We set out to characterize in depth the deneddylating activity of CSN by developing an in vitro
assay in which the conversion of neddylated Cul1 to Cul1 could be quantitatively measured. CSN was purified from HEK293 cells that stably express the Csn2 subunit modified with an N-terminal FLAG tag (27
). CSN recovered from an anti-FLAG affinity column was further enriched by gel filtration prior to being used for the experiments described here. All eight subunits were found to be present in the purified material, in apparently stoichiometric amounts (A
). Mass spectrometry analysis of purified CSN identified two co-fractionating proteins (Ddb1 and Hsp70) that could not be totally removed by conventional methods such as high salt or treatment with ATP and Mg2+
(data not shown).
FIGURE 1. Characterization of in vitro deneddylation assay components and enzymatic properties of human CSN.
A, purified CSN from HEK293 cells was fractionated by SDS-PAGE and analyzed by silver staining to check for purity and stoichiometry of enzyme subunits. (more ...)
To develop a quantitative, multiturnover assay for deneddylation, we first sought to generate a labeled form of Nedd8. To this end, we fused a sequence encoding eight histidines followed by a protein kinase A phosphorylation site to the N terminus of Nedd8 to generate His-PKA-Nedd8 (HPN8). When Cul1 was neddylated with HPN8 (HPN8-Cul1) and subsequently purified by gel filtration and nickel-nitrilotriacetic acid chromatography, the resulting CSN substrate was 98% neddylated as determined by SDS-PAGE (supplemental Fig. S1, A and B
). After radioisotope labeling with [32
P]ATP and cAMP-dependent protein kinase, >97% of the total signal from Nedd8 was attached to Cul1 as determined by phosphorimaging (B
; time = 0). When CSN (1 nm
) was added to HPN8-Cul1 (50 nm
) and the reaction progress analyzed, deneddylation proceeded at a linear rate (, B
Prior to performing kinetic analyses, we carried out a series of control experiments. First we confirmed that the phosphorylated His-PKA tag had little to no effect on the rate of deneddylation of Cul1 (supplemental Fig. S1C
). Second, we compared rates of deneddylation by our FLAG-tagged CSN prepared from 293 cells with untagged CSN expressed in insect cells from recombinant baculoviruses (supplemental Fig. S1D
) and untagged CSN expressed in E. coli
) (supplemental Fig. S1E
). The FLAG tag had no discernable effect on the activity of CSN isolated from eukaryotic cells. Meanwhile, the enzyme purified from E. coli
was ~2-fold less active, possibly because of removal of terminal sequences from some subunits to facilitate expression.
To obtain enzymological metrics for CSN-mediated deneddylation, we evaluated reaction rate as a function of substrate concentration. CSN was held constant at 0.8 nm
, and the initial rates of deneddylation at varying concentrations of HPN8-Cul1-Rbx1 were determined (supplemental Fig. S1F
). The rates for each concentration of substrate were plotted and fitted to the Michaelis-Menten equation, which yielded a Km
of 212 nm
and a kcat
of 1.1 s−1
) supplemental Table S1
Within cells, the fraction of Cul1 that is modified by Nedd8 is higher for Cul1 bound to F-box proteins (16
). We reasoned that this might arise from differential rates of deneddylation of Cul1, depending upon its assembly status. To test this possibility, we compared rates of deneddylation for HPN8-Cul1-Rbx1 versus
three different HPN8-conjugated SCF holoenzyme complexes. Deneddylation of HPN8-SCFFbw7
was assayed at 500 nm
. Strikingly, Fbw7-Skp1 had a major effect, reducing the rate of deneddylation by 5.8-fold (A
). β-TrCP-Skp1 had a weaker effect, reducing the rate by 2.2-fold. However, the recombinant β-TrCP used for this experiment lacks the N-terminal 138 amino acids, which were removed to facilitate efficient expression (30
). Interestingly, not all F-box proteins were inhibitory. Addition of Skp2-Cks1-Skp1 reduced the rate of deneddylation by <20% (B
). We do not understand the basis for this difference in behavior but note that endogenous Cul1 co-precipitated with transiently expressed F-box proteins was ≥50% neddylated in β-TrCP and Fbw7 immunoprecipitates but considerably less modified in Skp2 immunoprecipitates (supplemental Fig. S2, A and B
, and data not shown).
In other work we have shown that a mutant of Skp1 (Skp1ΔΔ) used for crystallography (31
) that lacks two acidic internal loops was able to bind Cul1-Rbx1 and assemble an active SCF complex but was unable to promote displacement of Cand1 from Cul1-Rbx1 (32
Because Fbw7-Skp1 had the most potent effect on deneddylation, we tested the impact of the Skp1 loops in this context. Interestingly, the loop deletions reduced the inhibitory effect of Fbw7-Skp1 by ~2.5-fold (supplemental Fig. S3
). However, the acidic loops of Skp1 were not sufficient to specify inhibition of deneddylation, because our Skp2-Cks1-Skp1 contained wild type Skp1.
Given the substantial effect of Fbw7-Skp1 on deneddylation, we next sought to test whether binding of substrate to Fbw7 might further influence deneddylation of the associated HPN8-Cul1. There is good reason to think this might be the case; Cul1 co-precipitated from cells with SCF substrates is essentially 100% neddylated (16
), implying that substrate might either increase the rate of neddylation or decrease the rate of deneddylation above and beyond the effect of the F-box protein. Consistent with this possibility, the addition of the Skp2-Cks1 substrate p27kip1
complexes in fractionated cell lysate decreases Cul1 deneddylation (33
). However, because this experiment was carried out with undefined protein fractions, a clear explanation for this phenomenon remains lacking.
To test the effect of substrate in a defined system, we compared the rate of deneddylation of HPN8-SCFFbw7
in the presence and absence of full-length phospho-cyclin E bound to Cdk2. Cyclin E must be phosphorylated on at least two sites (T-380 and S-384) to serve as a substrate for SCFFbw7
). The addition of phospho-cyclin E-Cdk2 further reduced the rate of deneddylation of HPN8-SCFFbw7
by ~2.5-fold (C
). The effect of substrate was specific, because phospho-cyclin E-Cdk2 had no effect on the rate of deneddylation of HPN8-Cul1 in the absence of Fbw7-Skp1 (D
). Together, Fbw7 plus phospho-cyclin E reduced deneddylation by >10-fold. Note that this experiment was done with 50 nm
substrate, in contrast to A
, which was done with 500 nm
. Thus, the inhibitory effect of Fbw7-Skp1 on deneddylation of HPN8-Cul1 was similar when CSN was either subsaturated or nearly saturated with substrate. To determine whether the effect of substrate applies to other SCF complexes, we tested the effect of phospho-p27-cyclin E-Cdk2 substrate on HPN8-SCFSkp2-Cks1
. The addition of substrate reduced the rate of deneddylation by ~2.3-fold (B
), similar to what was seen in C
. A similar magnitude of substrate-mediated inhibition was observed when SCFSkp2
complexes were assayed in the presence of phospho-p27-cyclin E-Cdk2 under conditions that were permissive (+Cks1) or not permissive (−Cks1) for substrate binding and ubiquitylation (E
). Taken together, these results imply that substrate reduced the rate of deneddylation equivalently regardless of whether or not it was undergoing ubiquitylation.
Cullin deneddylation in vivo
must occur in the presence of a substantial concentration of unmodified cullins as well as a large constellation of factors that bind cullins, any one of which might have an impact on the rate of deneddylation. To address this issue, we evaluated deneddylation of HPN8-Cul1-Rbx1 in the presence of different recombinant proteins purified from E. coli
and added at a fixed concentration of 1 μm
. Neddylated substrate was mixed with each potential regulator and allowed to interact for 5 min at room temperature before CSN was added, and the reaction progress was monitored. Interestingly, every single factor that was tested reduced the rate of deconjugation of HPN8 from substrate. The factors tested fell into two categories based on their ability to repress CSN deneddylase activity: moderate inhibitors (Ubc12, Dcn1, UbcH5C, and Nedd8), which repressed deneddylation by 2.2–4.8-fold, and strong inhibitors (Cul1, Ubxd7, Cdc34, and Cand1), which repressed deneddylation between 7.2- and 14.4-fold (A
). Supplemental Table S2
contains initial rates of deneddylation in the presence of each factor tested. Based on these results, we pursued in more detail the inhibition of deneddylation by Cand1 and unmodified Cul1-Rbx1.
Cand1 was previously reported to interact exclusively with unmodified cullins, including Cul1-Rbx1 (35
). Consistent with this, the co-crystal structure of Cul1-Rbx1-Cand1 showed that the Nedd8 conjugation site of Cul1 is partially obscured by Cand1 (32
). Moreover, the crystal structure of Nedd8 conjugated to the C-terminal domain of Cul5 showed how a Nedd8-induced conformational change blocks binding of the N-terminal domain of Cand1 (14
). Therefore, we were surprised to find that Cand1 was a potent inhibitor of deneddylation (A
). We investigated this property further in our in vitro
deneddylation assay by keeping the concentrations of HPN8-Cul1-Rbx1 substrate (150 nm
) and CSN enzyme (0.8 nm
) constant and varying the concentration of Cand1. This experiment yielded an apparent inhibition constant of 160 nm
). Two lines of evidence suggest that Cand1 inhibited deneddylation by binding substrate: the addition of Cand1 increased the Km
but did not affect the kcat
for deneddylation of HPN8-Cul1 (C
), and Cand1 exhibited no effect on the initial rate of deneddylation of HPN8-SCFSkp2-Cks1
). These data suggest that binding of the C-terminal domain of Cand1 to the N-terminal domain of Cul1 (which is blocked by Skp1-Skp2-Cks1) interferes with recruitment of CSN. Notably, in Aspergillus
, Cand1 is naturally split into two polypeptides, and the polypeptide corresponding to the C-terminal portion of human Cand1 can bind Cul1 in the absence of the N-terminal portion (37
CSN was also strongly inhibited by its reaction product, unmodified Cul1-Rbx1 (A
). We determined the IC50
for Cul1 to be 260 nm
when assayed at 50 nm
). This suggests that the Nedd8 modification must not confer a large amount of affinity for CSN, which is consistent with our observation that free Nedd8 was a weak inhibitor of deneddylation (A
). The surprisingly strong apparent affinity of unmodified Cul1-Rbx1 product for CSN is consistent with our original discovery that CSN associates with a mutant Cul1 that lacks the extreme C terminus including the Nedd8 conjugation site (10
) and raises the possibility that product dissociation might be rate-limiting for substrate deneddylation.
To conclusively demonstrate that CSN can form a stable interaction with unmodified SCF, we mixed purified CSN with purified SCFSkp2-Cks1 and fractionated the mixture on a Superdex 200 size exclusion column. SCFSkp2-Cks1 that was not mixed with CSN was used for comparison purposes. In the presence of CSN, a fraction of the SCFSkp2-Cks1 molecules was shifted to higher molecular weight fractions, corresponding to fractions that contained CSN (A).
FIGURE 4. CSN forms a stable complex with both neddylated and unmodified Cul1.
A, purified SCFSkp2-Cks1 (600 nm) was incubated for 15 min in either the presence (top panel) or the absence (bottom panel) of 300 nm purified CSN. Complexes were passed through a Sephadex (more ...)
To test whether unmodified full-length Cul1 (i.e.
, the product of deneddylation) exhibits significant binding to CSN in cells, we transiently transfected HA epitope-tagged wild type and K720R Cul1 expression constructs into HEK293 cells. K720R lacks the site on Cul1 to which Nedd8 is conjugated. Twenty-four hours post-transfection, the cells were lysed, and the HA
Cul1 was immunoprecipitated with HA antibody. The immunoprecipitates were then immunoblotted with antibodies to detect HA
Cul1 as well as the endogenous Skp1, Rbx1, Csn5, and Cand1 proteins (B
). Whereas wild type and K720R HA
Cul1 were expressed at similar levels and bound similar amounts of Rbx1 and Skp1, the K720R mutant actually retrieved more Csn5 than wild type. By comparison, a recent proteomic study reported equivalent association of CSN with wild type and K720R-Cul1 in HEK293T cells (38
). As independent confirmation of this result, we examined the interaction of endogenous Cul1 with transiently expressed FLAG-tagged Csn5. Cul1 co-immunoprecipitated with FLAG
Csn5 was exclusively in the unconjugated form (C
). Even if deconjugation occurred within the CSN-SCF complex in vitro
, this result emphasizes the point that unlike traditional enzymes, CSN did not rapidly let go of its substrate upon deconjugating it. To more directly compare the association of CSN with neddylated and unmodified Cul1, we repeated this experiment with FLAG
Csn5-ASA, which is mutated for two of the histidine residues that play a critical role in forming the active site that mediates deneddylation (12
). Although most of the endogenous Cul1 that co-immunoprecipitated with FLAG
Csn5-ASA was modified with Nedd8, a substantial pool of unmodified Cul1 was recovered, confirming that unmodified cullin substrate can associate stably with CSN. Finally, to explore structure-activity relationships in greater depth, we also evaluated binding of endogenous CSN to HA
Cul1 variants that could not bind to Skp1 and Rbx1. These mutants were generated by using the x-ray crystal structure of SCF as a guide (40
). Both of these mutants bound less Csn5 (B
). The failure of the RING-deficient mutant to bind CSN is consistent with the original finding that Csn2 binds Rbx1 in a yeast two-hybrid assay (10
). Taken together, our data suggest that CSN exhibits a complex mode of interaction with Cul1 that is not dependent on Nedd8 and involves both ends of the elongated CRL complex.
Stable binding to substrate and/or product is unusual for an enzyme and suggested to us that CSN might regulate CRLs by mechanisms other than deconjugation of Nedd8. This possibility is further supported by our observation that multiple factors that interact with Cul1, including the E2 enzymes UbcH5C and Cdc34, inhibited deneddylation of HPN8-Cul1-Rbx1. We therefore set out to test whether CSN can inhibit SCF activity, independently of its effects on Nedd8 conjugation. An ubiquitylation reaction was set up that contained unmodified SCFβTrCP
plus radiolabeled β-catenin peptide, ubiquitin, E1, and either UbcH5C or Cdc34. The ubiquitylation reaction was initiated by the addition of ATP and Mg2+
followed by the addition or omission of 300 nm
CSN (which is slightly less than the estimated in vivo
concentration of 500 nm
in 293 cells) (38
). The addition of CSN resulted in a 3.4–3.8-fold reduction in the rate of substrate conversion to products, independent of the E2 that was employed (). Inclusion of CSN affected both the extent of substrate conversion as well as the pattern of reaction products that were produced, indicating that CSN affected both ubiquitin chain initiation and elongation.
FIGURE 5. CSN inhibits ubiquitylation by unmodified SCF.
A, SCFβTrCP (100 nm) and ubiquitylation components (1 μm ubiquitin, 400 nm E1, 100 nm UbcH5C, 600 nm
32P-labeled-phospho-β catenin peptide) were incubated either in the presence or (more ...)
UbcH5 and Cdc34 catalyze SCF-dependent substrate ubiquitylation with Km
values that differ by approximately an order of magnitude (15
). Their equivalent sensitivity to inhibition by CSN suggested that CSN might not compete with E2 for binding to unmodified SCF. Consistent with this, 300 nm
CSN exerted a similar fold inhibition of ubiquitylation in reactions that contained either 1 or 10 μm
Cdc34 (supplemental Fig. S4A
). We also evaluated whether CSN might compete with substrate. Regardless of whether cyclin E peptide substrate was present at 0.1 or 1 μm
, 300 nm
CSN inhibited SCFFbw7
to a similar degree (supplemental Fig. S4B