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Department of Molecular and Cell Biology and Biochemistry,1 Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, Rhode Island 029032
Received 15 December 2004/ Returned for modification 14 January 2005/ Accepted 14 February 2005
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ABSTRACT |
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by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation. |
INTRODUCTION |
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domain, which is structurally and functionally homologous to the SOCS box (32). Numerous tumors are caused by mutations within the VHL domain. These mutations prevent Elongin-C recruitment to the VHL domain, which results in the loss of its activity in inducing proteolysis of its substrate, the transcription factor HIF-1 (15).
The ASB family is the largest among five groups of SOCS box-carrying proteins. Eighteen members of the ASB family have been identified in the National Center for Biotechnology Information human genome database. Recently, the SOCS box of ASB1 has been shown to interact with Elongins-B/C (14), implicating the potential of this group of proteins in mediating protein ubiquitination and degradation. While SOCS family members use the SH2 domain to recruit substrates with phospho-tyrosine motifs, ASB family members are expected to use the ankyrin (ANK) repeats to recruit substrates. The ANK sequence is loosely conserved at the amino acid level but has a tightly conserved secondary structure of approximately 33 amino acids that is found in various copy numbers. Consecutive repeats form V-shaped helix-turn-helix motifs and stack sequentially into bundles (32). As a functional domain specific for mediating protein-protein interactions, the ANK repeat domain has been found in proteins with a wide range of cellular functions. These include transcription factor regulators such as inhibitor of NF-
B (IB), p53-binding protein 2 (p53BP2), and GABP-ß as well as the cyclin-dependent kinase inhibitor family members (30).
To functionally characterize ASB proteins, we cloned ASB1, ASB3, and ASB4. Using an antibody array technology developed in our lab (33), we screened for ASB-interacting factors in cells. We report here that tumor necrosis factor receptor type 2 (TNF-R2) is one of the binding targets of ASB3. By binding to the C-terminal region of TNF-R2, ASB3 mediates TNF-R2 ubiquitination and degradation; ASB3 also inhibits TNF-R2-mediated JNK activation. Furthermore, modulation of asb3 levels by a small interfering RNA (siRNA) affected TNF-R2 protein stability and the extent of TNF-R2-mediated cytotoxicity in a stable cell line, 4E3, expressing TNF-R2. The data presented here therefore implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-.
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MATERIALS AND METHODS |
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was purchased from R&D Systems (Minneapolis, Minn.).
Plasmids and transient transfection.
ASB3 was cloned from 293 cells by reverse transcription-PCR (RT-PCR). The total RNA was isolated with the Trizol reagent per the manufacturer's protocol and subjected to reverse transcription using Superscript II reverse transcriptase (Invitrogen). ASB3 amplification was performed with the proofreading DNA polymerase PfuTurbo (Stratagene, La Jolla, Calif.) and the primers 5'-CCGGAATTCCATGGATTTTACAGAGGC-3' and 5'-CCGCTCGAGTTATCCATCTTGAATAGC-3'. ASB3 was cloned in frame with the c-Myc tag into EcoRI and XhoI sites of pcDNA3-c-Myc. c-Myc-ASB3
SB was constructed by first excising the insert by EcoRI and XhoI digestion followed by removal of the SOCS box domain of ASB3 by NcoI digestion. The insert was then subcloned in frame with the c-Myc tag into pcDNA3-cMyc by the use of EcoRI and blunt end ligation. TNF-R2 C-terminal-region deletion constructs were generated by PCRs using the proofreading Turbo Taq polymerase and were subcloned into the pcDNA vector. The primers used to generate TNF-R2S424 were 5'-ATGGCGCCCGTCGCCGTCTGG-3' and 5'-GGGGACCTGCTCGTCCTTCGG-3'; the primers used to generate TNF-R2364 were 5'-ATGGCGCCCGTCGCCGTCTGG-3' and 5'-GGCCTCCCCGGCCCCACTGGC-3'; and the primers used to generate TNF-R2331 were 5'-ATGGCGCCCGTCGCCGTCTGG-3' and 5'-CAGGGAGCTGCTGCTGGAGCT-3'. ASB3 ANK deletion mutants were generated by PCRs using the proofreading Turbo Taq polymerase and were ligated into the pcDNA3-Myc vector. The primers used for ANK deletion mutants were as follows: 5'-CCGGAATTCAGGATGGAACTCCTTGCACCAG-3' and 5'-CCGCTCGAGTTATCCATCTTGAATAGC-3' for ASB3-ANK1-4, 5'-CAGGAATTCTAAAGTAAGCCCTGTTTACTC-3' and 5'-CCGCTCGAGTTATCCATCTTGAATAGC-3' for ASB3-ANK1-8, and 5'-CAGGAATTCAGTTCCATCCCTGACCCATC-3' and 5'-CCGCTCGAGTTATCCATCTTGAATAGC-3' for ASB3-ANK12. K
R point mutant TNF-R2 constructs were generated by performing site-directed mutagenesis (Stratagene). All transient transfections were carried out in serum-free medium with the indicated amounts of plasmids by using either Lipofectamine or Lipofectamine 2000 (Invitrogen). Whole extracts from 293 cells (106) after transfection with expression constructs were prepared with RIPA lysis buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 0.1% sodium dodecyl sulfate [SDS], 1 mM dithiothreitol, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM NaF) containing a protease inhibitor cocktail (Roche, Indianapolis, Ind.).
RT-PCR. Cells (106) growing in six-well clusters were washed with ice-cold phosphate-buffered saline and lysed with Trizol (Invitrogen). Total RNAs were isolated according to the manufacturer's protocol and subjected to reverse transcription with Superscript II reverse transcriptase (Invitrogen). The primers for ASB3 amplification were the same as those used for ASB3 cloning. The primers used for ß-actin amplification were 5'-TGCGTGACATTAAGGAGAAG-3' and 5'-GCTCGTAGCTCTTCTCCA-3'. Semiquantitative RT-PCR primers used for determinations of siRNA knockout efficiencies were as follows: for ASB3 amplification, 5'-CCAGATAAATGAACTTCATTTGGC-3' and 5'CCGCTCGAGTTATCCATCTTGAATAGC-3'; and for ß-actin amplification, 5'-TGCGTGACATTAAGGAGAAG-3' and 5'-GCTCGTAGCTCTTCTCCA-3'.
Antibody array, immunoprecipitation, and immunoblot analysis.
Antibody arrays were prepared by spotting 300 commercial antibodies from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.) onto a polyvinylidene difluoride (PVDF) membrane according to a previously published protocol (14). Antibody arrays were preincubated for 4 h at room temperature with 5% milk in Tris-buffered saline with Tween 20 (TBST) followed by incubation with whole-cell extracts (1.5 mg) overnight at 4°C. Unbound proteins were removed by washing with TBST at room temperature for three times. The array was then blotted with a horseradish peroxidase (HRP)-conjugated c-Myc antibody (Santa Cruz Biotechnology Inc.) for 4 h at room temperature followed by TBST washes at room temperature. Signal detection was performed by enhanced chemiluminescence (ECL) detection (Amersham Biosciences, Piscataway, N.J.). For immunoprecipitations, cells were washed twice with ice-cold phosphate-buffered saline and lysed in RIPA lysis buffer. Cleared cell extracts were immunoprecipitated with the indicated antibodies, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting coupled with fluorescent signal detection with an Odyssey fluorescence scanner (LI-COR Biosciences, Lincoln, Nebr.). The following antibodies were used for this study. Anti-c-Myc (9E10), anti-TNF-R2 (N-18 and L-20), anti-ubiquitin (PD41), and anti-IB (C-20) were purchased from Santa Cruz Biotechnology Inc. Anti-hemagglutinin (anti-HA) (12CA5), anti-mouse-IRDye800, and anti-rabbit-Alexa fluor 680 were purchased from Roche, Rockland Immunochemicals (Gilbertsville, Pa.), and Molecular Probes (Eugene, Oreg.), respectively.
In vitro kinase assay.
Anti-HA precipitates were prepared from 293 cells that were transiently transfected with HA-JNK1. The HA precipitates were washed extensively in kinase assay buffer (20 mM Tris [pH 7.6], 10 mM MgCl2, 3 mM EDTA, 1 mM vanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM NaF, and protease inhibitor cocktail) and then incubated in kinase assay buffer containing 0.2 µCi/µl [
-32P]ATP (Amersham Biosciences) for 30 min at 30°C. Purified glutathione S-transferase (GST)-c-Jun was added as an exogenous substrate, and 1 µM ATP was added to initiate the reaction. The reactions were stopped by the addition of an equal volume of 2x SDS-PAGE sample buffer and then analyzed by SDS-PAGE followed by autoradiography.
In vitro ubiquitination assay. Whole extracts prepared from 293 cells transfected with c-Myc-ASB3 or TNF-R2 were incubated with agarose beads conjugated with anti-c-Myc or anti-TNF-R2 for immunoprecipitation. After being washed extensively with lysis buffer, the antibody-conjugated beads were resuspended in 50 µl of reaction buffer containing 20 mM HEPES (pH 7.3), 10 mM MgCl2, 1 mM dithiothreitol, 2 mM ATP, 5 µg/ml ubiquitin (Ub) or methyl-Ub, 50 µM ubiquitin-activating enzyme (E1), and 1 µM ubiquitin-conjugating enzyme (E2-UbcH5a) (Boston Biochem, Cambridge, Mass.). The reactions were incubated for 1.5 h at 37°C and were terminated by the addition of 2x SDS loading buffer. The reactions were then separated by SDS-PAGE and transferred to a PVDF membrane followed by anti-ubiquitin immunoblot analysis.
Luciferase reporter and cell death quantitation assays.
293 cells were cotransfected with 5x Gal4-luc, c-Jun-Gal4, a Renilla luciferase vector, and other constructs as indicated (1.5 µg of DNA in total). After 24 h, cells were harvested and luciferase activities were determined by use of a dual-luciferase assay kit from Promega (Madison, Wis.) according to the manufacturer's protocol and a Lumat luminometer from Berthold Technologies (Oak Ridge, Tenn.). The results shown are representative of three independent experiments performed in duplicate. Cell death was determined by the release of Renilla luciferase activity as described previously (17). 4E3 cells (2 x 106) were transfected with the pRL-TK Renilla reporter construct (1.5 µg) with either a control siRNA (200 nM) or the ASB3 siRNA (200 nM) by the use of Lipofectamine 2000. After 24 h of transfection, the cells were split into two wells and were either left untreated or treated with TNF- (10 ng/ml). The cells were lysed at the indicated times post-TNF- treatment, and the Renilla activity was measured by the addition of the Stop & Glow substrate (Promega, Madison, Wis.) with a Lumat luminometer. Cell death was calculated with the following formula: percent specific cytotoxicity = [100 – (experimental Renilla activity/control Renilla activity)] x 100.
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RESULTS |
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signaling factors in cells.
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treatment was repeatedly detected for cells expressing ASB3 (Fig. 2A). Membrane-associated tumor necrosis factor alpha (mTNF-) has been reported to preferentially bind to TNF-R2 (8, 20). Transient transfection of mTNF- reduced the TNF-R2 protein level and subsequent TNF-R2-ASB3 complex formation in 293 cells (Fig. 2D). Treatment of the cells with the 26S proteasome inhibitor MG132 reversed the negative effect of mTNF- on TNF-R2 and stabilized TNF-R2-ASB3 complex formation (Fig. 2D). These results strongly indicate that ASB3 can associate with TNF-R2 and affects TNF-R2's stability in a TNF- stimulation-dependent manner.
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ANK1-4) and ANK1 to ANK8 (ASB3ANK1-8) did not produce any detectable effect on ASB3-TNF-R2 complex formation (Fig. 3E). In contrast, further deletion of the remaining four ANK repeats (i.e., ASB3ANK1-12) completely disrupted the interaction between ASB3 and TNF-R2, suggesting that a partial ANK repeat domain is necessary and sufficient for ASB3 and TNF-R2 interactions. Furthermore, deletion of the SOCS box did not affect ASB3-TNF-R2 complex formation (Fig. 3E).
Primary and secondary structural analyses indicated that the ASB protein family and the SOCS family share important structural features within their SOCS box domains, such as conserved leucine and cysteine residues which are required for Elongin-B/C binding within the SOCS box 1-2-helix (Fig. 4A) (14). After profiling ubiquitinated proteins by using our antibody array technology, we found that multiple proteins, including TNF-R2, are ubiquitinated in 293 cells that have been transiently transfected with ASB3 and HA-ubiquitin (13), suggesting that ASB3 may function as a ubiquitin ligase. The SOCS box in SOCS family members has been proposed to be responsible for recruiting Elongins-BC/CUL-2/Rbx-1 for the formation of a ubiquitin E3 ligase complex (14). The interaction between ASB3 and Elongin-C was clearly detected in 293 cells (Fig. 4B), suggesting that ASB family members may also assemble ubiquitin E3 ligase complexes through their interactions with Elongin-C, thereby mediating the ubiquitination of their substrates (14, 18, 35).
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SB (Fig. 6B) nor ASB3ANK1-12 was able to induce TNF-R2 ubiquitination (data not shown). Likewise, TNF-R2424, which lacked the ASB3 binding site, was not ubiquitinated in the presence of ASB3 (Fig. 5C). Additionally, polyubiquitinated TNF-R2 accumulated when cells were pretreated with a proteasome inhibitor for 30 min, suggesting that ubiquitinated TNF-R2 may be targeted for proteasome-mediated degradation (Fig. 5B). To determine whether TNF-R2 or TNF-R2-associated proteins were ubiquitinated, we performed a two-step immunoprecipitation assay by boiling anti-TNF-R2 immunoprecipitates for 5 min, followed by a second round of immunoprecipitation with anti-TNF-R2 (17). After the removal of TNF-R2-associated proteins by boiling, the polyubiquitination pattern of TNF-R2 precipitates after reimmunoprecipitation was apparently not perturbed. In addition, the TNF-R2 ubiquitination intensity was enhanced by a TNF- treatment (Fig. 5D). We repeatedly observed TNF--dependent TNF-R2 ubiquitination following reimmunoprecipitation, presumably due to an enhanced sensitivity of detection of ubiquitinated TNF-R2 with this assay. Together, these data suggest that TNF-R2 is indeed ubiquitinated and that this modification is dependent on TNF-.
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The TNF-R2 C-terminal region has multiple lysine residues for ubiquitination and degradation.
Human TNF-R2 has four lysine residues (Lys417, Lys425, Lys447, and Lys459) in its C-terminal region, where ASB3 docks (Fig. 6A). To determine whether these lysine residues are responsible for TNF-R2 ubiquitination, we made TNF-R2 constructs with KR mutations and evaluated their ubiquitination profiles in 293 cells. Immunoblotting confirmed that all lysine point mutants were expressed at a level similar to that of wild-type TNF-R2 (Fig. 6B). TNF-R2 polyubiquitination, however, was markedly impaired for both TNF-R2K425R and TNF-R2K459R, whereas the ubiquitination levels of TNF-R2K417R and TNF-R2K447R appeared to be unaffected (Fig. 6B, top panel). A LysArg substitution in all four single point mutants (TNF-R2K417R, TNF-R2K425R, TNF-R2K447R, and TNF-R2K459R) did not affect ASB3 docking within the C terminus of TNF-R2 (Fig. 6B, middle panel), indicating that the reduction in polyubiquitination was not due to a loss of the interaction between TNF-R2 mutants and ASB3 or to gross conformational changes in the mutant proteins. These data suggest that residues K425 and K459 are both specific ubiquitin acceptor sites.
Polyubiquitin modification via K48-linked ubiquitin chains serves as the recognition signal for the 26S proteasome, targeting ubiquitin-modified proteins for proteasome-mediated degradation (9, 10). The TNF-R2 protein was steadily degraded over the course of a 6-h cycloheximide (CHX) chase in 293 cells when TNF-R2 was cotransfected with ASB3 but not with ASB3SB (Fig. 7A), indicating the requirement of the SOCS box for TNF-R2 degradation. Under the same conditions, the stability of TNF-R2-associated proteins such as TRAF2 (TNF receptor-associated factor 2) was unaffected by ASB3, suggesting that ASB3 specifically targets TNF-R2 for degradation (Fig. 7A, bottom panel). ASB3-mediated TNF-R2 degradation proceeds through a proteasome-mediated pathway, as its degradation was blocked by treatment of the cells with MG132, a reversible inhibitor of the 26S proteasome, but not by treatment with NH4Cl, a lysosomal inhibitor used for CHX chase experiments (Fig. 7B). Unlike wild-type TNF-R2, the TNF-R2 K425R and K459R mutants both remained relatively stable in the CHX chase experiment, corroborating our previous findings that both the TNF-R2K425R and TNF-R2K459R mutants are resistant to ubiquitination. In contrast, using TNF-R2K447R as an example of a mutant with a wild-type ubiquitination profile, we observed that this KR substitution did not alter the TNF-R2 degradation kinetics (Fig. 7C), consistent with our previous results showing that K447 is not a ubiquitination site (Fig. 6B).
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ASB3 inhibits TNF-R2-mediated JNK activation and apoptosis induction.
TNF-R2, in response to TNF-, activates the JNK pathway via the recruitment of TRAF2 (26). We examined the effect of ASB3 on JNK activation by performing in vitro JNK kinase assays. In a cell-free system, TNF-R2 mediated JNK activation in response to TNF- treatment (Fig. 8A). In addition, the coexpression of ASB-3 but not a mutant with the SOCS box deleted (ASB3SB) dramatically inhibited TNF-R2-dependent JNK activation (Fig. 8A). Consistent with our observations (Fig. 8B), the overexpression of TRAF2 alone has been shown to be sufficient for the induction of JNK activation, independent of a receptor association (24). In vitro JNK protein kinase activation by TRAF2 was not affected by ASB3 overexpression in 293 cells (Fig. 8B), indicating that ASB-3 likely functions upstream of TRAF2. We then evaluated the effect of ASB3 on JNK signaling as a function of c-Jun transcriptional activity measured by a c-Jun-Gal4- and Gal4-responsive luciferase reporter assay. While the expression of mTNF- or TNF-R2 alone could activate JNK, TNF-R2 and mTNF- together further enhanced JNK activation (Fig. 8C). The expression of ASB3, however, dramatically inhibited JNK activation by mTNF- in 293 cells (Fig. 8C). Interestingly, ASB3SB still partially inhibited JNK activation in both an in vitro kinase assay (Fig. 8A) and an in vivo reporter assay (Fig. 8B), even though ASB3SB had no detectable effect on TNF-R2 stability (Fig. 8D). Furthermore, analyses of TNF-R2 protein levels in the same cells revealed that TNF-R2 was down-regulated in the presence of ASB3 (Fig. 8D). Together, these findings provide further evidence that ASB3 is a TNF-R2-specific negative regulator which inhibits TNF-R2-JNK signal transduction, mainly by altering the TNF-R2 protein's stability.
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treatment (Fig. 9C). A knockdown of ASB3 in 4E3 cells attenuated TNF-R2 down-regulation in both untreated cells and cells treated with TNF- for 15 min and 4 h (Fig. 9D and E). JNK activation following TNF- treatment was markedly enhanced in 4E3 cells compared to that in Jurkat cells (Fig. 9B), consistent with previous findings that the overexpression of TNF-R2 can preferentially trigger JNK pathway activation (16, 20). In contrast, IB degradation by TNF- was moderately enhanced in 4E3 cells compared to that in parental Jurkat cells (Fig. 9A).
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-activated TNF-R2 has been shown to induce T-cell apoptosis via JNK activation (3, 5), we examined whether ASB3 can regulate TNF-R2-dependent apoptosis in 4E3 cells. In 4E3 cells, apparent cell death, as measured by a loss of Renilla luciferase activity (Fig. 9E), was induced by a TNF- treatment at 6 h, agreeing with previous reports that the overexpression of TNF-R2 sensitizes 4E3 cells to apoptosis in response to TNF- (3, 17). A knockdown of the asb3 mRNA in 4E3 cells resulted in stabilization of the TNF-R2 protein level (Fig. 9E) and further potentiated apoptosis induction in response to TNF- treatment (Fig. 9E). Collectively, these studies indicate that ASB3 can affect T-cell signaling via the polyubiquitination and degradation of TNF-R2 and can thereby regulate downstream signaling events in response to TNF-. |
DISCUSSION |
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. Our findings also delineate the putative mechanism underlying this regulation, including the recruitment of the ASB3/Elongin-BC/Cullin/Rbx-1 E3 ligase complex and the ubiquitination of TNF-R2 followed by its degradation. While several factors have been found to inhibit TNF-R2 signaling by targeting downstream TNF-R2 signaling molecules (17, 34), ASB3 does so by directly targeting TNF-R2.
The first described SOCS box protein, SOCS-1, was demonstrated to inhibit both interleukin-6-induced receptor phosphorylation and STAT activation (6, 23, 31) by targeting JAK (7). For SOCS family members, the C-terminal helical SOCS box has been shown to be responsible for E3 ligase complex formation by recruiting Elongins-B/C and Cul2 (35). The -helixes found in the C termini of ASB family members correspond to a typical SOCS box domain (ASB3 residues 460 to 518). A detailed sequence analysis indicated that the SOCS box regions of the SOCS and ASB families share conserved residues responsible for binding to Elongin-C (L462 and C466 in ASB3) (14). In addition, ASB3 has two arginine residues (R467 and R471) that are conserved among the 18 known family members and that may also contribute to the interaction with Elongin-C (Fig. 4A). The region within the SOCS box that has been demonstrated to associate with the ubiquitin ligase complex adapters Elongins-B/C (termed the BC box) has the consensus sequence A(P,S,T)LXXXCXXXA(I,L,V) (14). This consensus sequence was originally identified in Elongin-A and the VHL tumor suppressor protein (15) and is also present in ASB3 (S461LTHLCRLEI) and SOCS-1, both of which were shown here to bind to Elongin-C. TNF-R2 polyubiquitination is evident by both in vitro and in vivo methods and is dependent on ASB3 binding. The SOCS box domain of ASB3 is critical for this process, as demonstrated by the loss of TNF-R2 polyubiquitination when TNF-R2 was coexpressed with a mutant with a SOCS box deletion (ASB3SB). SOCS-1 has been shown to regulate the half-lives of the signaling molecules JAK and VAV by bridging them to the E3 ubiquitin ligase complex (5, 7). It appears reasonable that ASB3 acts similarly as the substrate recognition component of the E3 ubiquitin ligase complex through its interaction with Elongin-C in mediating the degradation of TNF-R2.
The amino acid sequence of ASB3 predicts a 518-amino-acid peptide with a short N-terminal sequence followed by 12 ANK repeats. ANK proteins, such as cyclin-dependent kinase (cdk) inhibitor family members (e.g., p16INK4a) and inhibitors of NF-B signal transduction (i.e., IB family members), have a wide range of biological activities (30). ANK repeats form helix-turn-helix motifs that are linked together by loops (30). Studies have shown that some loops serve as sites of protein-protein interactions, while the ANK repeats provide a stabilizing platform (1). Although ASB3 has 12 ANK repeats, a partial ANK repeat region (i.e., ANK9-12) was sufficient for its interaction with TNF-R2, suggesting that ASB3 may require only a partial recognition of its substrate for interaction. Similarly, both p16INK4a and IBß use partial ANK repeats for interaction with their substrates (1, 19). Additional ANK repeats may therefore simply function to further stabilize substrate binding or may be involved simultaneously in interactions with other substrates (30). Like the ANK repeats of p16INK4a and IBß (1, 19), the loop sequences of the ASB3 ANK repeats contain charged residues, which may be responsible for mediating ionic interactions with residues in the substrate. Although a conserved ANK repeat interaction motif has not yet been identified, Arg47 within the ANK repeats of p16INK4a has been shown to interact with Glu7 of an SXXE motif in the N terminus of its substrate, cdk4 (1). This interaction is primarily mediated by interactions between charged residues (1). The C terminus of TNF-R2 bears three such SXXE motifs, while TRAF2 has been shown to dock on one of these SXXE motifs (25). Whether SXXE motifs provide a modular binding motif for ANK repeats therefore becomes a very intriguing question.
The TNF-R2 C terminus also provides a docking site for TRAF2. The major known signaling factor downstream of TNF-R2 is TRAF2, which is a RING finger domain-containing protein (27). Reimmunoprecipitation experiments with TNF-R2 demonstrated that ASB3 mediates the ubiquitination of TNF-R2 itself (Fig. 5D). This excludes the possibility of misinterpreting the detection of polyubiquitinated TNF-R2 for that of TRAF2 or other TNF-R2-associated proteins. Additionally, our in vitro ubiquitination results indicated that TNF-R2 does not self-ubiquitinate and, in all likelihood, is not ubiquitinated by TRAF2. This is evidenced by the in vitro ubiquitination data shown in Fig. 5E (lane 1), which indicate that TRAF2 coimmunopurified with TNF-R2 from transfected 293 cells (data not shown) was unable to mediate a ubiquitination reaction. Only the addition of ASB3 to the in vitro ubiquitin reactions catalyzed TNF-R2 polyubiquitination.
Although TNF-R2 couples to the prosurvival TRAF2/NF-B signaling pathway, TNF-R2 activation has also been shown to induce apoptosis in T cells and several tumor cells (3, 4, 11, 21, 28, 36). In these cells, signals from TNF-R2 dramatically potentiate the magnitude and kinetics of caspase activation and apoptosis induction from TNF-R1, which presumably overcomes the induced NF-B antiapoptotic response (3). It was previously demonstrated that the overexpression of TNF-R2 in Jurkat cells overcomes the cells' innate resistance to TNF--induced apoptosis (3). This suggests that the cellular response to TNF- can be affected by the level of plasma-membrane-bound TNF-R2. Consistent with these findings, we observed that the overexpression of TNF-R2 in Jurkat cells rendered these cells susceptible to TNF--induced cell death. Additionally, a knockdown of endogenous asb3 in the 4E3 cell line stabilized TNF-R2 and potentiated TNF-R2-mediated cytotoxicity in response to TNF-. ASB3 modification of the surface availability of TNF-R2 may therefore be another cellular mechanism for altering cellular responses to TNF- stimulation.
TNF-R2 plays a crucial role in several inflammatory responses, and the up-regulation of this receptor has been linked to several diseases, including rheumatoid arthritis (2). Etanercept or Enbrel is a recombinant form of TNF-R2 used to treat rheumatoid arthritis via antagonizing TNF- actions (2, 22). Our data demonstrate that ASB3 attenuates TNF-R2 signaling in response to TNF- by mediating TNF-R2 proteolysis. These findings imply that targeting ASB3 may provide a novel means for the optimization of therapeutic efforts aimed at neutralizing the inflammatory effects of TNF-.
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ACKNOWLEDGMENTS |
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This work was supported by an RO1 grant (CA-82549) from the National Institutes of Health (NIH) (to Y.E.C.) and by a COBRE grant from the NIH to Brown University. A.S.C. was supported by an NIH predoctoral training grant (GM-07601).
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FOOTNOTES |
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