From the Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368
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ABSTRACT |
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Tumor necrosis factor receptor-associated factors
(TRAFs) associate with the CD40 cytoplasmic domain and initiate
signaling after CD40 receptor multimerization by its ligand. We used
saturating peptide-based mutational analyses of the TRAF1/TRAF2/TRAF3
and TRAF6 binding sequences in CD40 to finely map residues involved in
CD40-TRAF interactions. The core binding site for TRAF1, TRAF2, and
TRAF3 in CD40 could be minimally substituted. The TRAF6 binding site
demonstrated more amino acid sequence flexibility and could be
optimized. Point mutations that eliminated or enhanced binding of TRAFs
to one or both sites were made in CD40 and tested in quantitative
CD40-TRAF binding assays. Sequences flanking the core TRAF binding
sites were found to modulate TRAF binding, and the two TRAF binding
sites were not independent. Cloned stable transfectants of human
embryonic kidney 293 cells that expressed wild type CD40 or individual
CD40 mutations were used to demonstrate that both TRAF binding sites
were required for optimal NF- CD40 is a tumor necrosis factor
(TNF)1 receptor superfamily
member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells (1). CD40 signaling
is initiated by receptor oligomerization upon binding the trimeric
ligand CD154 (CD40 ligand/gp39). Numerous and different outcomes of
CD40 signaling occur in distinct cell types and imply a complex
regulation of CD40 signal transduction. Several different signal
transduction pathways have been demonstrated to be activated following
CD40 oligomerization. CD40-mediated signaling results in NF- The signaling functions of most of the six TRAF family members have
been characterized genetically and biochemically. A function for TRAF1
in regulating apoptosis has been suggested from transgenic mice
expressing a dominant negative TRAF1 transgene (16). Studies using
TRAF2 knockout mice and transgenic mice expressing a dominant negative
form of TRAF2 showed that TRAF2 is required for JNK activation but has
limited involvement in NF- The cytoplasmic domain of human CD40 interacts directly with TRAF1,
TRAF2, TRAF3, and TRAF6 (11-15). In previous studies, the consensus
sequence PXQX(T/S) (26) has been designated as a
TRAF1, TRAF2, and TRAF3 binding site. In the CD40 cytoplasmic domain, this sequence is 250PVQET. Mutations and deletions have
been made in the 250PVQET sequence of CD40 that eliminate
TRAF2 and TRAF3 binding (14, 27). However, specific mutations in the
CD40 cytoplasmic domain that individually affect TRAF1, TRAF2, or TRAF3
binding have not been identified. Although human TRAF5 does not
interact directly with the CD40 cytoplasmic domain, it can be recruited indirectly as a hetero-oligomer with TRAF3 (11). The TRAF6 binding site
has been mapped to a membrane-proximal region (231QEPQEINF)
(11, 15). However, it is not known which of these amino acid residues
in CD40 are critical for TRAF6 interaction. Whether the two TRAF
binding regions in the CD40 cytoplasmic domain are independent and can
simultaneously mediate binding of different TRAFs or whether binding of
a TRAF to one binding region would block binding of a second TRAF has
not been determined. Additionally, the role of individual TRAFs in
activating different CD40-dependent signaling pathways has
not been defined.
To gain a better understanding of the interactions between TRAFs and
their cognate binding sites on the CD40 cytoplasmic domain and define
the role of individual TRAFs in CD40 signaling, saturating peptide-based mutational analyses of the TRAF binding sites in the CD40
cytoplasmic domain were performed. TRAF1, TRAF2, and TRAF3 had similar
binding specificities for the core 250PVQET sequence with
few amino acid substitutions tolerated. The TRAF6 binding sequence
could be more freely substituted, and a TRAF6 consensus sequence is
proposed. Stable transfectants expressing selected CD40 mutations were
used to define the roles of individual TRAFs in CD40 signaling. Maximal
NF- Plasmids and Viruses--
The plasmid pGST-CD40c has been
described previously (11). Using oligonucleotides
5'-TCATCACTGTCTCTCCTGCACTGAGA-3'and
5'-TTTGGATCCATGGTTCGTCTGCCTCTGCAGT-3', the human CD40 gene was
amplified by RT-PCR from JY cells and ligated into pGem-T (Promega) to
create phCD40/GemT. The BstZI-SpeI fragment from
phCD40/GemT was ligated into pcDNA3.1+ digested with
NheI and NotI to make phCD40/cDNA. Amino acid
substitutions in CD40 were generated using complementary primers with
the desired base changes and pGST-CD40c or phCD40/cDNA as templates
using the QuikChange site-directed mutagenesis kit (Stratagene). All mutated genes were verified by automated DNA sequencing. The NF- Protein Expression and Purification--
Spodoptera
frugiperda (Sf21) cells were maintained and infected as
described previously (28) using medium supplemented with 5%
heat-inactivated fetal bovine serum (HyClone) and 50 µg/ml gentamicin
sulfate (Life Technologies, Inc.). Cytosolic extracts of
baculovirus-infected Sf21 cells were prepared as described (11,
28), frozen under liquid nitrogen, and stored at
The CA21 hybridoma producing a mouse IgG1 monoclonal antibody against a
peptide epitope derived from the cytoplasmic domain of human
L-selectin (31) was purified as described (32) and conjugated with N-hydroxysuccinimide
ester-XX-biotin (Calbiochem) as described (33). The 53-6 hybridoma producing a rat IgG2a against mouse CD8 Peptide Binding Assay--
Peptides C-terminally attached to
cellulose membranes were synthesized by Jerini BioTools, GmbH (Berlin,
Germany). Each peptide spot contained ~10 nmol of
peptide/3-mm2 circle (35, 36). Blocking and binding were
performed according to the manufacturer's protocol. Membranes were
probed with Sf21 cytosolic extracts diluted to contain
approximately 1 µg/ml CA21 epitope-tagged TRAF in binding buffer.
After electroblotting to PVDF membranes, TRAF proteins were detected
with biotin-CA21-epitope specific monoclonal antibody (31),
streptavidin-horseradish peroxidase (0.5 µg/ml; Jackson
ImmunoResearch), and horseradish peroxidase chemiluminescence blotting
substrate (Roche Molecular Biochemicals). The original membrane
containing the peptides was stripped sequentially according to the
manufacturer's protocol with four buffers: 500 mM lithium
perchlorate; 50 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM 2-ME; 50 mM glycine, pH 2.8; and 50 mM bis-Tris, pH 6.5, 1 M NaCl, 100 mM 2-ME.
TRAF Plate Binding Assay--
Serial dilutions of purified
GST-CD40c fusion protein were incubated in 96-well plates (Reacti-Bind,
glutathione-coated; Pierce) in 100 µl of 40 mM HEPES, pH
7.5, 100 mM NaCl, 0.05% Nonidet P-40, 1 mM
MgCl2, and 0.1 mM dithiothreitol (binding
buffer) for 1 h at room temperature. Plates were washed in binding
buffer and blocked in binding buffer containing 1% bovine serum
albumin for 1 h at room temperature. Plates were washed in binding
buffer and probed with TRAF-CA21 containing cytosolic extracts from
infected Sf21 insect cells diluted in binding buffer containing
0.1% bovine serum albumin. TRAF binding was detected with 1.0 µg/ml
biotin-conjugated CA21 antibody and streptavidin-horseradish peroxidase
(0.5 µg/ml; Jackson ImmunoResearch). Color development (37) was
stopped after 1-2 min by the addition of 100 µl of 0.2 M
citric acid/well. OD405-490 was quantitated on a 96-well
plate reader (Vmax; Molecular Devices).
Generation of Stably Transfected Cell Lines--
Ten micrograms
of phCD40/cDNA or plasmids expressing the indicated mutations of
CD40 were transfected into 2 × 106 human embryonic
kidney (HEK) 293 cells ((38), ATCC) using Superfect (Qiagen) according
to the manufacturer's protocol. Forty-eight h after transfection
medium was removed and replaced with medium containing 0.4 mg/ml
Geneticin (Life Technologies, Inc.). Geneticin-resistant cells were
expanded and stained with FITC mouse anti-human CD40 monoclonal
antibody (PharMingen). Narrowly gated CD40-expressing cells were sorted
into 96-well plates at 1 cell/well with a FACS Vantage (Becton
Dickinson). Clones were expanded, and surface CD40 levels were analyzed
by flow cytometry on a FACScan flow cytometer (Becton Dickinson) as
described above. Two independent clonal isolates expressing similar
levels of CD40 were chosen from each transfected cell line for
subsequent analysis. In all cases, the two independent clones of each
cell line responded similarly to CD40 stimulation. Representative
results from one isolate are shown for all experiments.
NF- JNK and p38 MAPK Activation--
One million HEK 293 cells or
stable transfectants were plated in 60-mm dishes and incubated
overnight at 37 °C. Cells were stimulated for 15 min with medium
alone or medium containing 10 µg/ml mouse CD8 Mutational Analysis of TRAF Binding Sites in CD40--
Minimal
binding sites for TRAF1, TRAF2, TRAF3, and TRAF6 in the human CD40
cytoplasmic domain were previously mapped using peptides from CD40
synthesized on cellulose membranes. Using overlapping peptides and
peptides with progressive deletions from the N terminus, C terminus, or
both termini, TRAF1, TRAF2, and TRAF3 bound optimally to the sequence
250PVQET, whereas TRAF6 bound to a more membrane-proximal
sequence: 231QEPQEINF (11). To more precisely define
residues critical for CD40-TRAF interactions within each binding site,
peptides singly substituted at each position by each of the 20 amino
acids were synthesized on cellulose membranes. Peptides were
synthesized with either single L-amino acid substitutions
or with single D-amino acid substitutions to test
requirements for amino acid side chain orientation. Insect cell
extracts containing recombinant TRAF1, TRAF2, TRAF3, or TRAF6 proteins
were individually tested for binding to the substituted immobilized peptides.
L-Amino acid or D-amino acid substitutions for
residues N-terminal to the 250PVQET sequence minimally
affected TRAF1, TRAF2, or TRAF3 binding (Fig.
1, A-C). This indicates these
residues were not involved in TRAF1, TRAF2, or TRAF3 interactions with
the peptides. Peptides with L-His substituted for
Pro250 supported TRAF1 and TRAF2 binding and appeared to
have enhanced TRAF3 binding (Fig. 1, A-C). All other
L-amino acid replacements for Pro250 eliminated
binding of TRAF1 and TRAF2. TRAF3 binding, but not TRAF1 or TRAF2
binding, was retained when Pro250 was replaced with
L-Gly. Peptides with Val251 replaced by
L-Ile still efficiently bound TRAF1, TRAF2, and TRAF3 (Fig.
1, A-C). Gln252 and Glu253 could
not be replaced by any other L-amino acids, indicating that
these residues were critical for TRAF1, TRAF2, and TRAF3 binding to the
peptides. Peptides with Thr254 replaced by
L-Ser still maintained TRAF1, TRAF2, or TRAF3 binding, but
all other substitutions for Thr254 eliminated binding (Fig.
1, A-C). Most L-amino acid or
D-amino acid substitutions of Leu255 had little
effect on TRAF1, TRAF2, or TRAF3 binding, indicating that this residue
was not required for TRAF interactions with peptides from the CD40
cytoplasmic domain. A summary of amino acids allowed at each position
and some amino acid replacements that resulted in reduced binding are
shown in Table I.
D-Amino acid replacements of most residues in the
250PVQET sequence eliminated TRAF1, TRAF2, and TRAF3
binding (Fig. 1, A-C). Peptides with D-Tyr
substituted for Pro250 could efficiently bind TRAF1.
Peptides with Pro250 replaced by several
D-amino acids still maintained TRAF2 binding. TRAF3, but
not TRAF1 or TRAF2, bound to peptides with Pro250 replaced
by numerous D-amino acids. The 251VQE sequence
could not be substituted with D-amino acids (Fig. 1,
A-C), indicating contributions of these side chains to
TRAF1, TRAF2, and TRAF3 interactions. Peptides with Thr254
replaced by D-Ser or D-Thr residues maintained
TRAF1, TRAF2, and TRAF3 interactions.
L-Amino acid substitutions in the TRAF6 binding peptide
indicated there were three amino acid residues critical for TRAF6 binding to the 14-amino acid peptide: Pro233,
Glu235, and Phe238 (Fig. 1D and
Table I). Pro233 could only be replaced by
L-Ala, and Phe238 could be replaced by
L-Trp or L-Tyr. Numerous L-amino
acid substitutions of the remaining residues of CD40 from positions 230 to 237 could be made without eliminating TRAF6 binding. This suggested
that those residues were not making essential contributions to TRAF6 binding to the peptides. Substitution of most residues C-terminal to
Phe238 with either L-amino acids or
D-amino acids minimally affected TRAF6 binding (Fig.
1D), indicating these residues were not required. Most
D-amino acid substitutions of the sequence
232EPQEINF eliminated TRAF6 binding to the peptides. These
results indicate that the core TRAF6 binding site may actually be
232EPQEINF. This is consistent with previous mapping
studies delineating Phe238 as the C-terminal end of the
TRAF6 binding site (11).
In our previous analyses of TRAF2 and TRAF3 binding sites in the CD40
cytoplasmic domain, sequences C-terminal to the 250PVQET
motif appeared to contribute to binding (11). To further investigate
the role of this region in TRAF2 and TRAF3 binding, a mutational
analysis on peptides containing the 250PVQET sequence with
11 additional C-terminal amino acids was performed. TRAF2 binding to
the longer peptide with single L-amino acid substitutions or D-amino acid substitutions was identical to that
observed for the mutational analysis in Fig. 1B (compare
Figs. 1B and 2A;
data not shown for D-amino acid substitutions). Similarly,
TRAF1 binding specificity for longer peptides in the mutational
analysis was not significantly different from the mutational analysis
on the shorter peptide (Fig. 1A; data not shown). In
contrast, numerous L-amino acid substitutions could be made
in the 250PVQET sequence without substantially affecting
TRAF3 binding when 11 amino acid residues C-terminal to
250PVQET were included in the peptide. Notably, most
L-amino acid substitutions for Glu253 had no
effect on TRAF3 binding, whereas no L-amino acids could be
substituted for Glu253 in the mutational analysis on the
shorter peptide in Fig. 1C (compare Figs. 1C and
2B). Peptides with several different L-amino acid substitutions for Pro250, Val251, or
Gln252 were also able to bind TRAF3 when 11 additional
amino acids C-terminal to 250PVQET were included. These are
summarized in Table I. Although the presence of amino acids C-terminal
to 250PVQET altered the ability of TRAF3 to bind to
peptides with mutated 250PVQET sequences, substitution of
residues within these C-terminal 11 amino acids did not affect TRAF3
binding (Fig. 2B). Binding of TRAF3 to longer peptides with
D-amino acid substitutions was also identical to results
with the shorter peptide (Fig. 1C) (data not shown). These
findings demonstrate that TRAF3 binding to the 250PVQET
sequence in the CD40 cytoplasmic domain was influenced by amino acid
sequences C-terminal to 250PVQET. Additionally, this
suggests that TRAF2 and TRAF3 interact with different amino acid
residues on CD40.
Relative Affinities of TRAFs for Altered CD40 Cytoplasmic
Domains--
Based on the peptide mutational analyses in Figs. 1 and
2, selected amino acid substitutions designed to differentiate binding of individual TRAFs were engineered in the CD40 cytoplasmic domain. The
mutations were tested for interactions with TRAF1, TRAF2, TRAF3, and
TRAF6 by expressing and isolating each as a glutathione S-transferase-human CD40 cytoplasmic domain (GST-CD40c)
fusion protein and quantitating TRAF binding in a solid-phase binding assay. In this assay, TRAF concentrations were kept constant, and each
GST-CD40c fusion protein was titrated over a wide concentration range.
Previous results with this assay have shown that receptors that bind
TRAFs weakly, such as OX40, only bind TRAF proteins at relatively high
GST-cytoplasmic domain concentrations. In contrast, receptors that bind
TRAFs strongly, such as CD40, give strong signals at 10-20-fold lower
concentrations of GST fusion
protein.3
To eliminate TRAF binding to the 250PVQET
(TRAF1/TRAF2/TRAF3 binding) sequence, the T254A mutation was produced.
Confirming and extending previous reports (14, 15), GST-CD40c T254A had
no significant binding of TRAF1, TRAF2, or TRAF3 (Fig.
3). This was confirmed in GST-CD40c
coprecipitation assays using glutathione-Sepharose beads (data not
shown). GST-CD40c T254A bound TRAF6 similar to wild type GST-CD40c
consistent with an earlier study (15). To target the
231QEPQEINF (TRAF6 binding) sequence, two mutations were
produced, one designed to eliminate and one designed to enhance TRAF6
binding. From the mutational analyses, a GST-CD40c fusion protein with the paired P233G/E235A substitutions was predicted to eliminate TRAF6
binding. As anticipated, GST-CD40c P233G/E235A did not interact with
TRAF6 (Fig. 3D). Interestingly, TRAF1, TRAF2, and TRAF3
exhibited increased binding to GST-CD40c containing the P233G/E235A
substitution. TRAF6 binding to GST-CD40c containing the second mutation
in the 231QEPQEINF sequence, N237D, was increased over
25-fold relative to wild type GST-CD40c (Fig. 3D). TRAF1,
TRAF2, and TRAF3 binding to the GST-CD40c N237D fusion protein was also
significantly increased (~3-4-fold). GST-CD40c fusion proteins that
contained combinations of mutations in the 250PVQET and the
231QEPQEINF TRAF binding sites were also produced. The
triple substitution P233G/E235A/T254A GST-CD40c exhibited no
significant binding to TRAF1, TRAF2, TRAF3, or TRAF6 (Fig. 3). This
assay was also used to verify that GST-CD40c fusion proteins containing
the T254A, P233G/E235A, N237D, and P233G/E235A/T254A mutations
exhibited no binding to TRAF5 (data not shown). GST-CD40c containing
combined N237D/T254A mutations had no significant binding of TRAF1,
TRAF2, and TRAF3, but still bound TRAF6 approximately 25-fold better than wild type GST-CD40c (Fig. 3). The GST-CD40c-TRAF binding results
demonstrated that although the sequences 231QEPQEINF and
250PVQET in CD40 appear to be independent binding sites for
unique subsets of TRAFs based on deletional (15, 27) and peptide scanning (11) analyses, point mutations in the 231QEPQEINF
binding site altered TRAF binding to the 250PVQET binding
site.
Based on the mutational analyses in Figs. 1 and 2, several other
mutations in the CD40 cytoplasmic domain were made and expressed as
GST-CD40c fusion proteins. These were purified and tested for TRAF
binding in the solid-phase binding assay. GST-CD40c containing a P250H
mutation had slightly increased TRAF3 and TRAF6 binding, but had almost
background levels of TRAF1 and TRAF2 binding (data not shown). Binding
of GST-CD40c containing a Q252E mutation to TRAF6 was identical to that
of wild type GST-CD40c. However, this mutation decreased binding of
GST-CD40c to TRAF3 approximately 3-fold and completely eliminated TRAF1
and TRAF2 binding (data not shown). These results with GST-CD40c
containing either P250H or Q252E alterations were consistent with the
mutational analysis in Fig. 2 and support the idea that amino acids
C-terminal to the 250PVQET sequence contributed to
CD40-TRAF3 interaction but not to CD40-TRAF1 or CD40-TRAF2
interactions. Based on the data in Fig. 1D, an I236W
substitution was also produced in GST-CD40c. As expected, GST-CD40c
I236W bound to TRAF6 approximately 4-fold better than to wild type
GST-CD40c; however, its binding to TRAF1, TRAF2, and TRAF3 was similar
to that of wild type GST-CD40c. The results with these mutations are
consistent with and support results obtained in the peptide-based
mutational analyses.
Signaling Functions of CD40 Mutations--
To study signaling
outcomes from altered TRAF binding to the CD40 mutations, each mutation
characterized in Fig. 3 was introduced into full-length human CD40. HEK
293 cells were demonstrated by RT-PCR (TRAF1, TRAF3, TRAF4, and TRAF5)
and immunoblot analyses (TRAF2 and TRAF6) to endogenously express all
six known TRAF molecules (data not shown). Stable transfectants of HEK
293 cells were generated that expressed the wild type human CD40
receptor or each of the five mutations characterized in Fig. 3. Cells
were cloned by flow cytometry, and clones that expressed similar levels
of CD40 on the cell surface were chosen for further studies (Fig.
4). To confirm in vitro
binding results with GST-CD40c, TRAF associations with CD40 in cells
were examined by coimmunoprecipitation. Coimmunoprecipitations of
TRAF2, TRAF3, and TRAF6 with wild type CD40 or the CD40 cytoplasmic domain mutations stably expressed in HEK cell lines were completely consistent with the specificity obtained using in vitro
CD40-TRAF binding assays (Fig. 3) (data not shown).
To measure CD40-dependent NF-
Previous studies demonstrated that JNK activation also results from
stimulation of CD40 signaling (4-7). The ability of each mutated CD40
receptor to mediate JNK activation was determined by stimulating each
of the stable transfectants with CD8
Another outcome of CD40 signaling in B lymphocytes is activation of p38
MAPK (8, 9). Therefore, activation of p38 MAPK in the CD40 HEK 293 transfectants was also examined. After 15 min of stimulation with
CD8 Previously, the sequence 250PVQET in the human CD40
cytoplasmic domain has been demonstrated to be necessary and sufficient
for interactions with TRAF1, TRAF2, and TRAF3 (11). Additionally, the
sequence 231QEPQEINF appeared sufficient for TRAF6 binding
(11). Our peptide-based mutational analysis around the
250PVQET sequence demonstrated that TRAF1, TRAF2, and TRAF3
have similar binding specificities for this core sequence (Fig. 1, A-C). This is interesting in view of the different
affinities of TRAF1, TRAF2, and TRAF3 for the CD40 cytoplasmic
domain4 (11). Since few
D-amino acid substitutions of the 250PVQET
residues were tolerated, the side chain orientation of the residues is
critical for TRAF1, TRAF2, and TRAF3 binding. From these studies a
consensus core binding site for TRAF1, TRAF2, and TRAF3 in the context
of CD40 can be defined as (P/H)(V/I)QE(T/S) (Table I). This extends the
previously proposed binding sequence, PXQX(T/S)
(26). Because in our mutational analyses only single amino acid
replacements were tested, other multiple combinations of amino acid
substitutions may also support TRAF1/TRAF2/TRAF3 binding. This is
likely since binding sites for TRAF1, TRAF2, and/or TRAF3 within other
receptors, for example TNFR2, ATAR, and a second site in CD30, do not
exactly fit the proposed consensus sequence.
Flanking sequences also modulated and generated some selectivity of
TRAF1/TRAF2/TRAF3 interactions. Sequences C-terminal to the
250PVQET sequence and previously implicated in facilitating
TRAF binding (11) were able to alter the binding specificity of TRAF3 but not TRAF1 or TRAF2 by increasing flexibility in the TRAF3 recognition sequence, 250PVQET. This suggests that although
sequences C-terminal to the 250PVQET are not sufficient to
support TRAF3 binding, they may alter the manner in which TRAF3
interacts with the 250PVQET sequence. Additionally, the
substitution P250H, which had little effect on TRAF1 and TRAF2 binding
in the context of peptides, eliminated binding of each to GST-CD40c. In
contrast, TRAF3 binding to GST-CD40c P250H was relatively unaffected.
Thus, each TRAF binding site in a receptor appears to be defined by a
core binding sequence in the context of flanking sequences.
Using the peptide-based amino acid substitutions to form a consensus
TRAF6 binding sequence, we predict the TRAF6 binding site in mouse CD40
to be 234RQDPQEMEDY (Fig. 8).
A region of mouse CD40 containing this sequence has been demonstrated
to be required for interaction with mouse TRAF6 (15). In this region,
the human and mouse CD40 sequences are only partially conserved, with
44% identical residues and a single amino acid insertion in the mouse
sequence. This lack of homology is consistent with the ability to
optimize this region for TRAF6 binding by two amino acid replacements
in human CD40 (N237D and I236W) that significantly increased TRAF6
binding. TRAF6 also interacts with interleukin-1 receptor-associated
kinase (IRAK) and receptor activator of NF-B and c-Jun N-terminal kinase
activation. In contrast, p38 mitogen-activated protein kinase
activation was primarily dependent upon TRAF6 binding. These studies
suggest a role in CD40 signaling for competitive TRAF binding and imply
that CD40 responses reflect an integration of signals from individual TRAFs.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
activation (2, 3), c-Jun N-terminal kinase (JNK) activation (4-7), and
p38 mitogen-activated protein kinase (MAPK) activation (8, 9).
Additionally, signaling through CD40 mediates activation of
protein-tyrosine kinases and phosphatases with an effect on protein
kinase C remaining controversial (reviewed in Ref. 10). The CD40
cytoplasmic domain binds directly to several TNF receptor-associated
factors (TRAFs), and this interaction is thought to initiate CD40
signaling (11-15). TRAF interactions with CD40 appear to require an
oligomerized receptor cytoplasmic domain and are thought to be
responsible for initiating activation of most of the CD40-mediated
signaling pathways.
B activation (17, 18). TRAF2 also mediates
p38 MAPK activation when transiently overexpressed (19). Although the
molecular function of TRAF3 is unclear, mice irradiated and bone marrow
reconstituted with fetal liver cells from TRAF3 knockout animals
exhibited impaired T cell priming (20). The outcome of TRAF4 signaling
is unknown. In transient overexpression studies, TRAF5 and TRAF6
mediated NF-
B and JNK activation (15, 21-25). Only TRAF6 has been
demonstrated to activate extracellular signal-regulated kinase
(21).
B and JNK activation through CD40 required both TRAF6 and
TRAF1/TRAF2/TRAF3 binding sites. In contrast, p38 MAPK activation
resulted primarily from TRAF6 binding to CD40. Competitive TRAF binding
and integration of signals from different TRAFs may contribute to a
diversity and complexity of CD40 signaling outcomes in different cell types.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B reporter plasmid pNF-
B-Luc was purchased from Stratagene.
Recombinant baculoviruses that express TRAF1-CA21, TRAF2-CA21,
TRAF3-CA21, and TRAF6-CA21 have been described previously (11).
80 °C. Expression
of GST-CD40c fusion proteins in Escherichia coli strain BL21
(DE3) was by induction with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
37 °C. Proteins were purified by affinity chromatography on
glutathione-Sepharose and gel filtration as described previously (11).
Purified proteins were quantitated as described (29), frozen in
aliquots under liquid nitrogen, and stored at
80 °C. Mouse
CD8
-human CD40L fusion protein was expressed in Sf21 insect cells2 (30) and purified from
concentrated supernatant medium by ion exchange chromatography on Poros
HS, followed by affinity chromatography on an anti-CD40L column. Fusion
protein was eluted from the affinity column with ImmunoPure Gentle
Ag/Ab elution buffer (Pierce), dialyzed against 25 mM
HEPES, pH 7.5, 150 mM NaCl, and frozen in aliquots at
80 °C.
was obtained from
the ATCC (34). Cells were grown in serum-free medium (Nutridoma-SP,
Roche Molecular Biochemicals) and the antibody was purified as
described (32).
B Reporter Assay--
Three micrograms of the reporter
plasmid pNF-
B-Luc (Stratagene) were transfected into 1 × 106 HEK 293 cells or stable CD40 transfectants of HEK 293 cells in 60-mm dishes using Superfect (Qiagen) according to the
manufacturer's protocol. Twenty-four h after transfection, medium was
removed and replaced with medium alone or with 10 µg/ml purified
mouse CD8
-human CD40L fusion protein and 15 µg/ml rat anti-mouse
CD8
monoclonal antibody (53-6). After 6 h, cell extracts were
prepared and assayed for luciferase activity using the Luciferase assay system (Promega) according to the manufacturer's protocol.
-human CD40L and 15 µg/ml rat anti-mouse CD8
. Cells were harvested and lysed in 2%
SDS, 50 mM Tris-HCl, pH 6.8, 2% 2-ME. After heating at
95 °C for 5 min, portions of the supernatant were subjected to
SDS-PAGE (12% polyacrylamide Tris-glycine; Novex) and transferred to a
PVDF membrane (Schleicher & Schuell) by electroblotting. Immunoblot
analysis of transferred proteins was performed by incubating membranes
with 1 µg/ml anti-p38 (N-20, Santa Cruz), 1 µg/ml anti-JNK (JNK1-FL, Santa Cruz), 1:2000 dilution of anti-Active p38 (Promega), or
1:5000 dilution of anti-Active JNK (Promega), followed by a 1:2000
dilution of Protein A-HRP (Bio-Rad). Visualization of total and
activated kinases was performed by chemiluminescence using Pierce
SuperSignal chemiluminescent substrate.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TRAF protein binding to CD40-derived peptides
with single amino acid substitutions. Peptides were synthesized in
spots on cellulose membranes, each containing a single L-
or D-amino acid substitution, as indicated. Membranes were
probed with insect cell extracts containing TRAF1-CA21 (A),
TRAF2-CA21 (B), TRAF3-CA21 (C), or TRAF6-CA21
(D) as described under "Materials and Methods." TRAF
proteins bound to peptides were visualized by chemiluminescent
detection and are shown as scans of the exposed films. The wild type
amino acid sequence of each TRAF binding peptide is indicated
vertically to the left of the peptide membrane.
The amino acid substituted for each position is indicated across the
top of each membrane. The first column
of peptide spots on the left side of each membrane contains
the wild type peptide sequence, as indicated by a dash ( ).
The same peptide membranes were used in A-C for sequential
TRAF1, TRAF3, and TRAF2 binding. Membranes were stripped after binding
each TRAF with four different buffers as described under "Materials
and Methods."
Summary of TRAF binding site mutations
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Fig. 2.
TRAF2 and TRAF3 binding to CD40-derived
peptides with single L-amino acid substitutions.
Peptides synthesized with single L-amino acid substitutions
were probed with insect cell extracts containing TRAF2-CA21
(A) or TRAF3-CA21 (B) as described under
"Materials and Methods" and the legend of Fig. 1. The wild type
amino acid sequence of the TRAF binding peptide is indicated
vertically to the left of the peptide membrane.
The amino acid substituted for each position is indicated across the
top of each membrane. The first column
of peptide spots on the left side of each membrane contains
the wild type peptide sequence, as indicated by a dash ( ).
The same peptide membrane was used in A and B for
sequential binding of TRAF2 and TRAF3. The membrane was stripped after
TRAF2 binding with the two 100 mM 2-ME-containing buffers
as described under "Materials and Methods."
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Fig. 3.
Solid-phase binding of TRAFs to wild type and
mutated GST-CD40c proteins. Serial dilutions of GST-CD40c fusion
proteins were bound to glutathione-coated 96-well plates as described
under "Materials and Methods." Insect cell extracts diluted in
binding buffer and containing approximately 1 µg/ml each TRAF1-CA21
(A), TRAF2-CA21 (B), TRAF3-CA21 (C),
or TRAF6-CA21 (D) were incubated in the wells and detected
with biotin-CA21 antibody as described under "Materials and
Methods." No signal was generated in the assay with biotin-CA21 when
wells were coated with GST-CD40c or not coated (data not shown). The
molecular mass of each mutated GST-CD40c fusion protein was verified by
electrospray ionization mass spectrometry. Each point is the mean of
duplicate wells. Figure is representative of three independent
experiments.
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Fig. 4.
CD40 expression on cloned stable
transfectants. Parental HEK 293 cells and clones of stable cell
lines were stained with either FITC-anti-hCD40 (gray
histograms) or FITC-isotype control antibody
(open histograms) and analyzed by flow cytometry.
Results for one of two independently isolated clones for each stable
transfectant are shown. Levels of CD40 expression remained constant
after over 6 months of passaging the wild type CD40 transfectants and
for over 3 months of passaging the mutated CD40 transfectants.
B activation, each clone
was transfected with an NF-
B-luciferase reporter plasmid and 24 h later stimulated with soluble CD8
-human CD40L and anti-CD8
. Luciferase activity was assessed 6 h after CD40L stimulation. HEK
293 cells and a stable cell line transfected with the empty expression
vector were used as negative controls and had similar basal levels of
NF-
B activity. There was no increase in NF-
B activity upon
stimulation of HEK 293 or vector-transfected cells with CD8
-CD40L
fusion protein (Fig. 5). Clones
expressing the wild type CD40 receptor displayed increased basal levels
of NF-
B activity when compared with HEK 293 cells and
vector-transfected clones. After stimulation with CD8
-CD40L, the
wild type CD40 transfectants showed a 4-fold increase in NF-
B
activity over their basal level (Fig. 5). In contrast, clones
expressing the CD40 T254A mutation had basal levels of NF-
B activity
similar to HEK 293 and vector-transfected clones. CD8
-CD40L
stimulation of the CD40 T254A clones resulted in a 150-fold enhancement
of NF-
B activity, although the absolute signal was 2-3-fold lower than in the wild type CD40 transfectants. Cells expressing the CD40
N237D receptor with increased TRAF6 binding exhibited the highest basal
and CD40L-stimulated levels of NF-
B activation (Fig. 5). The
increase in NF-
B activation after CD40L stimulation was 2.5-fold.
Cells expressing the CD40 N237D/T254A receptor showed increased basal
NF-
B activity when compared with cells expressing the CD40 T254A
mutation. The basal NF-
B activity was 2-3-fold lower than for the
wild type CD40 transfectants. Stimulation of the CD40 N237D/T254A
clones with CD40L resulted in a 5-fold increase in NF-
B activation
over basal levels. Cells expressing the CD40 P233G/E235A mutation with
no detectable TRAF6 binding had basal levels of NF-
B activity
approximately 2-fold lower than cells expressing wild type CD40. After
CD40L treatment, cells expressing the CD40 P233G/E235A receptor had
levels of NF-
B activation 3-fold lower than CD40L-treated wild type
CD40 transfectants. Cells expressing the CD40 P233G/E235A/T254A
mutation that gave no detectable TRAF1, TRAF2, TRAF3, or TRAF6 binding
showed basal levels of NF-
B activity similar to HEK 293 and vector
transfectants. Surprisingly, after CD40L treatment, there was an 8-fold
increase in NF-
B activity (Fig. 5). The identity of the CD40
P233G/E235A/T254A sequence in the transfectant was confirmed by RT-PCR
and DNA sequence analysis. This indicates that although TRAF1, TRAF2,
TRAF3, TRAF5, and TRAF6 did not bind to the CD40 P233G/E235A/T254A
receptor, CD40L-dependent activation of NF-
B was reduced
but not eliminated. Together, the NF-
B signaling results demonstrate
that both TRAF binding sites in CD40 contribute to NF-
B
activation.
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Fig. 5.
CD40L-stimulated
NF- B activation of stable HEK 293 transfectants. Clones were transfected with a NF-
B-luciferase
reporter plasmid as described under "Materials and Methods."
Lysates from mock (light gray) or CD8
-CD40L
(10 µg/ml) and rat anti-mouse CD8
(15 µg/ml) (dark
gray) stimulated cells were assayed for luciferase activity
6 h after treatment. Transfections were performed in triplicate,
and the results are shown as mean ± standard deviation. Initial
experiments were done with two independent clones of each transfectant
with similar results. CD40 P233G/E235A/T254A cDNA was PCR-amplified
from two independent clones and sequenced to confirm the identity of
the mutation. Figure is representative of two independent experiments
on one representative clone of each transfectant.
-CD40L fusion protein, and
anti-CD8
. After a 15-min stimulation, cell lysates were prepared and
subjected to immunoblot analysis using an antibody that recognizes
total JNK1 and JNK2 isoforms or an antibody that recognizes activated,
dual phosphorylated JNK1 and JNK2 isoforms. HEK 293 cells had very low
levels of constitutively activated JNK, and there was no increase in
activated JNK levels after stimulation with CD40L (Fig.
6A). The transfectant
expressing wild type CD40 had similar basal levels of activated JNK as
untransfected HEK 293 cells, and treatment with CD40L significantly
increased the levels of activated JNK1 and JNK2 (Fig. 6A).
Stimulation of the CD40 T254A-expressing cells with CD40L resulted in
significantly reduced JNK activation when compared with cells with wild
type CD40. Some residual JNK activation was still observed in the CD40 T254A cells. Cells expressing the CD40 N237D receptor with increased TRAF6 binding had elevated levels of JNK activation in the absence of
CD40L stimulation. After CD40L treatment, cells expressing CD40 N237D
showed the highest levels of activated JNK when compared with all of
the other cell lines (Fig. 6A). CD40L stimulation of cells
expressing the CD40 N237D/T254A receptor resulted in levels of JNK
activation similar to cells with wild type CD40. Cells expressing the
CD40 P233G/E235A receptor showed dramatically reduced JNK activation
after CD40L treatment. Consistent with a complete absence of TRAF1,
TRAF2, TRAF3, and TRAF6 binding by CD40 P233G/E235A/T254A, no JNK
activation was observed after CD40L treatment of CD40
P233G/E235A/T254A-expressing cells (Fig. 6A). Similar levels
of JNK1 and JNK2 isoforms were present in lysates from all the cell
lines (Fig. 6B). Together, these results demonstrate that
both TRAF binding sites in CD40 are involved in mediating JNK
activation. However, the TRAF6 binding site appeared to be more
critical for JNK activation.
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Fig. 6.
CD40-stimulated JNK activation in stable HEK
293 transfectants. Lysates from cells either mock ( ) stimulated
or stimulated with 10 µg/ml CD8
-human CD40L and 15 µg/ml rat
anti-mouse CD8
(+) for 15 min were separated by SDS-PAGE on a 12%
polyacrylamide gel and electroblotted to a PVDF membrane. Detection of
phosphorylated active JNK1 and JNK2 (A) and total JNK1 and
JNK2 isoforms (B) was performed by immunoblotting as
described under "Materials and Methods." Migration of JNK1 and JNK2
isoforms are indicated by arrows. A slight decrease in
mobility of phosphorylated forms of JNK1 could be seen with the
antibody that detected total JNK1 and JNK2 (B), confirming
the results obtained in A with the antibody that recognizes
only phosphorylated JNK1 and JNK2. Figure is representative of two
independent experiments on one representative clone of each
transfectant.
-CD40L, cell lysates were prepared and subjected to immunoblot
analysis using an antibody that recognizes total p38 MAPK or an
antibody that recognizes the activated, dual phosphorylated form of p38
MAPK. Similar low levels of active p38 MAPK were detected in
unstimulated or CD40L stimulated HEK 293 cells (Fig.
7A). Transfectants with wild
type CD40 showed a significant increase in active p38 MAPK following
CD40L treatment. After CD40L stimulation, cells expressing CD40 T254A
receptor showed levels of p38 MAPK activation similar to wild type CD40 transfectants (Fig. 7A). Cells expressing CD40 N237D and
CD40 N237D/T254A receptors also potently activated p38 MAPK after
stimulation with CD40L. In contrast, after CD40L treatment, cells
containing the CD40 P233G/E235A receptor showed a significant reduction
in the amount of p38 MAPK activated. No CD40L-dependent
increase in p38 MAPK activation occurred in cells expressing the CD40
P233G/E235A/T254A receptor. Similar levels of total p38 MAPK were
present in lysates from all the cell lines (Fig. 7B). These
results suggest that the TRAF6 binding site is critical for p38 MAPK
activation through CD40. These findings and the TRAF6 binding data in
Fig. 3 predict that p38 activation mediated by the CD40 N237D mutations
would be higher than that of wild type CD40. A reason this result was not observed in Fig. 7 may be that maximal p38 activation was already
achieved by signaling through wild type CD40, and it was therefore not
possible to observe increases in p38 activation. Results of TRAF
binding and signaling through NF-
B, JNK, and p38 MAPK pathways for
wild type CD40 and the five CD40 point mutants are summarized in Table
II.
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Fig. 7.
CD40-stimulated p38 MAPK activation in stable
HEK 293 transfectants. Lysates from cells either mock ( )
stimulated or stimulated with 10 µg/ml CD8
-human CD40L and 15 µg/ml rat anti-mouse CD8
(+) for 15 min were separated by SDS-PAGE
on a 12% polyacrylamide gel and electroblotted to a PVDF membrane.
Detection of phosphorylated active p38 MAPK (A) and total
p38 (B) protein was performed by immunoblotting as described
under "Materials and Methods." Figure is representative of two
independent experiments on one representative clone of each
transfectant.
Summary of TRAF binding and signaling for wild type and mutated CD40
receptors
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (RANK). Using the
mutational analysis on the 230KQEPQEINFPDDLP peptide (Fig.
1D) and a comparison of the mouse and human CD40 TRAF6
binding sequence (Fig. 8), we predict the TRAF6 recognition sequences
in human and mouse RANK and human IRAK (Fig. 8). For RANK, the
predicted TRAF6 binding site is located within a region demonstrated
previously to be involved in TRAF6 binding (39) and NF-
B and JNK
activation (39, 40). The predicted TRAF6 binding site in RANK is 78%
identical between mouse and human proteins (Fig. 8). A predicted TRAF6
binding site in IRAK is positioned C-terminal to the kinase domain (24)
(Fig. 8). Interestingly, RANK, IRAK, and mouse CD40 have a C-terminal Tyr residue instead of a Phe in the predicted TRAF6 binding site. It is
possible that tyrosine phosphorylation could modulate TRAF6 binding to
these other proteins.
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Fig. 8.
Predicted TRAF6 binding sites. The
binding site on human CD40 for TRAF6 is aligned by homology with
sequences of proteins known to interact with TRAF6. Conserved residues
from the mutational analysis on CD40 are highlighted in
black. Numbers indicate the amino acid residues
of each protein.
Selected amino acid substitutions were made in the context of the full-length CD40 cytoplasmic domain and characterized in vitro and in cells to confirm that the mutations individually eliminated TRAF interactions with either or both of the two TRAF binding sites (Table II). Two substitutions in the TRAF6 binding site, N237D and P233G/E235A, increased TRAF1, TRAF2, and TRAF3 binding to CD40 (Fig. 3). This suggests that although TRAF1, TRAF2, and TRAF3 do not independently bind to the TRAF6 site in CD40 (11), they may make contacts in the TRAF6 binding region. Thus, it is possible that the binding of a TRAF to either of the binding sites in CD40 could sterically prevent simultaneous TRAF interaction with the other site. The binding and competition of TRAF proteins to a more native multimerized form of CD40 engaged by CD40L remains to be tested. A competitive interaction of TRAFs with cross-linked CD40 suggests that the levels of individual TRAFs in different cell types could mediate specificity in outcomes of CD40 signaling. Alternatively, the TRAF6 binding region may be folded so as to be in close proximity to the TRAF1/TRAF2/TRAF3 binding region and form a tertiary structure that optimally interacts with TRAF1, TRAF2, and TRAF3.
Signaling by specific CD40 mutants was compared with wild type CD40 in
stably transfected HEK 293 clones (results summarized in Table II).
Since HEK 293 cells expressed all six identified TRAFs and little if
any CD40,2 it was possible to study CD40 signaling using
endogenous levels of signaling proteins. The high basal level of
NF-B activation in clones expressing wild type CD40 was reduced to
that of untransfected cells by expressing the T254A substitution that
eliminated TRAF1/TRAF2/TRAF3 binding. Due to high levels of CD40
expression in the transfectants, TRAF2 and TRAF3, which bind to CD40
better than TRAF1 and TRAF64 (11), may interact with CD40
and mediate NF-
B activation in the absence of receptor
cross-linking. Since TRAF2 appears dispensable for NF-
B activation
(17, 18), basal NF-
B activation could be mediated by TRAF5 that can
be recruited to CD40 as a hetero-oligomer with TRAF3 (11). The
cross-linking independent NF-
B activation also is consistent with
increased basal levels of NF-
B activity in cells expressing the CD40
N237D receptor with increased affinity for TRAF6 and TRAF2.
The dramatic increase in NF-B activity upon CD40L stimulation of the
CD40 T254A clones is consistent with previous findings (41), indicating
that TRAF6 is a potent activator of NF-
B through CD40. Reduced
levels of NF-
B activation were found in clones expressing CD40 with
TRAF6 binding eliminated (P233G/E235A). This further confirms an
important role for TRAF6 in mediating NF-
B activation through CD40.
Overall, the results suggested that NF-
B activation through CD40 is
mediated by TRAF6 as well as possibly TRAF2 and/or TRAF5. The
significant CD40-dependent NF-
B activation (yet the
complete absence of JNK and p38 MAPK activation) in clones expressing
the P233G/E235A/T254A mutation was unexpected. This suggests that there
may be another mediator of NF-
B activation through CD40 in addition
to the known TRAF proteins.
JNK activation through CD40 (4-9) could be mediated by TRAF2, TRAF5, and/or TRAF6 (17, 18, 21, 22, 42, 43). Both TRAF binding sites in CD40 were required for maximal JNK activation (Fig. 6 and Table II). Mutations that individually eliminated TRAF binding to either site in CD40 only partially reduced JNK activation after CD40 stimulation. Mutations that eliminated TRAF binding to both sites eliminated JNK activation. This suggests that all JNK activation through CD40 is mediated by CD40-TRAF interactions. In a previous study, TRAF2 activated p38 MAPK through TNFR1 in HEK 293 cells (19). In our analysis, a mutation that eliminated TRAF1, TRAF2, and TRAF3 binding (T254A) had little if any effect on p38 MAPK activation (Fig. 7). Instead, primarily TRAF6 binding appeared to be required for p38 MAPK activation through CD40. It is possible that differences in TRAF expression could account for these different findings since TRAF2 was overexpressed in the earlier study (19). If TRAF6 is of critical importance in mediating p38 MAPK-dependent events resulting from CD40 signaling, it would be expected that TRAF6 is essential for CD40-mediated events in B cells that are dependent upon p38 MAPK activation such as proliferation and ICAM-1 and CD40 induction (8).
Our studies are consistent with previous studies that used deletions
and selected substitutions to map regions within the CD40 cytoplasmic
domain required for signaling. Additionally, some confusing findings in
previous work are explained. A role for the 260PVTQED
sequence in contributing to TRAF3 binding to the 250PVQET
sequence may explain why deletion of the C-terminal 22 residues of
CD40, which includes the sequence 260PVTQED but not
250PVQET, caused a 50% reduction in NF-B activation
(41), reduced LFA-1 and ICAM-1 induction, and eliminated CD23, B7-1,
and Fas induction (44). This may reflect a role for TRAF3/TRAF5
hetero-oligomers in these events. The difference in the effects of this
deletion on different responses may indicate that a critical balance of TRAF1/TRAF2 versus TRAF3/TRAF5 interactions may be required
to mediate each outcome. Previously, removal of the C-terminal 32 residues of CD40, which includes the 250PVQET sequence,
eliminated all signaling through CD40 (41, 44). However, in that study,
human CD40 deletions were tested in a mouse cell line. It is possible
that, since the TRAF6 binding sites are not completely conserved in
mouse and human CD40 (Fig. 8), mouse TRAF6 may not interact efficiently
with human CD40. In contrast, in another study, CD40 with the
C-terminal 31 residues deleted still mediated NF-
B activation (45),
consistent with TRAF6 binding to the 231QEPQEINF sequence.
Previous signaling studies on the CD40 T254A receptor (41) are
confirmed by our finding of a reduction in NF-
B activation.
Additionally, the T254A substitution was found to reduce antibody
secretion in B cells, reduce LFA-1 and ICAM-1 induction, and eliminate
CD23, B7-1, and Fas induction (44, 46). The downstream outcomes of CD40
signaling that were partially inhibited by the T254A replacement are
most likely dependent upon events such as NF-
B, JNK, and p38 MAPK
activation that are also mediated by TRAF6. Because NF-
B and JNK
activation are also mediated through the 250PVQET sequence,
a certain threshold of signaling or combination of signals that can
only be achieved by signaling through both TRAF binding sites may be
required for some CD40-mediated effects.
This work demonstrates that outcomes of signaling through CD40 result
from a combination of the contribution of individual TRAFs interacting
with the CD40 cytoplasmic domain. It is not yet known whether the
extent of CD40 cross-linking could facilitate binding of some TRAFs but
not others. Diversity in the outcome of CD40 signaling in different
cell types could be achieved through differential regulation of levels
of different TRAFs as well as by regulating linkages to downstream
signaling pathways. Further studies will be necessary to determine the
distinct physiological outcomes of CD40 signaling using different cell
types that normally express CD40.
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ACKNOWLEDGEMENT |
---|
We thank Dan Everdeen for cloning human CD40,
Brian Castle for help purifying mouse CD8-human CD40L, Carol Stearns
for FACS analysis, Lee Frego and Walter Davidson for mass spectrometric analysis, and Dr. John Miglietta, Anthony Shrutkowski, and
Gale Hansen for DNA sequencing analysis.
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FOOTNOTES |
---|
* 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: Boehringer Ingelheim
Pharmaceuticals, Inc., 900 Ridgebury Rd., P.O. Box 368, Mail code
R6-5, Ridgefield, CT 06877-0368. Tel.: 203-791-6153; Fax: 203-791-6196; E-mail: mkehry{at}bi-pharm.com.
2 S. S. Pullen, D. S. Everdeen, and M. R. Kehry, unpublished results.
3 T. T. A. Dang, S. S. Pullen, J. J. Crute, and M. R. Kehry, manuscript in preparation.
4 S. S. Pullen, M. E. Labadia, R. H. Ingraham, J. J. Crute, and M. R. Kehry, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor necrosis
factor;
TRAF, TNF receptor-associated factor;
NF-B, nuclear factor
B;
JNK, c-Jun N-terminal kinase;
MAPK, mitogen-activated protein
kinase;
IRAK, interleukin-1 receptor-associated kinase;
GST, glutathione S-transferase;
GST-CD40c, GST fusion protein
with the CD40 cytoplasmic domain;
CD40L, CD40 ligand or CD154;
PCR, polymerase chain reaction;
RT, reverse transcription;
RANK, receptor
activator of NF-
B, also termed TNF-related activation-induced
cytokine receptor (TRANCE-R);
TNFR, TNF receptor;
ATAR, another
TRAF-associated receptor, also termed herpesvirus entry mediator (HVEM)
and TNFR-related 2 (TR2);
HEK, human embryonic kidney;
ICAM-1, intercellular adhesion molecule-1;
LFA-1, lymphocyte
functional-associated antigen-1;
PVDF, polyvinylidene difluoride;
WT, wild type;
2-ME, 2-mercaptoethanol;
bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane;
FITC, fluorescein
isothiocyanate;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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