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B, Elk-1, c-Jun N-Terminal Kinase, and p38 Mitogen-Activated Protein Kinase1
*
Laboratory of Experimental Endocrinology, Faculty of Medicine, University of Crete, Heraklion, Greece;
Institut National de la Santé et de la Recherche Médicale, Unité 131, Institut Paris-Sud sur les Cytokines, Clamart, France; and
Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 corresponding ligand BCMA transduces signals for cell
survival and proliferation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (11, 12, 13)
and activate the c-Jun N-terminal protein kinase (JNK)
(14). TRAF6 also mediates the activation of extracellular
signal-regulated 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 selectively expressed in mature B lymphocytes (19) and was therefore 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 conserved six-cysteine motif (17). A sensitive method of sequence analysis, hydrophobic cluster analysis (20), indicated that this conserved motif is similar to the six-cysteine repeat motif found in the extracellular part of TNFRs. There are two notable differences between 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 transiently
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 association.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 polyclonal anti-TRAF4 (N-16) and anti-goat HRP-conjugated Abs were purchased 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 Santé et de la Recherche Médicale, Unité 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 polyclonal 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).
Primers
The following primers were used in this study: BCMA5'ATG (5'-AAGCTTATGTTGCAGATGGCTGGGCA-3'), BCMA3'TAA (5'-GGATCCTTACCTAGCAGAAATTGATTTC-3'), 37 (5'-CCCAAGCTTATGGCTGGGCAGTGCTCC-3'), 43 (5'-CGCGGATCCTTATGGTTCAGAGCTTATCTTCCT-3'), AH7 (CGCGGATCCTTACCTAGCAGAAATTGATTTCT-3'), AH9 (5'-CCGCTCGAGGGCGCAACAGTGTTTCCACA-3'), AH10 (GGAAGATCTCTAACGACATCTAAAACACCAG-3'), BFL1 (5'-AACTGCAGCTGGGCAGTGCTCCCAAAA-3'), BFL2 (5'-CGGGATCCTTAATAGTCATTCGTTTTCGTGGTG-3'), BFL3 (5'-CGGGATCCTTAGCAATGGTCATAGTCGACCT-3'), BFL4 (5'-CGGGATCCTTAGCCTCTCGGAAGAATAATTTC-3'), and BFL5 (5'-CGGGATCCTTAGTTTTTAAACTCGTCCTTTAATG-3'). All primers used in this study were purchased from Genset (Paris, France).
Expression vectors
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, The Netherlands).
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 pcDNA3.
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-hBCMA144–184
(Fh143), BFL1 and BFL4 for FLAG-hBCMA119–184 (Fh118), and BFL1 and
BFL5 for FLAG-hBCMA92–184 (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 (TRAF26–86) (23),
pGEX-Jun1–79(1–79), pcDNA3-HA-JNK (28), and the
ß-galactosidase expression vector (pGK-ßgal), in which expression
is driven by the phosphoglucokinase promoter (22), have
been already described. pAT3FhTRAF4 encoding human FLAG-tagged TRAF4
was a generous gift from Dr. Catherine Regnier (Strasbourg,
France).
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 µg/ml of streptomycin and were grown at 37°C in 5% CO2. The 293EBNA cell line was purchased from Invitrogen and maintained in culture 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 µg/ml of streptomycin and grown at 37°C in 5% CO2. Adherent cells were seeded in six-well plates (5 x 105 cells per well) in 2 ml of complete medium, incubated at 37°C in 5% CO2 for 20–24 h, and transfected with lipofectamine according to the manufacturer’s instructions, using 1 µg of total plasmid DNA, for 6 h. BJAB cells were transfected by electroporation (960 µF, 210 V) in 400 µl 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 µg/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 ß-galactosidase assays
Transfected cells were washed twice with PBS and lysed with reporter lysis buffer (Promega, Madison, WI). The luciferase activity was measured using the reporter assay system (Promega). ß-galactosidase activity was measured using the luminescent ß-galactosidase reporter system (Clontech, Palo Alto, CA) in a Packard luminometer analyzer (Packard, Meriden, CT). Measurements of luciferase were normalized to ß-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 modifications. 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 protein-G Agarose beads, washed
three times in lysis buffer, once in kinase reaction buffer (12.5 mM
MOPS, pH 7.5, 12.5 mM ß-glycerophoshate, 7.5 mM
MgCl2, 0.5 mM EGTA, 0.5 mM NaF, 0.5 mM sodium
vanadate), and resuspended in 30 µl of the same buffer containing 2
µg of GST-Jun, 20 µM unlabeled ATP, and 5 µCi
[
-32P]ATP. After incubation at 20°C for 30
min, kinase reaction products were analyzed by SDS-PAGE and
autoradiography. Part of the immunoprecipitated material was resolved
by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and
immunoblotted 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 (ATF) 2 (p38) or Elk-1 (ERK) substrates. Analysis of phosphorylated substrates was performed by Western blotting using specific polyclonal phosphoantibodies.
Coimmunoprecipitation experiments
COS7 cells were cotransfected with one vector encoding a TRAF and one vector encoding one of the various FLAG-tagged BCMA constructs. Eighteen 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 µM FLAG peptide diluted in PBS. The eluate and the input were analyzed by PAGE and transferred onto a polyvinylidene difluoride membrane (Hybond-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 x 105 cells per condition
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
lymphocyte 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 efficiency (50–60%). On
the contrary, COS7 cell line does not express BCMA. The cell lines were
transfected with two vectors, one coding for a FLAG-tagged full-length
hBCMA (Fh184) and a second 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
secondary 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. 1
A). Full-length hBCMA
and the mutant hBCMA missing its cytoplasmic tail were similarly
distributed in both BJAB and COS7 cell lines. Both proteins displayed
an intracytoplasmic 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.
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and
indicate both surface and intracytoplasmic localization of HAm185.
Similar results have been obtained in two other clones tested (data not
shown).
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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 plasmid.
Overexpression of hBCMA (h184) induced a 12-fold activation 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 vectors
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 molecule, (h84), failed to
activate NF-B, confirming that the cytoplasmic tail of BCMA is
essential for transducing a signal and activating NF-B.
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B induction, we constructed a series of SV40 promoter-driven
vectors encoding N-terminal FLAG-tagged deletion mutants of hBCMA and
tested these mutants for NF-B activation (Fig. 3
C) 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 molecule, 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 hBCMA and mBCMA (Fig. 3B). BCMA activates the mitogen-activated protein kinase (MAPK) pathway
We next examined the activation of the nuclear factor Elk-1 using
a luciferase reporter system. Elk-1 is a substrate for the MAPKs: JNK,
p38, and ERK. Overexpression of hBCMA in 293 cells activated Elk-1 to a
level 4.5-fold higher than that obtained using the empty vector (Fig. 4A). Similar results were
obtained using mBCMA (3.5-fold activation). As expected, the mutant
hBCMA h84 failed to activate Elk-1. MAPK/ERK kinase (MEK) 1
overexpression 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
nuclear 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 factor Elk-1.
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A). 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
background 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 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 Western
immunoblotting and was found similar (Fig. 5C).
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.
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 cytoplasmic 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.
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B and Elk-1 nuclear factors
and the JNK and p38 MAPKs; furthermore, the cytoplasmic 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 intracytoplasmic tail
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 validate 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 immunoblotting with
corresponding anti-TRAF Ab. The hBCMA, under the experimental
conditions used, associated only with TRAF1, TRAF2, and TRAF3 (Fig. 7B).
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).
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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, consistent the TRAFs being involved
in these activations.
A dominant negative form of TRAF2 decreases BCMA-mediated NF-B
activation
The requirement of TRAF2 for BCMA-mediated NF-B activation 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, resulted 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-expressing 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 factors, 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. Analysis of the activation of JNK, NF-B,
and Elk-1 by deletion mutants of BCMA indicated that the same protein
segment of 25 aa residues (119–143) is indispensable for the
activation of these three proteins.
Coexpression of the different TRAF and BCMA evidenced association 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
association 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, major
(P/S/A/T)X(Q/E)E and minor PXQXXD TRAF2-binding consensus sequences
have recently been proposed (37). The major sequence motif
is present in the protein segment (amino acid positions 119–143) of
BCMA essential for both association of TRAFs and activation of JNK,
NF-B, and Elk-1, positions 122–125 (T122
V123 E124
E125). Therefore, we are trying actually to
verify whether this sequence motif is also necessary for the
association 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 corresponding ligand, BCMA
transduces signals for cell survival and proliferation.
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Acknowledgments |
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Footnotes |
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2 A.H., J.R., and M.-F.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Andreas Tsapis, Institut National de la Santé et de la Recherche Médicale Unité 131, Institut Paris-Sud sur les Cytokines, 32, rue des Carnets, 92140 Clamart, France.
4 Abbreviations used in this paper: TRAF, TNFR-associated factor; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-related kinase; BCMA, B cell maturation; 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.
Received for publication January 10, 2000. Accepted for publication May 18, 2000.
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 M. C. Ryan, M. Hering, D. Peckham, C. F. McDonagh, L. Brown, K. M. Kim, D. L. Meyer, R. F. Zabinski, I. S. Grewal, and P. J. Carter Antibody targeting of B-cell maturation antigen on malignant plasma cells Mol. Cancer Ther., November 1, 2007; 6(11): 3009 - 3018. [Abstract] [Full Text] [PDF]
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A. Chiu, W. Xu, B. He, S. R. Dillon, J. A. Gross, E. Sievers, X. Qiao, P. Santini, E. Hyjek, J.-w. Lee, et al. Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL Blood, January 15, 2007; 109(2): 729 - 739. [Abstract] [Full Text] [PDF]
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 Y.-T. Tai, X.-F. Li, I. Breitkreutz, W. Song, P. Neri, L. Catley, K. Podar, T. Hideshima, D. Chauhan, N. Raje, et al. Role of B-Cell-Activating Factor in Adhesion and Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment. Cancer Res., July 1, 2006; 66(13): 6675 - 6682. [Abstract] [Full Text] [PDF]
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 K. Yoshimoto, Y. Takahashi, M. Ogasawara, Y. Setoyama, K. Suzuki, K. Tsuzaka, T. Abe, and T. Takeuchi Aberrant expression of BAFF in T cells of systemic lupus erythematosus, which is recapitulated by a human T cell line, Loucy Int. Immunol., July 1, 2006; 18(7): 1189 - 1196. [Abstract] [Full Text] [PDF]
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P. K. A. Mongini, J. K. Inman, H. Han, R. J. Fattah, S. B. Abramson, and M. Attur APRIL and BAFF Promote Increased Viability of Replicating Human B2 Cells via Mechanism Involving Cyclooxygenase 2. J. Immunol., June 1, 2006; 176(11): 6736 - 6751. [Abstract] [Full Text] [PDF]
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M. Yang, H. Hase, D. Legarda-Addison, L. Varughese, B. Seed, and A. T. Ting B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells J. Immunol., September 1, 2005; 175(5): 2814 - 2824. [Abstract] [Full Text] [PDF]
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J. Moreaux, F. W. Cremer, T. Reme, M. Raab, K. Mahtouk, P. Kaukel, V. Pantesco, J. De Vos, E. Jourdan, A. Jauch, et al. The level of TACI gene expression in myeloma cells is associated with a signature of microenvironment dependence versus a plasmablastic signature Blood, August 1, 2005; 106(3): 1021 - 1030. [Abstract] [Full Text] [PDF]
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 L. He, A. C. Grammer, X. Wu, and P. E. Lipsky TRAF3 Forms Heterotrimers with TRAF2 and Modulates Its Ability to Mediate NF-{kappa}B Activation J. Biol. Chem., December 31, 2004; 279(53): 55855 - 55865. [Abstract] [Full Text] [PDF]
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 W Stohl A therapeutic role for BLyS antagonists Lupus, May 1, 2004; 13(5): 317 - 322. [Abstract] [PDF]
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D. R. Patel, H. J. A. Wallweber, J. Yin, S. K. Shriver, S. A. Marsters, N. C. Gordon, M. A. Starovasnik, and R. F. Kelley Engineering an APRIL-specific B Cell Maturation Antigen J. Biol. Chem., April 16, 2004; 279(16): 16727 - 16735. [Abstract] [Full Text] [PDF]
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J. Moreaux, E. Legouffe, E. Jourdan, P. Quittet, T. Reme, C. Lugagne, P. Moine, J.-F. Rossi, B. Klein, and K. Tarte BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone Blood, April 15, 2004; 103(8): 3148 - 3157. [Abstract] [Full Text] [PDF]
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B. He, A. Chadburn, E. Jou, E. J. Schattner, D. M. Knowles, and A. Cerutti Lymphoma B Cells Evade Apoptosis through the TNF Family Members BAFF/BLyS and APRIL J. Immunol., March 1, 2004; 172(5): 3268 - 3279. [Abstract] [Full Text] [PDF]
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C. Kern, J.-F. Cornuel, C. Billard, R. Tang, D. Rouillard, V. Stenou, T. Defrance, F. Ajchenbaum-Cymbalista, P.-Y. Simonin, S. Feldblum, et al. Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway Blood, January 15, 2004; 103(2): 679 - 688. [Abstract] [Full Text] [PDF]
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 G.-H. Cha, K. S. Cho, J. H. Lee, M. Kim, E. Kim, J. Park, S. B. Lee, and J. Chung Discrete Functions of TRAF1 and TRAF2 in Drosophila melanogaster Mediated by c-Jun N-Terminal Kinase and NF-{kappa}B-Dependent Signaling Pathways Mol. Cell. Biol., November 15, 2003; 23(22): 7982 - 7991. [Abstract] [Full Text] [PDF]
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K. Gee, M. Kozlowski, and A. Kumar Tumor Necrosis Factor-{alpha} Induces Functionally Active Hyaluronan-adhesive CD44 by Activating Sialidase through p38 Mitogen-activated Protein Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells J. Biol. Chem., September 26, 2003; 278(39): 37275 - 37287. [Abstract] [Full Text] [PDF]
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L.-G. Xu and H.-B. Shu TNFR-Associated Factor-3 Is Associated With BAFF-R and Negatively Regulates BAFF-R-Mediated NF-{kappa}B Activation and IL-10 Production J. Immunol., December 15, 2002; 169(12): 6883 - 6889. [Abstract] [Full Text] [PDF]
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 N. Feldhahn, I. Schwering, S. Lee, M. Wartenberg, F. Klein, H. Wang, G. Zhou, S. M. Wang, J. D. Rowley, J. Hescheler, et al. Silencing of B Cell Receptor Signals in Human Naive B Cells J. Exp. Med., November 18, 2002; 196(10): 1291 - 1305. [Abstract] [Full Text] [PDF]
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K. Gee, W. Lim, W. Ma, D. Nandan, F. Diaz-Mitoma, M. Kozlowski, and A. Kumar Differential Regulation of CD44 Expression by Lipopolysaccharide (LPS) and TNF-{alpha} in Human Monocytic Cells: Distinct Involvement of c-Jun N-Terminal Kinase in LPS-Induced CD44 Expression J. Immunol., November 15, 2002; 169(10): 5660 - 5672. [Abstract] [Full Text] [PDF]
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A. J. Novak, R. J. Bram, N. E. Kay, and D. F. Jelinek Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival Blood, September 26, 2002; 100(8): 2973 - 2979. [Abstract] [Full Text] [PDF]
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 L.-G. Xu, M. Wu, J. Hu, Z. Zhai, and H.-B. Shu Identification of downstream genes up-regulated by the tumor necrosis factor family member TALL-1 J. Leukoc. Biol., August 1, 2002; 72(2): 410 - 416. [Abstract] [Full Text] [PDF]
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S. Xu and K.-P. Lam B-Cell Maturation Protein, Which Binds the Tumor Necrosis Factor Family Members BAFF and APRIL, Is Dispensable for Humoral Immune Responses Mol. Cell. Biol., June 15, 2001; 21(12): 4067 - 4074. [Abstract] [Full Text]
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C. F. Ware April and Baff Connect Autoimmunity and Cancer J. Exp. Med., December 4, 2000; 192(11): f35 - f38. [Full Text] [PDF]
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