTCAGAGCTTA
TCTTCCT-3?), AH7 (CGCGGATCCTTACCTAGCAGAAATTGATTTCT-
3?), AH9 (5?-CCGCTCGAGGGCGCAACAGTGTTTCCACA-3?), AH10
(GGAAGATCTCTAACGACATCTAAAACACCAG-3?), BFL1 (5?-AACTG
CAGCTGGGCAGTGCTCCCAAAA-3?), BFL2 (5?-CGGGATCCTTAAT
AGTCATTCGTTTTCGTGGTG-3?), BFL3 (5?-CGGGATCCTTAGCAATG
GTCATAGTCGACCT-3?), BFL4 (5?-CGGGATCCTTAGCCTCTCGGAA
GAATAATTTC-3?), and BFL5 (5?-CGGGATCCTTAGTTTTTAAACTC
GTCCTTTAATG-3?). All primers used in this study were purchased from
Genset (Paris, France).
A full-length human BCMA (h184) was amplified by PCR from human
cDNA using the BCMA5?ATG and BCMA3?TAA primers; a fragment
encoding the N-terminal and transmembrane parts of hBCMA (h84) was
amplified by PCR using primers 37 and 43. The PCR fragments were
digested with BamHI and HindIII restriction enzymes and ligated into the
BamHI and HindIII sites of the vector pcDNA3 (Invitrogen, Groningen,
A full-length mouse BCMA was amplified by PCR from a mouse cDNA
library with the primers AH9 and AH10. The PCR fragment was digested
with XhoI and BglII and ligated into the XhoI and BglII sites of the vector
pDEB (21), giving rise to a fusion encoding a N-terminal HA-tagged
mouse BCMA (HAm185). The HA-tagged mBCMA was digested with
EcoRI and NotI and ligated into the EcoRI and NotI sites of the vector
N-terminal FLAG-tagged hBCMA deletion mutants were constructed
by PCR amplification using the following pairs of primers: BFL1 and AH7
for FLAG-hBCMA without deletion (Fh184), BFL1 and BFL2 for FLAG-
hBCMA?165–184 (Fh164), BFL1 and BFL3 for FLAG-hBCMA?144–
184 (Fh143), BFL1 and BFL4 for FLAG-hBCMA?119–184 (Fh118), and
BFL1 and BFL5 for FLAG-hBCMA?92–184 (Fh91). All PCR products
were digested with PstI and BamHI and ligated between the PstI and
BamHI sites of the vector pSG5-FLAG (22). All expression vectors were
constructed by standard recombinant DNA procedures. The sequence of
the plasmids constructed by PCR amplification were subsequently verified
by dideoxy sequencing.
The vectors pSG5hTRAF1 (10), pSG5hTRAF2, pSG5FLAGhTRAF2
(23), pSG5FLAGhTRAF1, pSG5hTRAF3, pSG5FLAGhTRAF3 (24),
pMEFLAGmTRAF5 (13), pMEFLAGmTRAF6 (25), pEBBhTRAF5 (26),
pcLMP1 (27), pcDNA3TRAF2.DN (TRAF2?6–86) (23), pGEX-Jun(1–
79), pcDNA3-HA-JNK (28), and the ?-galactosidase expression vector
(pGK-?gal), in which expression is driven by the phosphoglucokinase pro-
moter (22), have been already described. pAT3FhTRAF4 encoding human
FLAG-tagged TRAF4 was a generous gift from Dr. Catherine Regnier
Cell lines and transfections
Human embryonic kidney 293, 293T, and 293EBNA and simian kidney
COS7 cells were maintained in high-glucose DMEM supplemented with
10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml of penicillin, and
100 ?g/ml of streptomycin and were grown at 37°C in 5% CO2. The
293EBNA cell line was purchased from Invitrogen and maintained in cul-
ture according to the supplier’s instructions. The BJAB cell line is an
EBV-negative Burkitt lymphoma cell line (29) and was cultured in RPMI
1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100
U/ml of penicillin, and 100 ?g/ml of streptomycin and grown at 37°C in
5% CO2. Adherent cells were seeded in six-well plates (5 ? 105cells per
well) in 2 ml of complete medium, incubated at 37°C in 5% CO2for 20–24
h, and transfected with lipofectamine according to the manufacturer’s in-
structions, using 1 ?g of total plasmid DNA, for 6 h. BJAB cells were
transfected by electroporation (960 ?F, 210 V) in 400 ?l optimem medium
using a Bio-Rad Gene Pulser apparatus (Bio-Rad, Richmond, CA). Cell
extracts were tested for gene expression 24–48 h after transfection. To
establish cells stably expressing BCMA, 293 cells were transfected with
HAm185-expressing vector and were selected in high-glucose DMEM,
10% FCS, in presence of 400 ?g/ml geneticin. Geneticin-resistant clones
were screened by immunoblotting for BCMA expression.
Luciferase reporter system for NF-?B, Elk-1, and JNK
The NF-?B, Elk-1, and JNK activation assays were performed using the
corresponding luciferase reporter PathDetect Reporting systems purchased
from Stratagene (La Jolla, CA).
Luciferase and ?-galactosidase assays
Transfected cells were washed twice with PBS and lysed with reporter lysis
buffer (Promega, Madison, WI). The luciferase activity was measured us-
ing the reporter assay system (Promega). ?-galactosidase activity was mea-
sured using the luminescent ?-galactosidase reporter system (Clontech,
Palo Alto, CA) in a Packard luminometer analyzer (Packard, Meriden, CT).
Measurements of luciferase were normalized to ?-galactosidase activity
and are expressed as a ratio to values obtained from cells treated with
vector alone. The relative luciferase activities given are representative of
triplicate assays in three independent experiments.
Determination of JNK activity
JNK activity was determined as described previously (28) with minor mod-
ifications. Transfected cells were lysed in 10 mM Tris, pH 7.4, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% Nonidet P-40,
0.5 mM sodium vanadate, 0.2 mM PMSF, and 10% glycerol. Lysates were
clarified by centrifugation, and HA-tagged JNK was immunoprecipitated
using anti-HA mAb 12CA5. Immune complexes were collected on pro-
tein-G Agarose beads, washed three times in lysis buffer, once in kinase
reaction buffer (12.5 mM MOPS, pH 7.5, 12.5 mM ?-glycerophoshate, 7.5
mM MgCl2, 0.5 mM EGTA, 0.5 mM NaF, 0.5 mM sodium vanadate), and
resuspended in 30 ?l of the same buffer containing 2 ?g of GST-Jun, 20
?M unlabeled ATP, and 5 ?Ci [?-32P]ATP. After incubation at 20°C for
30 min, kinase reaction products were analyzed by SDS-PAGE and auto-
radiography. Part of the immunoprecipitated material was resolved by
SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and im-
munoblotted with anti-JNK polyclonal Abs to check that the same amount
of HA-JNK was used in each case.
Determination of p38 and ERK activity
The activity of these two kinases was assayed using the corresponding
assay kit purchased from New England Biolabs (Beverly, MA). Briefly,
transfected cells were lyzed, and the active phosphorylated kinase was
immunoprecipitated using specific mAbs. The immunoprecipitated protein
was assayed for its ability to phosphorylate activating transcription factor
1323The Journal of Immunology
(ATF) 2 (p38) or Elk-1 (ERK) substrates. Analysis of phosphorylated sub-
strates was performed by Western blotting using specific polyclonal
COS7 cells were cotransfected with one vector encoding a TRAF and one
vector encoding one of the various FLAG-tagged BCMA constructs. Eigh-
teen to 24 h after transfection, cells were lysed in lysis buffer (50 mM Tris,
pH 7.4, 150 mM NaCl, 1.5 mM EDTA, 10% glycerol, 0.1% Nonidet P-40,
and a protease inhibitor mixture) by incubation for 1 h at 4°C, and the
supernatant was then clarified by centrifugation. One-fortieth of this lysate
(input) was conserved to test the efficiency of transfection, and the rest was
incubated for 2 h, at 4°C, with M2 monoclonal anti-FLAG Ab, covalently
bound to agarose beads. The beads were washed three times with the lysis
buffer, and the bound proteins were eluted twice by addition of 250 ?M
FLAG peptide diluted in PBS. The eluate and the input were analyzed by
PAGE and transferred onto a polyvinylidene difluoride membrane (Hy-
bond-P; Amersham, Little Chalfont, U.K.). The presence of BCMA and of
the various TRAFs was tested by immunoblotting using M2 Ab to evidence
FLAG-tagged BCMA constructs and the corresponding anti-TRAF Ab for
each TRAF. HRP-conjugated anti-rabbit, anti-goat, and anti-mouse IgG
and SuperSignal Chemiluminescent substrate (Pierce, Rockford, IL) were
used to reveal the blots.
Immunofluorescence staining and FACS analysis
For immunofluorescence observation, transfected ells were stained with
M2 mAb then incubated with fluorescein-conjugated goat anti-mouse Ab
before analysis under a Leica DM microscope (Leica, Deerfield, IL) as
previously described (30). For FACS analysis, ?5 ? 105cells per condi-
tion were stained with saturating concentrations of Ab, then incubated with
PE-conjugated goat anti-mouse Ab before analysis in a FACScan flow
cytometer (Becton Dickinson, San Diego, CA), as previously described
(31). A minimum of 10,000 events per sample was analyzed. CellQuest
software (Becton Dickinson) was used for data analysis.
BCMA is present on both the surface of cells and in an
intracellular perinuclear structure
In a previous study of BCMA localization, in the human myeloma
cell line U266, we found most BCMA in a perinuclear Golgi-like
structure (30). We further analyzed the localization of BCMA by
transfection experiments in two cell lines: the human B lympho-
cyte BJAB cell line and the monkey kidney COS7 cell line. BJAB
cell line has been chosen because it expresses detectable amount of
BCMA mRNA, has nondetectable amounts of BCMA protein
(data not shown), and can be transiently transfected with high ef-
ficiency (50–60%). On the contrary, COS7 cell line does not ex-
press BCMA. The cell lines were transfected with two vectors, one
coding for a FLAG-tagged full-length hBCMA (Fh184) and a sec-
ond one coding for a FLAG-tagged hBCMA construct lacking the
entire intracellular cytoplasmic tail (Fh91), together with a green
fluorescence protein (GFP) expression vector. Eighteen hours after
transfection, cells were stained with M2 anti-FLAG Ab and a sec-
ondary PE-conjugated anti-mouse IgG. The GFP-expressing cell
population was gated, and the presence of FLAG-tagged proteins
on the surface and intracellularly was determined by two-color
cytofluorometry (Fig. 1A). Full-length hBCMA and the mutant hB-
CMA missing its cytoplasmic tail were similarly distributed in
both BJAB and COS7 cell lines. Both proteins displayed an intra-
cytoplasmic localization, but were also present on the cell surface.
The localization of full-length hBCMA was further examined by
fluorescence microscopy. Our results (Fig. 1B) confirmed that
BCMA was present on the cell surface. As previously observed in
the myeloma U266 cell line, intracellular BCMA was detected in
a perinuclear Golgi-like structure in both transfected BJAB and
COS7 cell lines.
The cell localization of BCMA was also examined in stably
transfected cells. To this end, 293 cell lines stably expressing HA-
tagged mBCMA (HAm185) were derived and tested by flow cy-
tometry for surface and intracytoplasmic expression of HAm185.
The results obtained for one clone (clone 12) are shown in Fig. 2
and indicate both surface and intracytoplasmic localization of
COS7 cell lines. The cell lines were transfected with two vectors, one
coding for a N-terminal FLAG-tagged full-length hBCMA (Fh184) and a
second one coding for a N-terminal FLAG-tagged hBCMA construct lack-
ing the entire intracellular cytoplasmic tail (Fh91), together with a GFP
expression vector. Eighteen hours after transfection, the cells were stained
with the M2 anti-FLAG mAb and a secondary PE-conjugated anti-mouse
IgG Ab, in the absence (for surface localization) and the presence (for
intracellular localization) of permeabilizing detergent and analyzed by two-
color flow cytometry (A). The GFP-expressing cell population was gated
and further analyzed for FLAG-tagged protein expression (Fig. 1A). The
intracellular and surface localization of Fh184 in BJAB and COS7 cells
was also analyzed by fluorescence microscopy (B).
Transient expression of hBCMA in transfected BJAB and
1324 BCMA ASSOCIATES WITH TRAFS AND ACTIVATES NF-?B, p38, AND JNK
HAm185. Similar results have been obtained in two other clones
tested (data not shown).
BCMA-mediated NF-?B activation
Most TNFRs, when overexpressed, activate NF-?B. To determine
whether BCMA overexpression also results in NF-?B activation,
293 cells were cotransfected with CMV promoter-driven BCMA
expression vectors together with a NF-?B luciferase reporter plas-
mid. Overexpression of hBCMA (h184) induced a 12-fold activa-
tion of NF-?B over the activation level obtained using the empty
vector (Fig. 3A). Similarly, overexpression of mBCMA (HAm185)
gave rise to a 10-fold activation of NF-?B. These activation levels
were in the range of that observed (12-fold) using LMP1, a known
activator of this nuclear factor. A dose-response curve was plotted
and showed that 100 ng of hBCMA or mBCMA expression vec-
tors were sufficient for maximal NF-?B activation (data not
shown). As expected, transfection of 293 cells with the deletion
mutant of hBCMA, lacking the intracytoplasmic tail of the mole-
cule, (h84), failed to activate NF-?B, confirming that the cytoplas-
mic tail of BCMA is essential for transducing a signal and acti-
To determine which sequence within BCMA cytoplasmic tail is
necessary for NF-?B induction, we constructed a series of SV40
promoter-driven vectors encoding N-terminal FLAG-tagged dele-
tion mutants of hBCMA and tested these mutants for NF-?B ac-
tivation (Fig. 3C) in 293T cells. The deletion mutants Fh164 and
Fh143, lacking the C-terminal 20 and 41 aa, respectively, had the
same NF-?B activation capacity as the full-length BCMA mole-
cule, Fh184. In contrast, mutants Fh118 and Fh91 did not activate
NF-?B. The level of expression of the deletion mutant proteins
was tested by immunoblotting and was found to be approximately
similar (Fig. 3D). Therefore, the protein segment between amino
acid residues 119 and 143 of BCMA is necessary for the activation
of NF-?B. Interestingly, this sequence is highly conserved in hB-
CMA and mBCMA (Fig. 3B).
BCMA activates the mitogen-activated protein kinase (MAPK)
We next examined the activation of the nuclear factor Elk-1 using
a luciferase reporter system. Elk-1 is a substrate for the MAPKs:
293 cell line was transfected with HA-tagged mBCMA pcDNA3 express-
ing vector (HAm185). The cells were selected with 400 ?g/ml of geneticin,
and seven clones were isolated. The expression of HAm185 protein was
tested in these clones by immunoblotting using the 12CA5 anti-HA mAb.
Nontransfected 293 cells were used as negative control. The 293cl12 cells
were stained with the 12CA5 anti-HA mAb and a secondary PE-conjugated
anti-mouse IgG Ab, in the absence (for surface localization) and the pres-
ence (for intracellular localization) of permeabilizing detergent and ana-
lyzed by flow cytometry.
Stable expression of mBCMA in transfected 293 cells. The
(vector), pcDNA3LMP1 (LMP1), pcDNA3-hBCMA (full-length hBCMA),
pcDNA3-HA tagged mBCMA185 (full-length mBCMA), and pcDNA3-
hBCMA84(h84), which contains only the extracellular and transmembrane re-
gions of hBCMA. B, Truncations of hBCMA cytoplasmic tail were obtained by
standard PCR in pSG5FLAG. A schematic representation of these mutants is
shown. Black blocks denote the transmembrane region (TM), gray blocks denote
positions 119 and 143 essential for the activation of NF-?B. The sequences of
human and mouse BCMA proteins in this region are shown to illustrate the sim-
ilarity. C, 293T cells were cotransfected with luciferase reporter plasmid and 100
ng of one of empty vector (F), Fh184, Fh164, Fh143, Fh118, and Fh91. NF-?B
activation was measured as described in Materials and Methods. The pGK-?-
to normalize transfection efficiencies. Forty-eight hours after transfection, cells
were harvested, lysed, and analyzed for luciferase and ?-galactosidase activity.
Luciferase values were normalized to ?-galactosidase activity, and the results are
displayed as a multiple of the induction by vector alone. A representative result of
three independent experiments is shown. Error bars denote SDs for triplicate sam-
ples. D, The level of production of deletion mutant proteins in 293T cells was
tested by Western blot using the M2 anti-FLAG mAb.
BCMA overexpression induces NF-?B activation. A, 293 cells
1325The Journal of Immunology
JNK, p38, and ERK. Overexpression of hBCMA in 293 cells ac-
tivated Elk-1 to a level 4.5-fold higher than that obtained using the
empty vector (Fig. 4A). Similar results were obtained using mB-
CMA (3.5-fold activation). As expected, the mutant hBCMA h84
failed to activate Elk-1. MAPK/ERK kinase (MEK) 1 overexpres-
sion was used as a positive control for Elk-1 activation (10-fold).
The activation of Elk-1 by the different deletion mutants of BCMA
was also studied in 293T cells (Fig. 4B). Fh184, Fh164, and Fh143
constructions activated Elk-1 3.5-, 5-, and 2-fold, respectively,
whereas Fh118 and Fh91 mutants gave no activation of this nu-
clear factor. The level of expression of the deletion mutant proteins
was tested by immunoblotting and was found similar (Fig. 4C).
Therefore the protein segment between amino acid residues 119
and 143 of BCMA is essential for the activation of the nuclear
To assess the ability of BCMA to activate JNK, we transiently
cotransfected 293 cells with an HA-tagged JNK vector together
with one of pcDNA vectors expressing h184, HAm185, h84,
pcDNA3, and pFRMEKK vectors. The activation of JNK was ex-
amined by measuring phosphorylation of its substrate, GST-Jun.
Overexpression of MEK kinase (MEKK) was used as a positive
control. The overexpression of either human or mouse BCMA sig-
nificantly increased the amount of phosphorylated GST-Jun, as
compared with that of the cells transfected with the empty vector
or with the h84 mutant of hBCMA, the mutant lacking the cyto-
plasmic tail (Fig. 5A). These data indicate that overexpression of
BCMA activates JNK. We also tested the level of activation of
c-Jun by the SV40 promoter-driven vectors expressing deletion
mutants of BCMA using a luciferase reporter system. As the back-
ground level was high in 293T cells, we have used 293EBNA cells
in which we have obtained a lower background. The expression of
Fh184, Fh164, and Fh143 mutants resulted in a 2-fold activation of
293 cells were cotransfected with the corresponding Pathfinder reporter
system plasmids and 100 ng of empty vector (vector), LMP1, full-length
hBCMA, HA-tagged full-length mBCMA, or h84, which contains only the
extracellular/transmembrane region of hBCMA. MEK1-encoding plasmid
was used as a positive control. B, 293T cells were cotransfected with the
corresponding Pathfinder reporter system plasmids and 100 ng of empty
pSG5FLAG (vector), Fh184, Fh164, Fh143, Fh118, or Fh91. Elk-1 acti-
vation was measured as described in Materials and Methods. The pGK-
?-galactosidase plasmid encoding ?-galactosidase was cotransfected in ev-
ery sample to normalize transfection efficiencies. Twenty-four (for 293
cells) and 48 h (for 293T cells) after transfection, cells were harvested,
lysed, and analyzed for luciferase and ?-galactosidase activities. Luciferase
values were normalized to ?-galactosidase activity, and the results are dis-
played as a multiple of the induction by vector alone. A representative
result of three independent experiments is shown. Error bars denote SDs
for triplicate samples. C, The level of expression of deletion mutant pro-
teins in 293T cells was tested by Western blot using the M2 anti-
BCMA overexpression activates Elk-1 nuclear factor. A,
cotransfected with 100 ng of HA-JNK plasmid and 100 ng of empty vector
(vector), pFRMEKK vector from Stratagene’s Pathfinder reporter system
(MEKK), full-length hBCMA, HA-tagged full-length mBCMA, or h84,
which contains only the extracellular/transmembrane region of human
BCMA. The immunoprecipitated HA-JNK was used in in vitro phosphor-
ylation experiments of GST-Jun substrate, and the radioactively phosphor-
ylated GST-Jun was analyzed by SDS-PAGE and autoradiography. Blot-
ting with anti-JNK polyclonal Abs confirmed that the same amount of JNK
was present in each of the phosphorylation mixtures. B, 293EBNA cells
were cotransfected with the corresponding Pathfinder reporter system plas-
mids and 100 ng of empty pSG5FLAG (vector), Fh184, Fh164, Fh143,
Fh118, or Fh91. c-Jun-dependent luciferase activity was measured 48 h
after transfection. The MEKK plasmid was used as a positive control for
JNK activation. Vector encoding ?-galactosidase was cotransfected in ev-
ery sample to normalize transfection efficiencies. Data are shown as the
mean ? SD of triplicate samples and represent one of the four independent
experiments, all of which gave similar results. C, The level of expression
of deletion mutant proteins in 293EBNA cells was tested by Western blot
using the M2 anti-FLAG mAb.
Overexpression of BCMA activates JNK. A, 293 cells were
1326BCMA ASSOCIATES WITH TRAFS AND ACTIVATES NF-?B, p38, AND JNK
c-Jun phosphorylation, whereas the Fh119 and Fh91 mutants gave
lower induction levels than the empty vector (Fig. 5B). The level
of expression of the deletion mutant proteins was tested by West-
ern immunoblotting and was found similar (Fig. 5C).
The ability of BCMA to activate p38 and ERK MAPKs were
assayed using a nonradioactive pull-down MAPK assay kit. 293T
cells were transiently transfected with no plasmid (negative con-
trol) and with 1 ?g of each pSG5FLAG empty vector, Fh91,
Fh184, and a human CD40-expressing vector (positive control).
The level of activation of the kinases was tested by measuring the
phosphorylation of ATF-2 (for p38) or Elk-1 (for ERK). The re-
sults obtained are shown in Fig. 6. The overexpression of Fh184
significantly increased the amount of phosphorylated ATF-2, as
compared with that of the cells transfected with no or empty vector
or with the Fh91 mutant of hBCMA, which is lacking the cyto-
plasmic tail. On the contrary, the overexpression of Fh184 did not
increase the amount of phosphorylated Elk-1. These data indicate
that overexpression of BCMA activates the p38 kinase and not the
ERK one. Cell lysates were immunoprecipitated for FLAG-tagged
proteins and assayed by Western blot using M2 anti-FLAG mAb;
the results showed that similar amounts of Fh91 and Fh184 have
been produced during transient transfection of the cells. Finally,
the amount of protein used for the experiment has been assessed by
Western blot using a rabbit polyclonal anti-PI3-Kp85 Ab and
found to be similar.
Overexpression of BCMA activates the NF-?B and Elk-1 nu-
clear factors and the JNK and p38 MAPKs; furthermore, the cy-
toplasmic protein segment comprised between positions 119 and
143 is essential for NF-?B, Elk-1, and JNK activation and is highly
conserved in hBCMA and mBCMA.
Functional and biochemical mapping of the BCMA
We studied the association of the six known TRAFs with BCMA.
COS7 cells were cotransfected with the mouse HAm185 plasmid
and one of the plasmids encoding FLAG-tagged human TRAF1,
TRAF2, TRAF3, F-tagged human TRAF4, or FLAG-tagged
mouse TRAF5 or TRAF6. The cells were lysed 48 h later, and
proteins were immunoprecipitated with the M2 anti-FLAG mAb
for TRAF1, TRAF2, TRAF3, TRAF5, or TRAF6 or with anti-F
mAb for TRAF4. Coimmunoprecipitated HAm185 was detected
by immunoblotting with anti-HA mAb (Fig. 7A). The mBCMA
associates strongly with TRAF1, TRAF2, and TRAF3 molecules,
weakly with TRAF5, and not with TRAF4 and TRAF6. To vali-
date these results, a second series of experiments was performed:
COS7 cells were cotransfected with the Fh184 plasmid and an
expression plasmid for either human TRAF1, TRAF2, TRAF3, or
TRAF5. The transfected cells were lysed 24 h later, and FLAG-
tagged hBCMA was immunoprecipitated with M2 anti-FLAG
mAb. Coimmunoprecipitated TRAFs were detected by immuno-
blotting with corresponding anti-TRAF Ab. The hBCMA, under
does not activate the ERK one. Two sets of 293T cells were transfected
with no plasmid (control) and 1 ?g of each empty pSG5FLAG vector
(vector), Fh91, Fh184, and a vector expressing full-length human CD40
(CD40). Twenty-four hours after transfection, the cells were lysed and 300
?g of lysate were assayed for p38 (phospho-ATF-2) and ERK (phospho-
Elk-1) activity, using a pull-down dual assay kit. Two hundred micrograms
of cell lysate was immunoprecipitated using M2 anti-FLAG mAb co-
valently bound to beads and assayed by Western blot using the M2 anti-
FLAG mAb for the expression of Fh184 and Fh91 proteins. To verify the
similar level of expression of proteins in 293T cells, 10 ?g of cell lysate
were assayed by Western blot using a rabbit polyclonal anti-PI3-Kp85 Ab.
Overexpression of BCMA activates the p38 MAPK and
BCMA. A, COS7 cells were cotransfected with HA-tagged full-length
mBCMA-HAm185 and plasmids encoding human FLAG-tagged TRAF1,
TRAF2, TRAF3, F-tagged human TRAF4, or FLAG-tagged mouse
TRAF5 or TRAF6. Cells were lysed, and the lysate was immunoprecipi-
tated with M2 anti-FLAG mAb, except for TRAF4, which was immuno-
precipitated with anti-F mAb. After washing, the bound proteins were
eluted by addition of FLAG peptide (TRAF4 eluted directly by addition of
gel loading buffer), and the eluate was electrophoresed, blotted onto a
membrane, and tested for the presence of coimmunoprecipitated TRAFs.
One-fortieth of the lysate (input) was electrophoresed, blotted, and tested
for the expression of the various TRAFs using anti-FLAG (TRAF1,
TRAF2, TRAF3, TRAF5, TRAF6), anti-F (TRAF4), and anti-HA
(mBCMA) Abs. The eluate of each immunoprecipitation was tested for the
presence of associated HA-tagged mouse BCMA. B, COS7 cells were co-
transfected with Fh184 and plasmids encoding human TRAF1, TRAF2,
TRAF3, or TRAF5. Cells were lysed, and the lysate was immunoprecipi-
tated with M2 anti-FLAG mAb. After washing, the bound proteins were
eluted by addition of FLAG peptide, and the eluate was electrophoresed,
blotted onto a membrane, and tested for the presence of coimmunoprecipi-
tated TRAFs. One-fortieth of the lysate (input) was tested for the expres-
sion of the various TRAFs and of the Fh184 (B). The eluate of each im-
munoprecipitation was tested for the presence of corresponding TRAFs.
TRAF1, TRAF2, and TRAF3 coimmunoprecipitate with
1327The Journal of Immunology
the experimental conditions used, associated only with TRAF1,
TRAF2, and TRAF3 (Fig. 7B).
To identify sequences necessary for the association between
BCMA and the various TRAF proteins, we studied the association
of the different FLAG-tagged mutants of hBCMA with human
TRAF1, TRAF2, and TRAF3. COS7 cells were cotransfected with
one of the Fh184, Fh164, Fh143, Fh118, or Fh91 expression vec-
tors and one of the TRAF1, TRAF2, or TRAF3 vectors. The
Fh184, Fh164, and Fh143 constructions associated with the
TRAF1, TRAF2, and TRAF3, whereas the Fh118 and Fh91 dele-
tion mutants did not bind any of the three TRAFs tested (Fig. 8).
The data presented indicate that the BCMA activates NF-?B,
Elk-1, and JNK and associates with TRAF1, TRAF2, and TRAF3.
The protein segment between the amino acid sequence positions
119 and 143 in the cytoplasmic tail of BCMA is required for both
TRAF association and NF-?B, Elk-1, and JNK activation, consis-
tent the TRAFs being involved in these activations.
A dominant negative form of TRAF2 decreases BCMA-mediated
The requirement of TRAF2 for BCMA-mediated NF-?B activa-
tion was tested using a vector that encodes the TRAF2 dominant-
negative mutant TRAF2.DN(?6–86). This mutant, lacking the N-
terminal RING finger domain, suppresses signaling of NF-?B by
interacting with the receptor and preventing activation of specific
endogenous TRAF2 molecules (11, 23, 32). Coexpression of
Fh184 and HAm185 expression vectors with increasing amounts
of TRAF2.DN expression vector, in transfected 293T cells, re-
sulted in a dose-dependent inhibition of NF-?B activation (Fig.
9A). The highest concentration of added TRAF2.DN expression
vector (150 ng) resulted in ?50% inhibition of NF-?B activation
for both hBCMA and mBCMA. The level of expression of either
Fh184 or HAm185 proteins was tested by immunoblotting and was
found unmodified until the addition of 150 ng of TRAF2.DN-ex-
pressing vector (Fig. 9B). We cannot answer the question whether
100% inhibition of NF-?B activation can be obtained, because
addition of higher amounts of TRAF2.DN vector resulted in a
decrease of expression of Fh184 and HAm185 proteins.
We addressed the issues of the localization of the BCMA protein
and its signal transduction. In a previous study, we have charac-
terized the BCMA gene and protein both in the human and the
mouse. We showed that, in the human myeloma U266 cell line, the
BCMA protein is mainly found in a Golgi-like perinuclear struc-
ture (30). Functional TNFR members are localized at the cell sur-
face, and therefore BCMA might also be found at the same loca-
tion. Indeed, production of FLAG-tagged BCMA in BJAB and
COS7 cell lines allowed us to demonstrate the presence of BCMA
on the cell surface, as well in a perinuclear Golgi-like structure. To
test whether the intracytoplasmic tail of BCMA is responsible for
the Golgi retention, as it has been reported for some proteins, we
have also studied the localization of the mutant BCMA construct
with its C terminal truncated. There were no differences in local-
ization of the full-length and mutant BCMA proteins. To answer
the question whether or not the surface localization of BCMA is
the result of its transient overexpression, we have established 293
clones stably expressing BCMA. We have found that these clones
have both a surface and intracytoplasmic localization BCMA. The
Golgi-like localization of a TNFR protein has been described, in
quired for the association with TRAF1, TRAF2, and TRAF3. COS7 cells
were cotransfected with one of the plasmids encoding human TRAF1,
TRAF2, and TRAF3 and with one of empty pSG5FLAG (vector), Fh184,
Fh164, Fh143, Fh118, and Fh91 plasmids. Cells were lysed, and the lysate
was immunoprecipitated with M2 anti-FLAG mAb. After washing, the
bound proteins were eluted by addition of FLAG peptide, and the eluate
was electrophoresed, blotted onto a membrane, and tested for the presence
of coimmunoprecipitated TRAFs. One-fortieth of the lysate (input) was
tested for the expression of the various TRAFs and the deletion mutants of
FLAG-tagged hBCMA. The input of FLAG-tagged mutants shown corre-
sponds to the coimmunoprecipitation experiments with TRAF1. The co-
expression of the two other TRAFs (TRAF2 and TRAF3) gave similar
The 25-aa protein segment (119–143) of hBCMA is re-
ated NF-?B activation. A, 293T cells were cotransfected with luciferase
reporter plasmid, 100 ng of one of empty vector (F), Fh184, HAm185, and
increasing amounts of a pcDNA3TRAF2DN expressing vector (50, 100,
and 150 ng). NF-?B activation was measured as described in Materials and
Methods. The pGK-?-galactosidase plasmid encoding ?-galactosidase was
cotransfected in every sample to normalize transfection efficiencies. Forty-
eight hours after transfection, cells were harvested, lysed, and analyzed for
luciferase and ?-galactosidase activity. Luciferase values were normalized
to ?-galactosidase activity, and the results are displayed as a multiple of the
induction by vector alone. A representative result of three independent
experiments is shown. Error bars denote SDs for triplicate samples. B, The
level of expression of Fh184 and HAm185 was assessed by Western blot
using the M2 anti-FLAG Ab for Fh184 and the 12CA5 anti-HA Ab for
Dominant negative TRAF2 protein inhibits BCMA-medi-
1328BCMA ASSOCIATES WITH TRAFS AND ACTIVATES NF-?B, p38, AND JNK
human endothelial cells, in which most TNFR1 is Golgi-associated
protein and little is found on the plasma membrane (33). Trans-
fection experiments in human monocyte U937 and human endo-
thelial ECV304 cell lines confirmed the Golgi localization of
TNFR1 (34). Furthermore, it has been reported that, in human
vascular smooth muscle cells, p53 activation transiently increased
surface Fas (CD95) expression by transporting the protein from the
Golgi complex to the plasma membrane (35). Therefore, it is pos-
sible that there is a mechanism modulating BCMA expression on
the cell surface of normal B lymphocytes by controlling its trans-
port from a Golgi-like structure.
Members of the TNFR superfamily associate either directly or
indirectly with TRAFs that recruit and activate downstream signal
transducers. TRAFs are adaptor proteins that further propagate the
signal elicited by TNF, which causes an activation of nuclear fac-
tors, namely the NF-?B, Elk-1, and JNK. We investigated whether
BCMA overexpression falls into the same signal propagation
scheme. Our results can be interpreted as follows.
The overexpression of BCMA activates the MAPK pathway,
especially JNK and p38 kinase, and the nuclear factors NF-?B and
Elk-1. As expected, a mutant BCMA lacking the cytoplasmic tail
failed to activate any of the factors studied. In this respect, BCMA
follows the scheme of other members of the TNFR family. Anal-
ysis of the activation of JNK, NF-?B, and Elk-1 by deletion mu-
tants of BCMA indicated that the same protein segment of 25 aa
residues (119–143) is indispensable for the activation of these
Coexpression of the different TRAF and BCMA evidenced as-
sociation of TRAF1, TRAF2, and TRAF3 adaptor proteins with
BCMA. Note that a faint association of mouse TRAF5 to mouse
BCMA was observed; this result was not confirmed when we
tested the association of either human TRAF5 with hBCMA or of
mouse TRAF5 with hBCMA. We further showed that the protein
segment (amino acid positions 119–143), which is essential for the
activation of JNK, NF-?B and Elk-1, was also necessary for the as-
sociation with TRAF1, TRAF2, and TRAF3, suggesting that the
activation is achieved through the association of TRAF proteins. We
have also showed that a dominant negative form of TRAF2 decreases
the NF-?B activation mediated by BCMA overexpression.
Several TRAF binding motifs such as PXQXT/S (10), EXGKE
(8), or VXX(T/S)XEE (36) have been identified in other TNFR
members as associating with TRAF1, TRAF2, TRAF3, and
TRAF5. None of these motifs is present in BCMA. However, ma-
jor (P/S/A/T)X(Q/E)E and minor PXQXXD TRAF2-binding con-
sensus sequences have recently been proposed (37). The major
sequence motif is present in the protein segment (amino acid po-
sitions 119–143) of BCMA essential for both association of
TRAFs and activation of JNK, NF-?B, and Elk-1, positions 122–
125 (T122V123E124E125). Therefore, we are trying actually to
verify whether this sequence motif is also necessary for the asso-
ciation of TRAF1 and TRAF3 with BCMA.
This study confirms that BCMA is a functional member of the
TNFR superfamily. Furthermore, as BCMA is lacking a “death
domain” and its overexpression activates NF-?B, p38, and JNK,
we can reasonably hypothesize that upon binding of its corre-
sponding ligand, BCMA transduces signals for cell survival and
We thank Drs. E. Kieff, G. Mosialos, and K. M. Kaye (Harvard Medi-
cal School, Boston, MA) for their generous gift of pSG5hTRAF1,
pSG5FLAGhTRAF1, pSG5hTRAF3, pSG5FLAGhTRAF3, pSG5hTRAF2
pSG5FLAGhTRAF2, and pcDNA3TRAF2.DN vectors, Dr. E. Hatzivassi-
liou (Harvard Medical School) for pSG5FLAG vector, Dr. C. Regnier
(Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 184) for
pAT3hTRAF4 plasmid, Dr. J Ghysdael (Institut Curie, Orsay, France) for
pDEB vector, Dr. G. Cheng (Molecular Biology Institute, University of
California, Los Angeles, CA) for pEBBhTRAF5 plasmid, and Dr. M.-C.
Rio (Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 184)
for anti-F mAb. We thank Dr. Y. Richard (Institut National de la Sante ´ et
de la Recherche Me ´dicale, Unite ´ 131) for fruitful discussions.
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