Reconstitution of Ubiquitin Ligase Activity of Human APC
To investigate the mechanism of APC, we coinfected Hi5 insect cells with multiple recombinant baculoviruses harboring 10 APC genes and the cofactors Cdc20 or Cdh1. Because the APC proteins were His6-tagged at their N termini, they were purified from the insect cell lysate with the use of Ni2+-NTA beads and assayed for their ubiquitination activity. As shown in Figure A, when Hi5 cells were infected with viruses encoding 10 APC subunits (APC1–11) and Cdc20, the expressed APC proteins contained a ubiquitin ligase activity that, in combination with UbcH10, efficiently ubiquitinated human cyclin B1. This activity also supported ubiquitination of human securin (our unpublished data; see below). To determine which subunits were required for this activity, each virus was omitted individually from the coinfection. Only APC2 and APC11 were required for the ligase activity (Figure A). Similar results were obtained when the Cdh1 baculovirus was used in the coinfection instead of Cdc20 (our unpublished data). The fact that omission of APC2 or APC11 alone caused the loss of the reconstituted activity indicated that the purified ligase activity was not due to the presence of the endogenous APC from insect cells.
Figure 1 Reconstitution of the ubiquitin ligase activity of APC. (A) Hi5 insect cells were coinfected with baculoviruses encoding the indicated APC subunits. The expressed His6-tagged APC proteins were isolated from the insect cell lysate with the use of the Ni (more ...)
Because Cdc20 and Cdh1 are positive regulators of APC, it was surprising to us that Cdc20 or Cdh1 were not required for the ubiquitin ligase activity of the reconstituted APC. We therefore compared the reconstituted APC activity with that of the intact APC from Xenopus
egg extracts. Nearly all APC substrates contain the destruction box (D-box) or the KEN-box motifs, which are required for the efficient ubiquitination and degradation of these substrates (King et al., 1996b
; Pfleger and Kirschner, 2000
). Cdc20 and Cdh1 have been shown to confer the D-box and KEN-box specificity of APC (Burton et al., 2001
; Hilioti et al., 2001
; Pfleger et al., 2001
). The activities of the intact APCCdc20
were assayed with cyclin B1 as the substrate. As shown in Figure B, purified Cdc20 and Cdh1 proteins greatly stimulated the ligase activity of the intact interphase Xenopus
APC with UbcH10 as the E2 enzyme and wild-type cyclin B1 as substrate. However, neither the intact APCCdc20
significantly ubiquitinated a D-box deletion mutant of cyclin B1 (ΔDB-cyclin B1). Similar results were obtained with Ubc4 as the E2 enzyme, although the patterns of cyclin B-ubiquitin conjugates formed by the two enzymes were different. Ubc4 appeared to be more processive than UbcH10 in the presence of either intact APCCdc20
Because Cdc20 and Cdh1 were not essential for the reconstituted APC activity, we tested whether the ligase activity obtained with overexpressed APC proteins conferred D-box specificity, similar to the intact APC. Not surprisingly, the reconstituted human APC ubiquitinated ΔDB-cyclin B1 equally efficiently, indicating that the reconstituted activity did not possess substrate specificity (Figure C). This activity also required the presence of APC2 and APC11. Therefore, we reconstituted the minimal ligase activity of APC, which lacked D-box dependency. At present, we do not know the exact cause for the lack of D-box dependency of our reconstituted APC. However, several factors might contribute to this. First, the reconstituted APC might not have the correct quaternary arrangement of all the relevant subunits. Second, the set of subunits used to reconstitute the APC is not complete. Additional human APC subunits are required for the proper function of the reconstituted APC. Finally, it is also possible that the high concentrations of the reconstituted APC and the substrates in our in vitro reactions may have eliminated the need for high-affinity interactions between APC and substrates. We are currently investigating these possibilities.
Heterodimeric Complex of APC2 and APC11 Is the Minimal Ligase Module of APC
Because both APC2 and APC11 were required for the reconstituted APC activity, we tested whether they interacted with each other in the absence of the rest of the APC subunits. To characterize the immediate binding partners of APC11 in the APC complex, the GST-APC11 virus was coinfected with each of the other viruses in a pairwise manner. GST-APC11 and its associated subunits were then purified with glutathione-Sepharose beads and analyzed by SDS-PAGE followed by Coomassie staining and immunoblotting. As shown in Figure A, APC11 binds tightly to APC2 and weakly to APC6. The identities of APC2 and APC6 were verified by immunoblotting (our unpublished data). It was not apparent from the GST-APC11 pull-down experiment whether GST-APC11 interacts with APC10 because the His6
-tagged APC10 (26 kDa) comigrated with proteolytic fragments of GST-APC11 on SDS-PAGE (our unpublished data). We therefore used a His6
-tagged APC10 virus to infect Sf9 cells together with the GST-APC11 virus. The APC10 protein was then purified with Ni2+
-NTA beads and analyzed by SDS-PAGE. APC10 binds tightly to GST-APC11 as revealed by Coomassie staining (Figure B) and immunoblotting with α-GST antibody (Figure C). Several large proteins contain the so-called DOC domains that are similar in sequence to APC10; some of these proteins also contain cullin homology domains or homologous to E6-AP C terminus (HECT) domains (Grossberger et al., 1999
). E6-AP, the founding member of a family of proteins containing HECT domains, mediates the HPV E6-dependent degradation of p53 (Scheffner et al., 1995
). It is likely that the DOC domains of these large multidomain proteins might also be involved in binding to yet unidentified RING-finger proteins.
Figure 2 APC11 interacts with APC2 and APC10. (A) GST-APC11 baculovirus was coinfected with APC1/8 (a single baculovirus encoding both APC1 and APC8), APC2/7, APC3/6, APC4/5, or Cdh1 viruses in a pairwise manner into Sf9 cells. GST-APC11 and its interacting proteins (more ...)
To identify subunits that interact with APC2, the His6-tagged APC2 virus was used to infect Sf9 cells together with other viruses. The APC2 protein was then purified with Ni2+-NTA beads and analyzed by SDS-PAGE. We confirmed that APC2 interacts with APC11, and found no other strong interactions between APC2 and the rest of APC subunits (our unpublished data). Therefore, APC2 and APC11 formed a complex in the absence of the other APC subunits.
We next tested whether the subcomplex of APC2 and APC11 (APC2/11) was sufficient to support ubiquitination of APC substrates. With the use of UbcH10 as E2, APC2/11 catalyzed the ubiquitination of human securin, whereas either APC2 or APC11 alone had no activity (Figure A). Consistent with previous reports, APC11 alone expressed either in bacteria or insect cells was sufficient to ubiquitinate human securin in the presence of Ubc4 (Figure B). Similar data were obtained with the use of human cyclin B1 as the substrate (our unpublished data). Therefore, APC2/11 represents the minimal ligase module of APC, because it supports the ubiquitination of APC substrates with the use of either Ubc4 or UbcH10 as E2s.
Figure 3 Heterodimeric complex of APC2 and APC11 possesses ubiquitin ligase activity. (A) Hi5 cells were either infected with His6-APC2 (lanes 2 and 7) and His6-APC11 (lanes 3 and 8) viruses individually, or coinfected with the His6-APC2 and His6-APC11 viruses (more ...)
Ubc4 Interacts with APC11, whereas UbcH10 Binds to APC2
Both Ubc4 and UbcH10 support the ubiquitination reactions catalyzed by APC in an additive manner (Yu et al., 1996
). It is unclear which enzyme is the physiological E2 of the APC pathway. Microinjection of a UbcH10 dominant-negative mutant protein into mammalian cells arrested cells in mitosis (Townsley et al., 1997
). In addition, mutation of the Schizosaccharomyces pombe
homolog of UbcH10, UbcP4, caused accumulation of cells in mitosis, similar to mutations of APC subunits (Osaka et al., 1997
). These findings suggest that UbcH10 might be involved in the mitotic degradation system in living cells. However, in budding yeast, mutations of either the Ubc4/5 family E2s or the UbcH10 homolog Ubc11 did not cause obvious mitotic phenotype (Townsley and Ruderman, 1998
). To further clarify this issue, we immunodepleted UbcX, the Xenopus
homolog of UbcH10, from the mitotic Xenopus
egg extract that contains active APC and degrades APC substrates with fast kinetics. As shown in Figure A, the α-UbcX antibody beads effectively depleted the UbcX protein from the mitotic extract. Although the mitotic extract depleted with a control antibody degraded cyclin B1 with a half-life of 5–10 min, cyclin B1 was stabilized in the UbcX-depleted extract. Because the concentration of UbcX in mitotic Xenopus
extracts was estimated to be 50 nM by semiquantitative immunoblotting (our unpublished data), we added 50 nM of recombinant purified UbcX expressed in bacteria back to the UbcX-depleted extract. Addition of the bacterially expressed UbcX restored the ability of the mitotic extract to degrade cyclin B1. Taken together, UbcX is required for proper degradation of cyclin B1, and possibly other APC substrates, in Xenopus
egg extracts. Unfortunately, we did not have access to an antibody that can immunodeplete Ubc4 from these extracts, and thus could not do similar experiments for Ubc4.
Figure 4 Ubc4 binds to the ring protein APC11 whereas UbcH10 binds to the cullin protein APC2. (A) Xenopus homolog of UbcH10, UbcX, was immunodepleted from mitotic Xenopus egg extracts with the use of a polyclonal α-UbcX antibody coupled to Affiprep protein (more ...)
We next examined why APC2 was not required for the ubiquitination reactions of Ubc4. As shown in Figure B, UbcH10 interacted strongly with APC2, whereas it did not bind to APC11. In contrast, Ubc4 associated directly with APC11. It did not exhibit significant binding toward APC2. This finding explains why APC2 is only required for UbcH10-catalyzed reactions. The two E2s may recognize different binding determinants within the APC2/11 ligase module. The fact that UbcH10 did not bind APC11 is consistent with previous structural studies on the interactions between the Cbl RING domain and UbcH7, an E2 of the Ubc4 subfamily (Zheng et al., 2000
). The two loops that are critical for binding to RING domains are conserved between Ubc4 and UbcH7 (Figure C). On the other hand, UbcH10 contains quite divergent amino acid sequences in these two loops. Therefore, Ubc4 and UbcH10 may be recruited to the intact APC with distinct mechanisms: Ubc4 recognize APC11 initially, whereas UbcH10 first interacts with APC2. However, it remains possible that, once they are bound to APC, Ubc4 and UbcH10 occupy a similar site on APC and use a similar mechanism for transferring ubiquitin.
Cullin Domain of APC2 Interacts with APC11 and UbcH10
We next mapped the regions within APC2 that interact with APC11 and UbcH10. A series of truncation mutants of APC2 were constructed and tested for binding to APC11 and UbcH10. A C-terminal fragment of APC2, APC2e, spanning residues 549–822, was sufficient for binding to APC11 and UbcH10 (Figure A). This region almost coincides with the cullin homology region of APC2 that includes residues 512–750 (Yu et al., 1998
). Interestingly, even although APC2 and APC11 formed an active complex when they were coexpressed in insect cells, the full-length APC2 protein did not bind to APC11 in this assay. This is consistent with the fact that, when APC2 and APC11 were expressed individually in insect cells and mixed after purification, no ubiquitin ligase activity was observed. Therefore, the full-length APC2 protein could not form a complex with APC11 post-translationally. However, smaller fragments of APC2 were able to bind to APC11 (Figure A).
Figure 5 Cullin domain of APC2 is sufficient for binding to both APC11 and UbcH10. (A) Purified His6-tagged APC11 and UbcH10 proteins were immobilized on Ni2+-NTA beads and incubated with 35S-labeled APC2 or various APC2 truncation mutant proteins. After (more ...)
Because APC2e binds to both APC11 and UbcH10, we examined whether the APC2e/11 complex was an active ubiquitin ligase. The APC fragments were coexpressed with APC11 in insect cells, and assayed for their ability to ubiquitinate cyclin B1 in the presence of UbcH10. The APC2b and APC2e fragments ubiquitinated cyclin B1 efficiently (Figure B); both of these fragments retained the ability to bind to APC11 and UbcH10 (Figure A). Therefore, a complex of the cullin domain of APC2 and the RING finger protein APC11 is sufficient to catalyze ubiquitination of APC substrates, albeit with decreased efficiency.
One potential caveat of reconstituting the APC activity in insect cells is that certain insect APC proteins might associate with the expressed human APC proteins and contribute to the observed ligase activity. To rule out this possibility, we purified the APC2e/11 complex to homogeneity (Figure C) and determined the native size of the APC2e/11 complex by gel filtration chromatography and dynamic light scattering experiments. APC2e/11 cofractionated as a single species with an apparent molecular mass of 50 kDa on the gel filtration column (our unpublished data). Based on the intensity of staining on SDS-PAGE (Figure C), we estimated that APC2e and APC11 formed a complex of 1:1 stoichiometry. The calculated molecular mass of the complex is thus 50 kDa. Based on the light scattering experiment, the APC2e/11 complex was mono-dispersed with an apparent molecular mass of 46 kDa. Therefore, it is extremely unlikely that the APC2e/11 complex contains any insect proteins.
Zn2+-binding of APC11 Is Essential for Its Ubiquitin Ligase Activity
Because other RING finger proteins are known to coordinate Zn2+
ions, APC11 may also bind Zn2+
. However, this has not been demonstrated experimentally. We thus performed a Zn2+
-binding assay on the purified APC2e/11 complex (Yu and Schreiber, 1995
). Surprisingly, based on four measurements, we found that APC2e/11 bound Zn2+
at a molar ratio of 3.2 ± 0.2. Similar results were obtained with purified GST-APC11 protein expressed in bacteria. Therefore, it appeared that, in addition to the two Zn2+
ions coordinated by the canonical RING finger motif, APC11 contained a third Zn2+
-binding site (Figure A). Sequence alignment of the APC11 and Rbx1 proteins from various organisms reveals that three cysteines (Cys 34, Cys 37, and Cys 44 in human APC11) and one histidine (His 58 in human APC11) are conserved among these proteins (Figure A). These conserved residues do not belong to the canonical RING-H2 finger motif, and thus are good candidates for coordinating the third Zn2+
Figure 6 One APC11 protein molecule binds three Zn2+ ions. (A) Sequence alignment of APC11 and Rbx1 from various organisms (Hs, Homo sapiens; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; and Sp, S. pombe). The residues that coordinate the (more ...)
To determine the residues that coordinate Zn2+ ions, all cysteines and histidines of human APC11 were individually mutated to serines and alanines, respectively. Mutations of the Zn2+-binding ligands of the RING finger motif markedly reduced the expression levels of these proteins in bacteria (our unpublished data). Similar results were obtained when Cys 34, Cys 37, Cys 44, and His 58 were mutated. In contrast, mutations of Cys 7, Cys 33, Cys 54, His 65, and His 72 had no effect on the expression levels of the APC11 protein (our unpublished data). These findings are consistent with the notion that Cys 34, Cys 37, Cys 44, and His 58 coordinate the third Zn2+ ion. It is possible that mutations of the Zn2+-binding ligands destabilized the tertiary structure of APC11, resulting in the reduced level of expression of APC11. Obviously, loss of expression of APC11 mutant proteins in bacteria can be caused by factors other than protein folding. Because the APC11 mutant proteins that involve the putative Zn2+-binding ligands could not be obtained at sufficient purity and quantity, we could not determine whether mutations of these ligands actually caused the loss of Zn2+-binding.
When coexpressed with APC2, the APC11 mutants that did not affect Zn2+-binding still possessed ubiquitin ligase activity toward cyclin B1 in the presence of UbcH10 (our unpublished data). Because the APC11 mutants that perturbed Zn2+-binding were not expressed well, we could not compare the ubiquitin ligase activities of these mutants with those of the wild type or mutants that did not affect Zn2+-binding. To circumvent this problem, we obtained all APC11 mutant proteins with the use of the in vitro transcription and translation system in rabbit reticulocyte lysate. Many RING finger-based ubiquitin ligases also autoubiquitinate in the presence of the proper E2 enzyme. We therefore tested the autoubiquitination activity of the APC11 mutants in the presence of Ubc4. The wild-type APC11 protein and the APC11 mutants that did not affect Zn2+-binding were ubiquitinated efficiently when Ubc4 was added, based on the appearance of APC11-ubiquitin conjugates and the percentage of APC11 conjugated to ubiquitin (Figure B). Mutations of the eight Zn2+-binding ligands of the RING finger motif greatly reduced the autoubiquitination activity of APC11 (Figure B). In contrast, mutations of Cys 34, Cys 37, Cys 44, and His 58 that coordinated the third Zn2+ only slightly reduced the autoubiquitination activity of APC11 (Figure B). Thus, unlike the two Zn2+ ions of the RING-H2 finger motif, the third Zn2+ of APC11 might not be involved in catalysis (Figure B). This Zn2+ ion may only be important for maintaining the structural integrity of APC11. We also tested the binding of all APC11 mutants to APC2e. None of the mutations affected the binding of APC11 to APC2 (our unpublished data). Because the N-terminal region of APC11 and Rbx1 proteins is conserved and this region is not present in other RING-finger proteins, we speculate that the N-terminal 20 residues of APC11 and Rbx1 proteins are involved in binding to APC2 and Cul1, respectively.
Zn2+ Ions Alone Stimulate Activity of Ubc4 to Ubiquitinate Cyclin B1
Recently, many RING finger proteins have been shown to possess ubiquitin ligase activities. Despite the relatively low sequence homology outside the RING finger motif, several RING finger proteins use the Ubc4/5 family of E2 enzymes in the ubiquitination reactions (Joazeiro et al., 1999
; Lorick et al., 1999
; Gmachl et al., 2000
; Leverson et al., 2000
). This suggested to us that the Zn2+
ions, the most obvious common feature of these RING finger proteins, might be directly involved in catalysis. Strikingly, we found that Zn2+
ions alone stimulated the ability of Ubc4 to ubiquitinate cyclin B1 (Figure A). Several other divalent cations, such as Cd2+
, and Ni2+
, also enhanced the activity of Ubc4, whereas Mn2+
, and Yb3+
had no effects (Figure , A and B). None of these cations stimulated the activity of UbcH10, which did not bind to RING proteins directly (our unpublished data). These findings further support the notion that Zn2+
may be directly responsible for the activity of the RING finger-containing ubiquitin ligases.
Figure 7 Zn2+ ions alone stimulate the ubiquitination activity of Ubc4. (A) Various concentrations of Zn2+, Cd2+, and other divalent cations were added to a reaction mixture containing E1, ubiquitin, Ubc4, ATP, and cyclin B1. Ubiquitination (more ...)
We next quantitatively compared the activities of the intact APCCdc20, the reconstituted APC, and the Zn2+ ions alone. Various concentrations of APCCdc20 were used in the in vitro ubiquitination assay with the use of cyclin B1 as the substrate. The ligase activity of APC was measured by the intensities of the cyclin-ubiquitin conjugates, which were normalized by the number of ubiquitin molecules in the conjugates (Figure C). The activity of the reconstituted APC at 5 μM was similar to that of the intact APCCdc20 at 90 nM, indicating that the intact APCCdc20 was 55 times more active than the reconstituted APC. Zinc ions at 100 μM exhibited ligase activity comparable to 25 nM of APCCdc20. Thus, the activity of zinc ions alone was 4000 weaker than that of the intact APC.