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UV-damaged DNA-binding Proteins Are Targets of CUL-4A-mediated Ubiquitination and Degradation*

Cul-4A, which encodes a member of the cullin family subunit of ubiquitin-protein ligases, is expressed at abnormally high levels in many tumor cells. CUL-4A can physically associate with the damagedDNA-binding protein (DDB), which is composed of two subunits, p125 and p48. DDB binds specifically to UV-damaged DNA and is believed to play a role in DNA repair. We report here that CUL-4A stimulates degradation of p48 through the ubiquitin-proteasome pathway, resulting in an overall decrease in UV-damaged DNA binding activity. The R273H mutant of p48 identified from a xeroderma pigmentosium (group E) patient is not subjected to CUL-4A-mediated proteolysis, consistent with its inability to bind CUL-4A. p125 is also an unstable protein, and its ubiquitination is stimulated by CUL-4A. However, the abundance of p125 is not dramatically altered byCul-4A overexpression. UV irradiation inhibits p125 degradation, which is temporally coupled to the UV-induced translocation of p125 from the cytoplasm into the nucleus. CUL-4A is localized primarily in the cytoplasm. These findings identify DDB subunits as the first substrates of the CUL-4A ubiquitination machinery and suggest that abnormal expression of Cul-4A results in reduced p48 levels, thus impairing the ability of DDB in lesion recognition and DNA repair in tumor cells.

Ubiquitin-dependent proteolysis plays an important role in controlling cell cycle, signal transduction, apoptosis, and a variety of other cellular processes. Through the action of a multienzyme system consisting of the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 1 ubiquitin-protein ligase, multiple ubiquitin moieties are delivered to the target protein to form a polyubiquitin chain through the isopeptide linkage between the ⑀-amino group of the lysine 48 residue of one ubiquitin and the carboxyl terminus of the adjacent ubiquitin. In turn, polyubiquitination serves as the signal for recognition and degradation by the 26 S proteasome (recently reviewed in Refs. 1 and 2).
The E3 component of the ubiquitin pathway is highly specialized in its ability to select specific cellular substrates for ubiquitination (reviewed in Ref. 3). The Rbx1-cullin subclass of RING E3s consists of multimeric protein complexes that are assembled around a core module composed of a cullin family member and the RING-H2 domain protein Rbx1/Roc1/Hrt1 (4 -7, and reviewed in Refs. 1, 2, and 8). There are at least six identified mammalian cullins (9), and they share extensive sequence homology in a region of ϳ200 amino acid residues designated as the cullin homology domain (CH) (10,11). Cullins interact with the RING-H2 domain protein Rbx1/Roc1/ Hrt1 through their CH domains to form core ubiquitin-protein ligase modules that connect to the E2 ubiquitin-conjugating enzymes and other E3 components and facilitate ubiquitin transfer to substrates (4,6,7,12,13). Among members of the cullin family, CUL-1, CUL-2, and CUL-3 have been demonstrated to mediate the selective degradation of regulators of cell cycle and signaling pathways (reviewed in Refs. 1 and 2 and the references therein). Kamura et al. (12) recently demonstrated that the CUL-5/Rbx1 module associates with the elongin BC complex and a novel elongin BC-box/leucine-rich repeat-containing protein MUF1 to form a functional ubiquitin-protein ligase. CUL-4A and CUL-4B are still poorly characterized because of the lack of known proteolytic substrates and components of the putative CUL-4 complex. The Cul-4A gene is amplified or overexpressed in human breast cancer and many other tumor types (14,15). Recent biochemical studies have identified a physical association between CUL-4A and the UVdamaged DNA-binding protein (DDB) (16), 2 which is involved in the repair of damaged genomic DNA and the non-transcribed strand of expressed genes (17)(18)(19). However, the biochemical consequences of the interaction between CUL-4A and DDB have not been elucidated.
DDB is a heterodimeric protein complex consisting of a 127-kDa subunit (p125 or DDB1) and a 48-kDa subunit (p48 or DDB2) (20 -23) that has high affinity for a variety of DNA lesions including UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts, as well as intrastrand cross-links by cisplatin and benzoapyrene adducts. (24 -26). For simplicity, the two DDB subunits are referred to as p125 and p48 from hereon. p125 is an abundant protein that is in excess of p48 (16,21), whereas the p48 subunit is the limiting factor for UV-DDB activity and functions to activate p125 binding to damaged DNA (19,27). UV irradiation induces p48 transcription in a * This work was supported by the Academic Medicine Development Company Foundation, the Mary Kay Ash Charitable Foundation, and the Dorothy Rodbell Cohen Foundation for Sarcoma Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  p53-dependent manner, resulting in the subsequent accumulation of p48 protein (18,28). p48 is localized to the nucleus, whereas p125 is primarily cytoplasmic but translocates into the nucleus upon UV irradiation by a mechanism that is partially p48-dependent (28,29). Mutations in the p48 subunit of DDB were identified in patients suffering from the autosomal recessive disease, xeroderma pigmentosium (complementation group E) (XP-E), which is characterized by defective nucleotide excision repair and predisposition to skin cancer (30 -32). Recent studies (18,27,33) indicate that the p48 mutant cells are deficient in repair of UV-damaged DNA, consistent with their impaired DDB activity (DDB Ϫ ). One such p48 mutant, 2RO (R273H), fails to associate with p125 and CUL-4A and is incapable of mediating nuclear accumulation of p125 (16,29). Mutations of the p125 subunit have not been identified in XP-E patients, however, a 50% decrease in the steady-state levels of p125 has been reported in several DDB Ϫ XP-E cells (34). These results suggest a requirement for the precise control of the amount of p125 and p48 inside the cell.
Here we report that CUL-4A stimulates the ubiquitination and degradation of the p48 subunit of DDB, resulting in an overall decrease of UV-DDB activity. The 2RO mutant of p48, which is defective in binding to p125 and CUL-4A, is resistant to degradation induced by Cul-4A overexpression. Ectopic expression of Cul-4A also resulted in accelerated p125 ubiquitination. These studies identify DDB as the first target of the CUL-4A ubiquitination machinery and suggest a possible link between the abnormal Cul-4A expression in tumor cells and the inhibition of DDB-dependent repair of UV-damaged DNA.
Indirect Immunolocalization-HeLa cells transfected with p125-HA were cultured on glass coverslips for 24 h and either untreated or irradiated with UV at 10 J/m 2 . At 0 and 8 h following UV irradiation, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized in 0.5% Triton X-100 for 10 min. Coverslips were blocked in 5% non-immune serum from the same species as the labeled secondary antibody. This was followed by incubation with affinity-purified anti-HA11 monoclonal antibody (1:1000 dilution) or affinity-purified anti-CUL-4A polyclonal antibody (1:200 dilution) in phosphate-buffered saline containing 5% non-immune serum from the same species as the labeled secondary antibody at room temperature for 1 h. The cells were washed with phosphate-buffered saline and incubated with secondary antibody-conjugated Cy-3 (1:3000 dilution) for 30 min. After washing in phosphate-buffered saline, coverslips were counterstained with DAPI and examined by fluorescence microscopy (Olympus). The localization of the endogenous CUL-4A in V79 -4 cells was examined as described above using either affinity-purified anti-CUL-4A polyclonal antibody or two commercial affinity-purified CUL-4 polyclonal antibodies (Santa Cruz Biotechnologies, Inc.). The localization of the endogenous CUL-4A in HeLa cells was further examined under a laser-scanning confocal microscope (Zeiss LSM510). The indirect immunofluorescence staining was performed as described above, with the exception that the secondary antibody was conjugated with Alexa Fluor ® 488 (Molecular Probes, Inc.), and cells were counterstained with propidium iodide.
Protein Turnover-HeLa cells were transiently transfected with indicated plasmids for 36 h by a calcium phosphate procedure. Cells were starved in DMEM without methionine and cysteine for 1 h, pulselabeled with a mixture of [ 35  ). The half-life of the endogenous p125 in HeLa cells or chromosomally integrated F-p48 in V79 -4 cells was measured similarly using the anti-p125 polyclonal antibody or anti-FLAG monoclonal antibody, respectively. To evaluate the effect of UV irradiation on the stability of p125 or p48, cells were irradiated with UV at 10 J/m 2 followed by recovery in DMEM for 8 h prior to pulse-chase analysis. In some instances, 100 M MG132 proteasome inhibitor (Peptide International) was included in the chase medium, and the stability of p125-HA was subsequently addressed. The pulse-chase experiments were repeated three or more times, and representative results are shown in Figs. 3

and 4.
In Vivo Ubiquitination Assay-5 ϫ 10 5 HeLa cells were transiently transfected with 10 g of F-p125 plasmid, 8 g of HA-Ub-expressing plasmid, and 4 or 8 g of myc-Cul-4A, F-Cul-4A(⌬), or myc-Cul-1, respectively. After 48 h, the cells were boiled in 1% SDS lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1% SDS, 1 mM dithiothreitol) for 10 min and were then diluted 10 times in Nonidet P-40 lysis buffer containing the protease inhibitor mixture (PharMingen) and immunoprecipitated with 3 g of the anti-HA antibody (Covance). For p48 ubiquitination assay, HeLa cells were similarly transiently transfected with 8 g of F-p48 plasmid, 8 g of HA-Ub-expressing plasmid, and 3 or 6 g of myc-Cul-4A for 40 h, treated with 50 M LLnL for 4 h, harvested as described above, and immunoprecipitated using the anti-HA affinity resin with the anti-HA antibody covalently coupled to the agarose beads (Roche Molecular Biochemicals). Immunoprecipitates were then subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with an anti-FLAG M2 monoclonal antibody to detect ubiquitinated F-p125 or F-p48. 100 g of the extracts were also directly subjected to SDS-PAGE and immunoblotting to detect expression of F-p125, F-p48, MYC-CUL-4A, F-CUL-4A(⌬), and MYC-CUL-1, respectively.
Electrophoretic Mobility Shift Assay (EMSA)-V79 -4 cells were transiently transfected with F-p48 alone or together with myc-Cul-4A or myc-Cul-1. 1 g of CD19 expression plasmid (pCMV-CD19) was included in each transfection for immunomagnetic selection of V79 -4 cells expressing the transfected DNA using the anti-CD19 monoclonal antibody conjugated to DYNALBEADS Pan Mouse IgG (DYNAL). V79 -4 cells cotransfected with F-p48 and myc-Cul-4A were also treated with 50 M proteasome inhibitor LLnL for 4 h before harvesting and immunomagnetic selection. CD19-expressing cells were lysed in EMSA buffer (700 mM NaCl, 1 mM EGTA, 1 mM EDTA, 10 mM ␤-glycerophosphate, 2 mM MgCl 2 , 10 mM KCl, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.1% Nonidet P-40, 10 g/ml each pepstatin, leupeptin, and aprotitin) and rotated at 4°C for 30 min followed by centrifugation at 13,000 rpm for 30 min at 4°C. 50 g of each extract was first subjected to immunoblotting with the anti-FLAG M2 antibody to detect the steady-state levels of F-p48 in response to myc-Cul-4A or myc-Cul-1 expression or to proteasome inhibitor treatment. For EMSA assays, the 148-bp DNA probe (f148) was isolated from the pSV2CAT plasmid by HindIII and PvuII digestion and was labeled with 32 P-dCTP by using the Klenow fragment of DNA polymerase I. 32 P-Labeled f148 probe was purified by a 4% polyacrylamide gel, and the eluted probe was damaged with UV at 5000 J/m 2 . Binding reactions were performed with 0.2 ng of the f148 probe, 1 g of lysates, and 1 g of salmon sperm DNA as nonspecific competitors in a 10-l reaction mixture at room temperature for 20 min (27,36). The DDB⅐[ 32 P]f148 complexes were resolved on 4% non-denaturing polyacrylamide gels and autoradiography.

RESULTS
CUL-4A Interacts with p125 Independent of p48 -To assess the function of CUL-4A, we searched for cellular proteins that could interact with human CUL-4A. Either F-CUL-4A or the pCDNA3 expression vector was transiently transfected into HeLa cells for immunoprecipitation by the anti-FLAG M2 monoclonal antibody, and the immunoprecipitates were analyzed by SDS-PAGE and silver staining. Two polypeptides with the molecular mass of ϳ125 and 45 kDa were specifically detected only from the CUL-4A immunoprecipitates (data not shown). While we were analyzing these polypeptides, Shiyanov et al. (16) reported the identification of CUL-4A as a T7-tagged p48-associated protein from the human osteosarcoma U2OS cell extracts. The two CUL-4A-interacting proteins we detected were subsequently confirmed to be p125 and p48.
To further examine how CUL-4A interacts with the individual subunits of DDB, we sought to analyze these interactions in the hamster V79 -4 lung fibroblast cells that lack p48 expression ( Fig. 1A) (19,27). Plasmids carrying p125-HA were transfected into V79 -4 cells along with a plasmid expressing F-Cul-4A or F-p48. The expression of each individual protein was verified by Western blotting (Fig. 1B). Coimmunoprecipitation was carried out using the anti-FLAG M2 antibody for F-p48 or F-CUL-4A and probed with anti-HA antibody to detect the presence of p125-HA in the immunoprecipitates. As shown in Fig. 1C, F-CUL-4A can form a complex with p125-HA in the absence of p48 in V79 -4 cells, indicating that p48 is dispensable for this interaction (Fig. 1C, lane 4).
CUL-4A Is Localized Primarily in the Cytoplasm-p125 is localized primarily in the cytoplasm in a variety of cell lines examined (Fig. 2B) (28,29). Given that UV irradiation stimulates p125 entry into the nucleus (28,29), we sought to determine the subcellular localization of CUL-4A in the absence or presence of UV treatment. We first examined the cellular distribution of endogenous CUL-4A in V79 -4 and HeLa cells using an affinity-purified anti-CUL-4A polyclonal antibody. In the absence of UV irradiation, CUL-4A, like p125, localized primarily in the cytoplasm in V79 -4 ( Fig. 2A) and HeLa cells (Fig. 2C, left panel). At 8 h after UV irradiation (10 J/m 2 ), p125 was found predominantly in the nucleus in HeLa cells (Fig. 2C, upper right panel), whereas CUL-4A remained largely in the cytoplasm (Fig. 2C, bottom right panel). In contrast, p48 was always localized in the nucleus (28, 29) (data not shown). These results indicate that unlike p125, CUL-4A is retained in the cytoplasm following UV irradiation. We further analyzed the subcellular distribution of CUL-4A by immunofluorescence confocal microscopy. As shown in Fig. 2B, a subpopulation of CUL-4A (approximately 2-3%) was indeed detected in the nucleus (Fig. 2B). We, therefore, conclude that CUL-4A is predominantly localized in the cytoplasm, whereas a small fraction also resides in the nucleus.
CUL-4A Regulates p48 Levels through Ubiquitin-dependent Proteolysis-Because the nuclear-localized p48 is the limiting factor for UV-DDB activity, we sought to determine whether p48 is subjected to CUL-4A-mediated proteolysis. First, we assessed the steady-state levels of p48 in response to ectopic Cul-4A expression. V79 -4 cells lacking endogenous p48 expression were transiently transfected with F-p48 along with the control vector pCDNA3, myc-tagged Cul-4A, or myc-tagged Cul-1. 1 g of pCMV-CD19 plasmid was also cotransfected for immunomagnetic selection of transfected V79 -4 cells, and the amounts of F-p48 were subsequently determined by immunoblotting using the anti-FLAG M2 monoclonal antibody. As shown in Fig. 3A, ectopic expression of myc-Cul-4A but not myc-Cul-1 induced a dramatic decrease in the steady-state levels of p48 (Fig. 3A, lanes 1-3). Treatment of myc-Cul-4A and F-p48 coexpressing V79 -4 cells with the proteasome inhibitor LLnL (Fig. 3A) or lactacystin (data not shown) dramatically inhibited the decrease of p48 levels, indicating that CUL-4A induced down-regulation of p48 through the proteasome (Fig.  3A, lane 4).
To assess whether CUL-4A reduces p48 levels through accelerating its ubiquitination, HeLa cells were transiently transfected with F-p48 alone (Fig. 3B, lane 1) or F-p48 and HA-tagged ubiquitin (Fig. 3B, lane 2) or F-p48, HA-Ub, and 3 or 6 g of myc-Cul-4A (Fig. 3B, lanes 3 and 4). Extracts were immunoprecipitated with the anti-HA affinity matrix and probed with the anti-FLAG M2 antibody against F-p48. In the absence of exogenous CUL-4A, there was no obvious modification of F-p48 by HA-Ub (Fig. 3B, lane 2). When myc-Cul-4A was FIG. 1. CUL-4A interacts with p125 independent of p48. A, total RNA was prepared from V79 -4, V79 -4/F-p48, and HeLa cells, and 10 g of RNA was subjected to Northern blotting to measure the levels of p48 mRNA using a 32 P-labeled probe encompassing the 792-bp EcoRV-EcoRI fragment of p48 cDNA. Similar levels of rRNA were detected by ethidium bromide staining of the agarose gel to verify equal loading of RNA samples (data not shown). B, the V79 -4 cells were transiently transfected with the indicated plasmids (in g). 2 g of pGREEN LANTERN-1 plasmid was included in each transfection to assess the transfection efficiency. 100 g of each cell extracts was analyzed by immunoblotting to verify the expression of p125-HA, F-CUL-4A, and F-p48. The levels of endogenous replication protein A (RPA) are indicated as an internal loading control. C, 1 mg of these lysates was immunoprecipitated with the anti-FLAG M2 monoclonal antibody and probed either with the same M2 antibody for F-p48 or F-CUL-4A or with the anti-HA monoclonal antibody for p125-HA.
transfected into F-p48-and HA-Ub-expressing cells, a series of slower migrating F-p48 species was readily detectable in the anti-HA immunoprecipitates (Fig. 3B, lane 3), and the amounts of HA-Ub-modified F-p48 increased dramatically with the elevated Cul-4A expression (Fig. 3B, compare lane 3 with lane 4).
These results indicate that p48 is specifically ubiquitinated by the CUL-4A ubiquitination machinery.
To evaluate whether CUL-4A-dependent down-regulation of p48 was the result of its accelerated turnover, we performed pulse-chase analysis to measure the half-life of p48 protein.
Because of the unavailability of the anti-p48 antibody to detect endogenous p48, we first generated a stable V79 -4/F-p48 cell line to monitor the turnover rate of chromosomally integrated FLAG-tagged p48 (Fig. 3C). In addition, we assessed the stability of transiently transfected F-p48 in HeLa cells, which exhibit high UV-damaged DNA binding activity (24). As shown by pulse-chase analysis in Fig. 3, C and D (upper panel), p48 is turned over in both V79 -4 and HeLa cells with a half-life of ϳ3.5 h. Ectopic expression of Cul-4A accelerated degradation of p48 (t1 ⁄2 of 1.2 h) (Fig. 3D, lower panel). The p48(R273H) mutant, which is defective for binding CUL-4A, was not subjected to CUL-4A-mediated p48 destruction (Fig. 3E). It is noted that the p48(R273H) mutant is still degraded, albeit independent of ectopic Cul-4A expression. The R273H mutation is within the WD40 domain of p48. One possible explanation is that the structural alteration resultant from the R273H mutation induces p48(R273H) degradation by an as yet unknown cellular proteolytic apparatus. Collectively, these results indicate that the CUL-4A machinery controls the stability of the p48 subunit of the UV-DDB complex through specifically enhancing the ubiquitination and degradation of p48.
CUL-4A Stimulates Ubiquitination of p125-The fact that CUL-4A also associates with the p125 subunit of the DDB complex independent of p48 ( Fig. 1C) and that both CUL-4A and p125 are localized in the cytoplasm in non-irradiated cells prompted us to examine whether CUL-4A is involved in p125 ubiquitination. We first assessed the stability of endogenous p125 in HeLa cells that exhibit high UV-damaged DNA binding activity (24). To measure the half-life of newly synthesized p125, exponentially growing HeLa cells were pulse-labeled with [ 35 S]methionine and [ 35 S]cysteine and chased for various periods of time points before immunoprecipitation with the anti-p125 polyclonal antibody. A rapid decrease of p125 protein levels was observed with a half-life of ϳ2 h (Fig. 4A, left panel). p125 was also degraded in V79 -4 cells with a half-life of ϳ2 h by pulse-chase analysis indicating that p48 is not required for p125 degradation (data not shown). Furthermore, ectopically expressed p125-HA was also rapidly turned over with a halflife of 2.5 h (Fig. 4B). When the proteasome inhibitor MG132 (Fig. 4B) or lactacystin (data not shown) was included in the chase medium, degradation of p125-HA was dramatically inhibited with a half-life of 9.5 h indicating that the 26 S proteasome plays a major role in the regulation of p125 stability (Fig.  4B, middle panel).
We next investigated whether the stability of endogenous p125 could be altered in HeLa cells 8 h after UV treatment (10 J/m 2 ) when the majority of p125 translocated from the cytoplasm to the nucleus (Fig. 2). As shown by pulse-chase, the half-life of both endogenous and transfected p125 proteins were dramatically prolonged upon UV irradiation (Fig. 4, A, right  panel, and B, lower panel). Because the majority of cytoplasmic p125 translocates into the nucleus in HeLa cells 8 h after UV irradiation and there is only a small fraction of CUL-4A in the nucleus (Fig. 2B), these experiments suggest that the CUL-4A might be limiting in the nucleus, and thus subcellular localization of p125 is a contributing factor that regulates p125 degradation within the cell.
Because the half-life of p125 was prolonged in the presence of the proteasome inhibitor, we further investigated whether p125 is directly modified by ubiquitin and whether CUL-4A plays a role in these processes. HeLa cells were transiently transfected with F-p125 alone (Fig. 5A, lane 1), F-p125 and HA-Ub (Fig. 5A, lane 2), or F-p125, HA-Ub, and increasing amounts of myc-Cul-4A (Fig. 5A, lanes 3 and 4). Expression of the transfected F-p125 was detected from 100 g of extracts by immunoblotting using the anti-FLAG (M2) monoclonal antibody (Fig. 5A, top panel). In addition to the 127-kDa F-p125, a series of slower migrating species was also observed that was immunoreactive with F-p125 (Fig. 5A, lane 2) and whose intensity increased significantly with exogenous expression of myc-Cul-4A (Fig. 5A, lanes 3 and 4). This finding suggested that F-p125 might be modified by multiple ubiquitin molecules.
To determine whether these slower migrating species are indeed derived from ubiquitin conjugation to F-p125, the same lysates were immunoprecipitated with the anti-HA monoclonal antibody against HA-Ub and probed with anti-FLAG antibody against F-p125. The slower migrating species were readily observed, and an elevated level of these species was detected with increased Cul-4A expression, indicating that p125 is indeed polyubiquitinated in a CUL-4A dose-dependent manner (Fig. 5, A, lower panel, and B, lanes 2-4). Two relatively constant ubiquitin-modified F-p125 species at molecular masses 135 and 143 kDa were specifically detected under these experimental conditions (Fig. 5, B and C, marked with an asterisk), which were consistent with the size of mono-and di-ubiquitinconjugated p125, and served as a sensitive readout of p125  4) (in g). A CD19 plasmid was included in each transfection for the subsequent immunomagnetic selection of V79 -4 cells, which received the transfected plasmids. 50 g of each extract was subjected to SDS-PAGE and immunoblotted with the ␣-FLAG antibody for F-p48, ␣-MYC (9E10) antibody for MYC-CUL-4A or MYC-CUL-1, or ␣-replication protein A antibody for replication protein A (loading control). B, HeLa cells were transfected with the plasmids as indicated (in g). In vivo ubiquitination assay was performed as described under "Materials and Methods." Ubiquitinated F-p48 species are indicated on the right. The migration position of unmodified F-p48 is indicated by an arrow on the right. The molecular mass markers are indicated on the left. 100 g of each extract was also subjected to immunoblotting with the anti-FLAG M2 antibody to detect F-p48 expression. C, pulse-chase analysis of F-p48 in the stable V79 -4/F-p48 cells. V79 -4/F-p48 cells were metabolically labeled and chased in cold medium. Lysates from indicated time points were immunoprecipitated using the anti-FLAG antibody and analyzed by SDS-PAGE followed by autoradiography. D and E, CUL-4A accelerates degradation of p48 but not the p48(R273H) mutant. HeLa cells were transfected with F-p48 or FLAG-tagged p48(R273H), either alone or together with myc-Cul-4A. Pulse-chase experiments were carried out as in C to determine the half-life of F-p48 or FLAG-tagged p48(R273H). Quantitative measurements of band intensities using the PhosphorImager scanning and analysis software are graphed on a logarithmic scale over time (in hours). ubiquitination for the following experiment in Fig. 5C. When 4 or 8 g of myc-Cul-4A plasmid was introduced into F-p125 and HA-Ub-expressing HeLa cells, we observed a CUL-4A dose-dependent increase in the level of these ubiquitinated p125 species (Fig. 5, B and C, lanes 3 and 4 marked by an asterisk on the  right). However, the expression of 4 or 8 g of either F-Cul-4A(⌬) or myc-Cul-1 plasmid DNA was incapable of stimulating p125 ubiquitination (Fig. 5C, lanes 5-8). The decrease of monoand di-ubiquitinated F-p125 in the presence of 8 g of myc-Cul-1 but not 8 g of F-Cul-4A(⌬) (Fig. 5C, compare lane 8 with lane 6) is likely because of the competition of high levels of CUL-1 with the endogenous CUL-4A for the endogenous Rbx1 and therefore reduces the ubiquitin-protein ligase activity of the CUL-4A machinery. Taken together, these results indicate that p125 is specifically ubiquitinated by CUL-4A, and this process requires the CH domain of CUL-4A, which is responsible for connecting to the E1 ubiquitin-activating enzyme-E2 ubiquitin-conjugating enzyme (E1-E2) ubiquitin transfer machinery. Interestingly, the overexpression of Cul-4A did not significantly alter the steady-state levels of transfected p125 (Fig. 1B, data not shown). The endogenous p125 is known to be a highly abundant protein (16,21). It is possible that CUL-4A is not the rate-limiting component of the p125 degradation machinery. Therefore, overexpression of Cul-4A alone was not capable of reducing the p125 levels. Alternatively, CUL-4Astimulated p125 ubiquitination may serve other roles than proteolysis. Further studies should be conducted to address the functional role of CUL-4A-mediated ubiquitination of p125.
Overexpression of Cul-4A Down-regulates UV-DDB Activity-DDB exhibits high affinity to UV-damaged DNA. We investigated whether CUL-4A-mediated proteolysis of DDB plays a role in controlling the UV-damaged DNA binding activity by the established EMSA (36). V79 -4 cells lack the UV-DDB activity because of the absence of p48 expression (Fig. 6, lane 2) (19,27). The binding of DDB to UV-damaged f148 DNA was restored by transient transfection of F-p48 into V79 -4 cells (Fig. 6, lane 3) or through stable F-p48 expression in V79 -4/ F-p48 cells (data not shown) (19). The DDB activity was characterized as two DNA protein complexes (Fig. 6, lane 3 marked  as B1 and B2) as was previously demonstrated (27). Hwang et al. (27) showed that B2 consists of f148 DNA in the complex with both p125 and p48, whereas B1 is primarily p125/f148. V79 -4 cells were transiently transfected with F-p48 alone (Fig.  6, lane 3) or together with myc-Cul-4A and treated either in the absence or presence of proteasome inhibitor LLnL (Fig. 6, lanes  4 and 5) or transfected with F-p48 and myc-Cul-1 (Fig. 6, lane  6). A CD19 expression plasmid was included in the transfection for enrichment of transfected V79 -4 cells by immunomagnetic selection prior to the preparation of cell extracts in EMSA analysis. In V79 -4 cells transfected with myc-Cul-4A and F-p48, a dramatic decrease of the B2 DNA protein complex was readily observed (Fig. 6, compare lane 4 with lane 3), consistent with the accelerated p48 degradation by CUL-4A (Fig. 3A). Moderate down-regulation of the B1 complex was also detected (Fig. 6, lane 4). Treatment of Cul-4A and F-p48 coexpressing cells with the proteasome inhibitor LLnL stabilized F-p48 (Fig.  3A, lane 4) and restored the DDB activity (Fig. 6, lane 5). In contrast, the expression of myc-Cul-1 did not inhibit the UV-DDB activity (Fig. 6, lane 6). Furthermore, in the repair-competent 293T cells (19,27), ectopic expression of Cul-4A also inhibited the UV-DDB activity (data not shown). These results provide functional evidence that CUL-4A-mediated proteolysis of p48 is involved in the regulation of UV-damage induced DNA binding activity. DISCUSSION Little is known about the proteolytic targets and the cellular processes controlled by the CUL-4A ubiquitination machinery. Recent biochemical studies indicate the association of CUL-4A with the DDB complex, however, the physiological role of this interaction is unknown. The results shown in this study provide compelling evidence that both subunits of the heterodimeric DDB complex are targets of CUL-4A-mediated ubiquitination. First, CUL-4A stimulated the accumulation of higher molecular weight species that were immunoreactive with both DDB subunits (p125 or p48) and HA-tagged ubiquitin ( Fig. 3B and Fig. 5). Second, the CUL-4A(⌬) mutant defective for binding Rbx1 3 was also impaired in ubiquitinating p125 and p48 (Fig. 5, data not shown). This indicates that the association between Rbx1 and the conserved CH domain of CUL-4A is essential for CUL-4A-mediated ubiquitination, similar to what has been demonstrated for the Rbx1/CUL-1 ma-3 L. Douglas, unpublished result. chinery (37). Third, ectopic expression of Cul-1 did not stimulate the ubiquitination and/or degradation of p125 or p48, suggesting that DDB proteins are specific targets of CUL-4A (Fig. 5, data not shown). Finally, the R273H (2RO) mutant of p48, which is defective for binding CUL-4A, is not subjected to CUL-4A-mediated degradation (Fig. 3E). These findings demonstrate that the CUL-4A ubiquitination machinery specifically targets the damaged DNA-binding proteins p125 and p48 for ubiquitination.
The CUL-4A-mediated degradation of DDB correlates with the cellular distribution of CUL-4A and the DDB subunits. Our immunofluorescence results indicate that CUL-4A is localized predominantly in the cytoplasm, and a small fraction (ϳ2-3%) of CUL-4A could be detected in the nucleus (Fig. 2). The presence of leucine-rich sequences within CUL-4A, which is similar to the human immunodeficiency virus rev-like nuclear export sequence (38), may account for its predominant cytoplasmic localization through nuclear export. 4 Because nuclear translocation of p125 upon UV irradiation correlates with a reduction of its turn over rate (Fig. 4), it is likely that the ubiquitinprotein ligase activity of CUL-4A is limiting in the nuclear compartment. This result is consistent with the observation that p48, which resides exclusively in the nucleus (28,29), was rapidly degraded when Cul-4A was overexpressed (Fig. 3). In accordance with p48 destabilization, we observed an overall reduction in damaged DNA binding activity by DDB (Fig. 6). Therefore, CUL-4A-induced degradation of p48 plays a critical role in restricting the UV-DDB activity.
p125 is a relatively abundant protein and is present in excess over p48 in unirradiated HeLa cells (16,21). Our studies indicate that p125 is metabolically unstable, suggesting a requirement for the precise control of p125 levels inside the cell. Interestingly, a previous study (34) reported that in at least three DDB Ϫ XP-E cells, p125 levels were at least 50% lower than that in the DDB ϩ cells. We have shown that CUL-4A induced a robust p125 ubiquitination, but a significant decrease in the overall levels of cellular p125 has not been observed under these experimental conditions (Fig. 1C, data not shown). One possibility is that CUL-4A may not be rate-limiting for degradation of p125, therefore, the overexpression of Cul-4A could not further reduce the steady-state levels of p125. 4 L. Douglas and P. Zhou, unpublished result. Lysates were subjected to immunoblotting to detect F-p125 or MYC-CUL-4A. B, 1 mg of lysate was immunoprecipitated with anti-HA monoclonal antibody and probed with anti-FLAG (M2) antibody to detect ubiquitinated F-p125. The mono-or di-ubiquitin-modified F-p125 species are marked with an asterisk. Polyubiquitinated p125 species are also indicated on the right. The position of migration of unmodified F-p125 is indicated by an arrow on the right. E, an unknown protein in HeLa cell extracts that was precipitated by the anti-HA antibody and reactive with the anti-FLAG antibody. C, in vivo ubiquitination assays were carried out using HeLa cells transfected with F-p125 and HA-Ub along with increasing amounts of myc-Cul-4A, F-Cul-4A(⌬), or myc-Cul-1 as in B (in g). The mono-or di-ubiquitinated F-p125, marked with an asterisk, was detected and served as a sensitive readout to assess the levels of p125 ubiquitination in response to increased expression of myc-Cul-4A, F-Cul-4A(⌬), or myc-Cul-1 (data not shown). F-p125 levels were determined from 100 g of extracts by immunoblotting using the anti-FLAG monoclonal antibody. Migration positions of molecular mass standards (in kilodaltons) are indicated on the left.

FIG. 6.
Overexpression of Cul-4A down-regulates the UV-DDB activity. V79 -4 cells were transiently transfected with the expression plasmids for F-48 along with myc-Cul-4A or myc-Cul-1 (in g) and either untreated (Ϫ) or treated (ϩ) with the proteasome inhibitor LLnL. A CD19 expression plasmid was included in each transfection for immunomagnetic selection of V79 -4 cells that received the transfected plasmids. Expression of myc-Cul-4A or myc-Cul-1 was detected by immunoblotting (see Fig. 3A). 1 g of the whole cell extracts from these transfectants was assayed for damaged DNA binding activity by electrophoretic mobility shift assay using the 32 P-labeled f148 DNA probe damaged at 5000 J/m 2 . The mobility of the two specific DDB⅐DNA complexes were marked as B1 and B2 on the right. The bands in bracket below the DDB⅐DNA complexes were nonspecific, because they were also observed for the free damaged probe alone (lane 1). F, free DNA probe migration. 50 g of each immunomagnetically selected extract was analyzed by immunoblotting using the anti-actin polyclonal antibody as a measurement of equal loading. Alternatively, p125 has been shown to participate in transcriptional regulation as well as other cellular functions (39 -43), and there might be only a fraction of cellular p125 that participates in DNA damage recognition and repair and is subjected to CUL-4A-mediated proteolysis. Because p125 is known to be a highly abundant protein inside the cell (ϳ10 5 molecules/cell) (21), it is likely that the endogenous p125 competes with the transfected p125-HA for the CUL-4A machinery. Alternatively, because proteolysis is not the only destination or immediate fate of an ubiquitinated protein (reviewed in Ref. 1), it is also possible that ubiquitination of p125 might have additional roles besides degradation. Non-proteolytic functions have been demonstrated for mono-ubiquitination or polyubiquitin chains linked through the Lys-63 residue of ubiquitin (44 -47). Intriguingly, the Ub(K63R) mutation leads to UV sensitivity and defective DNA repair in yeast (44,45). We have consistently observed the CUL-4A-stimulated formation of mono-and diubiquitin-modified p125 (Fig. 5, B and C). Future studies should address whether there is a degradation-independent function of CUL-4A-stimulated p125 ubiquitination, which specifies its distinct biological activities.
Microinjection of purified DDB into DDB Ϫ XP-E cells restored the in vivo DNA repair synthesis to normal levels, establishing the function of DDB in DNA repair (17). However, in vitro reconstitution studies indicated that DDB is not required for nucleotide excision repair of either naked or nucleosomal DNA (48 -50). Furthermore, Kazantsev et al. (49) observed an inhibitory rather than stimulatory effect of DDB on the excision repair of 6-4 photoproduct-containing DNA substrate in the in vitro nucleotide excision repair reactions. Thus, it has been suggested that DDB is involved in the initial DNA damage recognition step (28) and must subsequently be removed prior to the initiation of the nucleotide excision repair reactions. We propose that CUL-4A-stimulated ubiquitination and degradation of p48 may serve to restrict the steady-state levels of DDB inside the cell, therefore, ensuring the rapid dynamics of DDB at different stages of DNA damage recognition and repair.
The observation that abnormally high levels of CUL-4A are found in many human tumors suggests a potential role of CUL-4A in tumorigenesis. p48 has been shown to be the ratelimiting factor for UV-DDB activity and functions to suppress UV-induced mutations from genomic DNA and the non-transcribed DNA strand of expressed genes (19). "Loss of function" mutations in the p48 subunit of DDB, such as R273H, are responsible for the loss of UV-DDB activity and are associated with an increased risk for skin cancer. Our studies indicated that high level expression of CUL-4A down-regulates the steady-state levels of wild type p48 and, therefore, leads to the inhibition of UV-DDB activity. This will result in the accumulation of damage-induced lesions on DNA, a similar outcome as that resultant from the p48(R273H) mutation, and thus contribute to tumor development.