The von Hippel-Lindau Tumor Suppressor Gene Product Promotes, but Is Not Essential for, NEDD8 Conjugation to Cullin-2*

We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100–105). Cul-2 has also been reported to form a multiprotein complex, Cul-2·VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elonginsB and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2·VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.

The von Hippel-Lindau (VHL) 1 syndrome is a rare hereditary cancer syndrome characterized by the development of various tumors, including renal cell carcinomas, pheochromocytomas, and vascular tumors of the central nervous system (1,2). Mutation or transcriptional silencing of the VHL gene and the subsequent loss of the remaining VHL allele are associated with the tumorigenesis in patients with VHL syndrome (2)(3)(4). The VHL gene product (pVHL), composed of 213 amino acids, is expressed as a 30-kDa protein in all tissues (5) and forms a VBC complex with elongins B and C (6). Interestingly, tumorigenic pVHL mutants in VHL patients are unable to bind elongins B and C, resulting in the failure of VBC complex formation. Recently, the VBC complex has been shown to bind to human cullin-2 (Cul-2) to form a multiprotein complex, Cul-2⅐VBC (7,8).
Recently, we showed that a ubiquitin-like protein, NEDD8, covalently conjugates to Cul-2 (20). NEDD8 is a highly conserved 81-amino acid protein that is 60% identical and 80% homologous to ubiquitin. The message expression of NEDD8 is highly restricted to the heart and skeletal muscle in adult human tissues (21) and is developmentally down-regulated in mouse embryos (21,22). NEDD8 belongs to an expanding family of ubiquitin-like proteins such as UCRP (23), sentrin-1/ SUMO1 (24 -27), sentrin-2 (28), and sentrin-3 (29). These proteins share a common distinction in that the mature form of the proteins is always translated in precursor form, with one or more amino acids following a Gly-Gly dipeptide that forms the C terminus of the mature protein (30). In the NEDD8 conjugation process, the C-terminal tail of the precursor protein is cleaved off by C-terminal hydrolase such as UCH-L3 (31). The mature form has been shown to conjugate to a large number of nuclear proteins (21). The conjugation is thought to be catalyzed by NEDD8 activation enzyme (E1) and NEDD8 conjugation enzyme (E2) in the presence of ATP (32,33). Recently, we reported that all human cullins including Cul-2 are conjugated by a single molecule of NEDD8 (20). However, the precise role of NEDD8 conjugation to cullins has not been elucidated. In this paper, we investigate the relationship between NEDD8 conjugation to Cul-2 and the Cul-2⅐VBC complex.  1 The abbreviations used are: VHL, von Hippel-Lindau; pVHL, von Hippel-Lindau gene product; VBC, pVHL-elongin B-elongin C; Cul, cullin; SCF, Skip1-Cul-1-F-box protein; HIF, hypoxia-inducible factor; HA, hemagglutinin; mAb, monoclonal antibody.
Establishment of 786-0/VHL Cells-786-0 cells were transfected with pEC1214A containing a cDNA sequence for expression of HAtagged wild-type human pVHL, followed by selection with G418 (1 mg/ml) for 3 weeks. The expression level of HA-pVHL in selected clones was analyzed by Western blotting using anti-HA antibody 16B12. A stable clone with the highest expression was termed 786-0/VHL. This clone was maintained successfully in the absence of G418.
Plasmid Construction and Transfection-To express proteins tagged with an epitope at the N terminus in mammalian cells, pcDNA3/3HA-N, pcDNA3/RH-N (21), or pcDNA3/3RH-N was used as described previously (21). The mammalian expression vectors pcDNA3/3HA-N and pcDNA3/3RH-N were constructed by inserting a thrice-repeated epitope of HA or RH, respectively, into pcDNA3 (Invitrogen, San Diego, CA). Using pcDNA3/3HA-N or pcDNA3/3RH-N, proteins can be tagged with a thrice-repeated epitope at their N terminus and expressed in mammalian cells. This system allowed us to detect proteins expressed at low levels. The human cDNA of pVHL, elongin B, elongin C, Cul-2 (20), or NEDD8 (21) was amplified by polymerase chain reaction using appropriate primers from the cDNA library of human testis or brain (CLONTECH, Palo Alto, CA). These cDNAs were inserted into the aforementioned plasmid vectors. The sequence of each cDNA was confirmed by automated DNA sequencing. Plasmids were transfected into mammalian cells using Lipo-fectAMINE (Life Technologies, Inc.) or FuGENE 6 (Roche Molecular Biochemicals). The transfected cells were harvested for Western blotting or immunoprecipitation 20 h after transfection.
Site-directed Mutagenesis-A Cys-to-Phe or Arg-to-Trp substitution was generated in pVHL at Cys-162 or Arg-167, respectively. The cDNA of wild-type pVHL was mutated by polymerase chain reaction-based site-directed mutagenesis as described previously (34). The mutated cDNA was subcloned into pcDNA3/RH-N.
Immunoprecipitation-The immunoprecipitation method described by Kamitani et al. (34) was modified as follows. COS cells (5 ϫ 10 6 ) were cotransfected for expression of 3HA-Cul-2, RH-pVHL, RH-elongin B, and RH-elongin C, followed by trypsinization 20 h after transfection (see Fig. 2). In addition to COS cell transfectants, 2 ϫ 10 7 786-0 and 786-0/VHL cells were also used for immunoprecipitation (see Fig. 7). Cell pellets were transferred to liquid nitrogen and lysed in 1.25 ml of radioimmune precipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, ant 150 mM NaCl) containing 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 10 g/ml aprotinin, 1.5 M pepstatin, 1 mM Na 3 VO 4 , and 5 mM N-ethylmaleimide. Cell lysis was performed for 30 min on ice, and DNA in the sample was sheared with a 22-gauge needle. After centrifugation at 100,000 ϫ g for 30 min at 4°C, the supernatants were added to 10 g of mouse mAb coupled to 25 l of protein G-Sepharose beads (Amersham Pharmacia Biotech). Anti-HA mAb 12CA5 was used for samples from COS cell transfectants, and anti-Cul-2 or anti-Fas mAb was used for samples from 786-0 and 786-0/VHL cells. The bead suspensions were rocked for 3 h at 4°C. Beads were washed five times with RIPA buffer. The immunoprecipitates were treated with 30 l of 2% SDS treating solution containing 5% ␤-mercaptoethanol, and 3 l of solubilized samples were loaded onto 8% SDS-polyacrylamide gels. Then, Western blotting was performed. For samples from COS cell transfectants, rabbit anti-HA antibody HA.11 was used to detect 3HA-Cul-2 derivatives, and rabbit anti-NEDD8 antibody was used to detect 3HA-Cul-2 conjugated with NEDD8. For samples from 786-0 and 786-0/VHL cells, rabbit anti-NEDD8 antibody was used to detect endogenous Cul-2 conjugated with endogenous NEDD8, and rabbit anti-Cul-2 antibody was used to detect endogenous Cul-2 derivatives.
Western Blotting-Protein samples were treated at 45°C for 1 h in 150 l of 2% SDS treating solution containing 5% ␤-mercaptoethanol. After SDS-polyacrylamide gel electrophoresis, Western blotting was performed using the protocol provided by the ECL detection system (Amersham Pharmacia Biotech). Horseradish peroxidase-conjugated antibodies against mouse IgG or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were used as secondary antibodies.

RESULTS AND DISCUSSION
An 85-kDa Derivative of the Cul-2 Molecule Is Generated by Overexpression of pVHL in COS Cells-pVHL forms a VBC complex with elongins B and C (6). Recently, this VBC complex has been shown to assemble a heterotetramer with Cul-2 (7,8). Thus, it is of interest to examine the relationship between Cul-2 and the VBC complex. For this purpose, 3HA-Cul-2 was coexpressed with combinations of RH-tagged elongins B and C and pVHL in COS cells. As shown in Fig. 1A, 3HA-Cul-2 was clearly detected as an 80-kDa protein band (lanes 2-5). In addition, an 85-kDa protein band was clearly detected only when pVHL and elongins B and C (VBC) were coexpressed with 3HA-Cul-2 (lane 5). Although this protein band was also detectable in the sample transfected with 3HA-Cul-2 and RH-pVHL, it was extremely faint (lane 4). In this sample, a low level of 85-kDa protein might be generated by transfected pVHL and endogenous elongins B and C. Taken together, the 85-kDa Cul-2 derivative is highly generated by coexpression with pVHL and elongins B and C.
pVHL Generates an 85-kDa Derivative of Wild-type Cul-2, but Not of Cul-2(K689R)-Recently, we showed that Cul-2 is conjugated at Lys-689 by NEDD8 (20). Since NEDD8 monomer was detected as a 6-kDa band in SDS-polyacrylamide gel electrophoresis (21), we hypothesized that the 85-kDa protein was Cul-2 conjugated with endogenous NEDD8. To prove this hy- pothesis, RH-tagged VBC proteins were coexpressed with 3HA-Cul-2(K689R), which has a Lys-to-Arg substitution at the NEDD8 conjugation site and cannot be conjugated by NEDD8. As shown in Fig. 1B, overexpression of VBC led to the appearance of the 85-kDa derivative with wild-type Cul-2 (lane 2), but not with Cul-2(K689R) (lane 3). These results support the hypothesis that the 85-kDa protein is Cul-2 conjugated with endogenous NEDD8.
The 85-kDa Protein Is Cul-2 Conjugated with Endogenous NEDD8 -Next, the 85-kDa derivative was directly identified. 3HA-Cul-2 was coexpressed with RH-tagged VBC proteins in COS cells. 3HA-Cul-2 derivatives consisting of 80-and 85-kDa proteins were purified from total cell lysate by immunoprecipitation using anti-HA mAb. The immunoprecipitates were analyzed by Western blotting using anti-HA antibody to detect 3HA-Cul-2 derivatives and anti-NEDD8 antibody to examine the 85-kDa protein. As shown in Fig. 2, 80-and 85-kDa proteins were immunoprecipitated well (lane 2). The precipitated 85-kDa protein could be detected by anti-NEDD8 antibody, but the 80-kDa protein could not (lane 4). These results indicate that the 85-kDa protein is 3HA-Cul-2 conjugated with endogenous NEDD8 and that the 80-kDa protein is an unconjugated form of 3HA-Cul-2.
pVHL-(54 -197) Is Required for Promoting NEDD8 Conjugation to Cul-2-To identify the necessary domain of pVHL for promoting NEDD8 conjugation to Cul-2, we generated pVHL mutants with N-or C-terminal deletions. The mutants were tagged with the RH epitope and coexpressed with 3HA-Cul-2, RH-elongin B, and RH-elongin C in COS cells. The total cell lysates were analyzed by Western blotting using anti-HA mAb to detect Cul-2 derivatives. As shown in Fig. 4A hardly generated any 85-kDa protein, and the levels were not above the negative control (lane 1). The simplest interpretation of these data, summarized in Fig. 4B, is that pVHL-(54 -197) is required for promoting NEDD8 conjugation to Cul-2. pVHL-(54 -197) is almost the same region that is involved in complex formation with elongins B and C and Cul-2 (8). Thus, pVHL mutants with a tumorigenic substitution or deletion, which are unable to form a VBC complex with elongins B and C, do not possess the promoting activity for NEDD8 conjugation to Cul-2. These findings suggest that pVHL cannot promote NEDD8 conjugation to Cul-2 unless a Cul-2⅐VBC complex is assembled.
NEDD8 Conjugation Occurs in the pVHL-deficient Cell Line 786-0 -pVHL has an ability to promote NEDD8 conjugation to Cul-2 as described above. Is pVHL essential for NEDD8 conjugation to Cul-2 and/or other target proteins? To address this question, we employed a sporadic renal carcinoma cell line, 786-0, in which the VHL gene contains a 1-base pair deletion creating a frameshift from residue 104 in the remaining single allele (35). The conserved pVHL-(1-103) in 786-0 cells is not sufficient for promoting NEDD8 conjugation to Cul-2 (see Fig.  4). Using 786-0 cells, we examined whether NEDD8 conjugation could be still detected or not. For this purpose, 3HA-NEDD8 was transiently expressed by transfection in 786-0 cells and in positive control cells such as 293 and U-2OS. NEDD8 conjugation was analyzed by Western blotting using anti-HA mAb. As shown in Fig. 5, the unconjugated form of 3HA-NEDD8 (open arrowhead) and 3HA-NEDD8 conjugation to target proteins were clearly observed in 293 cells (lane 2) and U-2OS cells (lane 4). An 85-kDa protein (closed arrowhead) was predominantly detected in these cells, as previously reported (21). In 786-0 cells, an 85-kDa protein was also strongly detected (closed arrowhead), whereas the unconjugated form and  4) or without (lanes 1 and 3) 3HA-Cul-2. Total cell lysates were immunoprecipitated (IP) with mouse anti-HA antibody 12CA5. The immunoprecipitates were analyzed by Western blotting (WB) using rabbit anti-HA antibody HA.11 to detect Cul-2 derivatives (lanes 1 and 2) and rabbit anti-NEDD8 antibody to detect NEDD8-conjugated Cul-2 ( lanes  3 and 4). The identity of each band is indicated on the right. other conjugated forms of 3HA-NEDD8 were hardly detected (lane 6). These results indicate that NEDD8 conjugation occurs in 786-0 cells and that an 85-kDa protein is predominantly generated. Thus, pVHL is not essential for NEDD8 conjugation in human cells.
Endogenous Cul-2 and Cul-1 Derivatives in 786-0 and 786-0/VHL Cells-In Fig. 5, a prominent NEDD8-conjugated protein of 85 kDa was observed in 786-0 cells as well as in 293 and U-2OS cells. The size of this protein would be compatible with NEDD8-conjugated cullins (9). To examine whether cullins can be modified by NEDD8 in 786-0 cells, we performed Western blotting using total cell lysates of 786-0 and 786-0/VHL cells expressing wild-type pVHL (Fig. 6, lower panel). As shown in NEDD8 Conjugation to Cul-2 in 786-0 Cells-To characterize the doublet band of Cul-2 observed in Fig. 6 (upper panel), we immunoprecipitated endogenous Cul-2 in 786-0 or 786-0/VHL cells by mouse mAb against the N terminus of Cul-2 and performed Western blotting using rabbit polyclonal antibody against NEDD8 or the C terminus of Cul-2 to detect immunoprecipitates. As shown in Fig. 7 -6), or RH-pVHL containing C-terminal deletions (lanes 7-9). Total cell lysates were analyzed by Western blotting using anti-HA antibody 16B12 to detect the unconjugated and NEDD8-conjugated forms of 3HA-Cul-2. The identity of each band is indicated on the right. B, shown is a summary of the ability of pVHL mutants to promote NEDD8 conjugation to Cul-2. ϩ, pVHL mutants have the ability to promote NEDD8 conjugation to Cul-2 at levels similar to wild-type pVHL; Ϫ, the ability of the mutants is not above the negative control level; ϩ/Ϫ, the mutant has much less ability than wild-type pVHL, but the level is above the negative control. Shaded boxes indicate domains of pVHL that are involved in complex formation with elongins B and C and Cul-2 (8). Asterisks indicate sites of missense mutations.
FIG . 5. NEDD8 conjugation in 786-0 cells. 293 (lanes 1 and 2), U-2OS (lanes 3 and 4), and 786-0 (lanes 5 and 6) cells were transfected with empty vector (lanes 1, 3, and 5) or 3HA-NEDD8 (lanes 2, 4, and 6). Total cell lysates were prepared from transfectants, and NEDD8 conjugation was analyzed by Western blotting using anti-HA antibody 16B12. Unconjugated 3HA-NEDD8 is indicated by the open arrowhead, and the 85-kDa 3HA-NEDD8-conjugated protein is indicated by the closed arrowhead. NEDD8 conjugation to Cul-2 occurs more efficiently in 786-0/ VHL cells than in 786-0 cells. The differential expression ratio of the upper band to the lower band observed in Fig. 6 (upper panel) also suggested the inefficient NEDD8 conjugation to Cul-2 in 786-0 cells and the efficient NEDD8 conjugation to Cul-2 in 786-0/VHL cells. Thus, pVHL is a critical but not essential factor for NEDD8 conjugation to Cul-2. In pVHLdeficient 786-0 cells, other proteins might compensate for pVHL deficiency. Interestingly, there is a discrepancy between Figs. 6 (upper panel) and 7 (lanes 5 and 6) regarding the Cul-2 expression pattern. The unconjugated form of Cul-2 was poorly immunoprecipitated by anti-Cul-2 N terminus antibody under nondenaturing conditions, suggesting the possibility that the unconjugated form of Cul-2 might be folded and that the antigen determinant at its N terminus might be hidden. NEDD8 conjugation to Cul-2 might lead to a conformational change in the Cul-2 molecule and disclose the antigen determinant at the N terminus of Cul-2.
As shown in Figs. 6 (upper panel, lane 1) and 7 (lanes 3 and 5), endogenous Cul-2 in 786-0 cells was conjugated by endogenous NEDD8. In COS cells, however, transfected Cul-2 was not efficiently conjugated by endogenous NEDD8 in the presence of endogenous pVHL (Figs. 1A (lane 2), 3 (lane 1), and 4A (lane 1)). pVHL might be a major player for NEDD8 conjugation to Cul-2 in COS cells, but not in 786-0 cells. Alternatively, endogenous pVHL and elongins B and C (monkey origin) in COS cells might not be as functional against transfected human Cul-2 because of the species specificity of these molecules.
During the preparation of this article, Liakopoulos et al. (36) reported that NEDD8 conjugation to Cul-2 was stimulated by wild-type pVHL, but not by tumorigenic pVHL mutant. Using the in vitro translation method, they coexpressed Cul-2 with wild-type or mutant pVHL and showed that only wild-type pVHL stimulated NEDD8 conjugation to Cul-2. These in vitro findings support the in vivo results presented in this paper.
In summary, we used mammalian cells such as COS and 786-0 cells to show that wild-type pVHL has the ability to promote NEDD8 conjugation to Cul-2. This ability was not shown by deletion mutants or tumorigenic point mutants of pVHL, which were unable to form a VBC complex with elongins B and C. In addition, the promoting activity of pVHL for NEDD8 conjugation to Cul-2 was strongly detected only when the VBC proteins were coexpressed. These findings suggest that the complex formation of VBC is required for NEDD8 conjugation to Cul-2. Taken together, pVHL assembles a VBC complex and binds to Cul-2 to form Cul-2⅐VBC. Then, the VBC complex promotes NEDD8 conjugation to Cul-2. Thus, the VBC complex might catalyze the conjugation reaction as one of the "NEDD8 ligases."