Cul3–Cul3 Intermolecular Interactions Are Detected In Vivo
The COP-1 ubiquitin ligase has been established previously to function as a homodimer (Torii et al., 1998
); therefore, we investigated whether other ubiquitin ligases, such as the Cul3-based ubiquitin ligase, can also form functional dimers in vivo. To test this hypothesis, Cul3 fusion proteins CFP–Cul3 and YFP–Cul3 were coexpressed in HEK293 cells for FRET analysis. Cells were stimulated with the CFP excitation wavelength (436 nm), and the YFP emission wavelength (535 nm) was detected (A, d, i, and n). After subtracting FRET emissions from the negative controls, 44% of cells expressing both CFP–Cul3 and YFP–Cul3 showed high net FRET values, indicating that CFP–Cul3 binds to YFP–Cul3 (A, o). The intensity of net FRET values from A were quantified, normalized against the total FRET emission to control for differential protein expression in each cell, and compared with net FRET values from cells that expressed either CFP–Cul3 or YFP–Cul3 alone (B). No net FRET with YFP–Cul3 was seen when CFP–Cul3 was substituted with CFP–actin (Supplemental Figure 1).
Figure 1. Cul3–Cul3 intermolecular interactions are detected in vivo. (A) HEK293 cells expressing CFP-Cul3 (a–e), YFP-Cul3 (f–j), and CFP-Cul3 with YFP-Cul3 (k–o) were imaged for quantitative FRET analysis. Phase contrast (a, f, (more ...)
BTB Domain-containing Proteins Are Not Required for Cul3–Cul3 Binding
We performed coimmunoprecipitation assays to verify the Cul3–Cul3 binding interaction that was observed using FRET analysis. FLAG-tagged Cul3 and Myc-tagged Cul3 were coexpressed in HEK293 cells and tested for protein expression (C, bottom two lanes). Myc-tagged Cul3 was able to pull down FLAG-tagged Cul3 (C, lane 3), indicating potential interactions between two or more Cul3 molecules. Because BTB domain-containing proteins can form homodimers (Robinson and Cooley, 1997
), it has been proposed that the dimerization of BTB domain-containing proteins bound to each Cul3 partner could promote Cul3–Cul3 interactions (Stogios et al., 2005
). To test this hypothesis, a Cul3 mutant (Cul3L52AE55A) that cannot bind any BTB domain-containing protein (Zheng et al., 2002
; Xu et al., 2003
) (B, lane 3) was selected for immunoprecipitation assays. If Cul3 dimerization occurs through the BTB domain-containing protein, the Cul3L52AE55A mutant would not be able to sustain the Cul3–Cul3 interactions. We observed that the Myc-tagged Cul3L52AE55A pulled down FLAG-tagged Cul3L52AE55A with the same efficiency as the wild-type Cul3 pair (C, lane 4), suggesting that a BTB domain-containing protein is not required for the association of the Cul3–Cul3 complex.
Figure 4. The Cul3 heterodimer can bind a BTB domain-containing protein. (A) HEK293 cells were transfected with vectors encoding HA-tagged Nedd8, Myc-tagged SPOP, FLAG-tagged Cul3, and a non-BTB-binding Cul3 mutant (L52AE55A) as indicated. Protein expression was (more ...)
The Cul3–Cul3 Association Is Mediated by a Winged-Helix B (WH-B) Domain near the C-Terminus of Cul3
Based on observations that a ubiquitin molecule can mediate protein dimerization (Mueller and Feigon, 2002
), we investigated whether the conjugation of Nedd8 to the C-terminal domain of Cul3 participates in these Cul3–Cul3 interactions. To inhibit Nedd8 conjugation, the neddylation site lysine 712 near the C terminus of Cul3 was mutated to arginine, creating the Cul3K712R mutant (Wu et al., 2000
; Zheng et al., 2002
). Myc-tagged Cul3K712R was able to pull down FLAG-tagged Cul3K712R with a slight decrease in efficiency compared with the wild-type pair (D, lanes 3 and 4), showing that covalent attachment of Nedd8 is not necessary for these Cul3–Cul3 interactions. Because lysine 712 lies within the WH-B domain that contains a stretch of hydrophobic residues (Zheng et al., 2002
), we mutated these residues in helix 1 (patch A mutant) and helix 3 (patch B mutant) (D, bottom). All Cul3 mutants were able to bind Rbx1 and BTB domain-containing proteins (data not shown), suggesting that the mutation did not disrupt protein folding. Immunoprecipitation revealed that either the patch A or the patch B mutants alone reduce Cul3–Cul3 binding; however, when both mutations were made in the same molecule, Cul3–Cul3 interactions were completely disrupted (D, lanes 5–8).
The Availability of a Nedd8 Molecule Is Essential for Cul3–Cul3 Interactions
Because mutations in the WH-B domain inhibited Cul3–Cul3 interactions, we decided to further investigate the in vivo role of Nedd8 in this particular example of Cul3–Cul3 binding. FRET experiments were performed in a Chinese hamster ovary cell line with a temperature-sensitive mutation in the neddylation pathway (ts41 cells) to determine whether Nedd8 is required to facilitate these Cul3–Cul3 interactions. If the presence of Nedd8 is essential for the dimerization of Cul3, Cul3–Cul3 interactions should be disrupted at the nonpermissive temperature at which the Nedd8 conjugation pathway becomes nonfunctional and thus drastically reduces the amount of Nedd8 localized to Cul3 (Handeli and Weintraub, 1992
). However, if the interaction between Nedd8 and Cul3 is not required, Cul3–Cul3 binding should persist even at nonpermissive temperature. Ts41 cells expressing CFP–Cul3 and YFP–Cul3 were incubated at either permissive temperature (32°C) or nonpermissive temperature (39°C) before the FRET analysis. At permissive temperature (32°C), Cul3 fusion proteins yielded a high net FRET value, indicating strong Cul3–Cul3 interactions (A, lanes a–e). However, the net FRET values decreased at nonpermissive temperature (39°C) (A, lanes f–j), suggesting that Cul3–Cul3 interactions were minimized in the absence of a Nedd8 molecule. To confirm that Cul3 neddylation is inhibited at nonpermissive temperature (39°C), immunoprecipitation followed by immunoblotting of endogenous Cul3 protein was performed on ts41 cell lysates. Although the population of unneddylated Cul3 remained the same at both temperatures, the population of neddylated Cul3 was reduced drastically at nonpermissive temperature (39°C), confirming that the reduction of Cul3–Cul3 interaction was a direct result of the disrupted neddylation pathway (B). As an experimental control, CFP–Cul3 and YFP–Cul3 were expressed in CHO cells and subjected to the same conditions as the ts41 cells. Net FRET values detected from CHO cells are similar at 32 and 39°C (C), confirming that the decrease in Cul3–Cul3 interactions was due to the nonfunctional Nedd8 pathway and not other temperature-dependent factors. The results from this FRET analysis support our hypothesis that the presence of Nedd8 is essential for Cul3–Cul3 interactions.
Figure 2. The neddylation pathway is essential for Cul3–Cul3 interactions. (A) Ts41 cells expressing CFP–Cul3 and YFP–Cul3 proteins were used for quantitative FRET imaging. Cells were incubated at permissive temperature (32°C) (a–e) (more ...)
Nedd8 Mediates the Formation of a Cul3 Heterodimer Composed of One Neddylated and One Unneddylated Cul3
Ubiquitin-mediated protein–protein interactions depend on hydrophobic binding between the protein partners and the hydrophobic surfaces of the ubiquitin molecule; therefore, we investigated the type of interactions that Nedd8 requires for participating in Cul3–Cul3 binding. HA-tagged Nedd8 in HEK293 cell lysates was immunoprecipitated with anti-HA antibody, immunoblotted, and probed for endogenous Cul3. A Cul3 doublet containing both the Nedd8-conjugated and the unconjugated Cul3 bands of similar intensity was observed (A, left, lane 2). This ratio differed from the proportion of the total Cul3 population in cell lysates, in which the upper protein band (Nedd8-conjugated) was less than half as intense as the lower protein band (Nedd8-unconjugated) (A, left, lane 1). The same immunoblot was stripped and reprobed with anti-HA antibody to verify that the upper band of the Cul3 doublet is a neddylated form of Cul3 (A, left, lanes 3 and 4). To confirm that the Nedd8-bound Cul3 complex occurs endogenously, Nedd8 from untransfected HEK293 cells was immunoprecipitated with anti-Nedd8 antibody, immunoblotted, and probed for Cul3, revealing nearly equivalent precipitation of neddylated and unneddylated Cul3 (A, right, lane 2). These results demonstrate two significant findings. First, it is expected that Nedd8 immunoprecipitation would pull down the Nedd8-conjugated Cul3 based on previous observations that Nedd8 can covalently attach to the lysine residue near the C terminus of cullins (Osaka et al., 1998
; Hori et al., 1999
; Wada et al., 1999
). However, our observation that Nedd8 immunoprecipitated unconjugated Cul3 was unexpected and revealed a novel noncovalent interaction between Nedd8 and the Cul3 protein. Second, the Nedd8-bound Cul3 complex contains approximately equal amounts of neddylated and unneddylated Cul3, which is different from the proportion of neddylated and unneddylated Cul3 found in cell lysates.
Figure 3. Nedd8 mediates the formation of Cul3 heterodimers by interacting with the winged-helix B domain near the C termini of Cul3. (A) HEK293 cells were transfected with HA-tagged Nedd8 (left) or harvested without transfection (right). Cell lysates were immunoprecipitated (more ...)
To further investigate the observed noncovalent interaction between Nedd8 and Cul3, the binding between the two proteins was investigated in a two-hybrid assay. The relative strength of the interactions was determined using a β-galactosidase liquid culture assay with CPRG as a substrate. After subtraction of data from controls, we observed that the Cul3K712R mutant was able to bind to Nedd8 in cells although with reduced efficiency (89.7% for wild type and 30.3% for Cul3K712R) (). Together, these data confirm a second, noncovalent interaction between Nedd8 and Cul3 in both yeast and mammalian cells.
Relative binding between Nedd8 and Cul3 or a Cul3K712R mutant in S. cerevisiae
To determine the stoichiometry of the multimeric Cul3 complex, purified complexes were analyzed using a gel filtration column. FLAG-tagged Cul3 was pulled down by His-tagged Nedd8 expressed in HEK293 cells and used to determine the size of the Nedd8-bound Cul3 complex. Purified Cul3 monomers from E. coli were used as a control in this experiment. Because Nedd8 and the neddylation pathway do not exist in E. coli, the possibility of Nedd8-mediated Cul3 heterodimers in the control sample is eliminated. His-tagged Cul3 from E. coli was purified to obtain Cul3 monomers, analyzed by gel filtration, immunoblotted, and probed for Cul3 with anti-Cul3 antibody (B, bottom). Cul3 purified from E. coli eluted from the column close to the predicted molecular mass of the Cul3 monomer (90 kDa). In contrast, the Nedd8-bound Cul3 complex was eluted at ~216 kDa, which is approximately the same size as the predicted mammalian Cul3 dimer consisted of a single Nedd8 molecule, two Rbx1 proteins, and two Cul3 monomers (B, top). Therefore, these Cul3 complexes are dimers and do not represent a higher oligomeric state complex. These experimental observations led to the conclusion that Nedd8 mediates the formation of Cul3 heterodimers composed of one neddylated and one unneddylated Cul3.
Based on the proximity of the Rbx1 binding site to the site of Nedd8 attachment in the C-terminal domain of Cul3, Rbx1 was also tested for its ability to mediate the formation of Cul3 heterodimer. An immunoprecipitation of Rbx1 brought down neddylated and unneddylated Cul3 in the same ratio as that found in cell lysates (Supplemental Figure 2) and not the one-to-one ratio observed in the Nedd8 immunoprecipitation assays, suggesting that Rbx1 does not facilitate the formation of this Cul3 dimer.
Both an Isopeptide Linkage and Hydrophobic Binding between Nedd8 and the WH-B Domains of Cul3 Contribute to the Nedd8-mediated Cul3 Dimerization
Previous observations stating that the WH-B domain is essential for Cul3–Cul3 interactions and that Nedd8 is required for Cul3–Cul3 binding have led us to investigate whether the WH-B domain interacts directly with the Nedd8 molecule. To analyze the potential role of this domain in the formation of the Cul3 dimer, we immunoprecipitated HA-tagged Nedd8 and probed with anti-FLAG antibody for binding to FLAG-tagged Cul3 variants with mutations in the WH-B domain. As a negative control, cell lysates expressing FLAG-tagged Cul3 alone were immunoprecipitated with anti-HA antibody, proving that FLAG-tagged Cul3 was not pulled down nonspecifically by this immunoprecipitation (C, lane 1). When coexpressed with HA-tagged Nedd8, however, wild-type FLAG-tagged Cul3 and a FLAG-tagged Cul3L52AE55A mutant were pulled down as doublets with equal band intensities (C, lanes 2 and 3), confirming that a BTB domain-containing protein does not mediate the formation of Cul3 dimers. If an isopeptide bond between Nedd8 and Cul3 is the only interaction that holds the two molecules together, HA-tagged Nedd8 would not pull down a FLAG-tagged Cul3K712R mutant because it lacks the neddylation site (Supplemental Figure 3, lane 2). However, this study showed that HA-tagged Nedd8 was bound to the unneddylated Cul3K712R mutant, which further supports our observation that the second type of Nedd8–Cul3 interaction is noncovalent (C, lane 4). An immunoprecipitation of the Cul3 patch B mutant via Nedd8 shows a slightly greater amount of neddylated Cul3 than unneddylated Cul3 (C, lane 6), suggesting that hydrophobic residues in the WH-B domain of Cul3 participate in the dimerization. When a double mutant was made by combining K712R with the patch B mutations, only the unneddylated form of the Cul3 mutant was bound to Nedd8 (C, lane 7). A similar result was also found in the Cul3 patch A mutant, which has a partially disrupted hydrophobic surface and cannot be neddylated (C, lane 5). When both patch mutants were present on the same molecule, HA-tagged Nedd8 did not pull down any FLAG-tagged Cul3 mutants, indicating that both the neddylation site and the hydrophobic region in the WH-B domain are essential for the Nedd8–Cul3 interactions (C, lane 8; Supplemental Figure 3, lane 3).
Having demonstrated that Nedd8 mediates Cul3 dimerization, we suggest that two interactions contribute to the dimerization of Cul3: one Cul3 molecule interacts with Nedd8 via a combination of hydrophobic interactions and an isopeptide linkage between Nedd8 and lysine 712. The second Cul3 molecule uses hydrophobic binding between its WH-B domain and the surface of the Nedd8 protein (D).
The Cul3 Heterodimer Can Bind a BTB Domain-containing Protein
We suggest that the Cul3 heterodimer is an active form of the Cul3-based ubiquitin ligase complex. Based on the current model of an active Cul3 complex, Cul3 requires a BTB domain-containing protein to provide substrate specificity. A BTB domain-containing protein, SPOP, which is involved in X-chromosome inactivation (Hernandez-Munoz et al., 2005
; Kwon et al., 2006
; Zhang et al., 2006
), was tested for binding to the Cul3 dimer. We observed that HA-tagged Nedd8 did not bind Myc-tagged SPOP alone (A, top, lane 2); however, HA-tagged Nedd8 was able to pull down Myc-tagged SPOP in the presence of wild-type Cul3 (A, top, lane 3) but not in the presence of the Cul3L52AE55A mutant (A, top, lane 3). Nedd8 interactions with a Cul3 dimer and a BTB domain-containing protein suggest that a BTB domain-containing protein can be a member of the heterodimeric Cul3 complex.
To confirm the presence of a BTB domain-containing protein in the Cul3 dimer, we used the Cul3L52AE55A mutant that cannot bind a BTB domain-containing protein (Xu et al., 2002
; Zheng et al., 2002
). However, a Cul3 dimer consisting of a wild-type Cul3 and a Cul3L52AE55A mutant should enable the Cul3 mutant to bind a BTB domain-containing protein through its Cul3 partner. If a BTB domain-containing protein does not bind to a Cul3 dimer, a Cul3L52AE55A mutant would not form a complex with a BTB domain-containing protein regardless of the expression of wild-type Cul3.
To test this hypothesis, either a FLAG-tagged wild-type Cul3 or a FLAG-tagged Cul3L52AE55A mutant, Myc-tagged SPOP, and His-tagged wild-type Cul3 were coexpressed in HEK293 cells and subjected to immunoprecipitation. Myc-tagged SPOP was brought down by FLAG-tagged wild-type Cul3 but not by a FLAG-tagged Cul3L52AE55A mutant (B, top, lanes 2 and 3). Immunoprecipitation with anti-FLAG antibody could not coimmunoprecipitate Myc-tagged SPOP in cells that expressed only Myc-tagged SPOP and His-tagged Cul3 (B, top, lane 1). However, immunoprecipitation with anti-FLAG antibody coimmunoprecipitated Myc-tagged SPOP when FLAG-tagged Cul3L52AE55A, Myc-tagged SPOP, and His-tagged wild-type Cul3 were coexpressed (B, lane 4), suggesting that the FLAG-tagged Cul3L52AE55A formed a dimer with the His-tagged wild-type Cul3 that is bound to Myc-tagged SPOP. These results confirm that a Cul3 heterodimer can bind a BTB domain-containing protein.
A Cul3 Substrate Pulls Down a Heterodimeric Cul3 Complex
The proposed model that the Cul3 heterodimer is an active form of the Cul3-based ubiquitin ligase complex is supported by the ability of the Cul3 heterodimer to bind BTB domain-containing proteins. However, the role of a cullin-based ubiquitin ligase complex is to provide substrate specificity for the ubiquitination pathway by binding to its substrates and recruiting the E2 ubiquitin-conjugating enzyme. Therefore, the presence of protein substrates bound to the ubiquitin ligase complex is key to identifying active ubiquitin ligases. If the active Cul3 complex is a neddylated Cul3 monomer bound to a BTB domain-containing protein, immunoprecipitation of Cul3 substrates should only coimmunoprecipitate neddylated Cul3. However, if the Cul3 heterodimer is the active complex, Cul3 substrates would coimmunoprecipitate the Cul3 dimer with equal amounts of neddylated and unneddylated Cul3. We selected a known Cul3 substrate, cyclin E, to determine whether the Cul3 heterodimer is the active Cul3 complex. Immunoprecipitation of cyclin E brought down FLAG-tagged Cul3 as a doublet similar to the Cul3 doublet from HA-Nedd8 immunoprecipitation assays (, right lane). In contrast, immunoprecipitation and immunoblotting for FLAG-tagged Cul3 brought down the same proportion of Cul3 as that found in cell lysates (, left lane). The ability of the Cul3 heterodimer to bind its substrate supports the hypothesis that the Cul3 heterodimer is an active form of the Cul3-based ubiquitin ligase complex.
Figure 5. A Cul3 substrate, cyclin E, binds Cul3 in a heterodimeric form. FLAG-tagged Cul3 expressed in HEK293 cells was immunoblotted with anti-FLAG antibody (bottom). Cell lysates were subjected to immunoprecipitation with anti-FLAG (lane 1) or anti-cyclin E (more ...)
Coexpression of Two Nonfunctional Cul3 Mutants Rescues Its Ubiquitin Ligase Function
Although we demonstrated that the Cul3 heterodimer can bind a BTB domain-containing protein and its substrate, a functional assay is required to prove that Cul3 is active in its heterodimeric form. We have previously established that cyclin E is a substrate of the Cul3-based ubiquitin ligase complex and that coexpression of Cul3 with cyclin E increases the amount of ubiquitinated cyclin E in HEK293 cells (Singer et al., 1999
). Similar to the binding assay for a BTB domain-containing protein in B, we developed an in vivo rescue assay for cyclin E ubiquitination using two different Cul3 mutants that cannot individually ubiquitinate cyclin E. If Cul3 functions only as a monomer, cyclin E bound at the C terminus of Cul3 would only be ubiquitinated by an E2 bound at the C terminus of the same Cul3 molecule. However, if Cul3 can also function as a dimer, a substrate bound to the N terminus of one Cul3 molecule could potentially be ubiquitinated by the E2 bound at the C terminus of the second Cul3 molecule, leading to increased cyclin E ubiquitination when two mutants are coexpressed.
Myc-tagged cyclin E and HA-tagged ubiquitin were coexpressed with either wild-type Cul3 or Cul3 mutants. Immunoblotting was performed to determine relative expression levels of Cul3 proteins (A, bottom). Immunoprecipitation with anti-Myc antibody followed by immunoblotting that probes for Myc-tagged cyclin E revealed that coexpression of wild-type Cul3 results in the same high-molecular-weight cyclin E smear that could be induced by the addition of proteasome inhibitor MG132 (A, top and middle, lanes 2 and 3). For the rescue assay, two Cul3 deletion mutants were chosen: a Cul3 mutant with a deletion of the first 23 residues of the N terminus (Cul3ΔN) and a Cul3 mutant with a stop codon at residue 734 (Cul3stop734). Although a significant C-terminal portion has been deleted, Cul3 stop734 mutant can form a dimer by using an intact helix-1 in the WH-B domain. Individually, these Cul3 mutants did not induce cyclin E ubiquitination (A, lanes 4 and 5). However, cyclin E ubiquitination was rescued when the mutants were coexpressed, indicating that the Cul3 dimer is a functional form of the Cul3-based ubiquitin ligase complex. To confirm that the high-molecular-weight bands were polyubiquitinated cyclin E, the blot was stripped and reprobed with anti-HA antibody (B, top). Furthermore, the same lysates were subjected to immunoprecipitation with HA-ubiquitin and probed for cyclin E. The presence of a single band of cyclin E rather than a smear indicates that the most prominent ubiquitinated form of cyclin E is the monoubiquitinated form. Although the endogenous cellular machinery can catalyze the ubiquitination of cyclin E (B, middle, lane 1), overexpression of Cul3 protein increases the level of the ubiquitinated cyclin E close to that obtained by an addition of proteasome inhibitor, MG132 (B, middle, lanes 2 and 3). The Cul3ΔN and Cul3stop734 mutants yielded the two lowest amount of ubiquitinated cyclin E in this experiment, indicating that these mutants may function as dominant negative and prevent the cyclin E from being ubiquitinated by the endogenous proteins (B, middle, lanes 4 and 5). When the two mutants were coexpressed, the Cul3 ubiquitination activity was rescued (B, middle, lane 6), which is similar to the results seen in A. Quantitative measurements for the intensity of ubiquitinated cyclin E bands also support this observation (B, bottom).
Figure 6. The Cul3 heterodimer is an active form of the Cul3-based ubiquitin ligase complex. (A) HEK293 cells were transiently transfected with vectors encoding untagged Cul3, N-terminally truncated Cul3 mutant (ΔN), C-terminally truncated Cul3 mutant (stop734), (more ...)