The Ubiquitin-homology Protein, DAP-1, Associates with Tumor
Necrosis Factor Receptor (p60) Death Domain and Induces
Apoptosis*
Mei-Ling
Liou
§ and
Hsiou-Chi
Liou
¶
From the
Division of Immunology, Department of
Medicine, ¶ Graduate School of Medical Sciences, Cornell
University Medical College, New York, New York 10021
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ABSTRACT |
The tumor necrosis factor receptor, p60
(TNF-R1), transduces death signals via the association of its
cytoplasmic domain with several intracellular proteins. By screening a
mammalian cDNA library using the yeast two-hybrid cloning
technique, we isolated a ubiquitin-homology protein, DAP-1, which
specifically interacts with the cytoplasmic death domain of TNF-R1.
Sequence analysis reveals that DAP-1 shares striking sequence homology
with the yeast SMT3 protein that is essential for the maintenance of
chromosome integrity during mitosis (Meluh, P. B., and Koshland,
D. (1995) Mol. Biol. Cell 6, 793-807). DAP-1 is nearly
identical to PIC1, a protein that interacts with the PML tumor
suppressor implicated in acute promyelocytic leukemia (Boddy, M. N., Howe, K., Etkin, L. D., Solomon, E., and Freemont, P. S. (1996) Oncogene 13, 971-982), and the sentrin protein,
which associates with the Fas death receptor (Okura, T., Gong, L.,
Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M.,
and Yeh, E. T. (1996) J. Immunol. 157, 4277-4281). The in vivo interaction between DAP-1 and
TNF-R1 was further confirmed in mammalian cells. In transient
transfection assays, overexpression of DAP-1 suppresses NF-
B/Rel
activity in 293T cells, a human kidney embryonic carcinoma cell line.
Overexpression of either DAP-1 or sentrin causes apoptosis of
TNF-sensitive L929 fibroblast cell line, as well as TNF-resistant
osteosarcoma cell line, U2OS. Furthermore, the dominant negative
Fas-associated death domain protein (FADD) protein blocks the cell
death induced by either DAP-1 or FADD. Collectively, these observations
highly suggest a role for DAP-1 in mediating TNF-induced cell death
signaling pathways, presumably through the recruitment of FADD death effector.
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INTRODUCTION |
Tumor necrosis factor
(TNF)1 is one of the key inflammatory cytokines that
modulates normal and pathological immune responses due to its
pleiotropic activities on cell proliferation and apoptosis. Two types
of TNF receptors were identified: TNF-R1 and TNF-R2, with approximate
molecular masses of 60 and 80 kDa, respectively. TNF-R1 has diverse
biological activities including the induction of apoptosis and
NF-
B/Rel activation (5). These biological activities are mediated by
several TNF-receptor associated signaling proteins. Among these, the
TNF-receptor associated death domain protein (TRADD) (5, 6),
Fas-associated death domain protein (FADD) (7), and RIP (8) are
responsible for transducing cell death signals through the activation
of caspases. The TNF-receptor associated factors (TRAFs) transduce cell
proliferation and survival signals through the activation of c-Jun
NH2-terminal kinase, AP-1, and NF-
B/Rel transcription
factors (6, 9-13). In particular, TRAF2 was shown to be essential for
c-Jun NH2-terminal kinase, but not NF-
B/Rel, activation
and regulates lymphocyte proliferation and survival (14, 15). Whereas
RIP was found to be required for NF-
B/Rel activation and for
suppression of TNF induced cell death in vivo (16). TNF-R1
interacting proteins also include the TRAF-interacting protein (17),
PIP5K (18), FAN (19), and others (20-22). In addition, TNF-R1 induces
sphingomyelinase activity and ceramide generation, leading to
activation of cell death machinery (23, 24).
The ubiquitin-proteosome pathways are utilized in the regulation of
many intracellular functions. These include NF-
B/Rel activation (25,
26), cell cycle control (27), DNA repair (28), and protein localization
(29). Recently, many proteins with homology to ubiquitin were
identified. These ubiquitin-homology (UbH) proteins constitute an
expanding protein family with diverse biological activities. For
instance, RAD23 is involved in DNA excision repair (30), SIII p18
elongin protein is necessary for efficient transcription elongation
(31), PIC1 is present in a protein complex containing the PML tumor
suppressor protein (3), and sentrin is one of the Fas death receptor
associated proteins (4).
Another yeast UbH protein, Smt3p, is involved in centromere stability
during mitosis (1, 2). It was demonstrated by Johnson et al.
(2) that the activation of Smt3p can be activated by two enzymes with
homology to the ubiquitin-activating enzyme, E1. Activation of Smt3p by
the E1-like enzymes leads to cleavage and exposure of the diglycine
residues near the carboxyl-terminal, which permit conjugation of Smt3p
to the protein substrates. Although activation of the UbH protein
pathway occurs in a similar fashion to that of the ubiquitin-proteosome
pathways, they remain distinct in that activation of the UbH proteins,
such as Smt3p, results in substrate conjugation instead of
protein degradation. This distinction suggests that these parallel
pathways are likely to be regulated independently.
It remains obscure as to how these UbH proteins are regulated in
response to external stimuli and whether some of these UbH proteins are
associated with receptor-signaling mechanisms, such as the TNF receptor
system. Due to the diversity of TNF biological activities, it is
conceivable that multiple intracellular molecules are involved in its
signal transduction. Here, we report the identification of a novel UbH
protein, DAP-1, which specifically interacts with the death domain of
TNF-R1 and can trigger programmed cell death in a variety of cell lines.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs and HeLa cDNA Library--
All
TNF-R1-pEG202 constructs containing various portions of TNF-R1
cytoplasmic domain were generated by polymerase chain reaction and
subcloned into the EcoRI and NotI sites of the
pEG202 vector so that the TNF-R1 cytoplasmic fragment was in-frame with
the LexA DNA binding domain (32, 33). Constructs A, B, C, and D encode
peptides corresponding to amino acids 207-333, 207-403, 261-425, and
334-425 of the TNF-R1 cytoplasmic domain, respectively. The HeLa
cDNA library, kindly provided by Dr. Roger Brent, was generated by
cloning cDNA into the EcoRI and XhoI sites of
PJG4-5 vector in fusion with the LexA transcription activation domain (32, 33).
DAP-1, sentrin, and FADD cDNAs were subcloned into the
XbaI and HindIII sites of the CDM8 expression
vector with the hemagglutinin tag (HA tag) at the NH2
terminus of the cDNAs. The FADD-DN-CD2 and pBMN-LacZ expression
constructs were kindly provided by Dr. Yongwon Choi (Rockefeller
University) and Garry Nolan (Stanford University), respectively. The
pCMV-LacZ construct contains
-galactosidase gene under the
regulation of CMV promoter.
Yeast Two-hybrid Screening--
To evaluate any intrinsic
transcription activity of the four bait constructs, the bait plasmids
TNF-R1/pEG202(his+) were cotransfected with the reporter plasmid
PSH18-34/LacZ(ura+) into yeast strain EGY48, by Me2SO and
heat shock method (32, 33). Transfectants were plated on
Glu/CM-Ura,-His drop out plates, and incubated at 30 °C overnight.
For each construct, 12 colonies were streaked onto Glu/CM-Ura,-His
dropout plates. Colonies were lifted onto nylon filter, soaked in Z
buffer containing 1 mg/ml X-gal, and incubated at 30 °C for 10 min
to monitor for color changes. Only construct D gave rise to white
colonies (no intrinsic transcription activity). Constructs A-C gave
rise to light blue colonies.
TNF-R1-pEG202 construct D was utilized as the bait for screening
interacting proteins in HeLa cDNA library following the published protocol with the following modification. Thirty milligrams of library
plasmid, pJG4.5/cDNA(trp+), plus 2 mg of salmon sperm carrier DNA,
were transfected into the yeast strain, EGY48(leu+) cells, which were
already pretransformed with the bait construct, TNF-R1/pEG202(his+), and the reporter plasmid,
pSH18-34/LacZ(ura+). Transfection was facilitated by 40% polyethylene
glycol 4000, followed by 10% Me2SO. After incubation at
42 °C for 10 min, transfectants were plated on
Gal/CM-His,-Ura-Trp-Leu plates and further incubated at 30 °C for
48 h. A total of 4 × 106 colonies from library
transformations were screened on Glu/CM-His,-Ura-Trp-Leu plates. Next,
blue colonies were plated onto the four-criteria plates:
Glu/CM-His,-Ura-,Trp,-Leu; Gal/CM-His,-Ura-,Trp,-Leu; Glu/X-gal/CM-His,-Ura-,Trp; and Gal/X-gal/CM-His,-Ura-,Trp-plates. The
positive clones were selected based on their ability to grow and turn
blue on Gal/X-gal/CM-His-Ura-Trp plates, but not on
Glu/X-gal/CM-His,-Ura-,Trp plates.
Preparation of Yeast Extracts and Cell Lysates for Western Blot
Analysis--
The TNF-R1 interacting cDNA clones in yeast vector
(pJG4.5/cDNA) were transformed into bacteria strain, KC8, and
colonies were cultured in 10 ml of medium overnight at 30 °C prior
to lysate preparation. Bacterial or cell pellets were lysed in 300 µl
of 1 × RIPA buffer (Tris-HCl, pH 7.4, Triton X-100, SDS,
deoxycholate, EDTA, pH 7, NaCl) at 4 °C for 1 h. Protein
samples were resolved on 10% SDS acrylamide mini-gel (Bio-Rad mini-gel
system) with 150 volts for 1 h and transferred onto nitrocellulose
membrane by semi-drier apparatus (Bio-Rad) with 20 volts for 20 min.
Membranes were blocked with 1 × TBST (0.05% Tween 20, 0.15 M NaCl, 10 mM Tris, pH 8.0), 4% milk for
1 h, washed with 1 × TBST, and hybridized with anti-HA tag
antibody for 2 h at room temperature. Following 3 × 10-min
washes, the membranes were incubated with anti-mouse horseradish
peroxidase secondary antibody. The antibody-coated membrane was
developed by ECL reagent (Amersham Pharmacia Biotech) and analyzed by autoradiography.
Plasmid DNA Isolation and Sequencing Analysis--
Plasmid DNA
in yeast was extracted by using 0.45-mm acid-washed glass beads. The
soluble extracts were utilized to transform the bacteria strain, KC8,
competent cells and plated on LB/Amp+ plates. Colonies were further
streaked on LB/Amp±Trp dropout plates for plasmid DNA isolation using
the Qiagen plasmid mini-prep kit.
Sequencing analysis was performed by mixing 2 µg of plasmid DNA with
2 mM primer and 4.5 µl of premix sequencing reagents (containing Taq polymerase, nucleotides) included in the
Applied Biosystems Dye Terminator Cycle DNA sequencing kit
(Perkin-Elmer, Applied Biosystems 402080) and amplified for 28 cycles
by polymerase chain reaction. The final polymerase chain reaction
samples were applied to the Applied Biosystems automatic sequencer. A
sequence homology search was performed by accessing to the National
Institutes of Health BLAST data base.
TNF-R1 and DAP-1 Interaction Assay--
To generate the
TNF-R1/GST construct, the DNA fragment encoding amino acids 334-425 of
TNF-R1 region was subcloned in-frame with the GST fragment in the pEBG
vector (34). The TNF-R1/GST and DAP-1/CDM8(HA tag) plasmids were
cotransfected into 293T cells by calcium phosphate transfection
protocol described previously (34). Transfectants were plated on
six-well plates and incubated at 37 °C for 2 days. At that point,
cells were lysed with lysis buffer containing 0.1% of Nonidet P-40,
after which 100 µl of glutathione beads were added, and the mixture
was incubated at 4 °C in rotary motion for 2 h. The beads were
washed three times with 0.05% Nonidet P-40 buffer, boiled in SDS
sample loading buffer, and then subjected to SDS-polyacrylamide gel
electrophoresis and Western blot analysis using the anti-HA and
anti-GST antibodies.
NF-
B/Rel Activation Luciferase Assay--
The 293T cells were
transiently cotransfected, in triplicates, with either 0.6 or 0.9 µg of DAP-1/CDM8 plasmids with 0.3 µg of Ig
B-luciferase reporter
construct (34) and pBMN-LacZ. Transfectants were plates on six-well
plates and incubated at 37 °C for 48 h. Cells were then lysed
with 500 µl of luciferase cell lysis buffer. To normalize the
transfection efficiencies among samples, an aliquot of each lysate was
assayed for
-galactosidase activity by mixing 10 µl of lysate with
50 µl of LacZ buffer and 50 µl of
o-nitrophenol-
-D-galactoside in 500 µl total final
volume. Reactions proceeded at 37 °C for 10 min and were stopped
with 500 µl of 1 M Na2CO3 prior
to analysis by spectrophotometry at OD 420 nM. Normalized
amounts of lysates (50-70 µl) were then incubated with 20 µl of
luciferase substrate and analyzed by
counter.
Apoptosis Assay--
The test constructs, including DAP-1/CDM8,
sentrin/CDM8, FADD/CDM8, FADD-DN-CD2, or in combination, were
cotransfected with pCMV-LacZ reporter plasmid at 2:1 ratio into either
L929, 293T, or U2OS cell lines. All transfections were performed by
calcium phosphate method in triplicates in six-well plates with total 12 µg of DNA/well. Where indicated, some transfectants were treated with 10 ng of murine TNF-
at 3 h post-transfection. Cells were harvested at 48 h after transfection and fixed with 0.02%
glutardehyde in phosphate-buffered saline prior to incubation with the
X-gal staining solution. Cells were analyzed by phase contrast
microscopy. Several hundred to two thousand blue cells were counted
depending upon the transfection efficiency. Wrinkled blue cells with
condensed nuclei were scored as dead cells, whereas flat blue cells
with intact morphology were scored as live transfected cells. The
percentage of apoptotic cells was calculated as 100 × (dead cell
count/total transfected cell count). All transfections were repeated in
at least three independent experiments.
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RESULTS |
Cloning of a Novel TNF-R1 (p60)-binding Protein, DAP-1--
We
utilized the yeast two-hybrid system to isolate proteins that interact
with the TNF-R1 cytoplasmic domain (32, 33). In this system, the TNF-R1
DNA fragments were subcloned into the EcoRI and
NcoI sites of the pEG202 plasmid, in which it produced a
bait protein with LexA DNA binding domain (amino acids 1-222) at the
NH2-terminal in fusion with the TNF-R1 cytoplasmic peptide at the COOH-terminal. The HeLa cDNA library was cloned into the EcoRI site of the pJG4.5 vector, which produced fusion
proteins of the cDNA-derived peptide and the LexA transcription
activation domain. Initially, several constructs encompassing the
entire TNF-R1 cytoplasmic domain were tested for any potential
intrinsic transcription activity in the absence of the library plasmids (Fig. 1A). One of the
constructs containing amino acids 334-475 did not reveal basal
transcription activity and was utilized as the bait for the screening.
This bait contains the entire death domain and additional upstream 20 amino acids. Among 4 × 106 HeLa library cDNA
clones screened using the bait, 34 colonies were scored positive for
the two selection criteria: by their ability to grow in the leucine
deficient medium and to turn blue in the presence of X-gal substrate
(Fig. 1B). Using dot blot hybridization, these 34 colonies
were further categorized into seven distinct groups (data not shown),
which we named death domain-associated proteins or DAPs. Fifteen of
these 34 positive colonies (44%) correspond to DAP-1.

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Fig. 1.
Evaluation of various TNF-R1 receptor bait
constructs. A, TNF-R1 receptor bait constructs. Four
different DNA fragments encoding the mouse TNF-R1 cytoplasmic domain
corresponding to amino acids 207-333 (Construct A), amino
acids 207-403 (Construct B), amino acids 261-425
(Construct C), and amino acids 334-425 (COOH-terminal)
(Construct D) were subcloned into the pEG202 (His+) plasmid
in fusion with LexA DNA binding domain. The black box
represents the death domain. These constructs and the reporter plasmid
PSH18-34-LacZ (Ura+) were transfected into yeast to test for potential
intrinsic transcription activity. The transfected yeast were plated on
Glu/CM-Ura,-His drop out medium and subsequently tested for -galactosidase activity in
X-gal-containing Z buffer. Construct D exhibited no intrinsic
transcription activity and was subsequently utilized as the bait in
yeast two-hybrid screening. B, yeast clones interact with
TNF-R1 Construct D were identified in yeast two-hybrid system.
Construct D (see Fig. 1A) was utilized as a bait to identify
interacting proteins in HeLa cDNA library using the yeast two
hybrid screening method (see "Experimental Procedures").
Blue colonies represent a specific interaction between the
bait and the cDNA-derived peptides, whereas white
colonies indicate no direct physical interaction between the bait and
the cDNA-derived peptides.
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To estimate the molecular weight of DAP-1 protein, three clones with
variable DNA length, clone 3 (1.2 kb), clone 16 (1.5 kb), and clone 7 (1.7 kb), were further analyzed. In the original yeast extracts, DAP-1
clones encoded proteins with molecular masses of approximately 25-27
kDa (Fig. 2A). Since all these clones
contain 10 kDa of the LexA DNA binding domain, their actual molecular masses are about 15-17 kDa. To further confirm the protein size, DAP-1
was inserted into the CDM8 mammalian expression vector with HA tag
epitope attached at the 5' end of the cDNA. These constructs were
transfected into the human embryonic kidney carcinoma, 293T cell line.
Transfectant lysates were analyzed by Western blot analysis using
monoclonal anti-HA tag antibody. In mammalian cells, clone 3 produced
the protein of 15 kDa, whereas clones 7 and 16 express protein with
molecular mass of 17 kDa (Fig. 2B), consistent with previous
molecular mass estimation in yeast extract. It was later evident that
the molecular mass difference between clones 3 and 7/16 was the result
of a 14-amino acid deletion at the NH2 terminus of clone 3. The variation may be due to a cloning artifact or differential spliced
products from the DAP-1 gene.

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Fig. 2.
Protein and RNA expression of DAP-1
clones. A, Western blot analysis of DAP-1 protein in
yeast. Yeast lysates of DAP1 clones 3, 7, 16, and 48, as well as the
vector control, were analyzed by Western blot analysis using the
anti-HA antibody. For some clones, two independent colonies were
analyzed (a and b). All DAP-1 clones, but not the
vector control, express proteins with approximately size range of
25-27 kDa. Since the LexA transactivation domain is 10 kDa, the DAP-1
cDNA portion is estimated to encode peptide with molecular mass of
15-17 kDa. B, Western blot analysis of
DAP-1 in mammalian cells. DAP-1 cDNAs with HA tag were
subcloned into CDM8 mammalian expression vector and transfected into
293T cells, a human embryonic carcinoma cell line. Protein lysates were
further analyzed by Western blot analysis using the anti-HA antibody.
DAP-1 clone 3 expresses protein of 15 kDa, whereas clones 7 and 16 encode protein with molecular mass of about 17 kDa. C,
Northern blot analysis of DAP-1 mRNA expression.
Poly(A)+ RNA samples extracted from HeLa cells and WEHI231
(B cell lymphoma) cell line were hybridized with
32P-labeled DAP-1 cDNA probe. The size of DAP-1
mRNA is estimated to be 1.5 kb.
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To test that DAP-1 is a gene product and not derived from a random
sequence in the genome, Northern blot analysis was performed using
total RNA derived from HeLa and a murine B cell lymphoma cell line,
WEHI 231. A 1.5-kb transcript was detected by radioisotope-labeled DAP-1 clone 7 probe (Fig. 2C). This result confirms that
DAP-1 is indeed derived from a mammalian gene transcript.
Sequence Analysis of DAP-1 and Comparison with Ubiquitin-homology
Proteins--
DNA sequencing analysis of DAP-1 cDNA reveals an
open reading frame that encodes proteins of 122 amino acids (clone 3)
and 142 amino acids (clones 7 and 16), respectively (Fig.
3A). By searching BLAST and
EST GenBankTM data bases, we identified several sequences
with homology to DAP-1. DAP-1 is identical to the PML interacting clone
1 (or PIC1), which is part of the PML-containing multiprotein complexes
that are frequently disrupted in acute promyelocytic leukemia (Fig. 3B) (3). Except for the additional NH2-terminal
40 amino acids, DAP-1 and PIC1 are identical to the sentrin, a Fas
receptor-associated protein (4).

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Fig. 3.
Sequence analysis of DAP-1 cDNA.
A, sequences of DAP-1 cDNA clones and the LexA
transcription activation domain. The LexA transcription domain in the
PJG4-5 vector begins at the ATG start codon (amino acid 1) and ends at
the EcoRI cloning site (underlined; amino acid
106). This fusion domain also contains SV40 nuclear localization
sequence (PPKKKRKVA; underlined) and the HA epitope tag
(YPYDVPDYA; underlined). The DAP-1 clones contain an open
reading frame from amino acids 108-248. Clones 7 and 16 have an
additional 14 amino acids as compared with clone 3. B,
comparison of DAP-1 with homologous cDNAs. Using the BLAST
GenBankTM search, several cDNAs with homology to DAP-1
cDNA were identified. All of these proteins have high homology to
the ubiquitin-homologous proteins of various species. DAP-1 shares
about 50% identical (75% homology) to human SMT3A, SMT3B EST
sequences, and the yeast Smt3p protein. DAP-1 is 95% similar to the
PIC1 sequence. Except for the first 40 amino acids, it is identical to
sentrin.
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DAP-1 sequence is identical to the translated peptide sequence of SMT3C
in the human EST data base and also has 55% identity (75% homology)
to SMT3A and SMT3B EST sequences. DAP-1 reveals 52% identity (73%
homology) to the Smt3p protein in Saccharomyces cerevisiae
(Fig. 3B). The yeast Smt3p gene product has been implicated in the modulation of chromatin stability during mitosis by regulating a
centromere-binding protein, MIF2 (1, 2). Furthermore, DAP-1, PIC1 and
SMTs, share a significant degree of homology to ubiquitin and other
ubiquitin-homology proteins (see "Discussion"), but potential
ubiquitin-like activities are yet to be determined.
Direct in Vivo Interaction of DAP-1 and TNF-R1 in Mammalian
Cells--
In addition to an association in yeast, we also demonstrate
that DAP-1 can directly interact with TNF-R1 in mammalian cells. DNA
fragment encoding the cytoplasmic domain of TNF-R1 was subcloned into
the mammalian expression vector, pEBG-GST, which contains the
glutathione S-transferase (or GST) coding motif at the 5' end of the cloning site. After cotransfection of TNF-R1/pEBG-GST and
DAP-1/CDM8-HA tag into 293T cells, the cell lysates were applied to
glutathione-agarose beads, to which TNF-R1-GST fusion protein and its
associated proteins are expected to bind. The protein complexes
retained in the beads were further analyzed on SDS-polyacrylamide gel
electrophoresis gel and blotted with anti-HA antibody. As shown in Fig.
4, DAP-1 protein can coprecipitate with
TNF-R1-GST fusion protein. The interaction was specific to TNF-R1
cytoplasmic domain because DAP-1 protein did not coprecipitated with
other GST fusion proteins containing irrelevant peptide motifs (data not shown). These data confirm an in vivo interaction of
DAP-1 with TNF-R1 cytoplasmic domain in mammalian cells under
physiological condition.

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Fig. 4.
Interaction of DAP-1 with TNF-R1 in
vivo. TNF-R1 cytoplasmic domain (amino acids 334-425) was
subcloned in-frame with the GST gene in the pEBG vector. This construct
was cotransfected with DAP-1/CDM8 (with HA tag) plasmid into 293T cells
by calcium phosphate transfection method. Transfectants were lysed in
0.1% Nonidet P-40 lysis buffer and applied to glutathione-agarose
beads. Proteins bound to the beads were eluted with free glutathione
and analyzed by Western blot analysis using either anti-HA (upper
panel) or anti-GST (lower panel) antibodies. As shown,
DAP-1/HA tag proteins were specifically coprecipitated with the
TNF-R1/GST in the glutathione affinity column.
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Overexpression of DAP-1 Suppresses NF-
B/Rel Activity--
To
investigate the influence of DAP-1 protein on TNF-R1-mediated
NF-
B/Rel activation, we transfected DAP-1/CDM8, NF
B-luciferase reporter, and the pBMN-LacZ reporter constructs into 293T cells. An
aliquot of each lysate was initially assayed for
-galactosidase activity to normalize the transfection efficiencies among samples. Normalized amount of lysate samples were then assayed for luciferase activity as a measurement of NF-
B/Rel transcription activity. As
shown in Fig. 5, constitutive NF-
B/Rel
activity in 293T cells was significantly reduced by overexpression of
DAP-1. This result suggests that DAP-1 may play a negative role in the
activation of NF-
B/Rel.

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Fig. 5.
DAP-1 suppresses
NF- B/Rel activity in 293T cells. Various
DAP-1/CDM8 plasmids were cotransfected with Ig B-luciferase construct
(34) and pBMN-LacZ reporter plasmid into 293T cells in triplicates at
two different DNA concentrations. Transfected cell lysates were
analyzed by luciferase assay. Transient transfection efficiencies among
each sample were normalized by measuring -galactosidase
activity.
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We have attempted to address if DAP-1 has any effect on TNF-induced
NF-
B activity by transfecting DAP-1 into 293T cells that were
subsequently treated with TNF-
. As TNF-
induced nearly a 100-fold
increase in NF-
B activity, this activity was not diminished by
transient transfection of DAP-1 (data not shown). However, this is a
technically challenging experiment given the limitation of transfection
efficiency (less than 30%) but high inducibility of NF-
B by TNF in
nearly all 293T cells. Thus, the portion of cells that contain
B-luciferase construct, but not the DAP-1 cDNA, would contribute
to the surging luciferase activity which is not affected by DAP-1.
Overexpression of DAP-1 Induces Apoptosis--
To investigate
whether DAP-1 can modulate apoptosis function of TNF-R1, the DAP-1/CDM8
and pCMV-LacZ plasmids (ratio 2:1) were cotransfected into the mouse
fibroblast cell line, L929, which is extremely sensitive to
TNF-
-induced apoptosis. Half of the transfectants were treated with
TNF-
36 h postelectroporation. Cell death was quantified by
counting
-galactosidase positive (blue-colored) cells with condensed
nuclei under microscope after incubating in the X-gal buffer. DAP-1
alone induced 25-30% of cell death in L929 cells (Fig.
6A). Treatment of
DAP-1-transfected cells with TNF-
only slightly enhances apoptosis
to 35-40% (Fig. 6A). Since there is no significant synergy
between TNF-
and DAP-1, it suggests that DAP-1 and TNF-R1 may
converge at common cell death pathways. As the blue nuclei condensed
cells represent only a fraction of cells that are dying at the time of
analysis, we also counted the total transfected viable cells to better
estimate the effect of DAP-1 on cell death during the transfection
process. By counting the total blue cells, DAP-1-transfected plates had 50-70% reduced cell number as compared with the control transfectants (Fig. 6B). TNF-
treatment alone also resulted in similar
reduction in total cell number.

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Fig. 6.
DAP-1 induces apoptosis in L929 cells.
DAP-1/CDM8 plasmids and pCMV-LacZ reporter plasmid were transfected at
2:1 ratio in triplicate into the TNF-sensitive cell line, L929. Half of
the transfected cells were treated with TNF- (10 ng/ml) for 14 h prior to fixation and incubation in the X-gal solution. Blue cells,
indicative of plasmid DNA incorporation, were visualized by phase
contrast microscopy. Dead cells were determined by counting the blue
cells with dark and condensed nuclei (A) and the total
number of transfected viable cells assessed by counting blue cells on
each transfection plate (B).
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TNF induces the activation of caspases which eventually leads to
programmed cell death. We therefore tested if DAP-1-mediated cell death
is also due to the activation of caspase activity. DAP-1-transfected
L929 cells were treated with either the pan-caspase inhibitor, Z-VAD
peptide, or solvent (Me2SO) at 12 h posttransfection (35). While DAP-1 alone induced 47% cell death, treatment of these
transfectants with the Z-VAD peptide reduced the cell death to 22%
(Fig. 7A). This experiment
thus indicated that DAP-1-induced cell death involves the activation of
caspases.

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Fig. 7.
Caspase inhibitor blocked the apoptosis
induced by DAP-1. DAP-1/CDM8 or sentrin-CDM8 plasmids were
transfected into either L929 cells (A) or U2OS cells
(B) in the presence of pCMV-LacZ reporter plasmid at 2:1
ratio in triplicates. Where indicated, the DAP-1-transfected wells were
added with Z-VAD peptide (dissolved in Me2SO) to a final
concentration of 15 µM. As a solvent control,
Me2SO was added to the DAP-1-transfected wells to 0.025%
final concentration. Only the X-galactosidase-positive blue cells were
scored. Percentage of apoptotic cells were determined by ratio of
blue cells with condensed nuclei over total blue cells.
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Previously, it was shown that sentrin, a Fas receptor-associated
protein, protects BJAB cells from Fas-mediated apoptosis (4). Since
DAP-1 and sentrin share 99% identity except for the additional 40 amino acids in the NH2 terminus of DAP-1, we compared these
two proteins in our transfection assay system. As clearly demonstrated
in Fig. 7A, sentrin alone also induced significant percentage of cell
death (53%) in the TNF-sensitive L929 cell line. Further comparison
was performed in a TNF-resistant U2OS cell line. Consistent with our
previous observation, both DAP-1 and sentrin enhanced the percentage of
apoptotic cells in U2OS cells (Fig. 7B). Together, these
repeated experiments on several independent cell lines allowed us to
conclude that both DAP-1 and sentrin induced programmed cell death that
is mediated through the activation of caspases.
It has been demonstrated that TNF-R1 associates with the death
effector, TRADD and FADD, leading to the activation of FLICE and
downstream caspases (5-7). Since our data indicated that DAP-1
interacts with TNF-R1 and that both pathways result in the activation
of caspases, we tested whether DAP-1-induced apoptosis converges with
TNF-R1 signals at the death effectors, TRADD or FADD. To address this
issue, we cotransfected DAP-1 with the dominant negative FADD
(FADD-DN), which has been shown to block FADD mediated cell death into
L929 cells. As FADD-DN mutant clearly blocked FADD-induced cell death
(reduction from 50% to 24%), it also prevented DAP-1 induced
apoptosis (reduction from 39% to 16%) (Fig.
8). FADD-DN alone did not cause cell
death. Collectively, these transfection experiments strongly suggest
that DAP-1 converges with TNF receptor signaling pathway and that it
induces downstream caspase activities through the activation of FADD
death effector.

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|
Fig. 8.
FADD dominant negative mutant blocked the
apoptosis induced by DAP-1. Various expression plasmids that
contain DAP-1, sentrin, FADD, or FADD dominant negative mutant (FAD-DN)
were cotransfected into L929 cells, either alone or in combination,
along with pCMV-LacZ reporter plasmid. The amount of total DNA in all
transfectants was brought to the same level with CDM8 vector.
Percentage of apoptotic cells was determined by the ratio of blue cells
with condensed nuclei over total blue cells.
|
|
 |
DISCUSSION |
TNF-
-mediated receptor trimerization recruits a variety of
intracellular proteins that are crucial for signal transduction. Among
these are death domain-containing proteins and TRAF, which mediate
apoptosis or activation of NF-
B/Rel, respectively. Using yeast
two-hybrid system, we identified a protein, DAP-1, which specifically
interacts with the TNF-R1. Sequence analysis of DAP-1 indicated that it
is a member of the expanding ubiquitin-homology protein family. Our
studies demonstrated that DAP-1 can suppress NF-
B/Rel activity.
Furthermore, we provided evidence that overexpression of DAP-1 can lead
to programmed cell death, which can be blocked by caspase inhibitor and
the dominant negative FADD protein.
DAP-1 Suppresses NF-
B/Rel Activity--
Our data indicated that
one of the unique features of DAP-1 is its ability to suppress the
constitutive NF-
B/Rel activity in 293T cells. Recently, the
mechanism of NF-
B/Rel activation by TNF-R1 has been characterized.
It was shown that the association of TRAF or RIP with NF-
B-inducing
kinase activates the IKK
and IKK
(36-38). The IKK
/
heterodimer phosphorylates the I
Bs in the NF-
B/I
B complexes,
leading to the specific ubiquitination and degradation of I
Bs by
ubiquitin-proteosome pathways. Degradation of the I
Bs is necessary
for NF-
B/Rel activation and translocation to the nucleus.
Several possibilities may explain the nature of NF-
B/Rel suppression
by DAP-1. For instance, DAP-1 may interfere with TRAF- or RIP-mediated
NF-
B/Rel activation. Alternatively, since DAP-1 is a UbH protein, it
may modulate NF-
B/I
B complex activity by competing for common
ubiquitin substrates. One distinct feature of the UbH proteins is that,
unlike the ubiquitin-targeted proteolysis, some UbH proteins can
conjugate to other proteins without causing substrate degradation. The
diglycine residues at position 141 and 142 in DAP-1 protein can
potentially serve as the conjugation site for protein substrates,
similar to its yeast homologue, Smt3p (2). It will be of interest to
test whether NF-
B/Rel or I
B are the potential protein substrates
of DAP-1.
Association of DAP-1 with Apoptosis--
Our studies demonstrated
that expression of DAP-1 alone induced apoptosis in several cell lines,
regardless of their TNF sensitivity. It is clearly a programmed cell
death, rather than necrosis or nonspecific toxicity, because the cell
death induced by DAP-1 can be blocked by the caspase peptide inhibitor
as well as by the dominant negative FADD molecule. This raises an
interesting point, which suggests that DAP-1-induced cell death is
mediated through the interaction or recruitment of FADD death effector, which also associates with the TNF-R1. Given the unique ubiquitin-like conjugation activity of DAP1 (see below), DAP-1 may facilitate the
formation of the death-inducing signaling complex by serving as the
docking site for assembly of intracellular death domain containing proteins.
Our data, however, differ from the previous observation made by Okura
et al. (4) in that sentrin was shown to protect a B cell
lymphoma cell line from Fas-induced apoptosis as well as TNF-induced
cell death in L929 cells. There are several issues that this report
fails to resolve. First, given the percentage of cell survival
conferred by sentrin (55%) as compared with vector alone at 25 ng/ml
concentration of anti-Fas, it would indicate a nearly 30-50%
transfection efficiency of the BJAB lymphocytic cell line using
electroporation. To our knowledge and based on our experience with B
lymphocytes, it has been extremely difficult to achieve even more than
2-5% transfection efficiency in B cell lines using most existing
transfection methods.
Alternatively, the discrete activities of DAP-1 and sentrin could be
due to the NH2-terminal 40 amino acids that are present in
DAP-1 and PIC1, but absent in sentrin. In our repeated experiments, three independent DAP-1 constructs reproducibly induced cell death. Furthermore, a side by side comparison of sentrin and DAP-1 using the
same expression plasmid in several cell lines strongly supported the
similar apoptosis-inducing nature of both proteins.
The other relevant phenomenon was our observation that the yeast clones
expressing the DAP-1 protein had consistently slower growth rate than
other yeast colonies (data not shown). Furthermore, our attempts to
obtain stable cell lines expressing constitutive DAP-1 protein were
unsuccessful, presumably due to the toxicity of DAP-1 on the host
cells.2 The growth arrest
nature of this protein in yeast (smt3) and mammalian cells may be
related to its apoptosis-inducing activity as observed here by
transient transfection assays. Future studies will be necessary to
discern the molecular mechanism of DAP-1- and sentrin-induced apoptosis
in TNF-
- and Fas-mediated cell death signaling pathways.
A Potential Role of DAP-1 in Cell Cycle Control--
The other
intriguing possibility is that the apoptosis caused by DAP-1
overexpression may be an indirect effect of DAP-1 on cell cycle
checkpoint control. The supporting data came from the observation that
its yeast homologue, Smt3p, is involved in the regulation of MIF2
centromere binding protein (1). Thus, a potential function of DAP-1 may
be to modulate mitosis by influencing chromosome stability during
mitosis. It has been demonstrated that ubiquitin-proteosome systems are
involved in several major cell cycle transitions (39-41). For example,
the anaphase-promoting complex is a ubiquitin ligase complex that is
necessary for the degradation of proteins that inhibit separation of
chromosomes during mitosis. Interruption of the anaphase-promoting
complex function by a mutant E2 protein inhibits the destruction of
cyclins, arrests cells in M phase, and inhibits the onset of anaphase
(42). Since the DAP-1 homologue regulates the centromere-binding
protein, it is possible that DAP-1 may be involved in the modulation of anaphase-promoting complex function. Future studies on the effect of
DAP-1 on cell cycle checkpoint control will need to be established.
Recent studies by Johnson et al. provided the first
biochemical evidence that Smt3p could be attached to other proteins
posttranslationally through the functionally critical diglycine
residues, Gly97 and Gly98 (2). These diglycine
residues are crucial for the ability of Smt3p to conjugate with its
protein substrates and to complement the lethality of a smt3 mutant
strain. In several of our Western blot analyses, we also observed
similar high molecular mass bands (about 95 kDa) that were present in
samples transfected with DAP-1 but not in control samples (Fig. 2,
A and B). This observation may suggest similar
protein conjugation properties of the mammalian DAP-1 protein. The
protein substrates of Smt3p and DAP-1 are yet to be determined.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Roger Brent, Zheng-Sheng Ye,
Jackie Bromberg, Yongwon Choi, Garry Nolan, and Genetech for sharing
with us the reagents, plasmids, and protocol for yeast two-hybrid
system. We also thank Drs. Elaine Schattner, Sofija Andjelic, Yongwon Choi, and Edward Lin for critical review of this manuscript.
 |
FOOTNOTES |
*
This work was supported by a startup fund provided by
Cornell University Medical College (Charles Offin Charitable Trust and Tolly Vinik Trust) and National Institutes of Health Grant RO1CA68155.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Current address: ACCESS Graduate Program, University of California,
Los Angeles, CA 90034.
To whom correspondence should be addressed. Tel.:
212-746-4451; Fax: 212-746-8167; E-mail:
hcliou{at}mail.med.cornell.edu.
2
M.-L. Liou, unpublished observation.
 |
ABBREVIATIONS |
The abbreviations used are:
TNF, tumor necrosis
factor;
TRAF(s), TNF-receptor associated factor(s);
RIP, receptor-interacting protein;
UbH, ubiquitin-homology;
E1, ubiquitin-activating enzyme;
E2, ubiquitin carrier protein;
HA, hemagglutinin;
CMV, cytomegalovirus;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
GST, glutathione
S-transferase;
kb, kilobase pair(s);
PML, promyelocytic
leukemia gene;
EST, expressed sequence tag(s).
 |
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