The Ubiquitin-homology Protein, DAP-1, Associates with Tumor Necrosis Factor Receptor (p60) Death Domain and Induces Apoptosis*

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-κB/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.

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 NH 2 -terminal kinase, AP-1, and NF-B/Rel transcription factors (6, 9 -13). In particular, TRAF2 was shown to be essential for c-Jun NH 2 -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 mecha- were transfected into yeast to test for potential intrinsic transcription activity. The transfected yeast were plated on Glu/CM-Ura,-His drop out nisms, 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.

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 NH 2 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(hisϩ) were cotransfected with the reporter plasmid PSH18 -34/LacZ(uraϩ) into yeast strain EGY48, by Me 2 SO 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.
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 l of 1 ϫ 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 ϫ TBST (0.05% Tween 20, 0.15 M NaCl, 10 mM Tris, pH 8.0), 4% milk for 1 h, washed with 1 ϫ TBST, and hybridized with anti-HA tag antibody for 2 h at room temperature. Following 3 ϫ 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/Ampϩ plates. Colonies were further streaked on LB/AmpϮTrp dropout plates for plasmid DNA isolation using the Qiagen plasmid mini-prep kit.
Sequencing analysis was performed by mixing 2 g of plasmid DNA with 2 mM primer and 4.5 l 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 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. Using the BLAST GenBank TM 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. 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 l 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 g of DAP-1/ CDM8 plasmids with 0.3 g of IgB-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 l 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 l of lysate with 50 l of LacZ buffer and 50 l of o-nitrophenol-␤-D-galactoside in 500 l total final volume. Reactions proceeded at 37°C for 10 min and were stopped with 500 l of 1 M Na 2 CO 3 prior to analysis by spectrophotometry at OD 420 nM. Normalized amounts of lysates (50 -70 l) were then incubated with 20 l 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 g 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 phosphatebuffered 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 ϫ (dead cell count/total transfected cell count). All transfections were repeated in at least three independent experiments.

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 NH 2 -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 ϫ 10 6 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.
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 5Ј 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 NH 2 terminus of clone 3. The variation may be due to a cloning artifact or differential spliced products from the DAP-1 gene.
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 Ubiquitinhomology 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 Gen-Bank TM 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 NH 2 -terminal 40 amino acids, DAP-1 and PIC1 are identical to the sentrin, a Fas receptor-associated protein (4).
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 centromerebinding 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 5Ј 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 glutathioneagarose 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.
Overexpression of DAP-1 Suppresses NF-B/Rel Activity-To investigate the influence of DAP-1 protein on TNF-R1mediated NF-B/Rel activation, we transfected DAP-1/CDM8, NFB-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.
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.
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 (Me 2 SO) 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.
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 NH 2 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 TNFsensitive 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. 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 IBs in the NF-B/IB complexes, leading to the specific ubiquitination and degradation of IBs by ubiquitin-proteosome pathways. Degradation of the IBs 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/IB 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 IB 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 NH 2 -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. 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, Gly 97 and Gly 98 (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.