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Previous Article | Next Article 
Molecular and Cellular Biology, July 2007, p. 4708-4719, Vol. 27, No. 13
0270-7306/07/$08.00+0 doi:10.1128/MCB.02432-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520,1 Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan,2 Biomolecular Characterization, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan,3 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731044
Received 29 December 2006/ Returned for modification 12 February 2007/ Accepted 17 April 2007
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ubiquitin thioester intermediate. Finally, the ubiquitin-charged E2 cooperates with an E3 ligase to attach ubiquitin to a substrate protein. For those substrates targeted to the 26S proteasome, the initial ubiquitin attachment is followed by chain elongation (14). DET1 (de-etiolated 1) was originally isolated as a key regulator of light-activated development in Arabidopsis thaliana (6) and in tomato (known as HP2 for high pigment 2) (26). Det1 mutants have an altered gene expression pattern and are unable to undergo the etiolation developmental path in darkness (6, 24, 28, 33). In addition, the weak mutants are hypersensitive to light signals and exhibit an abnormal circadian rhythm. DET1 has been shown to negatively regulate ubiquitin-proteasome-mediated turnover of an important circadian clock regulator, LHY (late elongated hypocotyl) (34). DET1 forms a protein complex known as the CDD complex with the Arabidopsis homolog of DDB1 (damaged DNA binding protein 1) (33) and COP10 (41). COP10 is a ubiquitin E2 variant (UEV) that has the conserved catalytic core domain (UBC) but lacks the cysteine residue that forms the thioester linkage with ubiquitin and is therefore catalytically inactive (36). COP10 is distinct from other known UEVs, such as MMS2, UEV1, and TSG101, as it displays highest homology to the Saccharomyces cerevisiae Ubc4/5 family of canonical E2s (20, 36). The Arabidopsis COP10 belongs to the COP/DET/FUS class of loci that also includes conserved genes such as DET1, COP1, and most of the COP9 signalosome (CSN) genes. This group is defined by the characteristic photomorphogenic phenotype of the mutant plants (38, 42). Typically, cop/det/fus mutants, now including tomato hp1(ddb1), display a constitutive or hypersensitive light response (6, 22, 24). Recent studies have demonstrated that members of this group mediate their functions via the ubiquitin system (38, 42). Unlike DET1 and DDB1, the human counterpart of COP10, has not been identified to date by conventional bioinformatic approaches. Likewise, it remains unclear whether a homologous CDD complex exists in animals.
DDB1 functions as an adaptor for the Cul4A ubiquitin ligase, a member of the cullin-RING ubiquitin E3 ligase family (CRL). DDB1 forms a complex with WD domain-containing substrate receptors and assembles with Cul4A-Rbx1 (Roc1/Hrt1) into specific E3 ligase complexes (1, 10, 11, 17). The Cul4A-DDB1 E3 ligases regulate cell cycle progression, replication, and DNA damage responses (4, 13, 17, 31, 35). One of the Cul4A-DDB1 substrates is the DNA replication licensing factor CDT1, which is degraded in S phase and after DNA damage (13, 17).
Human DET1 has been shown to interact with human COP1, DDB1, and Cul4A and together mediate c-Jun ubiquitination and degradation (39). In addition, SCFFWD7 and Itch, which are CRL family E3 and HECT-domain E3, respectively, have also been demonstrated to mediate signaling-dependent ubiquitination of c-Jun in various biological contexts (9, 27). In this study, we report that DET1, which does not contain a WD40 domain, exhibits unique characteristics distinct from conventional Cul4-DDB1 substrate receptors, such as DDB2, CSA, and Cdt2. Our data show that DET1 interacts with multiple E3 ubiquitin ligases and negatively regulates Cul4A-mediated polyubiquitination in vitro. In addition, mammalian DET1 forms a stable DDD core complex with DDB1 and DDA (DET1, DDB1-associated 1). The DDD complex recruits a member of the UBE2E group of canonical E2s, forming a DDD-E2 complex. The UBE2E group shows highest sequence homology to COP10 in the Arabidopsis genome. Moreover, the DDD-associated UBE2E-3 is not charged with ubiquitin, while the free UBE2E-3 is. We suggest that the DDD-E2 complex is the mammalian counterpart of the Arabidopsis CDD complex.
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Purification of Flag-DET1 complex. To generate Flag-DET1 stable cell lines in HEK293 cells, pFlag-DET1 was cotransfected with pBABE, which confers puromycin resistance. One day later, cells were replated in selection medium containing 3 µg/ml puromycin (Sigma). A cell line with moderate expression of Flag-DET1 was chosen for purification of the complex.
Flag-DET1 cells from 40 15-cm dishes were collected 48 h after seeding. Cells were extracted by sonication in hypotonic buffer (20 mM HEPES pH 7.2, 5 mM KCl, 2.5 mM EDTA) supplemented with Complete protease inhibitors (Roche) and 0.4% NP-40. The extract was mixed with high-salt buffer (20 mM Tris pH 7.4, 1 M NaCl, 0.4% NP-40, 10% glycerol) to a final volume of 40% (vol/vol), incubated on ice for 10 min, and clarified by centrifugation (14,000 x g for 15 min at 4°C). M2 beads (Sigma) were incubated with the supernatant in a ratio of 80 µl slurry to 6 mg of total protein for 6 to 12 h on a roller at 4°C. The beads were washed in 50% high-salt buffer twice for 10 min at 4°C and then twice in elution buffer (25 mM Tris-HCL, pH 7.4, 150 mM NaCl, 10% glycerol). Finally, Flag peptide (1 mg/ml; Sigma) dissolved in elution buffer was incubated with the sample for 15 min at room temperature. To estimate the quantity of the complex, first the amount of associated E2 was determined based on silver staining intensity, and then the amount of the complex was calculated assuming that the E2s represented 10% of the total mass of the complex.
Gel filtration chromatography and immunoprecipitation. Cells were washed twice in phosphate-buffered saline (PBS) and collected in cold hypotonic buffer supplemented with 1 mM dithiothreitol (DTT), 0.1% NP-40, and protease inhibitor cocktail (Roche), with or without 10 mM ATP and 20 mM MgCl2. Cells were lysed by freeze-thaw in liquid nitrogen and a 37°C water bath or sonication. The samples were cleared by microcentrifugation for 10 min at 4°C. Supernatant was filtered through a 0.2-µm filter and applied to a prepacked Superose-6 gel filtration column (Amersham-Pharmacia). The equilibration and elution buffers contained 20 mM Tris-HCl pH 7.2, 200 mM NaCl, and 10% glycerol. Fractions of 0.5 ml each were collected starting from 7 ml and were mixed with sample buffer for immunoblot analysis.
For immunoprecipitation, cell extract was prepared in hypotonic buffer and sonicated as described above. The extract was then mixed 1:1 with PBS buffer containing 0.4% Triton X-100 and incubated on ice for 10 min. The sample was clarified by centrifugation (14,000 x g for 15 min at 4°C) twice, and the supernatant was incubated with antibody-linked agarose beads for 4 to 12 h at 4°C. The beads were washed four times with PBS buffer containing 0.1% Tween 20. For T7-Cul4A and Flag-DET1 complexes used for in vitro E3 assays, two additional washes with the ubiquitin reaction buffer were performed.
UV treatment. UV treatment of HeLa cells in 12-well or 6-well dishes was carried out 24 hours after transfection. Culture medium was removed, and the cells were washed once in PBS. The dishes were placed under UV-C lamps (254 nm) in the chamber of a UV Stratalinker 2400 model (Stratagene). After irradiation (40 J/m2), culture medium was added back and the cells were returned to the incubator for the specified times. To collect the samples, cells were washed with PBS and directly lysed with 3x sodium dodecyl sulfate (SDS) sample buffer, with about 80 µl per well for 12-well dishes and 160 µl for 6-well dishes. The samples were boiled for 6 to 10 min and cleared by centrifugation before SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
Antibodies and plasmids. To generate antibodies against UBE2-E1 and UBE2E2, N-terminal amino acid (aa) residues 1 to 44 of UBE2E-1 or aa residues 1 to 52 of the UBE2E-2 fragment were expressed as His-S-tagged fusion proteins in BL21 cells. Proteins were purified by Ni-nitrilotriacetic acid (NTA) affinity resin and introduced into rabbits for antibody production. Each antibody was then demonstrated to recognize only itself but not other homologous E2s. Antibodies specific to UBE2E-3 or UbcM2 were made against the specific N-terminal extension as described elsewhere (7, 30).
Anti-CDT1 (13), anti-COP1 (42), and anti-COP10 (36) have been described previously. Anti-Flag (M2; Sigma), anti-His (penta-His monoclonal; QIAGEN), antiubiquitin (UG9510; BioMol/Affinity), anti-UbcH5 and anti-UbcH12 (Boston Biochem), anti-elongin B (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA; sc-805; Santa Cruz), and anti-glutathione S-transferase (anti-GST; Amersham Bioscience) were purchased. The rabbit antibody against Cul4A was raised against the peptide ERDKDNPNQYHYVA (ProteinTech Group, Inc.). The rabbit antibody against human DDA was raised against the C-terminal peptide CAPPRKVARTDSPDMHEDT (ProteinTech Group, Inc.). Rabbit anti-DDB1 was raised against the human DDB1 C-terminal 384-aa fragment (aa 756 to 1140), which was produced in Escherichia coli from pET-DDB1C384 and purified by using Ni-NTA. Similarly, anti-DET1 antibody was raised against a C-terminal region of mouse DET1 expressed from pRSET-DET1C in E. coli.
Information for construction of plasmids used in this study is available upon request.
Purification of recombinant proteins and complexes. Recombinant proteins were expressed in the E. coli BL21(DE3) strain carrying the specified plasmids. The DDA1/DDB1 complex was obtained by expressing His-DDA1 and untagged DDB1 from the pET-Duet-HisDDA1-DDB1 vector. The DDA1/DET1 complex was obtained by expressing His-DDA1 and a T7-tagged DET1 from the pET-Duet-HisDDA1-T7DET1 vector (see Fig. 3A, below). The DDA1/DDB1/DET1 (DDD) complex was obtained by coexpressing pET-Duet-HisDDA1-DDB1 (Ampr) and pET28-HisT7DET1 (Kanr). All three complexes were purified via His tag (see Fig. 3A) as described below.
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E2ubiquitin thioester assay.
To detect the E2ubiquitin thioester linkage in vitro, 100 ng of recombinant GST-UBE2E-3 purified from E. coli was incubated with 50 ng E1 (Boston Biochem), 50 µM ubiquitin (Sigma) in the reaction buffer (25 mM Tris-HCl pH 7.4, 3 mM ATP, 5 mM MgCl2, 0.5 mM DTT) for 10 min at 30°C. The sample was incubated with an equal volume of nonreducing SDS sample buffer (8 M urea, 4% SDS, 15% glycerol, 50 mM Tris-HCl pH 6.8, and 0.001% bromophenol blue) for 30 min at 30°C. For reduced samples, a half volume of each of the nonreduced samples was mixed with DTT to a final concentration of 50 mM and boiled for 5 min before loading onto the SDS-PAGE, followed by immunoblotting with anti-GST or anti-UbcM2 antibodies. To detect the endogenous ubiquitin-charged form of UBE2E-3, cells were extracted in an ATP-containing buffer as described in "Gel filtration chromatography and immunoprecipitation," above. Samples were mixed in nonreducing or reducing sample buffer for immunoblotting.
In vitro E3 ligase activity assay. For the T7-Cul4A immunocomplex activity assay, various components as indicated in the figures were incubated at 30°C for 60 min in 40 µl of assay buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM DTT, and 3 mM ATP. Additional ingredients included 50 ng E1 (Boston Biochem), UbcH5c (Boston Biochem) in amounts specified in the figure legends, and 5 µg ubiquitin in a 1:5 ratio of N-terminal biotinylated ubiquitin (Boston Biochem) to unlabeled ubiquitin (Sigma). T7-Cul4A immunocomplex was obtained by immunoprecipitation from transfected 293 cells using anti-T7-conjugated resin (Novagen).
For the recombinant Cul4 and Cul1 complex experiment, reactions were performed in a total volume of 15 µl that contained 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM ATP, 50 ng yeast E1 (Boston Biochem), 200 ng UbcH5b (Boston Biochem), and 5 µg ubiquitin (Boston Biochem), together with either 300 ng GST-Roc1-Cul1(324-776) (a gift from Zhen-Qiang Pan, Mount Sinai School of Medicine) or 2 µg of DDB1/CUL4A/Rbx1 (a gift from Ning Zheng, University of Washington). Varying amounts of bacterially purified His-DDA1/DDB1, His-DDA1/T7-DET1, or His-DDA1/T7His-DET1/DDB1 (recombinant DDD complex [rDDD]) complexes and bovine serum albumin (Sigma) were added to the reaction mixtures as specified in the figure legends. Reaction mixtures were incubated for 1 h at 37°C before being terminated with DTT-containing SDS sample buffer.
Nucleotide sequence accession number. The sequence for DDA1 was submitted to GenBank (accession number DQ090952).
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A group of specific bands around 24 to 28 kDa were extensively analyzed. A peptide (UBE2E-3 aa 126 to 161) encompassing a conserved region of the UBC domain, identical among the UBE2E group of E2s, was repeatedly recovered (Fig. 1C). In addition, peptide sequences matching UBE2E-3 aa 68 to 94 and UBE2E-1 (or UbcH6) aa 54 to 80 were obtained, indicating that multiple E2s from the UBE2E family were in the complex. Each member of this family has a unique N-terminal extension, an identifying feature of class III E2s (25), and is highly homologous to yeast Ubc4/5 and Arabidopsis COP10 at the UBC domain (Fig. 1C). Importantly, because the peptide sequence data covered the region containing the active site cysteine-145 (UBE2E-3), we can definitively conclude that these proteins are canonical ubiquitin-conjugating enzymes rather than E2 variants.
Our data showed that stably expressed Flag-DET1 forms a stable protein complex consisting of DDB1, DDA1, and UBE2E-3 E2 enzyme. To verify that this complex exists in normal cells, we generated antibodies against DET1 and DDA1. Immunoprecipitation of endogenous DET1 or DDA1 from HEK293 cells readily pulled down DDB1, DDA1, and UBE2E-3 (Fig. 2A). In addition, DDA1, but not DET1, also coimmunoprecipitated CSN3 (Fig. 2A).
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Two different scenarios can be envisioned with regard to how multiple E2s are held in the complex. In one model, two or more E2 molecules are bound simultaneously as a dimer or through multiple E2-docking sites in one complex. Alternatively, each complex contains a single E2 molecule and different E2s are assembled into different DET1 complexes. These two models can be distinguished by asking whether the E2s can associate with each other. Each of the UBE2E group of E2s was immunoprecipitated from Flag-DET1 stable cells using specific antibodies for each enzyme (Fig. 2D). Each specific E2 antibody only precipitated itself and DDA1, but not the other E2 enzymes (lanes 5 to 7), while Flag-DET1 and DDA1 pulled down all of the E2s (lanes 2 and 4). These data conclusively show that UBE2E-1, UBE2E-2, and UBE2E-3 do not form stable heterodimers under these experimental conditions, nor are they present in a single DET1 complex. Therefore, each DET1 complex carries a single E2 enzyme, while different E2s are held in different complexes.
To distinguish different UBE2E-containing complexes, we hereafter refer to the complex composed of DET1, DDB1, and DDA1 as the DDD core complex. The DDD core recruits a specific UBE2E enzyme, for example, UBE2E-3, to assemble a specific DDD-UBE2E-3 holo-complex. Our data show that different UBE2E enzymes assemble with distinct DDD cores to form a heterogeneous population of DDD-E2 complexes in the cell.
DDA1 directly binds DET1 and DDB1 and is an integral subunit of the DDD core complex. We further characterized DDA1 with respect to its interactions with DDB1, DET1, and E2s. Although recombinant DDB1 and T7-DET1 were insoluble when expressed alone in E. coli, each formed a soluble complex when coexpressed with His-DDA1 and coeluted with it from the Ni-NTA resin (Fig. 3A). This result indicates that DDA1 is able to bind DDB1 and DET1 independently. In mammalian cells, anti-DDA1 antibody efficiently coprecipitated DDB1, DDA1, and UBE2Es (Fig. 2A and D), as well as Cul4A (Fig. 3B). Notably, overexpression of DET1 significantly increased the association of DDA1 with the E2, while its association with DDB1 or Cul4A remained unchanged (Fig. 3B, lanes 6 and 8). This result indicates that DET1 is the limiting factor for E2 binding.
DDB1 folds into a three-propeller structure (21). To understand how DDB1 binds DDA1, we expressed a series of DDB1 truncation mutants in cultured cells and tested their ability to bind DDA1. As shown in Fig. 3C, DDB1 N terminal aa 1 to 392, which form propeller A (BPA), is necessary and sufficient to bind DDA1. Thus, the DDA1 binding site resides in the BPA propeller of DDB1.
UBE2E-3 dynamic association with DET1 and role of its N-terminal extension. Gel filtration fractionation of cell extract showed that Flag-DET1 and DDA1 predominantly coeluted in the 250-kDa fractions as DDD-E2 complexes (Fig. 4A). A low-abundance shoulder of approximately 650 kDa was also detected. In addition, DDB1 displayed a wide elution profile, while the CSN1 blot served as an internal marker indicating the CSN peak position of approximately 500 kDa. Notably, UBE2E-3 was separated into two populations; about 10% or less cofractionated with DET1 and DDA1, while the majority eluted as "free UBE2E-3" in fractions corresponding to its monomer size (Fig. 4A). Other UBE2Es behave similarly to UBE2E-3 according to an anti-COP10 blot on these fractions (not shown). In addition, we found that association of UBE2E-3 with DET1 appeared to be more sensitive to ATP concentrations above 15 mM compared to DDA1 (Fig. 4B) and that knockdown of the UBE2E family of E2s does not detectably affect association of DET1, DDB1, and DDA1 (data not shown). We suggest that E2 association with DDD is a dynamic event, as illustrated in Fig. 4E.
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Association of uncharged UBE2E-3 with the DDD core complex.
The function of E2s involves the formation of an unstable E2Ub thioester intermediate via the catalytic site cysteine residue (ubiquitin loading or charging). We set out to determine whether the DDD core complex has a binding preference for the catalytic state of the E2. Toward this end, a recombinant complex comprised of HisT7-DET1, His-DDA1, and substoichiometric amounts of untagged DDB1 was isolated from E. coli (rDDD) (Fig. 5A). This complex could bind to GST-UBE2E-3 in vitro without ubiquitin charging (Fig. 5B, lane 6), indicating that ubiquitin charging is not required for the E2s to associate with DDD.
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ubiquitin thioester conjugates could be detected in total cell extracts and the unbound fraction, but not in DET1-bound samples (Fig. 5C). This result indicates that DET1 preferentially associates with the uncharged form of UBE2E-3. Further, we examined the distribution of ubiquitin-charged and uncharged UBE2E-3 in the gel filtration fractions. Remarkably, all of the ubiquitin-charged UBE2E-3 was found in the free E2 fractions, while the complex-bound UBE2E-3 was exclusively in the uncharged state (Fig. 5D). The relative ratio of the complex-associated and free UBE2E-3 did not significantly change with or without the 10 mM ATP supplement (Fig. 4A and 5D). An in vitro thioester assay was carried out to compare the ubiquitin-charging efficiency of recombinant GST-UBE2E-3 and DDD-UBE2E-3, the latter isolated as Flag-DET1 immunocomplex. Recombinant GST-UBE2E-3 could be detectably charged with ubiquitin by using as little as 20 ng of the protein (Fig. 5E, lanes 4 to 6). In contrast, the Flag-DET1 complex, which contained more than 40 ng of UBE2E-3 as judged by immunoblotting and silver staining, failed to produce a detectable level of ubiquitin thioester conjugates (Fig. 5E, lanes 1 and 2). Because the E2s do not carry thioester-linked ubiquitin in the DDD-E2 form, mammalian DDD-E2 complexes essentially resemble the Arabidopsis CDD complex, which is incapable of forming a ubiquitin thioester.
Association of DET1 with multiple E3 ubiquitin ligases. Substrate receptors for Cul4A-DDB1 E3s, such as DDB2, CSA, and Cdt2 WD40 proteins, can pull down approximately stoichiometric amounts of Cul4A and Rbx1, as well as abundant amounts of CSN components (10, 13, 23). Though DET1 resembles these WD40 proteins in binding to DDB1, we did not recover any peptides matching Cul4A or CSNs in the Flag-DET1 immunocomplex. Nonetheless, we detected COP1 and Cul4A in the Flag-DET1 complexes by immunoblotting (Fig. 6A) as previously reported (39). DET1 also coprecipitated a 75-kDa form of Mdm2. In addition, a transiently expressed ARA54, a RING E3 previously shown to bind the UBE2E family of E2s (16), could coprecipitate with DET1 (Fig. 6B). However, unlike CDT2, CSA1, and DDB2, the DET1 complexes did not contain detectable amounts of CSNs even by immunoblotting. In addition, DET1 showed no interaction with elongin B, a component of the Cul2 E3 complex (Fig. 6A), or Cul1 (not shown). Our data are consistent with the notion that DDD-E2 accumulates as a distinct protein complex that transiently interacts with Cul4A and other E3 ligases.
Free UBE2E-3, but not DDD-UBE2E-3, can act as an active E2 or an E2 enhancer for the Cul4A E3 ligase. Toward understanding the function of DDD-E2, we first tested whether this E2-containing complex can act as an E2 to support Cul4A E3 activity in vitro. T7-Cul4A immunocomplex was isolated from transfected cells, and Flag-DET1 immunocomplexes or UbcH5 were used as the sources of E2 (Fig. 6C). As widely used in combination with CRL E3s in vitro, UbcH5 was active in supporting T7-Cul4A immunocomplex, as indicated by the high-molecular-weight polyubiquitin products (lanes 4 and 5). These polyubiquitin products could be unanchored or could be attached to heterogeneous proteins in the T7-Cul4A immunocomplex. Since these polyubiquitin products were generated in a T7-Cul4A- and UbcH5-dependent fashion (Fig. 6C), we used it as a measure of Cul4A E3 activity. We found that the Cul4A complexes were active when supplied with UbcH5 (lane 4), but not with the DET1 complexes (lane 8). These data indicate that DDD-E2 complexes do not have classical E2 activities for Cul4A.
In the following series of experiments, we have dissected DDD-E2 complexes and addressed the activities of DDD and the associated E2s separately. With the same experimental setup as in Fig. 6C, we found that UBE2E-3 was an active E2 for Cul4A in vitro (Fig. 6D, lane 5). Curiously, the ubiquitin chains assembled by UBE2E-3 differed from those assembled by UbcH5, as the former generated shorter and more discrete ubiquitin conjugates in a Cul4A-dependent manner (Fig. 6D, lane 5, compared to E, lanes 1 and 3). The UBE2E-3(C/S) mutant, in which the catalytic cysteine residue was mutated to serine (30), was inactive on its own (Fig. 6D); however, it significantly enhanced UbcH5/Cul4A activity (Fig. 6E, lane 3, compared to lane 1). This observation echoes the E2-enhancing activity described for COP10 UEV (41). While UBE2E-3(C/S) enhanced the amount of polyubiquitin products, it did not change the characteristics of UbcH5-generated chains (Fig. 6E, lanes 1 and 3). Taken together, UBE2E-3 can act as an independent E2 or an E2 enhancer to support Cul4A ubiquitin ligase activity.
DET1/DDD inhibits Cul4A-mediated polyubiquitination in vitro. To investigate the role of DDD on Cul4A E3 activity, we first tested rDDD (Fig. 5A) in the Cul4A immunocomplex assay described above. Remarkably, addition of the rDDD complex at roughly double the molar amount relative to UBE2E-3 drastically inhibited all Cul4A-mediated polyubiquitination without affecting the level of Cul4A in the reaction mixtures (Fig. 6E, lanes 2, 4, and 6). The inhibition was observed in reaction mixtures containing UbcH5 (lanes 1 to 4) or combinations of UbcH5 and UBE2E-3 (lanes 5 and 6), suggesting that DDD-mediated inhibition of polyubiquitination is probably not specific for particular E2s.
We next addressed whether DDD exhibits any specificity at the level of E3s. Since DET1 interacts with Cul4A but not Cul1, we utilized a reconstituted Cul4A-DDB1-Rbx1 E3 complex (1) and a reconstituted Cul1-Rbx1 system comprised of the Cul1 C-terminal fragment (aa 324 to 776) in complex with Roc1 (Rbx1) (40). Both complexes were isolated from bacteria and were able to assemble polyubiquitin chains independent of substrate or substrate receptors (Fig. 7, lanes 3). In addition to rDDD complex, recombinant complexes containing DDA1/DDB1 and DDA1/DET1 were also tested. In Fig. 7A and B, recombinant complexes containing equal amounts of DDA1 were added to the reaction mixture, whereas in Fig. 7C and D, the complexes were normalized according to DET1. Addition of rDDD, but not partial complexes, robustly inhibited Cul4A-mediated polyubiquitination in both cases (Fig. 7A and C, lanes 8 and 9), while only slightly affecting Cul1 activity (Fig. 7B and D). The DDA1/DDB1 and DDA1/DET1 complexes were inactive, despite their containing equal or more DDA1, DDB1 (Fig. 7A), or DET1 (Fig. 7C and D) than DDD. This indicated that all three components were necessary for the inhibition. In addition, these results demonstrate that rDDD specifically inhibits Cul4A-dependent polyubiquitination, supporting the idea that direct interaction with the E3 is necessary for efficient inhibition. On the other hand, the identity of E2, which was UbcH5 in the above assays, appears not to be critical.
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Nevertheless, our data on DET1 are mutually compatible with a role of DET1 as an unconventional substrate receptor for Cul4A. This may apply to the case of c-Jun ubiquitination, in which DET1 works with COP1 (39). It remains unclear whether DET1 exerts a different effect on Cul4A in a substrate-dependent manner. Given that Arabidopsis DET1 has been shown to bind histones (2), it is an appealing possibility that DET1 may function in specific histone ubiquitination. This idea is further encouraged by recent reports that Cul4A-DDB1 ubiquitin ligases can mediate histone ubiquitination (19, 37) and that a member of the UBE2E family, UBE2E-1 (or UbcH6), participates in histone H2B monoubiquitination by RNF20/RNF40 (43).
DDD-E2 is the mammalian counterpart of Arabidopsis CDD despite distinctions between UBE2E enzymes and COP10. Arabidopsis COP10 is not homologous to any of the human UEVs but instead displays highest homology to the UBE2E family of E2s in humans (20). The obvious distinction of UBE2Es from COP10 is the fact that UBE2Es are canonical E2 enzymes that have important functions outside of the DDD-E2 complexes. For example, UBE2E-3 is an active E2 for RNF8 and ARA54 RING E3s (16). It also participates in HECT-domain E3 Nedd4-mediated ubiquitination of the Na+ channel protein EnaC (7). UBE2E-1 works with the RNF20/40 complex to mediate monoubiquitination of histone H2B (43). These differences may account for the observation that the majority of the UBE2E-3 population exists as a monomer, whereas the predominant population of COP10 is in the CDD complex (36). In addition, the mechanisms of nuclear translocation appear to differ. The UBE2E enzymes require ubiquitin loading to interact with importin-11 and to translocate into the nucleus (30), whereas COP10 localizes to the nucleus by piggybacking on the CDD complex (36).
Importantly, both UBE2Es and COP10 form stable complexes with DET1 and DDB1. Moreover, despite UBE2Es being functional E2s by themselves, once integrated into the DDD-E2 complex, they can no longer maintain ubiquitinthioesters. As a consequence, the DDD-E2 complex is equivalent to a UEV-containing CDD complex with respect to their biochemical capabilities. Based on these analogies, we suggest that the DDD-E2 complex is the mammalian counterpart of the Arabidopsis CDD complex.
Function of DDD-E2 complexes. Among the DDD components, DDA1 strongly binds DDB1 and DET1, and DDB1 provides a platform for interacting with Cul4A and WD40 proteins, while DET1 is the determinant factor in E2 binding and the key component in inhibition of Cul4A. We sometimes found weak polyubiquitination activities in the DET1 immunocomplexes, but the activities were highly variable, depending on the isolation conditions (not shown). Interpretation of these activities is further complicated by the tertiary association of various E3s with DET1.
All three UBE2E group E2s can independently assemble with DDD and together constitute a heterogeneous population of DDD-E2 complexes in the cell. We have shown that the DDD core can inhibit polyubiquitin chain assembly by Cul4A, while UBE2E-3 can facilitate Cul4A reactions in a capacity as either an E2 or an E2 enhancer. In a recent study of plant CDD (5), it was shown that a complex composed of COP10-DDB1-DET1 (without DDA1) enhanced Cul4A activity similar to COP10 alone (41). Still, the function of various DDD-E2 complexes remains elusive at present. It is conceivable that the DDD-E2 complexes may work with other E2s and promote unconventional ubiquitin chain assembly, similar to UbcH13/MMS2 (or UEV1) complexes (8, 15). Alternatively, it may facilitate specialized ubiquitination reactions, such as monoubiquitination.
The Arabidopsis COP/DET/FUS loci and the tomato HP (DDB1/DET1) loci (22, 26) define a class of regulators critical in plant light responses. This group includes CDD components, the RING-WD protein COP1, and the CSN components (38). Recently, Arabidopsis Cul4A has been shown to participate in light responses (3, 5). In this study, we have identified the mammalian DDD-E2 complex as a CDD homolog. It will be interesting to see whether the Arabidopsis DDA1 homolog is a component of CDD and whether a genetic mutant of DDA1 bears any resemblance to the phenotype for cop/det/fus. The mammalian counterparts of the COP/DET/FUS/DDB1 genes, along with Cul4A, are involved in cellular responses to UV irradiation (10, 38, 42). CSN is an established regulator of the Cul4A E3 complex. UV activation of Cul4A-DDB1-DDB2 ligase is believed to involve the dissociation of CSN from the E3 complex (10, 35). In this study, we found that DET1 also regulates Cul4A, revealing another level of complexity in the control of the Cul4A E3 ligase system.
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ACKNOWLEDGMENTS |
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E.P. is supported by a Yale University Brown Fellowship and by Yale University research funds to N.W. N.W. is supported by a grant from NIH (GM61812). T.T. is supported by a grant from MEXT-Japan (Grant-in-Aid for Scientific Research C-18570041). O.S.L. is supported by the Croucher Foundation and an NIH grant (GM47850) to X.W.D.
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FOOTNOTES |
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Published ahead of print on 23 April 2007.
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