(Received for publication, August 15, 1994; and in revised form, November 28, 1994)
From the
The yeast-based two hybrid has been used to identify a novel
protein that binds to the intracellular domain of the type 1 receptor
for tumor necrosis factor (TNFR-1IC). The TNF receptor-associated
protein, TRAP-1, shows strong homology to members of the 90-kDa family
of heat shock proteins. After in vitro transcription/translation and S labeling, TRAP-1 was
precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, showing that the two proteins
directly interact. The ability of deletion mutants of TNFR-1 to
interact with TRAP-1 was tested using the two hybrid system. This
showed that the amino acid sequences that mediate binding are diffusely
distributed outside of the domain in the C terminus of TNFR-1IC that
signals cytotoxicity. The 2.4-kilobase TRAP-1 mRNA was variably
expressed in skeletal muscle, liver, heart, brain, kidney, pancreas,
lung, and placenta. TRAP-1 mRNA was also detected in each of eight
different transformed cell lines. Identification of TRAP-1 may be an
important step toward defining how TNFR-1, which does not contain
protein tyrosine kinase activity, transmits its message to signal
transduction pathways.
Tumor necrosis factor (TNF) ()is produced
predominantly by macrophages activated by infections or
malignancies(1, 2, 3) . This potent
multifunctional cytokine was first characterized by its ability to
induce the hemorrhagic necrosis and regression of cancers in animals
and by the cytotoxic response that it can elicit from transformed cells in vitro. Subsequent studies have shown that through
interactions with virtually every type of cell, TNF also promotes
immunity, antiviral responses, inflammation, shock, and metabolic
alterations, including cachexia, which accompany disease
states(1, 2, 3) . Such diverse and profoundly
important functions have made TNF the subject of intense investigation.
The first step in TNF action is binding to specific receptors that are expressed on essentially all cells(4, 5, 6, 7) . Two TNF receptors have been identified as proteins of 55-kDa (the type 1 receptor, TNFR-1) and 75-kDa (the type 2 receptor, TNFR-2)(8, 9, 10, 11) , and their cDNAs have been cloned(12, 13, 14) . The extracellular domains of the TNF receptors share homologies with one another and with a group of cell surface receptors that include the FAS antigen, the low affinity NGF receptor, the murine cDNA clone 4-1BB from induced helper and cytolytic T cells, the B cell surface antigen CD40, the OX40 antigen of activated CD4-positive rat lymphocytes, and the T2 antigen of the Shope fibroma virus(15) . The intracellular domains do not display sequence similarities and couple to different signal transduction pathways. For this reason, the receptors induce distinct responses: TNFR-1 promotes cytotoxicity, fibroblast proliferation, antiviral responses, and the host defense against microorganisms and pathogens(10, 16, 17, 18, 19) ; TNFR-2 plays a role in cytotoxicity that is still being defined(20, 21, 22) , inhibits early hematopoiesis(23) , is involved in the proliferation of monocytes(24) , and promotes the proliferation of T cells(25) .
Neither TNF receptor contains intrinsic protein tyrosine kinase activity or any recognizable motif(12, 13, 14) , which suggests a mechanism through which the signal inherent in the hormone-receptor complex can be transmitted to signaling mechanisms. This may suggest that associated proteins, rather than the intracellular domain of either TNF receptor, act as essential elements in signal transmission. Such accessory molecules may be cytoplasmic tyrosine kinases, exemplified by JAK2, which interacts with the erythropoietin and other receptors(26) , or non-tyrosine kinases, such as gp130, which associates with the interleukin-6 and other receptors(27) . Consistent with the hypothesis that TRAPS may be important to TNF action are recent reports describing co-precipitation of TNFR-1 with a protein kinase activity(28, 29) . The present studies were initiated to identify proteins that associate with the intracellular domain of TNFR-1 (TNFR-1IC) and might then initiate signal transduction.
To address this problem, we used the yeast-based two hybrid system, a method for studying protein-protein interactions(30, 31) . This method is based on the properties of the yeast GAL4 protein, which consists of separable domains that mediate DNA binding and transcriptional activation. Plasmids encoding two hybrid proteins, one consisting of the GAL4 binding domain (GAL4-BD) fused to protein X and the other consisting of the GAL4 activation domain (GAL4-AD) fused to protein Y, are cotransformed into yeast. Interaction between these proteins permits transcriptional activation of an integrated copy of the GAL4-lacZ reporter gene.
To identify proteins that interact with TNFR-1IC, a plasmid in which TNFR-1IC was fused with GAL4-BD (pGBT-TNFR1IC) was cotransformed into yeast together with a pool of plasmids encoding GAL4-AD/HeLa S3 cDNA library fusion proteins. This has led to identification of a gene encoding a novel protein that binds TNFR-1IC. The protein was precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, demonstrating that it interacts directly with the TNF receptor. The protein shows significant homology to members of the 90-kDa family of heat shock proteins (hsp90) and is widely expressed in normal tissues and transformed cells. We also used the two hybrid system to assay the ability of deletion mutations of TNFR-1IC to interact with the newly discovered protein. These experiments show that the amino acid sequences necessary for interaction are diffusely distributed outside of the cytotoxicity domain in TNFR-1IC. Identification of this TNF receptor-associated protein, which we call TRAP-1, is an important step toward defining how TNFR-1 couples to signal transduction pathways and transduces TNF binding into responses.
Six primers were designed to amplify various regions within TNFR-1IC to make a series of deletion mutants. For oriented cloning, the 5` primers were linked to EcoRI, and the 3` primers were linked to BamHI. All constructs were sequenced at the fusion sites to confirm in frame fusion of TNFR-1IC and TNFR-1IC deletion mutants with GAL4-BD.
Using the N terminal 0.6-kb PCR product of the TRAP-1 gene from I-45 as a probe, a HeLa S3 cDNA library in lambda ZAPII (Stratagene) was screened using standard techniques. The longest 2.2-kb insert was rescued as a phagemid (pBluescript-TRAP1) by coinfection with the helper phage.
To identify proteins that interact with TNFR-1IC, plasmids
encoding TNFR-1IC fused with the GAL4-BD (pGBT-TNFR1IC) and HeLa S3
Matchmaker cDNA were cotransformed into HF7c. Double transformant
colonies were screened and selected for histidine prototrophy (Fig. 1a). Colonies isolated as His transformants were assayed for lacZ expression to
eliminate false positives, an effective procedure as the His3 and lacZ reporter genes in HF7c are under control of
dissimilar promoters. Activation domain library plasmids encoding
potential TRAPs were isolated from the double positive colonies, and
the GAL4-AD/TRAP gene fusion site was sequenced. We found two groups of
cDNAs that encode distinct proteins: TNF receptor-associated protein 1
(TRAP-1) and a second receptor associated protein (TRAP-2), which will
be described elsewhere. Two TRAP-1 clones, I-45 and I-57, were
sequenced at the fusion site; both were in frame with GAL4-AD, and I-57
was shorter than I-45 by 9 bases (Fig. 1b). Sequence
analysis of the cDNA insert of I-45 showed that the partial TRAP-1 gene
spans 1996 bases with an open reading frame ending at base 1827. From
the fusion site, this clone encodes a protein of 608 amino acids.
Figure 1:
a, two hybrid library
screen. pGBT9-TNFR1IC was used to screen a HeLa S3 cDNA library. Among
the interacting clones, I-45 and I-57 encode partial sequences of
TRAP-1 gene. b, schematic of two clones. Clone I-45 contains
the partial TRAP-1 gene that spans 1996 bases up to the beginning of
the poly(A) tail, which is in frame with GAL4-AD. Clone I-57 is shorter
by 9 bases, initiating at base 10. c, specificity of
interactions. Yeast strains transformed with combinations of plasmids
fused to the GAL4-BD and with clone I-45 were assayed for activation of
-galactosidase. The p53 gene (pVA3) and the SV40 large T antigen
(pTD1) associate strongly and serve as a positive control. pGBT-FAS is
a GAL4-BD/FAS-IC fusion; pGBT-TNFR2IC is a GAL4-BD/TNFR-2IC
fusion.
Experiments were conducted to establish that interaction of TNFR-1IC
with TRAP-1 is specific (Fig. 1c). I-45 by itself was
incapable of activating -galactosidase activity, which shows that
TRAP-1 does not contain a latent transcriptional activator. GAL4-BD
alone, lamin, TNFR-2IC, or FAS-IC, which contains a cytotoxicity domain
with homology to that found in TNFR-1IC, did not interact with TRAP-1.
Only when the TNFR-1IC gene was cotransformed with I-45 was activation
of the lacZ reporter gene detected. These observations rule
out the possibility that TRAP-1 nonspecifically interacts with other
proteins.
To identify the full-length size and distribution of expression of TRAP-1 RNA, we used a 0.6-kb N-terminal PCR product of I-45 as a probe to hybridize blots of multiple normal tissues and various transformed cell lines. As shown in Fig. 2, there was a single TRAP-1 transcript of about 2.4 kb. To obtain the full-length cDNA for TRAP-1, we used 5`-RACE PCR and also screened a lambda ZAPII cDNA library. The 5`-RACE PCR extended the known sequence of TRAP-1 by 180 bases. The combined sequence of I-45 with the 5`-RACE PCR product spans 2.2 kb (versus 2.4 kb, mRNA size) and is predicted to encode a protein of 661 amino acids (Fig. 3). A highly compressed GC-rich region of about 30 bases at the 5`-end of the PCR product may have prevented the reverse transcriptase from reading to the 5`-end of the message. Similarly, the longest phage clone isolated as a phagemid from the lambda ZAPII library search was 2.2 kb (pBluescript-TRAP1). However, it is most important that the sequence defined by the two hybrid screen and the 5`-RACE PCR encodes virtually the complete coding sequence of TRAP-1 and contains all of the elements necessary for binding to TNFR-1IC.
Figure 2:
Expression of TRAP-1 and human
hsp90-. Pre-blotted membranes containing mRNAs from human tissues (a, left) or cancer cell lines (b, right) were probed with a 0.6-kb random primed N-terminal PCR
product of the TRAP-1 gene in I-45 (top) and a specific probe
for human hsp90-
(bottom). The cancer cell lines from right to left were HL-60 promyelocytic leukemia
cells, HeLa S3 cells, k-562 chronic myelogenous leukemic cells, MOLT-4
lymphoblastic leukemia cells, Burkitt's lymphoma Raji, SW480
colorectal adenocarcinoma cells, A549 lung carcinoma cells, and G361
melanoma cells. Numbers to the left of the blots
indicate relative size in kb. The line to the right of the toppanel indicates the 2.4-kb TRAP-1
mRNA. The line to the right of the bottompanel indicates the 2.7-kb hsp90-
mRNA.
Figure 3: Sequence of TRAP-1 cDNA with predicted amino acid sequence. Clone I-45, which includes the poly(A) tail, and a cDNA segment obtained from 5`-RACE PCR were sequenced. The sequence spans 2156 bases without the poly(A) tail and encodes a protein of 661 amino acids.
To identify homologous proteins, the predicted amino acid sequence of TRAP-1 was used to search the National Center for Biotechnology Information (NCBI) data base using Blast command. TRAP-1 shares significant homology, 34% sequence identity and overall homology of about 60% when conserved substitutions are included, with members of the hsp90 family (33) (Fig. 4). Homologies between hsp90s and TRAP-1 are not restricted to a single sequence of amino acids but reside in at least six distinct domains. A striking difference between hsp90s and TRAP-1 is the absence of a highly charged domain in the latter, which is present in the former.
Figure 4:
Amino acid sequence alignment of rat
hsp90- with TRAP-1. Sequence alignment was obtained using Bestfit
command in Genetics Computer Group software . The uppersequence is TRAP-1, and the lowersequence is rat hsp90. Straightlines between amino acids
indicate identity, and dottedlines indicate
conserved substitutions. The highly charged segment in rat hsp90 is underlined, while obviously conserved domains in TRAP-1 and
hsp90 are boxed.
Northern blot analysis (Fig. 2, top) showed that TRAP-1 mRNA is variably present in eight different normal tissues and was in eight transformed cell lines. To rule out the possibility that the probe used to define the size and tissue distribution of TRAP-1 mRNA nonspecifically recognizes hsp90, the blots used for the experiments in Fig. 2were stripped and hybridized with a probe to hsp90 (Fig. 2, bottom). The hsp90 mRNA was distinguished from that of TRAP-1 by size (2.4 compared with 2.7 kb, respectively) and relative expression level in normal tissues and transformed cells.
An in vitro binding assay
was developed to demonstrate direct interaction between TRAP-1 and
TNFR-1IC and to establish that identification of TRAP-1 as a TNF
receptor-associated protein was not an artifact of the two hybrid
system. TRAP-1 expressed by in vitro translation and labeled
with [S]methionine was incubated with agarose
GST, and with GST fusion proteins containing full-length TNFR-1IC
(GST-TNFR1IC), the N-terminal half of TNFR-1IC
(GST-TNFR1NH
) and the C-terminal half of TNFR-1IC
(GST-TNFR1COOH) (Fig. 5). GST-TNFR1IC and GST-TNFR1NH
precipitated TRAP-1, whereas GST or GST-TNFR1COOH did not. These
observations show that TNFR-1IC binds TRAP-1 without the intermediacy
of third proteins and does so in a mammalian cell-free system as well
as in yeast. Thus, identification of TRAP-1 as a TNF
receptor-associated protein cannot be an artifact associated with the
use of the two hybrid system. These observations additionally show that
the binding site for TRAP-1 resides in the N-terminal half of TNFR-1IC.
Figure 5:
In vitro precipitation of TRAP-1. In vitro translated and
[S]methionine-labeled TRAP-1 was precipitated
using GST fusions of TNFR-1IC. Lane1, GST alone,
control; lane2, GST-TNFR1IC; lane3, GST-TNFR1NH
; lane4,
GST-TNFR1COOH. The arrow indicates TRAP-1 precipitated by
GST-TNFR1IC and GST-TNFR1ICOOH.
To further define the amino acid sequences important for interaction
of TNFR-1IC with TRAP-1, we constructed C- and N-terminal truncations
of TNFR-1IC. The deletion mutants were subcloned into the pGBT9 and
cotransformed into SFY526 together with I-45 to assay their
interactions using the two hybrid system. The relationship of these
mutants to TNFR-1IC and their ability to interact with TRAP-1 and
thereby activate -galactosidase is shown in Fig. 6a. The results obtained do not directly reflect
the affinity of the interaction between TNFR-1IC domains with TRAP-1
since expression of the GAL4-BD/TNFR-1IC fusion proteins in yeast,
assayed by Western blotting with an antibody directed against the
GAL4-BD (Fig. 6b), was somewhat variable. However, the
results can be used to determine whether any receptor domain contains
recognition elements essential to the interaction between TNFR-1IC and
TRAP-1, especially when interpreted together with observations made
using GST-TNFR1IC fusion proteins (Fig. 5). Peptides
encompassing amino acids 205-280 and 278-359 activated
-galactosidase, showing that the elements that promote interaction
of TNFR-1IC with TRAP-1 are diffusely distributed. The avidity with
which amino acids 205-359 bound TRAP-1 (123 Miller units of
-galactosidase activity) was more than predicted based on an
additive response to amino acids 205-280 and 278-359 (45
Miller units), suggesting that the conformation of the peptide and
perhaps the accessibility of crucial binding domains plays a role in
regulating interaction. The inability of residues 352-426 to
produce
-galactosidase activity shows that the binding sites
reside largely, if not entirely, outside of the cytotoxicity domain, a
result consistent with the ability of GST-TNFR1NH
but not
GST-TNFR1COOH to precipitate TRAP-1 (Fig. 5). A larger
C-terminal peptide (amino acids 278-426) was also unable to
activate
-galactosidase, despite the capacity of the peptide
encompassing residues 278-359 to do so. One explanation for this
result is that the cytotoxicity domain negatively regulates interaction
of TNFR-1IC with TRAP-1.
Figure 6:
Mapping the TRAP-1 binding sites in
TNFR-1IC. a, plasmids encoding GAL4-BD/TNFR-1IC deletion
mutants were cotransformed with I-45, and -galactosidase activity
was assayed. The topbar illustrates the structure of
TNFR-1IC. The blackenedhorizontalbars represent the relative size and location of each deletion mutant
in TNFR-1IC. b, expression of GAL4-BD/TNFR-1IC deletion
mutants was assayed by Western blotting using antisera directed to
GAL4-BD. Lanea, full-length TNFR-1IC (amino acids
205-426); laneb, TNFR-1IC amino acids
205-280; lanec, TNFR-1IC amino acids
205-359; laned, TNFR-1IC amino acids
278-359; lanee, TNFR-1IC amino acids
278-426; lanef, TNFR-1IC amino acids
352-426, which consistently appeared to overlap a nonspecific
band. The arrow to the right of each lane in
the Western blot indicates the position of the fusion
protein.
Studies taking advantage of the strong species specificity of
TNFR-2 for murine compared with human TNF (17) or employing
agonist antibodies specific to either TNF receptor(10, 16, 17, 18, 19, 20) have
shown that signaling through TNFR-1 promotes growth inhibition and
cytotoxicity in transformed cells, antiviral activity, proliferation of
fibroblasts, induction of NF-kB, accumulation of c-Fos, interleukin-6,
and manganous superoxide dismutase mRNAs, prostaglandin E synthesis, and HLA class I and II cell surface antigen
expression(1, 2, 3, 10, 16, 17, 18, 19, 20) .
Mice deficient in TNFR-1 are severely impaired in the ability to clear
the bacterial pathogen Listeria monocytogenes and die rapidly from
infections; however, these animals are resistant to
lipopolysaccharide-mediated septic shock(19) . Thus, TNFR-1
plays a decisive role in the host's defense against
microorganisms and their pathogenic factors. The significant role of
this receptor in TNF action led us to initiate our search for TRAPs
using TNFR-1IC as bait in a two hybrid screen of a cDNA library.
The
two hybrid system detects proteins capable of interacting with a known
protein by transcriptional activation of a reporter
gene(30, 31) . This method has demonstrated
interactions between Bcl-2 and R-ras p23(34) , Sos 1
and GRB2(35) , complex formation between Ras and Raf and other
protein kinases(36) , binding of the human immunodeficiency
virus type 1 GAP protein with cyclophilins A and B(37) , and
also identified proteins that interact with the type 1 receptor for
TGF- (38) and the type 2 receptor for TNF(39) .
Previously, we used the two hybrid system to demonstrate
self-association of TNFR-1IC(40) . Evidence for such
aggregation was obtained from a screen of a HeLa S3 cDNA library using
TNFR-1IC as bait; this yielded a clone encoding a protein encompassing
the death domain at the C-terminal of TNFR-1IC(41) .
Aggregation is independently suggested by the ability of non-functional
TNFR-1 deletion mutants to suppress signaling by non-defective
endogenous TNF receptors(42) . Thus, our previous studies (40) demonstrated that the two hybrid system can successfully
identify proteins that interact with TNFR-1, one of which is TNFR-1IC
itself.
In this and another study that describes a second TNF
receptor-associated protein, TRAP-2, ()the two hybrid system
was used to search for additional proteins that bind TNFR-1IC, leading
to the discovery of TRAP-1 and TRAP-2. Both proteins interact
specifically with TNFR-1 and not with unrelated proteins. mRNAs for
both TRAPs are highly expressed in numerous transformed cell lines and
are detected at different levels of expression in the heart, brain,
placenta, lung, liver, skeletal muscle, kidney, and pancreas.
Fig. 7a summarizes the domain structure of TNFR-1IC,
showing the relationships of the death/aggregation domain to the
binding sites for TRAP-1 and TRAP-2. Each TRAP binds to sites in
TNFR-1IC that reside outside of the death (41, 42) and/or aggregation (40) domain.
Mutations in this domain disrupt the ability of TNFR-1 to signal not
only cytotoxicity but antiviral activity and induction of nitric oxide
synthase as well. These observations should not lead one to conclude
that TRAP-1 and TRAP-2 are not involved in TNF action. While the
cytotoxicity domain is undoubtedly important for induction of some
responses mediated by TNFR-1, it is not the only domain involved in
signaling. Recent observations show that the ability of TNF to activate
an endosomal acidic sphingomyelinase is abrogated by C-terminal
deletions encompassing all or part of the cytotoxicity domain in
TNFR-1, whereas activation of a membrane-associated neutral
sphingomyelinase is unaffected(43) . Ceramide produced by the
neutral sphingomyelinase is important for activation of a
proline-directed serine threonine kinase and phospholipase
A. The acidic sphingomyelinase activates NF-kB. Thus,
different domains in TNFR-1IC control important and distinguishable
second messenger pathways and cellular responses.
Figure 7: a, map of TRAP binding sites in TNFR-1IC defined using the two hybrid system and by in vitro precipitation. b, relationship of hsp90, TRAP-1, and TRAP-2. Six domains that share homology in hsp90 and TRAP-1 are indicated by speckledboxes and Romannumerals. The blackboxes delineate a highly charged segment in hsp90 and TRAP-2. The length of each bar represents the relative size of each protein.
TRAP-1 shows strong homology to members of the hsp90 family. Fig. 4illustrates the presence of at least six domains of high amino acid identity and conservation that relate TRAP-1 and hsp90s. Interestingly, a well conserved, highly charged sequence of amino acids in hsp90 is absent from TRAP-1 but identified in TRAP-2. Fig. 7b illustrates the structural relationship of hsp90 to TRAP-1 and TRAP-2. While the significance of the relationship of the TRAPS to one another and hsp90 is not presently known, a basis exists for believing that such stress proteins are important to defining cellular responses to TNF. First, induction of the heat shock response renders transformed cells resistant to TNF and macrophage-mediated cytotoxicity(44) . Second, transfection of hsp70s into otherwise responsive cells induces a state of resistance(45) , and TNF-induced phosphorylation of hsp28 stress proteins is also associated with protection against cytotoxicity (46) . Third, the elements in TNFR-1IC necessary for binding to TRAP-1 and TRAP-2 reside outside of the cytotoxicity/aggregation domain. Large deletions outside of this domain(41) , where TRAP-1 and TRAP-2 bind, do not impair the ability of mutant receptors to induce cytotoxicity or antiviral activity.
Heat shock proteins are likely candidates for service as TRAPs as they act as cofactors or chaperones in cell growth-associated processes, protein folding and transport, and cell division and membrane function (47) . Hsp90s associate with steroid receptors, which are thereby stabilized in a non-DNA binding conformation, and also with protein kinases(47) . Hsp90s also modulate signaling through transduction cascades. Mutations in a member of the hsp90 family, hsp83, impairs signaling by the sevenless receptor tyrosine kinase, which is required for differentiation of the R7 photoreceptor neuron in Drosophila(48) . Also, lethal expression of vSrc in Saccharomyces cerevisiae is suppressed by a reduction in the level of Hsp82(49) . The diverse properties of hsps suggest that the discovery of TRAP-1, and also TRAP-2, is important and likely to provide insight into the mechanistic basis for TNF action.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U12595[GenBank], U12596[GenBank].