CD40−Tumor Necrosis Factor Receptor-Associated Factor (TRAF) Interactions: Regulation of CD40 Signaling through Multiple TRAF Binding Sites and TRAF Hetero-Oligomerization
Identifieur interne : 002231 ( Istex/Corpus ); précédent : 002230; suivant : 002232CD40−Tumor Necrosis Factor Receptor-Associated Factor (TRAF) Interactions: Regulation of CD40 Signaling through Multiple TRAF Binding Sites and TRAF Hetero-Oligomerization
Auteurs : Steven S. Pullen ; Heather G. Miller ; Daniel S. Everdeen ; Thu T. A. Dang ; James J. Crute ; Marilyn R. KehrySource :
- Biochemistry [ 0006-2960 ] ; 1998.
Abstract
CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3−TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.
Url:
DOI: 10.1021/bi981067q
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<front><div type="abstract">CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3−TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.</div>
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<profileDesc><abstract><p>CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand
initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain.
Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and
used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2,
TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40
interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers.
TRAF3−TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited
to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used
to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region.
The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with
progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET
sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence
QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping
TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one
site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40
binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.
</p>
</abstract>
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<metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration>
<istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id>
<journal-id journal-id-type="coden">bichaw</journal-id>
<journal-title-group><journal-title>Biochemistry</journal-title>
<abbrev-journal-title>Biochemistry</abbrev-journal-title>
</journal-title-group>
<issn pub-type="ppub">0006-2960</issn>
<issn pub-type="epub">1520-4995</issn>
<publisher><publisher-name>American Chemical Society</publisher-name>
</publisher>
<self-uri>pubs.acs.org/biochemistry</self-uri>
</journal-meta>
<article-meta><article-id pub-id-type="doi">10.1021/bi981067q</article-id>
<article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>CD40−Tumor Necrosis Factor Receptor-Associated Factor (TRAF) Interactions:
Regulation of CD40 Signaling through Multiple TRAF Binding Sites and TRAF
Hetero-Oligomerization</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Pullen</surname>
<given-names>Steven S.</given-names>
</name>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Miller</surname>
<given-names>Heather G.</given-names>
</name>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Everdeen</surname>
<given-names>Daniel S.</given-names>
</name>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Dang</surname>
<given-names>Thu T. A.</given-names>
</name>
</contrib>
<contrib contrib-type="author"><name name-style="western"><surname>Crute</surname>
<given-names>James J.</given-names>
</name>
</contrib>
<contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Kehry</surname>
<given-names>Marilyn R.</given-names>
</name>
<xref rid="bi981067qAF1">*</xref>
</contrib>
<aff>Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368
</aff>
</contrib-group>
<author-notes><corresp id="bi981067qAF1">
To whom correspondence should be addressed at Boehringer
Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., P.O. Box 368,
Mail code R6-5, Ridgefield, CT 06877-0368. Telephone: 203-791-6153. Fax: 203-791-6196. EMail: mkehry@bi-pharm.com.</corresp>
</author-notes>
<pub-date pub-type="epub"><day>7</day>
<month>08</month>
<year>1998</year>
</pub-date>
<pub-date pub-type="ppub"><day>25</day>
<month>08</month>
<year>1998</year>
</pub-date>
<volume>37</volume>
<issue>34</issue>
<fpage>11836</fpage>
<lpage>11845</lpage>
<history><date date-type="received"><day>08</day>
<month>05</month>
<year>1998</year>
</date>
<date date-type="rev-recd"><day>30</day>
<month>06</month>
<year>1998</year>
</date>
<date date-type="asap"><day>7</day>
<month>08</month>
<year>1998</year>
</date>
<date date-type="issue-pub"><day>25</day>
<month>08</month>
<year>1998</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 1998 American Chemical Society</copyright-statement>
<copyright-year>1998</copyright-year>
<copyright-holder>American Chemical Society</copyright-holder>
</permissions>
<abstract><p>CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand
initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain.
Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and
used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2,
TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40
interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers.
TRAF3−TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited
to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used
to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region.
The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with
progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET
sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence
QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping
TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one
site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40
binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.
</p>
</abstract>
<custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name>
<meta-value>bi981067q</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><sec id="d7e153"><title></title>
<p>Tumor necrosis factor (TNF)<xref rid="bi981067qX00001" ref-type="bibr"></xref>
receptor superfamily members regulate cellular proliferation, differentiation, and apoptosis in inflammatory and immune responses. Signaling
through TNF receptor superfamily members is initiated by
oligomerization of the receptors with trimeric ligands,
bringing intracellular domains in close proximity. Signal
transduction through many of these receptors is mediated in
part by a recently identified family of proteins termed TNF
receptor-associated factors (TRAFs). Six TRAF family
members have been identified (<italic toggle="yes">1</italic>
−<italic toggle="yes">8</italic>
). Subsets of TRAF
proteins have been shown to interact with the TNF receptor
family members TNFR2, CD40, CD30, LTβR, ATAR, OX-40, and 4-1BB (<italic toggle="yes">2</italic>
−<italic toggle="yes">5</italic>
, <italic toggle="yes">8</italic>
−<italic toggle="yes">18</italic>
). However, the mechanisms
mediating receptor specificity and cell-type selectivity of
receptor recognition by TRAF proteins and linkages to
downstream signal transduction pathways are poorly understood. TRAF1 may be involved in the regulation of
apoptosis (<italic toggle="yes">19</italic>
). TRAF2, TRAF5, or TRAF6 overexpression
in mammalian cells induces nuclear factor κB (NF-κB) and
c-Jun N-terminal kinase (JNK) activation (<italic toggle="yes">20</italic>
−<italic toggle="yes">24</italic>
). TRAF6
additionally mediates extracellular signal-regulated kinase
(ERK) activation (<italic toggle="yes">25</italic>
). Targeted disruption of the TRAF2
gene in mice results in increased sensitivity to TNF-induced
apoptosis and generates severe defects in JNK activation
through TNFR1 (<italic toggle="yes">23</italic>
). Targeted disruption of the TRAF3
gene in mice causes impaired immune responses to T-dependent antigens and results in early postnatal lethality
(<italic toggle="yes">26</italic>
).
</p>
<p>The conserved C-terminal region of TRAFs (TRAF-C)
binds to receptor cytoplasmic domains and mediates interactions with the signaling proteins NF-κB inducing kinase
(NIK) and I-TRAF/TANK (<italic toggle="yes">2</italic>
, <italic toggle="yes">27</italic>
−<italic toggle="yes">29</italic>
). A less conserved
region N-terminal to the TRAF-C domain (TRAF-N) is a
predicted coiled-coil region that mediates TRAF homo- and
hetero-oligomerization (<italic toggle="yes">1</italic>
−<italic toggle="yes">4</italic>
, <italic toggle="yes">30</italic>
). With the exception of
TRAF1, each TRAF protein contains a predicted N-terminal
zinc RING finger and either five or seven predicted zinc
fingers. These N-terminal domains are required for TRAF2-mediated NF-κB and JNK activation (<italic toggle="yes">20</italic>
−<italic toggle="yes">22</italic>
, <italic toggle="yes">30</italic>
). When
overexpressed, the TRAF-NC domains of TRAF2, TRAF3,
TRAF5, and TRAF6 act as dominant negative inhibitors of
NF-κB activation (<italic toggle="yes">1</italic>
, <italic toggle="yes">8</italic>
, <italic toggle="yes">10</italic>
, <italic toggle="yes">16</italic>
, <italic toggle="yes">31</italic>
, <italic toggle="yes">32</italic>
). The mechanism of
inhibition is unclear and could be either competition for
binding to receptor cytoplasmic domains or functional
inactivation by hetero-oligomerization with full-length TRAF
molecules.
</p>
<p>The CD40 receptor is expressed constitutively on professional antigen-presenting cells (B lymphocytes, macrophages,
dendritic cells), and its expression can be induced with
cytokines on fibroblasts, epithelial cells, and endothelial cells
(<italic toggle="yes">33</italic>
). CD40 signaling in B cells activates multiple signal
transduction pathways, including the NF-κB, JNK, and ERK
pathways, that result in B cell proliferation and differentiation
(<italic toggle="yes">25</italic>
, <italic toggle="yes">34</italic>
−<italic toggle="yes">37</italic>
). Multimerization of CD40 induced by trimeric
CD40 ligand initiates signaling, presumably by recruitment
and interaction of TRAF proteins with the oligomerized 62
amino acid cytoplasmic domain (<italic toggle="yes">38</italic>
). Previous reports have
demonstrated that CD40 associates with TRAF2, TRAF3,
TRAF5, and TRAF6 (<italic toggle="yes">2</italic>
, <italic toggle="yes">4</italic>
, <italic toggle="yes">5</italic>
, <italic toggle="yes">9</italic>
, <italic toggle="yes">10</italic>
). Limited deletional and
mutational analyses of the CD40 cytoplasmic domain have
broadly mapped regions involved in binding of individual
TRAF proteins. However, it remains unclear how TRAF
proteins may compete or cooperate in mediating signal
transduction and how the observed cellular selectivity in
CD40 signaling is generated. Fine-structure mapping of
TRAF binding sites and a detailed biochemical analysis of
the interactions have been limited by low levels of endogenous TRAF proteins.
</p>
<p>To gain a detailed understanding of the biochemistry and
selectivity of CD40−TRAF interactions, the six human
TRAF proteins were expressed in insect cells with recombinant baculoviruses. We demonstrate that TRAF1, TRAF2,
TRAF3, and TRAF6 interacted directly with the human
CD40 cytoplasmic domain in vitro. Oligomerization studies
indicated that only TRAF1 and TRAF2 efficiently hetero-oligomerized through the TRAF domain, but that full-length
TRAF3 and TRAF5 interacted, presumably through their
predicted isoleucine zippers. Peptide-based mapping analyses precisely defined the binding sites for TRAF1, TRAF2,
TRAF3, and TRAF6. TRAF1, TRAF2, and TRAF3 recognized the same amino acid sequence motif whereas TRAF6
interacted with a nonoverlapping membrane proximal region
in CD40. These studies provide insight into mechanisms
that regulate activation of multiple CD40-dependent signal
transduction pathways through TRAF proteins.
</p>
</sec>
<sec id="d7e283"><title>Materials and Methods</title>
<p><italic toggle="yes">Plasmids</italic>
<italic toggle="yes">and</italic>
<italic toggle="yes">Viruses.</italic>
To generate the GST fusion protein
with the human CD40 cytoplasmic domain, amino acids
216−277 of CD40 (<italic toggle="yes">39</italic>
) were PCR-amplified using oligonucleotides 5‘-CCCGGGCCATGGCCAAAAAGGTGGCCAAGAAGCCAACC-3‘and 5‘-CCCGGGAATTCTCATCACTGTCTCTCCTGCACTGAGATGCG-3‘and ligated into
pCR2.1 (InVitrogen) to generate pCD40c. pCD40c was
digested with <italic toggle="yes">Eco</italic>
RI, and the 217-bp fragment was ligated
into pGEX-4T-3 (Amersham Pharmacia Biotech) to generate
pGST−CD40c. Full-length human TRAF1, TRAF2, TRAF3,
and TRAF6 were PCR-amplified from a PHA-stimulated
human peripheral blood leukocyte cDNA library (Clontech)
using oligonucleotide pairs 5‘-GGATCCATGGCCTCCAGCTCAGGGAGCAGTCCT-3‘ and 5‘-GCGGCCGCCTAAGTGCTGGTCTCCACAATGCACTTGA-3‘, 5‘-AAAAGGAAAAGCGGCCGCTTATTAGAGCCCTGTCAGGTCCA-3‘
and 5‘-TTGGTTGGATCCTATAAATATGGCTGCAGCTAGCGTGA-3‘, 5‘-TTGGTTGGATCCTATAAATATGGAGTCGAGTAAAAAGATGGACTC-3‘ and 5‘-GCGGCCGCTCATCAGGGATCGGGCAGATCCGA-3‘, and 5‘-GGATCCATGAGTTCTGCTAAACTGTGAAAACAGCTGTGGCTCCAGCCAGTCT-3‘ and 5‘-GCGGCCGCTATACCCCTGCATCAGTACTTCGTGGGTGA-3‘ and ligated into
pGem-T (Promega) to make pTRAF1/GemT, pTRAF2/GemT, pTRAF3/GemT, and pTRAF6/GemT, respectively.
A phage (HF-2) containing the N-terminus (amino acids
1−59) and plasmid (pHF16-8) encoding the C-terminus
(amino acids 18−557) of human TRAF5 were a kind gift
from Dr. Hiroyasu Nakano. Using oligonucleotides 5‘-CCATGGCTTATTCAGAAGAGCATAAAGGT-3‘ and 5‘-GTCTGGTGGGGGTTGTGAAGCA-3‘ with phage DNA as
a template, an N-terminal fragment of TRAF5 was PCR-amplified and ligated into pGem-T to create pN-termTRAF5/GemT. After digestion of pHF16-8 with <italic toggle="yes">Eco</italic>
RI, the fragment
containing the C-terminus of TRAF5 was ligated into pN-termTRAF5/GemT at <italic toggle="yes">Eco</italic>
RI to make pTRAF5/GemT.
pTRAF2/GemT, pTRAF3/GemT, and pTRAF6/GemT were
digested with <italic toggle="yes">Bam</italic>
HI and <italic toggle="yes">Not</italic>
I, and the TRAF-containing
fragments were ligated into the transplacement vector
pVL1393 (InVitrogen) to generate pTRAF2/1393, pTRAF3/1393, and pTRAF6/1393, respectively. An <italic toggle="yes">Nco</italic>
I linker
(CCCATGGG) (New England Biolabs) was ligated into
pVL1393 digested with <italic toggle="yes">Sma</italic>
I to create pVL1393/NcoI<sup>+</sup>
. The
plasmids pTRAF1/GemT and pTRAF5/GemT were digested
with <italic toggle="yes">Nco</italic>
I and <italic toggle="yes">Not</italic>
I, and the TRAF-containing fragments
were ligated into pVL1393/NcoI<sup>+</sup>
to make pTRAF1/1393
and pTRAF5/1393, respectively.
</p>
<p>Oligonucleotides 5‘-GATCCGATGGACTACAAAGACGACGATGACAAAGCGTCGGCGTCGGGCATGG-3‘ and
5‘-AATTCCATGGCGCCCGACGCCGACGCTTTGTCATCGTCGTCTTTGTAGTCCATCG-3‘ were annealed and
ligated into pVL1393 at the <italic toggle="yes">Bam</italic>
HI and <italic toggle="yes">Eco</italic>
RI sites to
generate pFlag/1393. The TRAF1-NC domain (amino acids
185−416), TRAF2-NC domain (amino acids 272−501),
TRAF3-NC domain (amino acids 354−568), TRAF5-NC
domain (amino acids 329−557), and TRAF6-NC domain
(amino acids 274−511) were PCR-amplified from pTRAF1/GemT, pTRAF2/GemT, pTRAF3/GemT, pTRAF5/GemT,
and pTRAF6/GemT using oligonucleotides 5‘-CCATGGCCCTGGCTGAGCTGGAGGGGAAGCT-3‘, 5‘-CCATGGCCTGCGAGAGCCTGGAGAAGAAGACGGCCACTTTTGA-3‘, 5‘-CCATGGTGGAGTCCCTCCAGAACCGCGTGACCGAGCT-3‘, 5‘-CCATGGCCTTATTACAAATGGTTAACCAGCAACA-3‘, and 5‘-CCATGGCCCAGGCTGTTCATAGTTTGAGCGTTATACCCGA-3‘ with the respective C-terminal primers mentioned above (except for TRAF5 where
the oligonucleotide 5‘-GCGGCCGCCTAGAGATCCTCCAGGTCAGT-3‘ was used) and ligated into pGem-T to
create pTRAF1-NC/GemT, pTRAF2-NC/GemT, pTRAF3-NC/GemT, pTRAF5-NC/GemT, and pTRAF6-NC/GemT,
respectively. The <italic toggle="yes">Nco</italic>
I to <italic toggle="yes">Not</italic>
I TRAF-NC domain-containing
fragment from each of these plasmids was ligated into pFlag/1393 at the same sites to make pFlagTRAF1-NC/1393,
pFlagTRAF2-NC/1393, pFlagTRAF3-NC/1393, pFlagTRAF5-NC/1393, and pFlagTRAF6-NC/1393, respectively. Full-length and TRAF-NC domain constructs of TRAF1, TRAF2,
TRAF3, TRAF5, and TRAF6 were C-terminally tagged with
the nine amino acid epitope (SKRSMNDPY) recognized by
the CA21 monoclonal antibody (<italic toggle="yes">40</italic>
) by PCR methods to
generate TRAF1-CA21, TRAF2-CA21, TRAF3-CA21,
TRAF5-CA21, TRAF6-CA21, TRAF1-NC-CA21, TRAF2-NC-CA21, TRAF3-NC-CA21, TRAF5-NC-CA21, and TRAF6-NC-CA21 in pVL1393. Recombinant baculovirus stocks
were generated by standard methods (<italic toggle="yes">41</italic>
) from the transplacement vectors described above. Viruses Flag-TRAF4-NC and TRAF4-NC-CA21 were described previously (H.
G. Miller, H. E. White, and J. J. Crute, unpublished results).
</p>
<p><italic toggle="yes">Protein</italic>
<italic toggle="yes">Expression</italic>
<italic toggle="yes">and</italic>
<italic toggle="yes">Purification.</italic>
<italic toggle="yes">Spodoptera</italic>
<italic toggle="yes">frugiperda</italic>
(Sf21) cells were maintained and infected as
described previously (<italic toggle="yes">42</italic>
) using medium supplemented with
5% heat-inactivated fetal bovine serum (Hyclone) and 50
μg/mL gentamicin sulfate (Life Technologies, Inc.). All
purification procedures were performed at 4 °C. Cytosolic
extracts of baculovirus-infected Sf21 cells were prepared as
described (<italic toggle="yes">42</italic>
), without the addition of ATP or MgCl<sub>2</sub>
, frozen
under liquid nitrogen, and stored at −80 °C. Expression of
GST and GST−CD40c fusion proteins in <italic toggle="yes">E.</italic>
<italic toggle="yes">coli</italic>
strain BL21(DE3) was by induction with 1.0 mM IPTG for 3 h at 37
°C. Harvested cell paste was resuspended in 2 volumes of
lysis buffer (20 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM
DTT, 1 mM EDTA, 1 mM EGTA, 10% v/v glycerol, 1 mM
PMSF, 4 μg/mL leupeptin, 4 μg/mL pepstatin A), frozen
under liquid nitrogen, and stored at −80 °C. Thawed cell
paste was resuspended in an equal volume of lysis buffer,
and cells were disrupted by nitrogen cavitation. Extracts
were clarified by ultracentrifugation for 75 min at 100000<italic toggle="yes">g</italic>
and applied to a glutathione−Sepharose 4B column (Pharmacia Biotech) equilibrated in buffer A (20 mM HEPES,
pH 7.5, 150 mM NaCl, 1 mM DTT, 10% v/v glycerol, 0.1
mM EDTA, 0.1 mM EGTA, and 1 mM PMSF). The column
was washed with buffer A containing 400 mM NaCl, and
protein was eluted with 50% ImmunoPure Gentle Ag/Ab
Elution Buffer (Pierce)/50% buffer A containing 400 mM
NaCl without DTT. Peak fractions were pooled and chromatographed through Sephacryl S-300 HR (Amersham
Pharmacia Biotech) equilibrated in buffer A containing 300
mM NaCl. Purified proteins were quantitated as described
(<italic toggle="yes">43</italic>
), frozen in aliquots under liquid nitrogen, and stored at
−80 °C.
</p>
<p>The CA21 cell line producing a mouse IgG1 monoclonal
antibody against a peptide epitope (<italic toggle="yes">40</italic>
) was a gift from A.
Wayne (Boehringer Ingelheim) and was grown and purified
as described (<italic toggle="yes">40</italic>
, <italic toggle="yes">42</italic>
). Purified CA21 antibody was conjugated with <italic toggle="yes">N</italic>
-hydroxysuccinimide ester-XX-biotin (Calbiochem) as described (<italic toggle="yes">44</italic>
).
</p>
<p><italic toggle="yes">GST</italic>
<italic toggle="yes">Coprecipitation</italic>
<italic toggle="yes">Assays</italic>
. Purified GST−CD40c (20
μg; 590 pmol), Sf21 cytosolic extracts containing CA21
epitope-tagged TRAF, and 10 μL of glutathione−Sepharose
4B were combined in 500 μL of 40 mM HEPES, pH 7.0,
100 mM NaCl, 0.1% Nonidet P-40, 1 mM MgCl<sub>2</sub>
, and 1
mM DTT (binding buffer). Samples were rotated at 4 °C
overnight, washed 5 times in ice-cold binding buffer, and
eluted in 40 μL of 2% SDS, 50 mM Tris-HCl, pH 6.8, 2%
2-ME (sample buffer). After heating at 95 °C for 5 min,
portions of the supernatant were analyzed by SDS−PAGE
(12% polyacrylamide Tris−glycine; Novex) followed by
staining with Coomassie Brilliant Blue R350 or immunoblotting analysis with anti-Flag or CA21 antibody (see
below).
</p>
<p><italic toggle="yes">Immunoprecipitation</italic>
<italic toggle="yes">Assays.</italic>
Immunoprecipitations using
Sf21 cytosolic extracts containing CA21 epitope-tagged
TRAFs were performed in RIPA buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50
mM Tris, pH 8.0) with 20 μL of a 50% slurry of CA21
antibody coupled to Sepharose 4B. Samples were rotated
overnight at 4 °C, washed twice in ice-cold RIPA buffer and
3 times in ice cold RIPA buffer containing 500 mM NaCl,
and eluted as described above in sample buffer. Portions of
the supernatant were analyzed by SDS−PAGE (12% polyacrylamide Tris−glycine; Novex) and either stained with
Coomassie Brilliant Blue R350 or transferred to a PVDF
membrane (Schleicher & Schuell) by electroblotting. Immunoblot analysis of transferred Flag-tagged proteins was
performed by incubating with 0.5 μg/mL anti-Flag BioM2
monoclonal antibody (Kodak) followed by 0.5 μg/mL
streptavidin−horseradish peroxidase (Jackson ImmunoResearch) and developed with 3-amino-9-ethyl-carbazole
(Sigma) and hydrogen peroxide (0.01%). Antisera specific
for TRAF3 and TRAF6 were generated by immunizing
rabbits with the peptide CPVFVAQTVLENGTYIKDDTIFIKVIV conjugated to maleimide-activated ovalbumin (Pierce)
and peptides CHEKMQRNHLARHLQENTQSH, CDQSEAPVRQNHEEIMDAKPE, and CSTRFCMGSLRREGFQPRSTD conjugated to maleimide-activated keyhole limpet
hemocyanin (Pierce), respectively. TRAF2-specific rabbit
antiserum was purchased from Santa Cruz Biotechnology
(antiserum N-19). Detection of rabbit antisera on PVDF
membranes was with horseradish peroxidase conjugated
Protein A (BioRad) as described above for the anti-Flag
BioM2 antibody.
</p>
<p><italic toggle="yes">Peptide</italic>
<italic toggle="yes">Binding</italic>
<italic toggle="yes">Assay.</italic>
Peptides C-terminally attached to
cellulose membranes were synthesized by Jerini BioTools,
GmbH (Berlin, Germany). Each peptide spot contained ∼10
nmol of peptide/3 mm<sup>2</sup>
circle (<italic toggle="yes">45</italic>
, <italic toggle="yes">46</italic>
). Blocking and binding
were performed according to the manufacturer's protocol.
Membranes were probed with Sf21 cytosolic extracts containing 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 (<italic toggle="yes">40</italic>
), streptavidin−horseradish
peroxidase (0.5 μg/mL; Jackson ImmunoResearch), and POD
chemiluminescence blotting substrate (Boehringer Mannheim). Quantitation was performed by laser densitometry
with a Molecular Dynamics Personal Densitometer SI using
local averages for background values.
</p>
</sec>
<sec id="d7e457"><title>Results</title>
<p><italic toggle="yes">Recombinant</italic>
<italic toggle="yes">TRAF1,</italic>
<italic toggle="yes">TRAF2,</italic>
<italic toggle="yes">TRAF3,</italic>
<italic toggle="yes">and</italic>
<italic toggle="yes">TRAF6</italic>
<italic toggle="yes">Associate</italic>
<italic toggle="yes">with</italic>
<italic toggle="yes">CD40</italic>
<italic toggle="yes">in</italic>
<italic toggle="yes">Vitro.</italic>
Previous studies describing the
interactions between CD40 and various TRAF proteins used
yeast two-hybrid analysis and coprecipitation assays with
extracts from transfected cells or in vitro translated TRAF
proteins (<italic toggle="yes">2</italic>
, <italic toggle="yes">4</italic>
, <italic toggle="yes">5</italic>
, <italic toggle="yes">10</italic>
). To study direct CD40 interactions
with a complete panel of all TRAFs and finely map the
TRAF binding sites in the CD40 cytoplasmic domain,
recombinant baculoviruses overexpressing each of the six
human TRAF proteins were generated. Isolated glutathione
<italic toggle="yes">S</italic>
-transferase−human CD40 cytoplasmic domain (GST−CD40c) fusion protein was tested for the ability to coprecipitate insect cell-expressed full-length, CA21 epitope-tagged TRAF proteins. TRAF1, TRAF2, TRAF3, and
TRAF6 each coprecipitated with GST−CD40c (Figure <xref rid="bi981067qf00001"></xref>
A,
lanes 1−5). The specificity of these interactions was
demonstrated by the inability of TRAF proteins to interact
with GST alone as detected by immunoblot analysis (data
not shown). The interaction of TRAF2, TRAF3, and TRAF6
with CD40 has been described previously (<italic toggle="yes">2</italic>
, <italic toggle="yes">4</italic>
, <italic toggle="yes">5</italic>
, <italic toggle="yes">9</italic>
, <italic toggle="yes">10</italic>
).
An interaction with human TRAF5 was not detected, even
after immunoblot analysis (data not shown), although the
recombinant TRAF5 was demonstrated to be functional by
its interaction with the ATAR cytoplasmic domain (H. G.
Miller and J. J. Crute, unpublished results). TRAF4 interacted equally well with GST−CD40c and GST, precluding
the ability to assess CD40 interactions with full-length
TRAF4 (data not shown). Since an approximately equivalent
amount of TRAF was present in each Sf21 cell extract
(Figure <xref rid="bi981067qf00001"></xref>
A, lanes 6−10), the relative amount of each TRAF
protein coprecipitated by GST−CD40c could be directly
compared to give an indication of the relative binding
affinity. The majority of the TRAF2 or TRAF3 in the
extracts was coprecipitated by GST−CD40c. However, only
a fraction of TRAF1 or TRAF6 coprecipitated with GST−CD40c. Although it appeared that more TRAF1 bound to
GST−CD40 than TRAF6, TRAF1 expression in insect cell
extracts was high relative to the other TRAFs, and the relative
strength of TRAF1 and TRAF6 interactions with CD40 could
not be determined. These results indicate that the TRAF2
and TRAF3 interactions with CD40 were considerably
stronger than the TRAF1 and TRAF6 interactions.
<fig id="bi981067qf00001" position="float" orientation="portrait"><label>1</label>
<caption><p>In vitro association of TRAF proteins with the CD40 cytoplasmic domain. (A) GST coprecipitation assays and immunoprecipitations were performed as described under Materials and Methods. Lanes 1−5, GST−CD40c coprecipitations using insect cell extracts containing the indicated full-length TRAF-CA21. Lanes 6−10, immunoprecipitations with CA21 monoclonal antibody from the same cell extracts used in the coprecipitations. Precipitated proteins were separated by SDS−PAGE on a 12% polyacrylamide gel and visualized by staining with Coomassie Brilliant Blue. Molecular mass markers listed in kilodaltons. The asterisk indicates the position of immunoglobulin heavy chain from the CA21 antibody. Predicted molecular masses (in kilodaltons) are TRAF1, 47; TRAF2, 57; TRAF3, 66; TRAF5, 66; TRAF6, 61. (B) GST coprecipitation assays were performed as described under Materials and Methods. Lanes 1−6, GST−CD40c coprecipitations using insect cell extracts containing Flag-TRAF-NC domains. Lanes 7−12, 50% of input insect cell extracts used in lanes 1−6 containing Flag-TRAF-NC domain proteins. Proteins were separated by SDS−PAGE on a 12% polyacrylamide gel, electroblotted to a PVDF membrane, and detected using anti-Flag antibody by immunoblot analysis. Predicted molecular masses (in kilodaltons) are Flag-TRAF1-NC, 28; Flag-TRAF2-NC, 28; Flag-TRAF3-NC, 26; Flag-TRAF4-NC, 27; Flag-TRAF5-NC, 28; Flag-TRAF6-NC, 30. Quantitation of the immunoblot in (B) had a smaller dynamic range than quantitation of the Coomassie-stained gel in (A). Relative binding of TRAFs to GST−CD40 is more accurate in (A). Data are representative of the results of two independent experiments.</p>
</caption>
<graphic xlink:href="bi981067qf00001.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p>The specificity of the TRAF-CD40 interactions was
confirmed by testing the ability of GST−CD40c to precipitate the TRAF-NC domain of each TRAF. Consistent with
the results using full-length TRAF proteins, the TRAF-NC
domains of human TRAF1, TRAF2, TRAF3, and TRAF6
interacted with GST−CD40c (Figure <xref rid="bi981067qf00001"></xref>
B, lanes 1−6).
TRAF4-NC domain and TRAF5-NC domain did not interact
with CD40. None of the TRAF-NC domains coprecipitated
with GST (data not shown).
</p>
<p><italic toggle="yes">Hetero-oligomerization</italic>
<italic toggle="yes">of</italic>
<italic toggle="yes">TRAF</italic>
<italic toggle="yes">Proteins.</italic>
TRAF1, TRAF2,
TRAF3, and TRAF6 have been demonstrated to homo-oligomerize through the TRAF domain (<italic toggle="yes">1</italic>
−<italic toggle="yes">4</italic>
). TRAF1 and
TRAF2 hetero-oligomerize (<italic toggle="yes">3</italic>
, <italic toggle="yes">30</italic>
); however, the ability of
other TRAFs to hetero-oligomerize has not been systematically addressed. Since hetero-oligomerization of TRAFs
could result in activation of additional signaling pathways
through CD40, the ability of all pairwise combinations of
TRAFs to hetero-oligomerize was examined. Immunoprecipitations with CA21 antibody were performed using
extracts from insect cells coinfected with two recombinant
baculoviruses, one expressing a CA21-tagged TRAF domain
and the other expressing a Flag-tagged TRAF domain.
Associated TRAFs were detected with anti-Flag antibody.
Each TRAF domain homo-oligomerized, and TRAF1-NC
and TRAF2-NC hetero-oligomerized as efficiently as each
homo-oligomerized (Figure <xref rid="bi981067qf00002"></xref>
). This confirms and extends
previous reports (<italic toggle="yes">3</italic>
, <italic toggle="yes">30</italic>
). TRAF1-NC and TRAF2-NC each
hetero-oligomerized with TRAF3-NC to a lesser extent.
Weaker interactions were detected between TRAF4-NC or
TRAF6-NC and each of the other TRAFs (Figure <xref rid="bi981067qf00002"></xref>
). This
indicates that TRAF hetero-oligomers do not form efficiently
through the TRAF domains of TRAF3, TRAF4, TRAF5, and
TRAF6.
<fig id="bi981067qf00002" position="float" orientation="portrait"><label>2</label>
<caption><p>TRAF-NC domain-mediated oligomerization. Insect cells were coinfected with all possible pairwise combinations of recombinant baculoviruses expressing Flag (listed on top) and CA21 (listed on the left) epitope-tagged TRAF-NC domains. Numbers designate the TRAF protein number. Immunoprecipitations were performed with insect cell extracts and the CA21 monoclonal antibody; proteins were separated by SDS−PAGE on a 12% polyacrylamide gel, electroblotted to a PVDF membrane, and detected with anti-Flag antibody. Data are representative of the results of two independent experiments.</p>
</caption>
<graphic xlink:href="bi981067qf00002.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p>TRAF3 and TRAF5 are the only TRAF proteins that
contain a predicted isoleucine zipper N-terminal to the TRAF
domain. Since isoleucine zipper sequences have been shown
to mediate protein homo-oligomerization (<italic toggle="yes">47</italic>
), it was possible
that full-length TRAF3 and TRAF5 might interact through
the isoleucine zipper. To test this, insect cells were infected
with individual baculoviruses or with pairwise combinations
of baculoviruses expressing TRAF5-CA21 and either TRAF2,
TRAF3, or TRAF6. Only full-length TRAF3 and TRAF5
coimmunoprecipitated (Figure <xref rid="bi981067qf00003"></xref>
A, lane 8). Since the TRAF
domains of TRAF3 and TRAF5 did not interact (Figure <xref rid="bi981067qf00002"></xref>
),
and TRAFs that lack the predicted isoleucine zipper (TRAF2
and TRAF6) did not interact with TRAF5, this indicates that
the TRAF3−TRAF5 interaction is most likely mediated
through the isoleucine zipper domains. Additionally, the
TRAF3−TRAF5 hetero-oligomer coprecipitated with GST−CD40c (Figure <xref rid="bi981067qf00003"></xref>
B, lane 6). Thus, although human TRAF5
did not interact directly with the CD40 cytoplasmic domain,
TRAF5 was capable of being recruited indirectly to CD40
as a hetero-oligomer with TRAF3.
<fig id="bi981067qf00003" position="float" orientation="portrait"><label>3</label>
<caption><p>Hetero-oligomerization of TRAF3 and TRAF5. (A) Insect cells were infected with various combinations of recombinant baculoviruses expressing TRAF5-CA21, TRAF2, TRAF3, or TRAF6 as indicated by “+” over the lanes. Proteins from whole cell extracts (IP, indicated by “−”) or immunoprecipitations of TRAF5-CA21 using the CA21 monoclonal antibody (IP, indicated by “+”) were separated by SDS−PAGE on a 12% polyacrylamide gel, electroblotted to a PVDF membrane, and detected with antisera specific for TRAF2 (lanes 1−4), TRAF3 (lanes 5−8), or TRAF6 (lanes 9−12). The internal controls on the immunoblots demonstrate the specificity of the antisera to TRAF2, TRAF3, and TRAF6. (B) Insect cell extracts in lanes 6−8 in panel A were used for GST−CD40 coprecipitation assays as described under Materials and Methods. Precipitated proteins were separated by SDS−PAGE on a 12% polyacrylamide gel, electroblotted to a PVDF membrane, and detected using anti-TRAF3 antisera (lanes 1−3) or CA21 antibody (lanes 4−6). Data are representative of the results of two independent experiments.</p>
</caption>
<graphic xlink:href="bi981067qf00003.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p><italic toggle="yes">Mapping</italic>
<italic toggle="yes">the</italic>
<italic toggle="yes">TRAF</italic>
<italic toggle="yes">Binding</italic>
<italic toggle="yes">Sites</italic>
<italic toggle="yes">on</italic>
<italic toggle="yes">CD40.</italic>
To map the
TRAF1, TRAF2, TRAF3, and TRAF6 binding sites on the
CD40 cytoplasmic domain, a linear peptide binding approach
was employed. Overlapping peptides of 14 and 10 amino
acid residues scanning across the CD40 cytoplasmic domain
(with an overlap of 13 and 9 residues, respectively) were
synthesized on cellulose membranes (Figure <xref rid="bi981067qf00004"></xref>
A,B). Insect
cell extracts containing individual TRAF proteins were tested
for binding to the immobilized peptides. TRAF1 bound to
peptides in two separate regions of the CD40 cytoplasmic
domain (Figure <xref rid="bi981067qf00004"></xref>
C,D). Binding to the membrane distal
region (Figure <xref rid="bi981067qf00004"></xref>
C, peptides 30−34) correlated with peptides
containing the sequence PVQET that has been implicated
as being important for TRAF2 and TRAF3 binding to CD40
(<italic toggle="yes">11</italic>
, <italic toggle="yes">27</italic>
). Optimal TRAF1 binding in this region occurred
when the PVQET sequence was positioned toward the
N-terminus of the peptides. TRAF1 interaction with a
membrane proximal region (Figure <xref rid="bi981067qf00004"></xref>
C, peptides 1−5)
correlated with the presence of positively charged residues
in the peptides (see Figure <xref rid="bi981067qf00004"></xref>
A,B). Similar interactions with
this region were observed for TRAF2 and TRAF3 when 20-mer peptides were used (data not shown). This binding to
the membrane proximal region may be due to nonspecific
ionic interactions of the TRAFs with the numerous positively
charged residues in these peptides (Figure <xref rid="bi981067qf00004"></xref>
A).
<fig id="bi981067qf00004" position="float" orientation="portrait"><label>4</label>
<caption><p>TRAF protein binding to peptides scanning the CD40 cytoplasmic domain. Overlapping 14 amino acid residue (A) and 10 amino acid residue (B) peptides synthesized in spots on cellulose membranes were probed with TRAF1, TRAF2, TRAF3, or TRAF6 as described under Materials and Methods. TRAF proteins bound to 14-mer (C) and 10-mer (D) peptides were visualized by chemiluminescent detection and are shown as scans of the exposed films. Peptide spot numbers correspond to the peptide sequences in panels A and B. Relative levels of binding in (C) and (D) were quantitated and the relative integrated areas indicated in (A) and (B). Numbers were normalized within each binding assay to give the darkest spot a value of 100. Relative binding below 3% is indicated by a dash (−). Data are representative of the results of two independent experiments for TRAF1 and TRAF6 and four independent experiments for TRAF2 and TRAF3.</p>
</caption>
<graphic xlink:href="bi981067qf00004.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p>TRAF2 and TRAF3 bound strongest to a single region
defined by the 14 and 10 amino acid residue peptides (Figure
<xref rid="bi981067qf00004"></xref>
C,D). TRAF2 and TRAF3 bound the best to the same 10
amino acid peptides with the PVQET sequence at the
C-terminus of the peptide (Figure <xref rid="bi981067qf00004"></xref>
D, peptides 30, 31).
Binding decreased as the PVQET sequence was positioned
toward the N-terminus of the peptide. For TRAF3, low-level binding to 14-mers was also detected when a similar
sequence (PVTQE) was positioned near the C-terminus of
the peptides (Figure <xref rid="bi981067qf00004"></xref>
A,C, peptides 36, 37). TRAF2 and
TRAF3 binding to the PVTQE sequence was stronger when
20-mer peptides were used. An increased binding was
observed with PVTQE in conjunction with the PVQET
sequence (data not shown). TRAF6 interacted with a series
of 14 amino acid residue peptides corresponding to a region
N-terminal to the PVQET sequence (Figure <xref rid="bi981067qf00004"></xref>
C, peptides 10,
12−16). A single 10 amino acid peptide interacted weakly
with TRAF6 (Figure <xref rid="bi981067qf00004"></xref>
D, peptide 14). This TRAF6 binding
sequence was positioned within a larger region previously
identified as required for TRAF6 binding to CD40 (<italic toggle="yes">10</italic>
).
</p>
<p><italic toggle="yes">Defining</italic>
<italic toggle="yes">Minimal</italic>
<italic toggle="yes">TRAF</italic>
<italic toggle="yes">Binding</italic>
<italic toggle="yes">Sites</italic>
<italic toggle="yes">in</italic>
<italic toggle="yes">CD40.</italic>
To more
finely map the TRAF1, TRAF2, TRAF3, and TRAF6
binding sites in CD40, peptides containing the TRAF1,
TRAF2, TRAF3, or TRAF6 binding regions were synthesized on cellulose membranes with progressive single amino
acid deletions from either the N-terminus, the C-terminus,
or both termini. Progressive removal of residues from the
C-terminus of a 14 amino acid long TRAF1 binding region
peptide increased TRAF1 binding. Removal of Thr<sup>254</sup>
nearly
abolished binding (Figure <xref rid="bi981067qf00005"></xref>
A, peptides 1−12). When amino
acids were deleted from the N-terminus, TRAF1 binding was
eliminated once Pro<sup>250</sup>
was removed (Figure <xref rid="bi981067qf00005"></xref>
A, peptides
13−24). When amino acids were deleted simultaneously
from both ends of the peptide, TRAF1 bound weakly to the
sequence VQETLHGC (Figure <xref rid="bi981067qf00005"></xref>
A, peptides 25−29). This
indicates that the sequence PVQET is required for optimal
TRAF1 binding to CD40, but that Pro<sup>250</sup>
and Thr<sup>254</sup>
may not
be essential.
<fig id="bi981067qf00005" position="float" orientation="portrait"><label>5</label>
<caption><p>TRAF1, TRAF2, TRAF3, and TRAF6 protein binding to peptides with progressive deletions. Based on the TRAF binding peptides in Figure <xref rid="bi981067qf00004"></xref>
, peptides with progressive deletions from the C-terminus, N-terminus, or both termini were synthesized on cellulose membranes and probed for TRAF1 (A), TRAF2 or TRAF3 (B), or TRAF6 (C) binding as in Figure <xref rid="bi981067qf00004"></xref>
. TRAF proteins bound to the membranes were visualized by chemiluminescent detection, were scanned, and are shown to the right of each set of peptides. Numbers indicate the peptide sequence corresponding to individual peptide spots. Peptide strips that were bound to TRAF2 in (B) containing peptides 1−10 and 21−26 were exposed for shorter times relative to the peptide strip containing peptides 11−20. Although peptides 1 and 21 appear less positive than peptide 11, the TRAF2 binding on equal exposures was equivalent for peptides 1, 11, and 21.</p>
</caption>
<graphic xlink:href="bi981067qf00005.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
<p>Progressive removal of amino acids from the C-terminus
of a 13 amino acid long TRAF2 and TRAF3 binding region
peptide increased the ability of TRAF2 or TRAF3 to bind.
When Thr<sup>254</sup>
was deleted, TRAF2 and TRAF3 binding to
the peptide was eliminated (Figure <xref rid="bi981067qf00005"></xref>
B, peptides 1−10).
Deletions from the N-terminus had no effect on TRAF2 and
TRAF3 interactions until Pro<sup>250</sup>
was removed. The deletion
of Pro<sup>250</sup>
reduced TRAF3 binding and abolished TRAF2
binding (Figure <xref rid="bi981067qf00005"></xref>
B, peptides 11−20). TRAF2 and TRAF3
did not bind to peptides once Val<sup>251</sup>
was removed. Simultaneous deletion of residues from both ends of the peptide
demonstrated that the sequence PVQET was sufficient for
nearly optimal levels of TRAF2 and TRAF3 binding (Figure
<xref rid="bi981067qf00005"></xref>
B, peptide 25).
</p>
<p>The ability of TRAF6 to bind to a 14 amino acid long
peptide was eliminated once Phe<sup>238</sup>
was removed by progressive C-terminal deletions (Figure <xref rid="bi981067qf00005"></xref>
C, peptides 1−12).
Removal of residues from the N-terminus had no effect until
Gln<sup>231</sup>
was removed. The deletion of Gln<sup>231</sup>
nearly eliminated TRAF6 binding (Figure <xref rid="bi981067qf00005"></xref>
C, peptides 13−24). Simultaneous deletions from both ends of the peptide supported
the importance of Gln<sup>231</sup>
for optimal binding (Figure <xref rid="bi981067qf00005"></xref>
,
peptides 25−30). The sequence QEPQEINF may be a core
TRAF6 binding sequence; however, adjacent residues appeared to contribute to optimal TRAF6 binding. Together
with the scanning results in Figure <xref rid="bi981067qf00004"></xref>
, the peptides with
progressive amino acid deletions define the minimal TRAF1,
TRAF2, TRAF3, and TRAF6 binding regions in the CD40
cytoplasmic domain (summarized in Figure <xref rid="bi981067qf00006"></xref>
).
<fig id="bi981067qf00006" position="float" orientation="portrait"><label>6</label>
<caption><p>Minimal TRAF binding sites in the CD40 cytoplasmic domain. The minimal binding site for each TRAF protein is indicated by a solid bar over the human CD40 cytoplasmic domain sequence.</p>
</caption>
<graphic xlink:href="bi981067qf00006.eps" position="float" orientation="portrait"></graphic>
</fig>
</p>
</sec>
<sec id="d7e717"><title>Discussion</title>
<p>A subset of TNF receptor family members including
CD40, TNFR2, CD30, LTβR, ATAR, OX40, and 4-1BB
signal by directly interacting with TRAF proteins following
receptor cross-linking. Previous studies have addressed the
interactions of individual TRAFs with subsets of TNF
receptors, but no complete and comparative biochemical
analysis of TRAF interactions with CD40 has been performed. By testing each of the six recombinant TRAF
proteins for interaction with the CD40 cytoplasmic domain,
we found that, consistent with previous studies, CD40
interacted with TRAF2, TRAF3, and TRAF6 (<italic toggle="yes">2</italic>
, <italic toggle="yes">4</italic>
, <italic toggle="yes">5</italic>
, <italic toggle="yes">10</italic>
).
Additionally, a direct TRAF1 interaction with CD40 was
demonstrated. A hierarchy of CD40−TRAF interaction
could be proposed from coprecipitation experiments. CD40
interactions with TRAF2 and TRAF3 were considerably
stronger than interactions with TRAF1 and TRAF6. Thus,
the inability to detect an interaction between TRAF1 and
CD40 in earlier reports (<italic toggle="yes">11</italic>
, <italic toggle="yes">31</italic>
) may be due to a significantly
lower affinity of TRAF1 for CD40 when compared to
TRAF2 and TRAF3. In support of our findings, transiently
expressed TRAF1 can be coimmunoprecipitated with CD40
in a CD40 ligand-stimulated B cell line (R. J. Noelle,
personal communication). Two studies have shown conflicting results on the ability of TRAF5 to bind CD40 (<italic toggle="yes">8,</italic>
<italic toggle="yes">9</italic>
).
Although these studies used mouse TRAF5 and CD40, our
results using human proteins consistently show no direct
binding of TRAF5 to CD40. Additionally, no evidence for
direct binding of TRAF4 to CD40 was obtained.
</p>
<p>Homo-oligomerization of TRAF1, TRAF2, TRAF3, and
TRAF6 has been reported to be mediated by the TRAF
domain (<italic toggle="yes">1</italic>
−<italic toggle="yes">4</italic>
, <italic toggle="yes">30</italic>
). We confirmed that the TRAF domains
of TRAF1, TRAF2, TRAF3, and TRAF6 homo-oligomerize
and have extended these results to demonstrate homo-oligomerization of TRAF4 and TRAF5. TRAF1 is recruited
to the TNFR2 cytoplasmic domain indirectly by hetero-oligomerizing with TRAF2 (<italic toggle="yes">3,</italic>
<italic toggle="yes">30</italic>
). This TRAF domain-mediated hetero-oligomerization allows for indirect recruitment of TRAF proteins to receptors. In coexpression
experiments in insect cells, we found that only TRAF1 and
TRAF2 hetero-oligomerized efficiently through the TRAF
domain. TRAF1 and TRAF2 each interacted weakly with
TRAF3, but the physiological significance of this interaction
is unclear considering the weakness of the interaction and
the low endogenous levels of TRAF proteins in cells. No
other TRAF domain hetero-oligomers formed to an appreciable extent. The limited ability of TRAFs to hetero-oligomerize through the TRAF domain restricts the potential
combinations of TRAFs recruited to receptors. Thus,
extensive hetero-oligomerization of TRAFs would not be
expected to play a role in the complexity of TNF receptor
superfamily signaling.
</p>
<p>Despite an inability to hetero-oligomerize through the
TRAF domain, full-length TRAF3 and TRAF5 interacted
(Figure <xref rid="bi981067qf00003"></xref>
). TRAF3 and TRAF5 TRAF domains did not
interact, and full-length TRAF5 did not interact with full-length TRAF2 or TRAF6 that have similar zinc RING and
zinc finger motifs. This suggests that the predicted isoleucine
zipper regions in TRAF3 and TRAF5 may mediate their
interaction. It is possible that TRAF3 and TRAF5 may form
higher order homo- and hetero-oligomers when compared
with oligomers formed through the TRAF domain alone.
Consistent with this possibility, TRAF3/TRAF5 hetero-oligomers were found to coprecipitate with GST−CD40c.
Consequently, TRAF5 could be recruited to CD40 and other
receptors through interactions with TRAF3. These findings
suggest that TNF receptors which interact with TRAF3
would be capable of indirectly recruiting TRAF5. Additionally, the mechanism by which overexpressed TRAF3 acts
as a negative modulator of NF-κB activation (<italic toggle="yes">16</italic>
) could be
a down-modulation of TRAF5 recruitment to receptors.
</p>
<p>Obtaining cell-type selectivity in activating CD40-dependent signal transduction pathways (<italic toggle="yes">37</italic>
) through TRAFs has
been difficult to understand because all TRAFs except
TRAF6 appear to recognize the same site on CD40. It is
not clear whether TRAFs compete for CD40 binding in cells.
By fine structure peptide-based mapping, the sequence
PVQET was demonstrated to be sufficient for optimal
binding of TRAF1, TRAF2, and TRAF3. The accuracy of
mapping TRAF binding sites using peptides covalently linked
to membranes has been supported by preliminary studies with
soluble peptides. The ability of soluble versions of the
peptides in Figure <xref rid="bi981067qf00005"></xref>
to compete for CD40−TRAF2 and
CD40−TRAF3 binding correlated well with TRAF2 and
TRAF3 binding to the cellulose-linked peptides (M. R. Kehry
and J. J. Crute, unpublished results). Since the binding
affinity of TRAF2 and TRAF3 to CD40 in coprecipitation
experiments appeared similar, it is thus likely that, depending
on the relative concentrations of TRAF2 and TRAF3 in the
cell, these two TRAFs could compete for binding to CD40.
This additionally may account for some of the similar cellular
effects of dominant-negative TRAF2 and TRAF3 proteins.
Interestingly, a 30-fold decrease in the density of peptides
coupled to the cellulose membranes dramatically weakened,
although did not eliminate, the signal for TRAF3 binding
but left TRAF2 binding intact (S. S. Pullen and M. R. Kehry,
unpublished results). Thus, the extent of receptor cross-linking might also contribute to a hierarchy of TRAF binding
in activated cells. A binding hierarchy of TRAFs to CD40,
combined with possible differential levels of TRAF expression in different cell types, suggests that some cellular
selectivity in CD40-dependent signaling (<italic toggle="yes">37</italic>
) could be
established by competition of TRAF1, TRAF2, and TRAF3
for binding to cross-linked receptors.
</p>
<p>The interaction of TRAF1 with the PVQET sequence was
weaker when compared with interactions of TRAF2 and
TRAF3 (Figure <xref rid="bi981067qf00004"></xref>
C,D). Sequences similar to the PVQET
sequence of CD40 that support binding of TRAF1, TRAF2,
and TRAF3 are found in LMP1 (PQQAT), CD30 (PEQET),
and OX40 (PIQEE) (<italic toggle="yes">11</italic>
, <italic toggle="yes">13</italic>
, <italic toggle="yes">17</italic>
). Our finding that TRAF1
binds to CD40 is therefore not surprising. Interestingly, the
TRAF binding motifs in LMP1, CD30, and CD40 [previously
designated PXQXT/S (<italic toggle="yes">48</italic>
)] have different hierarchies of
affinity for TRAF1, TRAF2, and TRAF3 (<italic toggle="yes">11</italic>
, <italic toggle="yes">17</italic>
). For
example, TRAF3 interacts strongest with the LMP1 sequence, and TRAF2 interacts the weakest with LMP1 (<italic toggle="yes">17</italic>
).
Therefore, sequences flanking PXQXT/S or the amino acids
at the second and fourth positions in the motif may increase
or decrease the relative affinities of TRAF1, TRAF2, and
TRAF3 binding. Thus, a distinct hierarchy of affinities of
TRAF1, TRAF2, and TRAF3 for the PXQXT/S motif in
different receptors may alter the outcome of receptor cross-linking to provide receptor specificity in signal transduction.
We are currently testing this idea using the CD40 and OX40
cytoplasmic domains.
</p>
<p>TRAF2 and TRAF3 also interacted with a membrane distal
region distinct from the PVQET sequence. Peptides 14
amino acid residues in length containing the sequence
PVTQE interacted weakly with TRAF3, but not with TRAF2.
However, 20 amino acid residue peptides containing the
PVTQE sequence interacted well with both TRAF2 and
TRAF3. This may indicate that CD40 contains two distinct
TRAF2 and TRAF3 binding sites. This possibiltiy is
analogous to the two binding sites for TRAF1 and TRAF2
in the CD30 cytoplasmic domain (<italic toggle="yes">11</italic>
, <italic toggle="yes">13</italic>
). Alternatively,
CD40 may possess two separate regions that form contacts
with the same TRAF subunit.
</p>
<p>In contrast to TRAF1, TRAF2, and TRAF3, TRAF6
interacted with a distinct nonoverlapping sequence in the
CD40 cytoplasmic domain (amino acids 231−238; Figure
<xref rid="bi981067qf00006"></xref>
) that is contained within a larger region previously defined
as a TRAF6 binding site (<italic toggle="yes">10</italic>
). It is thus possible that
multimerized CD40 may be able to interact simultaneously
with TRAF6 and either TRAF1, TRAF2, or TRAF3. Additionally, the TRAF6 binding region in CD40 shares limited
sequence similarity with the TRAF1, TRAF2, and TRAF3
interaction site (Figure <xref rid="bi981067qf00006"></xref>
), although similar amino acids are
present (P, Q, and E). CD40 is the only TNF receptor family
member demonstrated to directly interact with TRAF6, which
may impart unique signaling outcomes to this receptor.
</p>
<p>After multimerization of TNF receptor family members
by their respective trimeric ligands, the initial event in
signaling is thought to be mediated by a transient recruitment
of TRAF proteins (<italic toggle="yes">38</italic>
). Since there is a report of constitutive
association of TRAF2 and CD40 prior to CD40 cross-linking
(<italic toggle="yes">49</italic>
), it is possible that some stable CD40−TRAF interactions
could form during coprecipitation assays. Preliminary
biochemical studies on a monomeric form of the 62 amino
acid CD40 cytoplasmic domain suggest that it is unstructured
and is a poor competitor of GST−CD40c−TRAF interactions in coprecipitation assays (S. S. Pullen and J. J. Crute,
unpublished results). These data suggest that the non-cross-linked CD40 receptor on unstimulated cells would be a poor
binding site for TRAFs. These studies support a model
where the cytoplasmic domains of multimerized CD40 form
binding sites with differing affinities for individual TRAFs
depending upon the extent of CD40 cross-linking. Additionally, the relative in vivo concentrations of different TRAF
proteins in different cell types would establish a dynamic
equilibrium of TRAF binding and an overall integrated
signaling response. Defining differences in the downstream
signaling capacities of TRAF2, TRAF5, and TRAF6 and the
outcomes of CD40 interactions with TRAF1 and TRAF3
remain to be determined. Although it is thought that TRAFs
are released from CD40 shortly after initiation of signaling
(<italic toggle="yes">38</italic>
, <italic toggle="yes">49</italic>
), the mechanism of this release, the involvement of
protein kinases, and molecular links to downstream signaling
pathways require further elucidation.
</p>
</sec>
</body>
<back><ack><title>Acknowledgments</title>
<p>We thank A. Shrutkowski for cDNA library DNA, H. E.
White for oligonucleotide purification and assistance in
cloning TRAF4, G. Bell for purification of CA21 antibody,
and J. Pelletier for TRAF3 and TRAF6 antisera preparation.
</p>
</ack>
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<mods version="3.6"><titleInfo><title>CD40−Tumor Necrosis Factor Receptor-Associated Factor (TRAF) Interactions: Regulation of CD40 Signaling through Multiple TRAF Binding Sites and TRAF Hetero-Oligomerization</title>
</titleInfo>
<titleInfo contentType="CDATA"><title>CD40−Tumor Necrosis Factor Receptor-Associated Factor (TRAF) Interactions: Regulation of CD40 Signaling through Multiple TRAF Binding Sites and TRAF Hetero-Oligomerization</title>
</titleInfo>
<name type="personal"><namePart type="family">PULLEN</namePart>
<namePart type="given">Steven S.</namePart>
<affiliation>Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368</affiliation>
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<name type="personal"><namePart type="family">MILLER</namePart>
<namePart type="given">Heather G.</namePart>
<affiliation>Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368</affiliation>
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<name type="personal"><namePart type="family">EVERDEEN</namePart>
<namePart type="given">Daniel S.</namePart>
<affiliation>Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368</affiliation>
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<name type="personal"><namePart type="family">DANG</namePart>
<namePart type="given">Thu T. A.</namePart>
<affiliation>Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368</affiliation>
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<name type="personal"><namePart type="family">CRUTE</namePart>
<namePart type="given">James J.</namePart>
<affiliation>Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368</affiliation>
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<name type="personal" displayLabel="corresp"><namePart type="family">KEHRY</namePart>
<namePart type="given">Marilyn R.</namePart>
<affiliation>Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368</affiliation>
<affiliation> To whom correspondence should be addressed at BoehringerIngelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., P.O. Box 368,Mail code R6-5, Ridgefield, CT 06877-0368. Telephone: 203-791-6153. Fax: 203-791-6196. EMail: mkehry@bi-pharm.com.</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<typeOfResource>text</typeOfResource>
<genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre>
<originInfo><publisher>American Chemical Society</publisher>
<dateCreated encoding="w3cdtf">1998-08-07</dateCreated>
<dateIssued encoding="w3cdtf">1998-08-25</dateIssued>
<copyrightDate encoding="w3cdtf">1998</copyrightDate>
</originInfo>
<language><languageTerm type="code" authority="iso639-2b">eng</languageTerm>
<languageTerm type="code" authority="rfc3066">en</languageTerm>
</language>
<abstract>CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3−TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.</abstract>
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