By
From the * Howard Hughes Medical Institute, New York 10021; and The Rockefeller University,
New York 10021
Through their interaction with the TNF receptor-associated factor (TRAF) family, members
of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and
characterized a novel component of the receptor-TRAF signaling complex, designated TRIP
(TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction
with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-B activation
that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a
receptor-proximal regulator that may influence signals responsible for cell activation/proliferation
and cell death induced by members of the TNFR superfamily.
Members of the TNF receptor (TNFR)1 superfamily
play important roles in the induction of diverse signals leading to cell growth, activation, and apoptosis (1).
Whether the signals induced by a given receptor leads to
cell activation or death is, however, highly cell-type specific and tightly regulated during differentiation of cells. For
example, the TNFRs can exert costimulatory signals for proliferation of naive lymphocytes but also induce death signals required for deletion of activated T lymphocytes (1).
The cytoplasmic domains of these receptors lack intrinsic
catalytic activity and also exhibit no significant homology
to each other or to other known proteins. Exceptions to
this include Fas(CD95) and TNFR1 that share a significant
homology within an 80-amino acid region of their cytoplasmic tails (called the "death domain"; 2, 3). Therefore, it
is suggested that the TNFR family members can initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor
complex (1).
Indeed, several types of intracellular signal transducers
have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily (4). Recent biochemical and molecular studies
showed that a class of signal-transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death
domains (2, 3, 6, 12, 17, 20). For example, Fas(CD95) and
TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD
(MORT1)/RIP through the interactions of their respective death domains (2, 3, 6, 12, 17, 20, 21). Clustering of
these signal transducers leads to the recruitment of FLICE/ MACH, and subsequently, to cell death (13, 14).
The TNFR family members can also recruit a second
class of signal transducers called TRAFs (TNFR-associated
factor), some of which are responsible for the activation of
NF- Several effector functions of TRAFs were revealed by
recent experiments based on a transfection system. TRAF2,
first identified by its interaction with TNFR2 (4), was subsequently shown to mediate NF- Here we report the isolation and characterization of a
novel component of the TNFR superfamily/TRAFs signaling complex, named TRIP (TRAF-interacting protein).
TRIP associates with the receptor/TRAF signaling complex, and inhibits the TRAF2-mediated NF- Yeast Two-Hybrid Screening.
A bait plasmid pEG202-TRAF1
(27), which encodes the LexA-DNA binding domain fused to
TRAF1(183-409), was used for a yeast two-hybrid screening of a
mouse thymocyte cDNA library (provided by F. Alt, Harvard
Medical School, Cambridge, MA). The plasmids and yeast strains
for the two-hybrid system were provided by R. Brent (Harvard
Medical School). The isolation of positive clones and subsequent
analyses were carried out as previously described (27). The interaction of proteins in the two-hybrid assay was scored by the cDNA Cloning and Northern Blot Hybridization.
The TRIP cDNA
insert of ~1.0 kb isolated by two-hybrid screening was used as
probe to screen mouse thymocytes and T cell hybridoma cDNA
libraries in Reverse Transcriptase-PCR Assay.
For the stimulation of lymphocytes, lymph node cells were isolated from BALB/c mice (4-6
wk old) and cultured on plates coated with anti-TCR Ab (10 µg/ml) and anti-CD28 Ab (1 µg/ml) for 48 h as described (37).
Total RNA was prepared from unstimulated and stimulated lymph
node cells (total RNA isolation kit; Strategene Corp.). Firststrand cDNA was synthesized from 10 µg of total RNA using
M-MLV reverse transcriptase and random hexanucleotides following the protocols provided by the supplier (GIBCO BRL, Gaithersburg, MD). Quantitative PCR was performed in the linear phase
of amplification by testing PCR products from different dilutions
of first-strand cDNA products. PCR amplification was performed
for 35 cycles using 1 of 1,000 of the first-strand cDNA synthesized above. PCR products were then electrophoresed in a 2%
agarose gel and subjected to Southern blot analysis as described previously (37). The following primers used for quantitative PCR
analysis were: TRAF1 (sense), 5 Reagents and Cell Lines.
Rabbit polyclonal antisera recognizing mTRIP were prepared by Animal Pharm Services, Inc.
(Healdsburg, CA) using bacteria-produced glutathione-S-transferase (GST)-TRIP fusion proteins. Polyclonal antisera were negatively selected with purified glutathione-S-transferase (GST) proteins before use. Anti-TRAF1 and anti-TRAF2 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
The monoclonal Ab against the HA epitope (12CA5) was purchased from BabCo (Berkeley, CA). 293 cells were obtained
from E. Spanopoulou (Mount Sinai School of Medicine, NY).
Recombinant human TNF and IL-1 were purchased from R & D
Sys. Inc. (Minneapolis, MN).
Recombinant Plasmids.
Eukaryotic expression vectors for wildtype or mutant forms of TRAF1 and TRAF2, CD8-TNFR2, CD8CD30, GST-TNFR2, and GST-CD30 have been described (27,
30). Expression vectors for TRADD was made by cloning the
PCR-amplified murine TRADD cDNA into pH Transfection and Reporter Assays.
Transfection of 293 cells were
performed in 6-cm dishes by calcium phosphate precipitation as
described previously (27). Each transfection maintained an equal
amount of total DNA by adding appropriate amount of the control vector, pcDNA3.1 (Invitrogen). 48 h after transfection,
luciferase activity was determined and normalized relative to
Precipitation of GST Fusion Proteins and Western Blot Analysis.
293 cells were transfected with various combinations of expression vectors as indicated. 36 h after transfection, cells were harvested in phosphate-buffered saline/1 mM phenylmethylsulfonyl fluoride, pelleted, and lysed in lysis buffer (20 mM Hepes [pH 7.9],
100 mM KCl, 300 mM NaCl, 10 mM EDTA, 0.1% NP-40, plus
protease inhibitors) as described (30). After lysis, aliquots of cell lysates were incubated with glutathione-Sepharose (Pharmacia, Piscataway, NJ) for 2 h at 4°C. The beads were then washed five times with the lysis buffer, followed by an additional wash with the lysis buffer lacking NP-40. The proteins were then recovered by boiling in SDS-PAGE sample buffer. The eluted proteins were separated on 10% SDS-PAGE and transferred to Immobilon P
(Millipore Corp., Bedford, MA). The blot was subjected to Western analysis using enhanced chemiluminescence system (Amersham Corp., Arlington Heights, IL) as described (30).
Previous
experiments have suggested that TRAF1 may function as
an adapter protein to recruit additional signaling proteins such as c-IAPs to the receptor complex (7), or that TRAF1
may initiate an as yet uncovered signaling pathway. To determine the potential role of TRAF1 in the receptor signaling complex, we searched for additional TRAF1-interacting
proteins using the yeast two-hybrid system. By screening
cDNA libraries derived from mouse thymocytes, multiple
cDNA clones representing several distinct proteins were
isolated (data not shown). Among these, one set of cDNAs
encoded proteins which could interact with both TRAF1 and TRAF2 in the two-hybrid assay.
Analysis of the DNA sequence of the TRAF1- and
TRAF2-interacting cDNA clones revealed that they were
derived from a single novel gene named TRIP. Since
TRIP interacted strongly with both TRAF1 and TRAF2
in the two-hybrid assay, we tested whether these proteins
interacted in mammalian cells. Expression vectors encoding TRAF1 or TRAF2 were coexpressed in 293 cells in the
presence of an expression vector encoding either GST
alone or GST-TRIP fusion protein. Cell lysates were precipitated with glutathione-Sepharose beads, and analyzed
by Western blot analysis with anti-TRAF1 or anti-TRAF2
antibodies. Consistent with the yeast two-hybrid assay, GST-TRIP coprecipitated both TRAF1 and TRAF2,
demonstrating that TRIP can interact directly with TRAF1
and TRAF2 in human cells (Fig. 1).
Full-length sequence of TRIP was derived from sequence analysis of multiple cDNA clones from both thymocyte and T cell cDNA libraries (Fig. 2 A). TRIP mRNA
is predicted to encode proteins of 470 amino acids. Using a
murine TRIP as a hybridization probe, we also isolated several human TRIP cDNA clones from a human thymocyte cDNA library. Human TRIP encodes a 469-amino acid
protein that is overall 76% identical to murine TRIP (Fig.
2 A). The amino acid sequence identity between the NH2terminal half of mTRIP and hTRIP (residues 1-270) is
even higher (87% identical). A homology search of the
TRIP amino acid sequence revealed that TRIP is a novel
protein with an NH2-terminal RING finger sequence motif (Fig. 2, A and B) (38). The NH2-terminal RING finger
motif of TRIP is followed by an extended putative coiledcoil domain (Fig. 2 A). The putative coiled-coil domain
of TRIP can be further devided into the NH2-terminal
coiled-coil structure, similar to the rod-like tails of myosin
heavy chains (residues 56-150; reference 39) and the
COOH-terminal leucine zipper-like coils (residues 221- 260; reference 40), both of which are implicated in protein-protein interactions. A helical representation of the
putative leucine zipper (Fig. 2 C) shows that the position
next to the zipper is always hydrophobic or uncharged,
whereas other sides of the wheel contain many charged but
few hydrophobic residues, suggesting an amphipathic structure that can interact with another helix.
To characterize TRIP further, its expression pattern was
examined. Northern blot analysis of various mouse tissue
RNA samples revealed that TRIP-specific probes detected
a ~2.1-kb mRNA species present in various tissues, but
most abundant in testes, thymus, and spleen (Fig. 3 A). To
characterize further the expression of TRIP in lymphocytes, we analyzed its expression during lymphocyte proliferation by semiquantitative PCR. The expression of TRIP was significantly reduced when lymphocytes were stimulated to proliferate via antigen receptors (Fig. 3 B). This is
in contrast to that of other components of the TNFR-
TRAF signaling complex. For example, the expression of
TRAF1 and c-IAP1 was upregulated upon lymphocyte
proliferation (Fig. 3 B). These results suggest that the repertoire of signal transducers available in a given cell type can
change depending on the state of the cell.
The yeast twohybrid assay was used to determine the structural requirements for the interaction of TRIP with TRAF1 or TRAF2. In the yeast two-hybrid assay, a mutant TRIP comprising
the NH2-terminal half of the protein (TRIP[1-275]) interacted with TRAFs, whereas a mutant TRIP lacking the
NH2-terminal RING finger and the coiled-coil domain
(TRIP[275-470]) failed to interact with TRAFs (Fig. 4 A). Further deletion analysis suggested that the putative coiledcoil region of TRIP mediates the interaction with TRAFs,
since a mutant TRIP lacking the NH2-terminal RING finger motif still interacted with TRAFs (TRIP[56-275])
(Fig. 4 A). In addition, both TRIP(56-185) and TRIP
(186-275) interacted with TRAFs, suggesting that TRIP contains two independent TRAF binding sites within the
long coiled-coil domain of the protein (Fig. 4 A).
To delineate a region in TRAF that is required
for TRIP binding, the interaction of TRIP with various
truncation mutants of TRAF1 or TRAF2 was determined
by the yeast two-hybrid assay or by a transfection-based coprecipitation assay in 293 cells. TRIP interacted with an
NH2-terminal deletion mutant of TRAF1 expressing the
entire TRAF domain (TRAF1[183-409]), but failed to interact with an NH2-terminal deletion mutant of TRAF1
expressing only the TRAF-C domain (TRAF1[252-409])
(Fig. 4 B). TRIP did not interact with a COOH-terminal
deletion mutant of TRAF1 lacking the TRAF-C domain
(TRAF1[1-251]), suggesting that the interaction of TRIP with TRAF1 requires the entire TRAF domain (Fig. 4 B).
Mutational analysis of TRAF2 also showed that TRIP interacts with TRAF2 through the TRAF domain (Fig. 4 B).
TRIP did not directly interact with the cytoplasmic
domains of TNFR2 or CD30 in the yeast two-hybrid assay
(data not shown). However, since the interaction of TRAFs
with the cognate members of the TNFR superfamily is
mediated through the TRAF-C domain rather than the
entire TRAF domain (4, 5, 10, 11, 15, 24, 26, 27), it was
important to determine whether TRIP can indirectly interact with the receptors through TRAFs. To test this,
HA-epitope-tagged TRIP and GST fusion proteins with
the cytoplasmic domains of TNFR2 (GST-TNFR2) or
CD30 (GST-CD30) (30) were coexpressed in 293 cells in
the presence or absence of TRAFs. Cell lysates were precipitated with glutathione-Sepharose beads, and analyzed
on Western blots with anti-HA, anti-TRAF1, and antiTRAF2 antibodies. Consistent with the yeast two-hybrid
assay, TRIP was not coprecipitated by the GST-TNFR2
or GST-CD30 (Fig. 5). When TRAF2 was coexpressed,
however, TRIP was readily coprecipitated by the GSTTNFR2 (Fig. 5). GST-TNFR2, which does not strongly
interact with TRAF1 oligomer (4), did not readily coprecipitate the TRAF1-TRIP complex (Fig. 5). Coexpression
of both TRAF2 and TRAF1 did not increase the amount
of TRIP coprecipitated with GST-TNFR2 (Fig. 5). Similar to GST-TNFR2, GST-CD30 also coprecipitated TRIP efficiently in the presence of TRAF2 (Fig. 5). Although
TRAF1 homooligomer can interact with CD30 or TRIP
efficiently in 293 cells, only low level of TRIP was coprecipitated by GST-CD30 in the presence of TRAF1 alone
(Fig. 5). Taken together, these results suggest that TRIP
can be recruited to the TNFR2 or CD30 through the
TRAF2 homooligomer. However, whether TRIP is also
recruited to the receptor via TRAF2-TRAF1 heterooligomer cannot be excluded.
The ability of TRIP to bind the receptor-TRAF
signaling complexes raised the possibility that TRIP may
regulate the receptor-mediated signal transduction. In particular, the association of TRIP with TRAF2 suggests that
TRIP expression may regulate TRAF2-mediated effector
function such as NF-
Since TRIP associates with the receptor complex, we
also tested the effect of TRIP on NF- Since TRAF2 also mediates NF- Stimulation of members of the TNFR superfamily activates signaling cascades leading to the regulation of cell activation/growth or death (1). The recent identification of
distinct families of receptor-associated signal transducers have
provided insight into how members of the TNFR superfamily may induce pleiotropic effects on cells (4). Many
of these signal transducers contain either TRAF or death
domains, which mediate protein-protein interactions. The
TRAF family proteins interact directly with some members
of the TNFR family (TNFR2, CD40, LT- In this study we have carried out experiments to study
how TRAF proteins contribute to the signal transduction
pathway triggered by the TNFR superfamily because
TRAFs do not contain any domains of known signaling
function despite their importance as signal transducers. It
has been previously suggested that TRAFs might work as
adapters to recruit different types of effector proteins to the
receptor complex, which will induce specific signals (28, 29). For example, TRAF2 may interact with downstream
signal transducers which phosphorylate IkB to activate
NF- We now report the identification and characterization of
a novel signal transducer of the TNFR superfamily. TRIP
interacts with the receptor-TRAF signaling complex and
inhibits the TRAF2-mediated NF- TRIP is recruited to the receptors TNFR2 or CD30 via
its interaction with TRAF proteins. The recruitment of
TRIP to these receptors was efficient in the presence of
TRAF2 oligomer. Although only TNFR2 and CD30 have
been tested in this study, TRIP may affect the signaling
pathway mediated by many other members of the TNFR superfamily because TRAF2 is expressed ubiquitously and
interacts with most of the TRAF-binding members of the
TNFR superfamily. This suggestion is supported by the
fact that TRIP also inhibits the induction of NF- This specificity of TRIP makes it unique among other
signal transducers (I-TRAF and A20) which inhibit TRAF2mediated NF- Apart from TRIP, c-IAPs are the only other proteins
which have been shown to be recruited to the receptor-
TRAF complex (7). In contrast to TRIP, however, c-IAPs
do not exert a negative effect on the activation of NF- The studies in this paper have identified TRIP as a novel
signaling component of the TNFR superfamily and also
shown that TRIP works as a receptor-proximal negative
regulator of NF-
In this model, the balance of proactivation/growth or
procell death signals mediated by the receptor-TRAF complex may be controlled by the particular set of signal transducers (e.g., c-IAPs or TRIP) which are recruited to the
receptor complex. When c-IAPs are recruited to the receptor complex, TRAF2-mediated NF- The choice of which type of signal transducers (c-IAPs
or TRIP) is to be recruited to the cognate receptors may be
determined by their availability and also by the presence of
different TRAF proteins (e.g., TRAF1). This idea is consistent with several observations. First, the expression of
TRAF1 is tissue-specific, whereas that of TRAF2 is not (4).
Second, when lymphocytes are stimulated to proliferate via
their antigen receptors, the expression of c-IAP1 or TRAF1
is upregulated, whereas TRIP expression is decreased (Fig.
3 B). In contrast, TRAF2 expression is not significantly affected during lymphocyte proliferation (27). During antigen-stimulation of lymphocytes, therefore, the formation
of TRAF2-TRAF1-c-IAP complex will be favored and
recruited to the cognate TNFR family members, which
may exert costimulatory signals for lymphocyte proliferation (1). Third, TRAF1 overexpression which may antagonize the formation of TRAF2 homooligomer in cells, impairs the activation-induced cell death of mature CD8+ T
cells (data not shown) which is partly mediated by the
TNFR2 signaling complex (32). Lastly, TRIP expression is
most abundant in thymocytes, most of which are destined
to die during clonal deletion, which is in part mediated by
CD30 (36). In addition to TRAF1, the repertoire of other
TRAF proteins present in a particular cell and the repertoire of downstream signal transducing molecules expressed
in a given cell type may control the switch between cell activation/growth and cell death triggered by the receptor- TRAF signaling complex. Future studies will be directed
towards identifying downstream signal transducers which
are responsible for cell activation/growth (e.g., a protein
which directly activates NF-B or JNK (9, 20, 22). TRAF proteins were identified
by their biochemical ability to interact with TNFR2,
CD40, CD30, or LT-
R (4, 5, 10, 11, 15, 23). These
receptors interact directly with TRAFs via a short stretch of
amino acids within their cytoplasmic tails, but do not interact with the death domain containing proteins (4, 5, 15,
24). To date, five members of the TRAF family have
been identified as signaling components of the TNFR family members. All TRAF members contain a conserved
TRAF domain, ~230 amino acids in length, that is used
for either homo- or heterooligomerization among the
TRAF family, for interactions with the cytoplasmic regions of the TNFR superfamily, or for interactions with downstream signal transducers (4, 5, 8, 10, 11, 19, 23, 28). In
addition to the TRAF domain, most of the TRAF family
members contain an NH2-terminal RING finger and several zinc finger structures, which appear to be important for
their effector functions (4, 5, 10, 11, 23).
B activation induced by
two TNF receptors, CD40 and CD30 (9, 28). TRAF5
was also implicated in NF-
B activation mediated by LT
R (10), whereas TRAF3 (also known as CRAF1, CD40bp, or LAP1; references 5, 11, 24, and 25) was shown to be involved in the regulation of CD40-mediated CD23 upregulation in B cells (5). The role of other TRAF members in
the TNFR family-mediated signal transduction is not clear.
They may possess some effector functions as yet to be revealed, or work as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members
of the cellular inhibitor of apoptosis protein (c-IAP) family
to the TNFR2-signaling complex (7). In addition to the signal transduction by the TNFR family members, TRAFs
may regulate other receptor-mediated signaling pathways.
For example, TRAF6 is a component of IL-1 receptor (IL1R)-signaling complex, in which it mediates the activation
of NF-
B by IL-1R (31). Since TRAFs form homo- or
heterooligomers, it is suggested that the repertoire of TRAF
members in a given cell type may differentially affect the
intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs
with a specific set of downstream signal transducers. Although many aspects of TRAF-mediated effector functions
leading to cellular activation have been defined, it needs to
be determined whether TRAF proteins will also mediate
the apoptotic signals induced by the "death-domain-less" members of the TNFR superfamily (1, 27, 32).
B activation. Biochemical studies indicate that TRIP associates with the
TNFR2 or CD30 receptor complex via its interaction with
TRAF proteins, suggesting a model which can explain why
the ligation of these receptors can promote different cell
fates: proliferation or death.
-galactosidase activity of yeast transformants containing both activators
and baits upon galactose induction as previously described (30).
In brief, yeast cells were permeabilized with 0.0025% SDS and
5% chloroform, and the cell debris was removed by centrifugation. The
-galactosidase assay was performed at 25°C and OD420
was measured.
ZAP (Strategene Corp., La Jolla, CA) as previously described (37). A human thymocyte cDNA library in
gt10
(Clontech, Palo Alto, CA) was similarly screened using fulllength mouse TRIP (mTRIP) cDNA. For sequence analysis of
mTRIP and human TRIP (hTRIP), several cDNA clones were
sequenced using the Sequenase Kit (United States Biochemical
Corp., Cleveland, OH). Northern analysis of mouse tissue RNA
was performed as described (37).
-AACGAATTCATGGCCTCCAGCTCAGCCCCT-3
; TRAF1 (antisense), 5
-CTTGGATCCCTACTGAGCCAGCAGCTTCTCCTT-3
; c-IAP1 (sense),
5
-GGCGAATTCATGGACAAAACTGTCTCCCAG-3
; c-IAP1
(antisense), 5
-TAGCTGCAGGGATCCATCCTTGAT-3
; TRIP
(sense), 5
-AGTGAATTCATCATGCCTATCCTCTCTCTGTG-3
; TRIP (antisense), 5
-CTGGGATCCTCACATGTCTCGAATCATCTCCTC-3
; actin (sense), 5
-ATGAA GATCCTGACCGAGCG-3
; actin (antisense), 5
-TACTTGCGCTGAGGAGGAGC-3
.
Apr-1-neo. An epitope tagged TRIP was made by PCR with the 5
primer
(5
-AGTGAATTCATCATGCCTATCCTCTCTCTG-3
) and
the 3
primer (5
-GGAGTTAACTGACATAAGAAGGTATCCAGC-3
), which was subsequently cloned into 5
-EcoRIHpaI-3
sites in the pBlue-HA vector carrying nucleotides coding
for the sequence LTGGGSGFYPYDVPDYA* as described previously (37). The HA epitope is underlined and * indicates the stop
codon. Epitope-tagged mTRIP cDNA was cloned into pcDNA3.1
(Invitrogen, San Diego, CA). Deletion mutants of TRIP was similarly generated by PCR as described (27, 30). The reporter constructs, p(
B)3-IFN-LUC and pCMV-
-galactosidase (
gal) (provided by K. Saksela [Rockefeller University, NY] and E. Spanopoulou [Mount Sinai School of Medicine, NY]), were previously described (28, 30). To generate eukaryotic expression
vectors for GST-wild-type TRIP or mutant TRIPs, various
TRIP cDNAs were generated by PCR and in-frame cloned into
5
-BamHI-NotI-3
sites in pEBG vector as described (27).
-galactosidase activity as described previously (30).
Isolation of TRIP as a TRAF-interacting Protein.
Fig. 1.
Interaction of TRIP with TRAF1 and TRAF2. An expression vector encoding TRAF1 or TRAF2 was co-transfected into 293 cells with an expression vector encoding either GST alone or GST-TRIP
fusion proteins. After 36 h, cell lysates were prepared and subjected to purification with glutathione-Sepharose beads. Proteins coprecipitated with
GST fusion proteins were analyzed by Western blot analysis with antiTRAF1 or anti-TRAF2 polyclonal antibodies. (Pre) Cell lysates before
precipitation with glutathione beads were analyzed by Western blot analysis to show that similar amounts of TRAF1 or TRAF2 are present in
each sample. (GST beads) Proteins coprecipitated with GST-fusion proteins were analyzed. TRAF1 or TRAF2 are indicated with arrows.
[View Larger Version of this Image (24K GIF file)]
Fig. 2.
Predicted amino
acid sequences of mTRIP and
hTRIP. (A) The full length
mouse sequence is shown and
numbered. The human sequence has one less amino acid than that
of the mouse (indicated with a
dot at position 302). Dashes indicate positions in the human sequence which are identical to
those in the mouse. Cysteine and histidine residues defining the
RING finger motif are marked
by boxes. Brackets indicate the
potential coiled-coil region of
TRIP. Within the brackets,
amino acids that form the putative coiled-coil structures are
marked by overlying dots, and
those that form leucine-zipper structures are indicated in bold.
The accession numbers for the mouse and human TRIP sequences reported in this paper are U77844 and U77845, respectively. (B)
Comparison of amino acid sequences from various proteins that contain
RING finger motifs. The RING finger domains of mTRIP and hTRIP
are aligned with those of TRAFs, c-IAP1, human protooncogene c-cbl,
human RING1, human ribonucleoprotein SS-A/Ro, chicken C-RZF,
and Drosophila neuralized gene (38). Residues corresponding to the consensus sequence are indicated in bold. (C) Helical wheel representation of
residues 225 to 260 of TRIP. The wheel starts with the inner residue
Leu225 at position d and finishes with the outer residue Ala260 at position d.
[View Larger Versions of these Images (46 + 33 + 16K GIF file)]
Fig. 3.
TRIP mRNA expression in mouse tissues and its regulation
during cell activation. (A) Northern blot analysis of TRIP mRNA in mouse tissues. Total RNA isolated from various tissues was hybridized with
a TRIP-specific probe. The TRIP probe hybridized to an ~2.1-kb mRNA, indicated by the arrow. Positions of 18S and 28S ribosomal RNA are indicated. The amount of total RNA loaded in each lane was
similar based on the intensity of EtBr-stained rRNAs (data not shown).
(B) Expression of TRAF, c-IAP1, and TRIP during lymphocyte stimulation. cDNAs were prepared from lymph node cells stimulated with antiTCR Ab plus anti-CD28 Abs for 0 (cont.) and 48 (activ.) h. The cDNAs
were then subjected to semiquantitative PCR using primers specific for
mTRIP, mTRAF1, and m-c-IAP1 as described in Materials and Methods.
[View Larger Versions of these Images (34 + 47K GIF file)]
Fig. 4.
Mapping of TRIP-TRAF interaction domains. (A) Interaction of TRAF1 or TRAF2 with the NH2- and COOH-terminal domains of
TRIP. Expression vectors encoding wild-type TRIP or the indicated deletion mutants of TRIP fused to the transcription activation domain were cotransformed into yeast with plasmids expressing LexA DNA binding domain-TRAF1 or -TRAF2 fusion proteins. Interactions between fusion proteins
were scored by measuring -gal activity of yeast transformants. (+) Average
-gal activity of three independent yeast transformants was higher than 1,000 Miller units; (
) average
-gal activity of three independent yeast transformants was about 100 Miller units, which was similar to that of negative controls
(bait plasmid alone). (B) Interaction of TRIP with TRAFs. Expression vectors encoding the NH2-terminal deletion mutants of TRAF fused to the LexA
DNA binding domain were co-transformed into yeast with plasmids expressing TRIP fused to the transcription activation domain. Interactions between
fusion proteins were scored by measuring
-gal activity of yeast transformants as described in Fig. 4 A. For the analysis of the COOH-terminal deletion
mutants of TRAFs, a transient transfection-based coprecipitation experiment was used. The indicated COOH-terminal deletion mutants of TRAFs were coexpressed with GST-TRIP fusion proteins in 293 cells. Cell lysates were subjected for purification with glutathione-Sepharose beads, followed by
Western blot analysis with anti-TRAF1 or anti-TRAF2 polyclonal antibodies as described in Fig. 1.
[View Larger Versions of these Images (13 + 23K GIF file)]
Fig. 5.
TRAF2-mediated interaction of TRIP with CD30 or
TNFR2. 293 cells were transiently transfected with the indicated combinations of equal amounts of HA-TRIP, TRAF1, TRAF2, GST-CD30,
or GST-TNFR2 expression vectors for 36 h. Aliquots of cell lysates were
subjected for purification with glutathione-Sepharose beads as described in Materials and Methods. Proteins coprecipitated with GST fusion proteins were analyzed by Western analysis with an anti-HA mAb (12CA5),
and anti-TRAF1 or anti-TRAF2 polyclonal antibodies. In control experiments, GST proteins did not coprecipitate any of the proteins tested (data
not shown). Pre, cell lysates before purification with glutathione-Sepharose
beads were analyzed by Western analysis with anti-TRIP polyclonal antibodies to show that equal amounts of TRIP were expressed in each case.
The positions of molecular mass markers are shown on the left. Arrows
indicating the positions of TRAF1, TRAF2, or TRIP are also shown on
the left.
[View Larger Version of this Image (64K GIF file)]
B Activation.
B activation (9, 28). Therefore, we tested the effect of TRIP expression on TRAF2-mediated NF-
B-dependent reporter gene activation using a
transient transfection assay in 293 cells. When overexpressed, TRIP significantly inhibited TRAF2-mediated
NF-
B activation (Fig. 6 A). This inhibition was similar to
that exerted by overexpression of a dominant negative form of TRAF2 (TRAF2[241-501]; Fig. 6 A; references
28). The inhibition of NF-
B activation by TRIP required the same domains of TRIP which mediate the interaction. An NH2-terminal deletion mutant of TRIP which
lacks the TRIP-TRAF interaction domain (TRIP[275- 470]) failed to inhibit TRAF2-mediated NF-
B activation
(Fig. 6 A). Moreover, a COOH-terminal deletion mutant
of TRIP containing the NH2-terminal RING finger motif
and the putative coiled-coil domain (TRIP[1-185]) was
sufficient to inhibit TRAF2-mediated NF-
B activation
(Fig. 6 A). However, further deletion analysis showed that
the RING finger motif of TRIP was not required for inhibition of TRAF2-mediated NF-
B activation because a
mutant TRIP containing only the putative coiled-coil domain (TRIP[56-275]) was sufficient to inhibit TRAF2mediated NF-
B activation (Fig. 6 A). Overexpression of a
mutant TRIP expressing only the NH2-terminal RING
finger motif failed to inhibit NF-
B activation (data not shown). These results suggested that the coiled-coil domain
of TRIP (amino acids 56-275) is required for TRIP-
TRAF interaction and also for inhibition of TRAF2-mediated NF-
B activation.
Fig. 6.
Inhibition of TRAF2-mediated
NF-B activation by TRIP overexpression. (A, left) A dose-dependent effect of
TRIP expression on TRAF2-mediated NF
B activation. 293 cells were transfected with
0.5 µg of TRAF2 expression vector together with 0.5 µg of p(
B)3-IFN-LUC
(28) in the presence of the indicated amount of TRIP expression vectors. Controls were
transfected with 0.5 µg of pcDNA3.1 control vector and 0.5 µg of p(
B)3-IFN-LUC.
All the transfections included 0.25 µg of
pCMV-
-gal plasmids. 48 h after transfection, cell lysates were prepared and used for luciferase assay. All values represent luciferase activities normalized to
-gal activities and are shown as means with their
respective SEMs for representative experiments performed in duplicate. (A, right) The
putative coiled-coil domain of TRIP is required to inhibit TRAF2-mediated NF-
B
activation. 293 cells were transfected with 0.5 µg of TRAF2 expression vector together
with 0.5 µg of p(
B)3-IFN-LUC in the
presence of 5 µg of plasmids expressing a dominant negative form of TRAF2
(TRAF2[241-501]), or expressing the indicated TRIP mutants. For the control experiment, cells were transfected with 0.5 µg of
pcDNA3.1 control vector and 0.5 µg of
p(
B)3-IFN-LUC. All the transfections included 0.25 µg of pCMV-
-gal plasmids.
48 h after transfection, cell lysates were prepared and used luciferase assay. All values
represent luciferase activities normalized to
-gal activities and are shown as means with
their respective SEMs for representative experiments performed in duplicate. Luciferase activity of the control experiments is shown A, left. (B) Dose-dependent inhibition of TNFR2- or CD30-mediated NF-
B activation by TRIP.
293 cells were transfected with 1 µg of plasmids expressing the chimeric receptors, CD8-TNFR2 or CD8-CD30 (30), together with 0.5 µg of p(
B)3IFN-LUC in the presence of the indicated amount of TRIP expression vectors. For the control experiment, cells were transfected with 0.5 µg of
pcDNA3.1 control vector and 0.5 µg of p(
B)3-IFN-LUC. All the transfections included 0.25 µg of pCMV-
-gal plasmids. All values represent luciferase activities normalized to
-gal activities and are shown as means with their respective SEMs for representative experiments performed in duplicate.
(C) TRIP overexpression inhibits TNF-induced NF-
B activation. 293 cells were transfected with 0.5 µg of p(
B)3-IFN-LUC in the presence or absence of 5 µg of plasmids expressing a dominant negative form of TRAF2 (TRAF2[241-501]), or TRIP. For the control experiment, cells were transfected with 0.5 µg of pcDNA3.1 control vector and 0.5 µg of p(
B)3-IFN-LUC. All the transfections included 0.25 µg of pCMV-
-gal plasmids. 36 h
after transfection, cells were treated for 6 h with 100 pg/ml of either TNF or IL-1. All values represent luciferase activities normalized to
-gal activities and are shown as means with their respective SEMs for representative experiments performed in duplicate. (D) TRIP overexpression inhibits TRADDmediated NF-
B activation. 293 cells were transfected with 0.5 µg of plasmids expressing TRADD together with 0.5 µg of p(
B)3-IFN-LUC in the
presence of the indicated amounts of TRIP expression vectors. For the control experiment, cells were transfected with 0.5 µg of pcDNA3.1 control vector and 0.5 µg of p(
B)3-IFN-LUC. All the transfections included 0.25 µg of pCMV-
-gal plasmids. All values represent luciferase activities normalized
to
-gal activities and are shown as means with their respective SEMs for representative experiments performed in duplicate.
[View Larger Version of this Image (35K GIF file)]
B activation induced via TNFR2 or CD30. As previously shown, overexpression of chimeric receptors with the extracellular domain of CD8 fused to the cytoplasmic domain of TNFR2
(CD8-TNFR2) or CD30 (CD8-CD30) induced NF-
B
activation in 293 cells without further cross-linking (Fig. 6
B; reference 30). This is similar to the activation of NF-
B
induced by overexpression of wild-type TNFR2, CD40,
or other chimeric receptors in 293 cells, which will trigger
the clustering of signal transducers without additional crosslinking by cognate ligands or antibodies (19, 28, 29, 34,
41). When TRIP was coexpressed, the receptor-mediated NF-
B activation was significantly inhibited (Fig. 6 B). Because NF-
B activation by TNFR2 and CD30 is mediated
by TRAF2 (29, 30), the results are consistent with the fact
that TRIP works as a proximal negative regulator of TRAF2mediated NF-
B activation by members of the TNFR superfamily.
B Activation Induced by TNF, but Not
by IL-1.
B activation
triggered by the TNFR1-TRADD complex (9), we tested
the effect of TRIP overexpression on TNF-induced NF-
B
activation in 293 cells, which is mediated by TNFR1 (29).
Overexpression of TRIP in 293 cells inhibited TNF-
induced NF-
B activation (Fig. 6 C). Consistent with this, TRIP overexpression also inhibited NF-
B activation mediated by TRADD overexpression in 293 cells (Fig. 6 D).
While TRAF2 is required for TNF- or TRADD-induced
NF-
B activation, it is not required for NF-
B activation
induced by IL-1 in 293 cells (9, 29). To test whether TRIP
affects TRAF2-mediated NF-
B activation specifically, the
effect of TRIP overexpression on NF-
B activation by IL-1 was also tested. In contrast to TNF-induced NF-
B activation, IL-1-induced NF-
B activation was not inhibited by
TRIP overexpression (Fig. 6 C). Recent experiments have
shown that IL-1-induced NF-
B activation is mediated by
another member of the TRAF family, TRAF6 (31). These
results suggest that TRIP is a specific inhibitor of TRAF2mediated NF-
B activation, rather than a general inhibitor of NF-
B activation.
R, or CD30)
and play pivotal roles in the activation of signaling pathways induced by these receptors (4, 5, 10, 11, 15, 23).
For example, the activation of NF-
B triggered by these
receptors is mediated by TRAF2 or TRAF5 (9, 10, 28).
Proteins containing death domains like FADD/MORT1,
RIP, or TRADD interact with Fas(CD95) or TNFR1 (6,
12, 17, 20). Once associated with the receptors, these proteins recruit downstream signal molecules that act to initiate cascades leading to cell death or activation (2, 3, 6, 9,
12, 17, 20, 21). For example, TRADD recruits FADD/
MORT1 or TRAF2 to the TNFR1 complex to initiate
the signal transduction required for cell death or NF-
B
activation, respectively (9).
B. Other classes of signal transducers are also likely to
be recruited to the receptor complex to regulate various biological effects exerted by the TNFR superfamily. For example, members of the c-IAP family (c-IAP1 and c-IAP2)
are recruited to the TNFR2 signaling complex by their interaction with TRAFs (7). The role of c-IAPs in the signal transduction pathway of the TNFR superfamily is not
clear, but some members of the c-IAP family are involved
in the protection of cells from apoptosis (42). Since
members of the TNFR superfamily can interact with different sets of TRAF proteins, a diverse collection of downstream signal transducing molecules are likely to be recruited to the receptor complex.
B activation. The structural features of TRIP include an NH2-terminal RING
finger motif followed by a long putative coiled-coil structure. The putative coiled-coil domain of TRIP is divided
into two subdomains. Amino acid sequences of the NH2terminal half of the coiled-coil domain of TRIP shows
~50% similarity to the rod-like coiled-coil structure of myosin heavy chain (39), while those of the COOH-terminal half of the coiled-coil domain of TRIP are characteristic of
a leucine zipper (40). The putative coiled-coil domain of
TRIP was shown to be required not only for TRIP-TRAF
interactions, but also for the inhibition of TRAF2-mediated NF-
B activation by TRIP. Although the RING finger domain of TRIP has not been implicated in the regulation of NF-
B activation in this study, it may play some
other regulatory role based on analogy to other RING-finger proteins (38). The COOH-terminal half of TRIP distal to the coiled-coil domain does not show any significant
homology to other proteins but contains several potential
phosphorylation sites, suggesting that TRIP may be regulated by kinases.
B activation mediated by TNFR1, which indirectly interacts with
TRAF2 via TRADD (9). When the role of TRIP was examined by a transient transfection assay in 293 cells, TRIP inhibited NF-
B activation induced by TNFR2, CD30,
and TNFR1, and also by TRADD, all of which activates
NF-
B via TRAF2 (9, 29, 30). However, TRIP did not
inhibit the activation of NF-
B by IL-1R which is mediated by TRAF6 (31), suggesting that a negative effect of
TRIP on NF-
B activation was specific to a TRAF2-
mediated pathway.
B activation (8, 19, 45). In contrast to
TRIP, both I-TRAF and A20 inhibit the activation of NF
B induced by IL-1R as well as by TNFRs (8, 19). In addition to its specificity, TRIP differs from I-TRAF or A20
in several additional aspects. First, TRIP is recruited to the
cognate receptor-TRAF signaling complex, but I-TRAF is
not (8). Whereas TRIP can be recruited to the cognate receptors via its interaction with TRAF2 homooligomer, A20 interacts only with TRAF2-TRAF1 heterooligomer
(19). Second, the inhibitory mechanism acting on NF-
B
activation by I-TRAF, A20, and TRIP appears to be different. I-TRAF inhibits TRAF2-mediated NF-
B activation by blocking the recruitment of TRAF2 to the receptor complex, which would normally initiate the clustering
of TRAF proteins (8). In contrast, TRIP is recruited to the
receptor complex by its association with TRAF2. Although A20 interacts with TRAFs, its inhibitory effect on
TRAF2-mediated NF-
B activation does not require direct protein-protein interaction in a transfection assay in
293 cells (19). TRIP, however, inhibits TRAF2-mediated NF-
B activation only when its coiled-coil domain required for TRIP-TRAF interaction is intact. TRAF3 also
inhibits TRAF2-mediated NF-
B activation when overexpressed in 293 cells. However, TRAF3 does not interact
with TRAF1 or TRAF2 in the yeast two-hybrid assay (29),
suggesting that the inhibitory mechanism regulating TRAF2
effector function by TRAF3 is different from that by
TRIP. Therefore, a unique property of TRIP that distinguishes it from other inhibitors of TRAF2 function is that
TRIP is a component of the receptor-TRAF complex and
inhibits proximal events necessary for TRAF2-mediated
NF-
B activation. However, the mechanism of TRIP's inhibitory effect is difficult to predict. Future identification of
signal transducers required for TRAF2-mediated NF-
B
activation will be required to understand how TRIP might
negatively regulate the function of TRAF2.
B
induced by these receptors (7), rather, they have been implicated in the inhibition of cell death (42). In addition
to their functional differences, TRIP and c-IAPs are recruited differently to their cognate receptors. c-IAPs are recruited to TNFR2 only through the TRAF2-TRAF1
heterooligomer (7), but TRIP can be recruited to the cognate receptors (TNFR2 or CD30) in the presence of the
TRAF2 homooligomer. Therefore, the level of TRAF1
expression which is controlled differently among various
tissues (4) may influence whether the cognate receptor will
recruit c-IAPs or TRIP.
B activation. In addition, the studies of
the regulation of TRIP suggested a model of how the signals mediated by the TNFR2- or CD30-TRAF signaling
complex can initiate such seemingly opposing effects on
cells, namely cell activation/growth or cell death (Fig. 7).
Fig. 7.
A model of interrelationship of TRAFs, c-IAP, and TRIP,
and the switch of the TRAF-mediated signals between cell activation and
cell death. The upper part of the diagram (shaded) describes how the receptor-TRAF signaling complex will inhibit cell death and promote cell
activation/growth (7, 8, 19, 28, 29), in which A20 can work as a negative
feedback regulator for TRAF2 (19). In this model, the members of the
TNFR family which do not contain the death domains (e.g., TNFR2 or
CD30) are postulated to trigger the induction of cell death by yet to be
identified mechanism which is indicated by question mark. The lower
part of the diagram explains how TRIP inhibits the TRAF-mediated cell activation/growth and contributes to the promotion of signals for cell
death. For simplicity, the model does not include the receptor-TRAF2-
TRAF1-TRIP complex, the presence of which cannot be excluded.
However, the signals from this complex may be similar to those from the
receptor-TRAF2-TRIP complex. All indicated protein association may
represent dimers or higher oligomers. Three types of proximal signal transducers (TRAFs, c-IAP, or TRIP) are described. TRAFs are kept inactive
in the cytoplasm due to their association with I-TRAF (also known as
TANK) (8). For simplicity, a costimulatory role of I-TRAF/TANK is
not included (28).
[View Larger Version of this Image (44K GIF file)]
B activation proceeds unaffected. The activation of NF-
B induces the expression of various genes and also suppresses cell death (22, 46-
48) which drives the cells towards the proactivation/growth
state. In addition, c-IAPs themselves may contribute to antiapoptotic signals (42). For example, c-IAP1 (also known
as MIHB and HIAP-1) was shown to inhibit the apoptosis
triggered by IL-1
converting enzyme overexpression or
serum deprivation (42, 44). Manganese superoxide dismutase or A20 induced by NF-
B activation will also contribute to the survival of cells during cell proliferation/growth
(45, 49). When TRIP is recruited to the receptor complex,
however, TRIP inhibits NF-
B activation which is required for antiapoptotic signals. In addition, the contribution of antiapoptotic proteins like c-IAPs, manganese superoxide dismutase, or A20 will be diminished. Therefore,
the signals by the receptor-TRAF-TRIP complex will drive cells toward the antiactivation/procell death state.
Consistent with this idea and also with recent findings solidifying the anti-apoptotic role of NF-
B during TNFmediated apoptosis (22, 46), TRIP overexpression enhanced TNF-mediated cell death in HeLa cells (data not
shown).
B) or those which are responsible for cell death in this signaling pathway.
Address correspondence to Dr. Yongwon Choi, HHMI, The Rockefeller University, 1230 York Ave., Box 295, New York, NY 10021.
Received for publication 7 January 1997 and in revised form 3 February 1997.
Y. Choi, is an assistant investigator of the Howard Hughes Medical Institute.We would like to thank Drs. Philippa Marrack, Dan Littman, Moses Chao, Eugenia Spanopoulou, Konstantina A. Alexandropoulos, Kalle Saksela, and Brian Wong for helpful discussions and also for critically reading the manuscript. We wish to thank F. Alt, R. Brent, E. Spanopoulou, and K. Saksela for supplying plasmids and various reagents used in this study. We like to give special thanks to Angela Santana for her excellent technical help.
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