The Unique N-terminus of the UbcH10 E2 Enzyme Controls the Threshold for APC Activation and Enhances Checkpoint Regulation of the APC

doi:10.1016/j.molcel.2008.07.014

Mol Cell. Author manuscript; available in PMC 2010 January 28.

Published in final edited form as:

Mol Cell. 2008 August 22; 31(4): 544.

doi: 10.1016/j.molcel.2008.07.014

PMCID: PMC2813190

NIHMSID: NIHMS67427

The Unique N-terminus of the UbcH10 E2 Enzyme Controls the Threshold for APC Activation and Enhances Checkpoint Regulation of the APC

Matthew K. Summers,¹ Borlan Pan,² Kiran Mukhyala,³ and Peter K. Jackson^1,⁴^*

¹Department of Cellular Regulation, Genentech Inc., South San Francisco, CA 94080, USA

²Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080, USA

³Department of Bioinformatics, Genentech Inc., South San Francisco, CA 94080, USA

⁴Department of Pathology, Stanford University School of Medicine, Palo Alto, CA 94308, USA

Corresponding Author: Dr. Peter K Jackson, Ph.D. Email: pjackson/at/gene.com (Hall et al., 2008) 650-255-7894

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Summary

In vitro, the Anaphase Promoting Complex (APC) E3 ligase functions with E2 ubiquitin conjugating enzymes of the E2–C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2–C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2–E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N-terminus increases substrate ubiquitination, the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction-boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N-terminus to UbcH5 restricts ubiquitination activity, but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2–E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control.

Keywords: Mitosis, Emi1, Ubiquitin-Protein Ligases, UbcH10, Anaphase Promoting Complex, Spindle Assembly Checkpoint

Introduction

The Anaphase Promoting Complex (APC) is an E3 ubiquitin (Ub) ligase, which following the sequential actions of E1 and E2 enzymes catalyzes the transfer of Ub to a host of substrates, targeting them for destruction by the 26S proteasome. Despite central roles in meiosis and the cell cycle, and more recently discovered roles in senescence, differentiation, and synaptic maturation, surprisingly little is known about the molecular mechanisms employed by the APC to ubiquitinate substrates (Kim and Bonni, 2007; Peters, 2006). The complexity of the holoenzyme, composed of at least twelve core subunits, several of which are essential for cell viability, has hindered examination of APC enzymology. Two essential components, the RING domain subunit, APC11, and the cullin-like subunit, APC2, form a subcomplex, which comprises the Ub ligase activity of the enzyme (Tang et al., 2001b). The roles of other subunits are less clear although some are implicated in activator binding, substrate recognition, or regulatory functions.

The substrates of the APC, notably mitotic cyclins, Securin, and Geminin contain conserved APC-targeting sequence elements, primarily the KEN-and Destruction-box (D-box) (Peters, 2006). Recognition of these degrons is mediated, at least in part, by the activator proteins Cdc20 and Cdh1 (Peters, 2006). Direct recognition of the D-box by the APC was also demonstrated and may involve the APC10/Doc1 subunit (Passmore et al., 2003). Substrate ubiquitination requires the activator protein, but the mechanism is poorly understood (Kramer et al., 1998). Phosphorylation of the APC increases its affinity for Cdc20, while phosphorylation Cdc20 and Cdh1 negatively regulates their activity (Keck et al., 2007; Peters, 2006; Tang et al., 2004).

Several additional mechanisms for inhibiting the APC have also been described. Of these, the best characterized are the Spindle Assembly Checkpoint (SAC) and the Emi1/Emi2 proteins (Peters, 2006). The SAC effector proteins Mad2 and BubR1/Mad3 restrain APC^Cdc20 activity during mitosis until all chromosomes are properly prepared for segregation (Diaz-Martinez and Yu, 2007). Mad2 binds Cdc20 and presumably prevents it from recognizing substrates and/or activating the APC. In addition, Mad2 facilitates the interaction of BubR1 with Cdc20. BubR1 binds Cdc20 in a KEN and D-box dependent manner, inhibiting the APC as a pseudosubstrate (Burton and Solomon, 2007; King et al., 2007). Similarly, Emi1 antagonizes APC^Cdh1 activity during interphase and early mitosis by binding the APC core via its D-box, preventing the association of substrates and further inhibiting ligase activity via its zinc-binding region (Ban et al., 2007; Hsu et al., 2002; Miller et al., 2006). In a similar fashion, Emi2 antagonizes the APC during meiosis maintaining oocytes at metaphase II (Tung et al., 2005).

The APC also requires an E2 enzyme for activity and has been demonstrated to function, in vitro, with the Ubc4/5 and E2–C families of E2 enzymes, specifically UbcH5 and UbcH10 in humans (Yu et al., 1996). E2-C enzymes are class III E2s possessing a unique N-terminal extension in addition to the catalytic domain of class I E2s, such as the Ubc4/5 family members. Although both enzymes catalyze robust APC activity in vitro, it is unclear if these activities are biologically relevant. Genetic evidence from S. pombe and Drosophila supports that the E2-C family members, Ubc11 and Vihar, are critical for APC function (Mathe et al., 2004; Osaka et al., 1997). Mutants of these E2s recapitulate a loss of APC activity. Inactivation of UbcH10 in mammalian cells using a catalytically inactive mutant gives a similar phenotype, also mimicking the loss of APC activity, while mutant UbcH5 does not (Bastians et al., 1999; Townsley et al., 1997). However, strong enzymological evidence explaining the selectivity of the APC for the E2–C family and elucidation of why this E2 family is critical for APC function is lacking. Moreover, we know very little about what defines the E2-E3 interface.

A recent study has again highlighted the importance of E2 activity for APC function in Saccharomyces cerevisiae and introduced a third E2, budding yeast Ubc1, as an E2 that extends Ub-chains following monoubiquitination by a “priming” E2 (Rodrigo-Brenni and Morgan, 2007). However, whether a similar two-step mechanism using a pair of APC-directed E2 enzymes is also important in higher eukaryotes is unclear. Surprisingly, although the UbcH10 E2 enzyme is not present in S. cerevisiae, it is highly conserved, even in related yeasts.

We have employed a biochemical approach to examine E2 how enzymes contribute to APC activity. Comparison of E2–E3 interactions in multiple in vitro, in extracto, and in vivo experiments indicates that UbcH10 is preferentially used by the APC. Importantly, the use of UbcH10 is critical for proper regulation of APC activity. The UbcH10 N-terminus sets a threshold for APC activation by UbcH10 and requires proper substrate engagement for ubiquitination to occur. The threshold enhances the fidelity of substrate selection and ubiquitination, and in doing so, is critical for regulation of the APC by mitotic checkpoints.

Results

UbcH10 is the cognate E2 of the APC

The APC exhibits high activity with both UbcH10 and UbcH5 in reconstituted in vitro assays (Figure 1A). To understand the relevance of this activity in a setting better reflecting the in vivo state, we generated extracts from nocodazole-arrested HeLa cells. We tested the ability of these extracts to mediate mitotic destruction events by monitoring the stability of ³⁵S-labeled, in vitro translated substrates. As in nocodazole-arrested cells, Cyclin A is readily destroyed and this destruction was dependent upon APC-mediated ubiquitination and subsequent degradation by the proteasome (Figure S1A). Securin, however, remained stable (Figure S1B). In cells, the mitotic stability of Securin is maintained by SAC activity. SAC function is also recapitulated by the mitotic extracts, as addition of either the Mad2 antagonist p31^Comet or a dominant-negative fragment of BubR1 induced Securin destruction in a dose-dependent fashion (Figure S1C) (Tang et al., 2001a; Xia et al., 2004).

Figure 1

UbcH10 is sufficient and required for APC mediated destruction events and its N-terminus regulates ubiqutination activity

We then asked whether the addition of specific E2 enzymes was sufficient to activate the APC and cause Securin destruction. Only UbcH10 was sufficient to induce Securin destruction (Figure 1B, upper panels). UbcH10 was also able to induce the destruction of endogenous substrates, notably Cyclin B (Figure S1D). We confirmed that the UbcH10 effect was mediated by the APC by rescuing Securin stability by simultaneous addition of BubR1 (Figure S1E). The contrasting effects of UbcH10 and UbcH5 on APC activity is due to specific activation of the APC by UbcH10 and not a differential ability of these E2s to displace spindle assembly checkpoint components. BubR1 and Mad2 remain associated with the APC in both UbcH10 and UbcH5 treated extracts (Figures S1F). Consistent with APC activation, addition of UbcH10, but not UbcH5, accelerated the destruction of Cyclin A in extracts (Figure S1G). Thus, UbcH10 has a greater capacity than UbcH5 to activate the APC.

Surprisingly, the simultaneous addition of excess Ub allowed UbcH5 to catalyze Securin destruction as well (Figure 1B, lower panels). These data suggest that UbcH10 is specific for the APC in more physiological settings, but that E2 selectivity is lost at high levels of Ub. We reasoned that UbcH10 selectivity would reflect a requirement for providing the proper amount of charged E2 (E2~Ub) to the APC. Consistent with this model, increasing the concentration of UbcH10 restored Securin stability (Figure S2A). As a dramatic demonstration of the importance of E2~Ub balance, the block in APC activity caused by excess UbcH10 was reversed by the addition of Ub (Figure S2B). The ability of Ub to stimulate the activity of UbcH5 with the APC does not reflect limiting E1 activity as increasing the amount of E1 in extracts did not allow UbcH5 to trigger Securin destruction (Figure S2C). Stimulation of UbcH5-APC activity requires conjugation of Ub to substrate, as addition of Ub lacking the C-terminal diglycine (ΔGG), which cannot form isopeptide bonds, did not promote Securin destruction by UbcH5 (Figure S2D).

Titrating Ub addition to UbcH5 in extracto caused a dose-dependent increase in Securin-Ub observed, upon prolonged exposure of autorads. (Figure S2D). Therefore, we reasoned that the difference in the two E2s was in their ability to ubiquitinate substrate and not in the type of Ub-conjugates formed. To examine the Ub-conjugates, we took advantage of the ability of Securin bearing N-terminal Myc epitope-tags to be ubiquitinated, but not destroyed. Consistent with our previous destruction results, UbcH10 addition resulted in the ubiquitination of Myc-Securin, whereas no detectable ubiquitination was observed with UbcH5 (Figure S2E, upper panels). Increasing the Ub concentration both increased the ubiquitination of Myc-Securin generated by UbcH10 and allowed UbcH5 to catalyze Myc-Securin ubiqtuination (Figure S2E, lower panels). These data demonstrate that the amount of E2~Ub encountered by the APC must reach a threshold level to produce Securin-Ub. For exogenous UbcH10, the pool of Ub in the extract is sufficient to achieve this level and increasing the available Ub increases the amount of Securin-Ub (Figure S2F (1) and (2)). The documented promiscuity of UbcH5 for E3-enzymes (Brzovic and Klevit, 2006) effectively dilutes the amount of E2~Ub, generated by the extract Ub, so that it is below the threshold required to activate the APC and Securin remains stable. Addition of Ub raises the amount of E2~Ub above the threshold for Securin ubiquitination (Figure S2F (3) and (4), S2G). Taken together, our results support that UbcH10 and the APC constitute a specific and tightly regulated E2–E3 pair.

To confirm the relevance of this E2–E3 pair, we took advantage of our ability to induce Securin destruction by adding p31^Comet to extracts and used this assay to ask which E2(s) was required for APC activity. We challenged p31^Comet-induced Securin destruction with a panel of catalytically inactive E2 enzymes. Only UbcH10_cs blocked APC-mediated Securin destruction (Figure 1C). The dominant negative effect was observed at 2µM, ~4-and 7-fold greater than the concentration of UbcH5 and UbcH10, respectively, present in extracts. Even at 20µM UbcH5_cs exhibited only a weak effect (Figure S3A).

UbcH10_cs also robustly prevented Cyclin A destruction at 2µM, whereas 20µM UbcH5_cs was required to stabilize Cyclin A (Figure S3B). At 3–10µM UbcH5_cs did block the in extracto destruction of substrates of other E3s, such as SCF^β-TrCP-mediated destruction of Emi1 (data not shown), demonstrating that UbcH5_cs is capable of dominant-negative function in extracto.

We next titrated UbcH10 in the APC reaction and found strong activity from 0.3µM-10µM E2 (Figure S4A). We then tested the ability of mutant E2s to inhibit APC reactions driven with 2.5µM UbcH10 or UbcH5. In vitro, UbcH10_cs was a potent antagonist of both E2s. UbcH5_cs had little effect on UbcH10 driven reactions and was less effective than UbcH10 at antagonizing UbcH5 driven reactions (Figure S4B). UbcH2_cs had no effect on either E2. These data confirm the specific pairing of UbcH10 and the APC.

We also examined the contribution of UbcH1, the human homolog of S. cerevisiae Ubc1, to APC function. In a previous study, UbcH1 elongated Ub-chains on UbcH10-primed substrates in vitro and was suggested to mediate a chain elongating step for the APC (Rodrigo-Brenni and Morgan, 2007). UbcH1_cs did not effect p31^Comet-induced Securin destruction or Cyclin A degradation (Figure 1C and S3A–B). Furthermore, although UbcH10 was required for and synergized with p31^Comet-induced MT-Securin ubiquitination in extracto, no effect was observed for UbcH1 or UbcH1_cs in this assay (Figure S3C). Although we cannot exclude that there is an elongating E2, UbcH1 is unlikely to be required for human APC activity.

The N-terminus of UbcH10 regulates APC Ubiquitination Activity whereas APC Specificity is Mediated by the E2 Core Domain

The E2-C family members are class III E2 enzymes, characterized by the presence of a N-terminal extension in addition to the catalytic core domain. As UbcH10 and UbcH5 exhibit ~40% identity over the E2 core, the unique N-terminus of UbcH10 was a likely mediator of the specificity of UbcH10 for the APC. Intriguingly, the N-terminal extension contains regions of high conservation from yeast to humans (Figure 1D). Although the N-terminus is not present in the putative S. cerevisiae ortholog, Ubc11p, the genomes of the related Saccharomycete, Yarrowia, and other Ascomycetes, such as Aspergillus, encode E2s bearing the highly conserved QNP motif (not shown). To further examine our biochemical results and test the ability of the UbcH10 N-terminus to direct APC specificity, we expressed FLAG-tagged UbcH5, UbcH10, or mutants with either the highly conserved residues Q4, N5, and P8 mutated to alanine (UbcH10 QNP-AAA) or the N-terminal 27 residues deleted (UbcH10 ΔN) in HeLa cells and examined the ability of the spindle checkpoint to arrest these cells. Transfected cells were subject to a single thymidine block and released into Taxol-containing media. Cells were harvested at various time points after release and the mitotic index of FLAG-positive cells was determined by staining for phospho-Histone H3 (pH3) (Figure 1E and 1F). Control, UbcH10, QNP-AAA, and UbcH5 expressing cells accumulated in mitosis with similar kinetics. However, UbcH10 and QNP-AAA expressing cells did not maintain the arrest and the mitotic index subsequently dropped. ΔN expressing cells even more strongly failed to accumulate in mitosis compared to other E2 expressing cells. To confirm that these cells were entering mitosis, the percentage of G2 cells (identified as pH3-negative, Securin-positive) was also determined following thymidine release. Similar percentages of G2 cells accumulated by 6h and decreased with comparable kinetics as cells entered mitosis in both control and E2-expressing populations (Figure S5). Consistent with the G2 data, we also observed similar S-phase profiles for both control and E2-expressing populations (data not shown). In contrast to UbcH5, the UbcH10 core domain functions with the APC even more strongy than wild-type, indicating that APC specificity is mediated by the E2-core domain while the N-terminus provides a regulatory function.

To begin to understand how UbcH10 ΔN enhanced checkpoint bypass, we used recombinant UbcH10, UbcH5, and UbcH10 ΔN in reconstituted APC ubiquitination assays. Like wild type, UbcH10 ΔN exhibited robust activity with the APC holoenzyme in these reactions. Unexpectedly, the mutant E2 produced a higher molecular weight form of substrate-Ub conjugates than the wild type protein, similar to those observed with UbcH5, suggesting the loss of an intrinsic inhibitory mechanism (Figure 1G).

Despite exhibiting similar activity with holoenzyme in vitro, APC activity with UbcH10 ΔN and UbcH5 is strikingly different in vivo (Figure 1F–G), indicating that the UbcH10 core domain (UbcH10_core) mediates APC specificity and we examined this possibility using APC subcomplexes in vitro. UbcH5, but not UbcH10, functions with APC11 alone (Gmachl et al., 2000; Tang et al., 2001b). Like full-length UbcH10, but unlike UbcH5, UbcH10 ΔN did not form substrate-independent polyUb chains (Figure 2A) or catalyze the ubiquitination of Securin in the presence of APC11 (data not shown). The autoubiquitination of UbcH10 observed in Figure 2A is independent of APC11 (data not shown). Both UbcH10 and UbcH5 were shown to function with the APC2-APC11 subcomplex (Tang et al., 2001b). However, we and others find only UbcH5 to function in this reaction (Vodermaier et al., 2003). Consistent with the results above, neither UbcH10 nor ΔN were able to ubiquitinate Securin in the presence of APC2-APC11 (Figure 2B). Lastly, we asked how the mutation of the N-terminus affected UbcH10 function in extracto. Similar to the in vitro assays above, both N-terminal mutants behaved like wild type UbcH10 in extracto, triggering Securin ubiquitination and destruction (Figure 2C, upper panels and S2E). These data indicate that the UbcH10 N-terminus is not the major determinant of UbcH10-APC specific interaction. We conclude that this interaction is mediated by determinants in UbcH10_core and by a specific UbcH10 receptor/site in the 10 APC holoenzyme. This result also argues that the lack of APC activation by UbcH5 is due to competing interactions with other E3 enzymes and not a consequence of a difference in Ub-conjugates. Together, these data suggest that UbcH10_core retains APC-specific activity not found in UbcH5, while the N-terminus regulates activity.

Figure 2

UbcH10-APC specificity and activity are determined by UbcH10_core and the N-terminal extension, respectively

The N-terminus Confers Selectivity for Substrate Lysine Selection and Substrate Engagement (D-box)

Surprisingly, the QNP-AAA mutant recapitulates the deletion of the entire N-terminus in vitro, converting the pattern of Ub-conjugate formation to higher molecular weight species (Figure 2C, lower panels). Two factors may contribute to the ability of the N-terminus to inhibit UbcH10: the N-terminus itself and the interaction of the QNP motif with APC2. To first confirm that this change in the form of Ub-conjugates was mediated by the N-terminus, we created a chimeric E2, fusing the N-terminal extension of UbcH10 to the N-terminus of UbcH5. As predicted, the presence of the UbcH10 N-terminus resulted in the shifting of reaction products from the high molecular weight Ub-conjugates typical of UbcH5 to a lower molecular weight forms typical of UbcH10 (Figure 2D). In contrast to UbcH10, introducing the QNP-AAA mutation into the chimeric UbcH5 did not alleviate the effect of the N-terminal fusion on UbcH5 activity (Figure S6). This result confirms that the QNP motif specifically interacts with the APC and is required for regulation of UbcH10_core-APC activity, but is irrelevant for the non-specific interaction of UbcH5 with the APC. The N-terminus alone is sufficient to inhibit UbcH5.

The products of APC catalyzed reactions consist predominantly of multiple monoUb additions rather than polyubiquitination chains (Kirkpatrick et al., 2006), suggesting that the difference between Ub-conjugates formed by wild type and ΔN may not reflect a difference in chain length, but in the number of single Ub additions to substrate. Comparison of the products from APC reactions reconstituted with either wild type Ub or a mutant lacking lysine residues were used to test this idea. The reaction products formed with the mutant Ub consist of monoUb additions and the number of different Ub-conjugates observed reflects the number of substrate lysines utilized. Deletion of the N-terminus increased both the number of lysines conjugated to Ub (Figure 3A, 1^st and 3^rd panels) and the polyubiquitination (Figure 3A compare 1^st and 2^nd panels with 3^rd and 4^th panels) of Securin. Similar results were obtained for the Cyclin B1 N-terminus and Geminin (Figure S7A).

Figure 3

The N-terminus of UbcH10 restricts the number of substrate lysines targeted by the APC and enhances D-box selectivity

The increase in the number substrate lysines ubiquitinated by ΔN suggests the N-terminus may limit either: 1) the orientation or recruitment of substrate to the active site; and/or 2) the rate of ubiquitination, altering the timing of catalytic events and restricting the use of the additional lysines. As a result of this increased Ub transfer, ΔN and QNP-AAA may have an increased potential for ubiquitinating substrates that are not properly engaged by the APC. Consistent with this prediction, UbcH10 N-terminal mutants ubiquitinated D-box mutant (Db-) Securin much more efficiently than the full length E2, while neither ubiquitinated a double KEN and D-box mutant Securin (Figure 3B). Similar results were obtained for wild type versus a (Db-) Geminin (Figure S7B). Similarly, the chimeric UbcH5 also lacked activity toward the mutant substrate (Figure S7B). Importantly, this result was recapitulated in in extracto degradation assays. UbcH10 and ΔN both efficiently triggered the destruction of WT-Securin, whereas ΔN more efficiently triggered the destruction of Securin (Db-) (Figure 3C). As in vitro, neither E2 affected the stability of the double mutant. Thus, the N-terminus enforces the D-box dependent ubiquitination of APC substrates.

The UbcH10 N-terminus Confers Sensitivity to APC Inhibitors

A mechanism common to specific APC inhibitors, including Emi1 and BubR1, is the perturbation of substrate-APC interactions by binding to the D-box receptor (pseudosubstrate inhibition) (Burton and Solomon, 2007; King et al., 2007; Miller et al., 2006). Thus, the prerequisite of proper substrate engagement for APC-UbcH10 activity would be predicted to confer enhanced regulation of this enzyme pair by these inhibitors. We tested the effect of pseudosubstrate inhibition both in vitro and in extracto. First, we challenged in vitro APC ubiquitination of the Cyclin B1 N-terminus catalyzed by either UbcH10 or ΔN with increasing concentrations of Emi1 and monitored the production of Ub-conjugates. Emi1-dependent inhibition of UbcH10 rapidly reached a maximum at 0.3µM, drastically decreasing the amount of product formed. In contrast, for ΔN this level of inhibition was only approached at 3µM (Figure 4A). We then tested the effect of E2 concentration on the inhibition of the APC by the psuedosbustrate BubR1 in extracto. Consistent with our in vitro results, both E2s efficiently triggered Securin destruction at 2µM (Figure 4B). UbcH10 rapidly lost the ability to induce Securin destruction as the E2 concentration decreased, with diminished kinetics at 670nM and very weak activity at 222nM and 74nM. Conversely, ΔN efficiently triggered destruction of Securin at all concentrations albeit with reduced kinetics as the concentration decreased (Figure 4B). These data illustrate that by enforcing D-box dependency, then N-terminus enhances the activity of pseudosubstrate inhibitors.

Figure 4

Constraints imposed by the N-terminus confer enhanced regulation of APC activity in vitro

Structural Insights into Regulation by the UbcH10 N-terminus

Our biochemical evidence shows that both the N-terminus and core E2 domains of UbcH10 contribute to its specific pairing with the APC. We propose that the interaction of UbcH10_core with the APC requires an E2 binding site on the APC, which may require additional determinants beyond the E2 interaction with the RING and Cullin domains (see Discussion). Future studies will address that interaction. Here, we focused on the N-terminal domain and performed three analyses to better understand its structural role.

First, we examined the hypothesis that the N-terminus might interact with UbcH10_core to regulate its activity. Previous crystallographic studies of UbcH10 and goldfish E2-C found the E2 core domain to assume a fold typical of E2s (Jiang and Basavappa, 1999; Lin et al., 2002). The N-terminal extension was not observed in the crystal of either protein, leading to the prediction that the N-terminus is disordered. To characterize the solution structure of the N-terminal domain, we compared the ¹H-¹⁵N HSQC spectra of UbcH10 and ΔN (Figure 5A). The spectra of UbcH10 showed approximately 25 additional backbone amide cross-peak resonances compared to the spectra of ΔN, 158 and 133 resonances, respectively. The presence of additional and shifted peaks in the UbcH10 spectra (Figure 5A, Table S1) are consistent with the insertion of the N-terminus between the vector-derived linker sequence and UbcH10_core (see Supplementary Results) and there was no clear evidence for the N-terminus interacting with UbcH10_core. However, we cannot completely exclude this possibility. In addition, the majority of the additional cross-peaks in the wild-type spectra display limited chemical shift dispersion suggesting that the residues corresponding to these peaks are generally disordered.

Figure 5

NMR analysis of the UbcH10 N-terminus

Second, we tested whether the N-terminus is critical for binding APC2. UbcH10 interacts with both APC2 (Tang et al., 2001b) and presumably the APC11 RING domain. The APC11 interaction is likely mediated by UbcH10_core. We reasoned that the APC2 interaction might be mediated by the N-terminus. We tested this interaction in an in vitro binding assay using recombinant E2s to capture ³⁵S-labeled APC2. As expected, UbcH10 captured APC2 and, consistent with our hypothesis, the interaction was abolished by the N-terminal mutations (Figure 5B). Taken together these data indicate that the UbcH10 N-terminus does not clearly make any meaningful contacts with UbcH10_core and that interaction with the E3 mediates the effect of the N-terminus on APC regulation. Consistent with this, we were unable to detect an interaction (physical or functional) between UbcH10 or ΔN and peptides encompassing the N-terminus in vitro (data not shown).

Third, we modeled the interaction between the UbcH10 N-terminus and the APC. To this end, we examined the existing crystal structures of the Cul1-Rbx1-Skp1-F box^Skp2 Ub-ligase complex, the c-Cbl-UbcH7 complex, and UbcH10 (Lin et al., 2002; Zheng et al., 2002; Zheng et al., 2000). APC11 and Rbx1 are members of a family of RING-H2 proteins and interact with cullin-like proteins, suggesting that these complexes will share structural homology as well (Ohta et al., 1999; Yu et al., 1998). We modeled APC2 and APC11 onto the Cul1-Rbx1 structure. Docking UbcH10_core onto the APC2-APC11 model was guided by alignment of UbcH10 and APC11 with the UbcH7-c-Cbl E2-E3 structure. We then added the N-terminal extension, modeled after the structure of Ubc12 N-terminus with placement guided by the clam E2-C structure (Huang et al., 2004; Jiang and Basavappa, 1999). The resultant APC2-APC11-UbcH10 model (Figure 5C) does not allow prediction of the exact location or conformation of the N-terminus nor whether this might affect UbcH10_core positioning or catalysis (see below), but the positioning of the N-terminus, extending from helix1 would be sufficient to place the QNP motif in contact with APC2.

Discussion

The importance of regulating APC activity for cell cycle control and checkpoint regulation is well recognized, but our understanding of APC regulatory mechanisms at a molecular level is relatively poor. Notably, there was little mechanistic data explaining how a specific E2 enzyme is paired with a specific E3. Here, we have confirmed UbcH10 as the cognate E2 for APC function. Extending this biological result, we find that the highly conserved N-terminus of UbcH10 mediates several critical functions of APC activity, ultimately enhancing the regulation of the APC. Surprisingly, the regulation of APC activity by the N-terminal domain of UbcH10 is not simply important for efficiency of substrate ubiquitination, but critically important for the control of substrate selection. These results demonstrate that substrate selection by E2–E3 enzymes may require a controlled rate of Ub transfer to substrate to ensure proper substrate regulation and biological control.

UbcH10 as the E2 partner of the APC

Genetic data from organisms that possess a clear UbcH10 homolog supports the E2-C family as the E2 partners of the APC, rather than the Ubc4/5 family members. Ubc11 (S. pombe) and Vihar (Drosophila) mutants, as well as experiments using catalytically inactive UbcH10, result in mitotic arrest and stabilization of APC substrates, e.g. cyclins, recapitulating a loss of APC activity (Mathe et al., 2004; Osaka et al., 1997). The sufficiency of UbcH10 for APC activation was suggested by work demonstrating that cyclin A requires greater E3 activity than Securin to achieve comparable ubiquitination and degradation (Rape et al., 2006). Cyclin A is stable at low levels of UbcH10, but Securin is destroyed (Rape and Kirschner, 2004). The authors found UbcH5 and UbcH10 to be interchangeable in activation of the APC and interpreted the results to show that destruction of cyclin A, but not Securin, required UbcH10. We note that Ub was added to all extracts in this study, masking the differential ability of these two E2s to activate the APC we describe here and suggest that low, but regulated levels of UbcH10 activity, and not UbcH5, cause Securin destruction in these experiments.

By relying on the existing Ub in mitotic extracts to charge the exogenous E2 enzymes, we observe a clear functional difference in APC activation by these E2s. This is not dependent on more efficient charging of UbcH10 by the E1 as increasing the E1 concentration did not allow UbcH5 to activate the APC with endogenous levels of Ub. Furthermore, we examined the kinetics of E2~Ub thioester formation and find the N-terminus to be a negative regulator of this reaction as well, indicating that UbcH5 charging is more efficient than that of UbcH10 (M.K.S. and P.K.J., unpublished data, (Huang et al., 2008). Only addition of excess Ub allows UbcH5 to activate the APC. UbcH5 is a promiscuous E2 and functions with a broad range of RING-domain E3 enzymes in vitro (Brzovic and Klevit, 2006). This promiscuity is reflected in the need to increase the amount of UbcH5~Ub in the extract in order for a functional interaction with the APC to occur. In support of specificity for UbcH10, mass spectrometry analysis of products from APC reactions harboring both UbcH10 and Ubc4 (an E2 nearly identical to UbcH5), shows cyclin-Ub conjugate patterns specific for UbcH10 to predominate even when Ubc4 is in 5-fold molar excess (D. Kirkpatrick, personal communication). This lack of specificity for the APC questions the relevance of UbcH5 as a component of the APC pathway.

Rodrigo-Brenni and Morgan have demonstrated the ability of the S. cerevisiae E2 enzyme, Ubc1, to build K48-linked chains on substrates initially ubiquitinated by APC-Ubc4. In vitro, the human homolog, UbcH1, was also shown to function with the human APC. In our assays neither UbcH1 nor UbcH1_cs had an effect on APC activity. We cannot conclude that a two-step E2 pathway does not exist in human cells only that it is unlikely to use UbcH1. In the absence of a UbcH10 homolog, budding yeast may require a second E2 to achieve efficient ubiquitination. Alterniatively, other organisms may have adopted the use of different E2 pairs, as suggested for fission yeast (Seino et al., 2003). The finding that multi-monoubiquitinations catalyzed by the APC are efficiently destroyed by the proteasome, together with our data, suggests that UbcH10 activity is potentially both necessary and sufficient for destruction of APC substrates (Kirkpatrick et al., 2006).

A Receptor/Co-Activator of the UbcH10 Core on the APC

The interaction of the N-terminus of UbcH10 with the APC is not the determinant of APC specificity. We have attempted to determine the residues within UbcH10_core that target it to the APC holoenzyme. We initially mutated residues on the backside (relative to the catalytic cysteine) of UbcH10, identified in the crystal structure of UbcH10_core (Lin et al., 2002), and conserved among E2-C family members, to the corresponding residues from UbcH5, but were unable to convert the enzyme to UbcH5-like activity in extracto.

Although we have not mapped the exact determinants within UbcH10_core, the interaction with the holoenzyme is likely mediated by the APC1, APC4, or APC5 subunits. These subunits, along with APC2 and APC11, form a complex that is sufficient for triggering the ubiquitination activity of the Xenopus UbcH10 homolog (Vodermaier et al., 2003). However, this subcomplex is deficient in Ub transfer to substrates. In contrast, while the APC2–APC11 subcomplex is capable of interacting with UbcH10, this subcomplex is not able to activate ubiquitination by the E2. The RING domain of APC11 may not be sufficient for activating UbcH10_core, thus requiring these additional subunits. In addition, the lack of activity toward substrates by UbcH10 in the APC1, 2, 4, 5,11 subcomplex is consistent with its stringent requirement for proper substrate engagement, as neither the putative substrate receptor APC10/Doc1 nor Cdh1 and Cdc20 associate with this complex (Vodermaier et al., 2003). It is not clear if these APC subcomplexes exist in vivo. If this subcomplex exists in cells (perhaps as a transient intermediate), it is intriguing that recruiting an E2 that cannot ubiquitinate substrates in its presence would intrinsically inhibit it as this complex also lacks the ability to interact with APC inhibitors.

Regulation of Ubiquitination Activity by the N-terminus of UbcH10

Overall, our data support a model in which UbcH10_core is recognized by APC subunits in addition to the ligase module, APC2–APC11. The N-terminus of UbcH10 then binds APC2 engaging the regulatory mechanism (Figure 6). Upon proper engagement, the substrate lysines achieve proper binding, orientation, or sufficient dwell time with the active site, and are efficiently ubiquitinated. In the absence of proper substrate engagement, such as a D-box mutant substrate or in the presence of a pseudosubstrate inhibitor, the criteria for ubiquitination are not met and the substrate remains unmodified. Without the interaction between the N-terminus and APC, the increased Ub transfer by the E2 is able to overcome the unmet criteria resulting in the inappropriate ubiquitination and destruction of substrates. In addition, deletion of the N-terminus also results in unregulated autoubiquitination of the mutant E2 in conjunction with the APC (Rape and Kirschner, 2004).

Figure 6

Model of the N-terminal regulation of UbcH10-APC activity

Consistent with a need for substrate orientation, mapping of the ubiquitination sites on cyclin B1 by mass spectrometry revealed that UbcH10 preferentially uses lysines near the D-box (D. Kirkpatrick, personal communication). This suggests that a primary role of the D-box is in orienting the substrate lysines near the E2 active site. The location of Ub-chains upon a substrate can affect the rate of destruction by the proteasome (Petroski and Deshaies, 2003). It is tempting to speculate that lysines targeted by UbcH10 may be in the optimal confirmation for interacting with the proteasome. The requirement for substrate lysines to be properly positioned by the D-box for ubiquitination by UbcH10 would make the APC more efficient by conjugating only the lysines critical for proteasomal targeting.

Several mechanisms may explain regulation by the N-terminus. First, analogous to the NEDD8 E2, Ubc12, which is positioned for optimal charging on the NEDD8 E1 enzyme by the Ubc12 N-terminal extension (Huang et al., 2004), the N-terminus-APC2 interaction may constrain UbcH10 orientation restricting the number of substrate lysines with access to the E2 active site. Alternatively, the N-terminus-APC2 interaction could form a channel and cooperate with the D-box receptor to optimally orient the substrate. Relaxation of substrate positioning by removal of the N-terminus would then expose different substrate lysines to the E2 while positioning in the channel would be severely impaired by D-box mutations, preventing ubiquitination. The addition of peptides encompassing the N-terminus to in vitro APC reactions would potentially allow us to address these two models. Unfortunately, the addition of UbcH10 N-terminal peptides did not show a strong effect on APC activity with either WT or UbcH10 ΔN (M.K.S. and P.K.J, unpublished data). However, solubility of the peptides was a problem, making these experiments difficult to interpret. We also detected no interaction between UbcH10 and APC substrates nor did UbcH10 addition affect cyclin B-APC binding (M.K.S. and P.K.J., unpublished data). As a third possibility, perturbing the interaction of the UbcH10 N-terminus with APC may result in enhanced Ub transfer, allowing additional substrate lysines to accept Ub from the mutant E2. Supporting this possibility, long range allostery between the E3-binding and active sites of E2s has been proposed as a mechanism of E2 activation by RING-E3s and mutations causing minor alterations in E2–E3 interactions or minor structural alterations within the active site have strong effects on E2 activity (Ozkan et al., 2005; Yunus and Lima, 2006).

We, and more recently others, have described the regulation of the APC by pseudosubstrate inhibition, employed by Emi1 family proteins, BubR1/Mad3, and Amc1 in yeast (Burton and Solomon, 2007;Enquist-Newman et al., 2008; Hall et al., 2008; King et al., 2007; Miller et al., 2006; Ostapenko et al., 2008). As demonstrated for Emi2/XErp1 and proposed for the spindle assembly checkpoint, pseudosubstrate inhibitors function via a dynamic inhibitor-APC interaction with the inhibitor reversibly engaging and disengaging, but ultimately functioning by having a higher affinity for APC than the substrates (Diaz-Martinez and Yu, 2007; Hansen et al., 2007). If a substrate gains access to the APC during a period of inhibitor activity, the inhibitor could rapidly displace it. Importantly, by partnering with an E2 that has gated activity, it is unlikely that a substrate would fully engage the APC and remain engaged long enough for Ub transfer before being displaced by the inhibitor. This is indeed a very robust mechanism of inhibition and appears to be a critical mode of APC regulation. Intriguingly, while the mechanisms of inhibition for other APC regulators are less well defined, pseudosubstrate inhibition is a likely mechanism as Rae1, Nup98, Mes1 and Mnd2 all contain D-boxes.

Is Intrinsic Regulation a Common Theme in non-Class I E2s?

The regulation of E2 activity by the C-terminal extensions of class II E2s, including polyUb linkage specificity, pathway specificity, and activity towards substrates, is long known (Kolman et al., 1992; Silver et al., 1992; Sung et al., 1988). However, with the exception of the NEDD8 E2, U bc12, little is known about the function of the N-terminal extensions of class III E2s. Our present study demonstrates a critical role in the regulation of the Ub pathway for the N-terminus of UbcH10. Similar differences are observed in the Ub-conjugates formed by a Cullin4 ligase complex with the class III E2, UBE2E3, compared to those formed with UbcH5, suggesting that this type of regulation is a general property of E2 N-terminal extensions (Pick et al., 2007).

Examinimg E1-E2 interactions in the Ub and NEDD8 pathways demonstrates that in addition to facilitating charging with NEDD8, the Ubc12 N-terminus also limits charging with Ub, as does the N-terminus of UbcH10 (Huang et al., 2008). The authors thus suggest that E2 enzymes are optimized for specific E1–E2–E3 pathways, rather than for E1–E2 or E2–E3 paired interactions alone. Our data validate this concept. UbcH10 has maintained both negative regulation of the E1-E2 reaction and a critical determinant for the tight regulation of downstream E2–E3 activity, ensuring the precise performance of the entire enzymatic cascade. In sum, regulating the Ub-flux through the pathway, mediated here by two structural determinants within UbcH10, is an integral balancing point between catalysis and regulation. The significance of this balance is accentuated by the autonomous regulation of UbcH10 levels and the correlation between UbcH10 overexpression and cancer (Okamoto et al., 2003; Rape and Kirschner, 2004). We expect that as the biochemical details of more E3 pathways are discovered, the importance of the E2 enzymes in regulating ubiquitination activity will become increasingly apparent.

EXPERIMENTAL PROCEDURES

Mitotic HeLa Extract Assays (in extracto)

HeLa cells, were maintained in Dulbecco's modified Eagle's medium (GibcoBRL) according to standardized procedures. Mitotic cells were collected by shake-off after 20h in 300ng/ml nocodazole and flash frozen in N₂. Thawed pellets were resuspended in lysis buffer (20mM Tris-HCl, pH 7.2, 2mM DTT, 0.25mM EDTA, 5mM KCl, 5mM MgCl₂) on ice and subjected to 1500psi N₂ in a nitrogen disruption chamber. The lysate was spun for 15min at 15, 000g. Supernatants were divided into single use aliquots and flash frozen in N₂.

For assays, extracts, on ice, were supplemented with an energy regenerating system (30U/ml rabbit creatine phosphokinase type I, 7.5mM creatine phosphate, 1mM ATP, 1mM MgCl₂, 0.1mM EGTA), non-destructible cyclin B, cycloheximide. Proteins or drugs were then added in a final volume of 14µl and a final extract concentration of 6µg/µl. ³⁵S-labeled substrate (1µl) was added; aliquots were made and shifted to 30°C. Samples were quenched at the indicated times by the addition of sample buffer, resolved by SDS-PAGE and imaged on a Typhoon phosphorimager (GE Healthcare). Densitometry was performed with Imagequant software (GE Healthcare).

Immunofluorescence and in vivo Mitotic Checkpoint Bypass

HeLa cells were transfected with pCS2 plasmids with FuGene6 (Roche) as per the vendor protocol. Twenty-four hours post-transfection, cells were blocked in 2mM thymidine for 24h and released into fresh medium containing 30nM Taxol. The cells were fixed with 4% paraformaldehyde, permeablized with methanol, and stained with antibodies against phospho-Histone H3 Ser10 (Upstate), FLAG (Sigma), Securin (Zymed) and FITC, Cy3, and Cy5 antibodies (Jackson Immunoresearch). The DNA was counterstained with Hoechst 33342. Image analysis was performed using a Zeiss AX10 microscope and Slidebook 4.1 software. For mitotic checkpoint bypass analysis the cells were imaged on an ImageXpress Micro system (Molecular Devices) and analyzed with MetaXpress software (Molecular Devices).

in vitro Ubiquitination Assays and APC2 Binding Assay

The APC assay was performed as described (Miller et al., 2006) except that human APC was used for experiments in Figure 2, Figure 3, Figure 4, Figure S4 and Figure S6. The total substrate per lane was determined using ImageQuant and the percent ubiquitinated substrate was then calculated. For Figure 3B, the percentage of wildtype Securin ubiquitinated by an E2 was set as 100% activity. The percent of this activity for the E2 towards mutant substrates was then calculated.

APC11 and APC2–PC11 reactions were carried out in 50mM Tris-HCl, pH 7.5, 5mM MgCl₂, 0.6mM DTT, 2mM ATP and incubated at 30°C. Samples were taken at the indicated times and quenched by the addition of sample buffer. For APC11 reactions, 4ng E1, 140ng E2, and 32ng FLAG-Ub were mixed in a final volume of 10µl. Reactions products were analyzed by anti-FLAG immunoblotting following SDS-PAGE. For APC2–APC11 reactions 100ng E1, 500ng E2, and 6ug Ub were mixed with ³⁵S-labeled substrate in a final volume of 5µl and analyzed as above.

APC2 binding assays were performed as described (Tang et al., 2001b).

Supplementary Material

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Acknowledgments

We thank Marc Kirschner for the UbcH10 construct; Hongtao Yu for p31^Comet and BubR1 reagents and advice; Adam Eldridge, Don Kirkpatrick, and Danny Huang and Brenda Schulman for communicating unpublished results and helpful discussions; Jorge Torres, Ingrid Wertz, Lisa Belmont, and Monica Venere for critical reading of the manuscript; Ivan Bosanac and members of the Jackson lab for discussions and advice. This work was supported, in part, by NIH Post-Doctoral Training Grant T32 CA09151 to M.K.S. and NIH grants RO1 GM054811 and RO1 GM063023 to P.K.J. All authors are employees of Genentech and may own Genentech shares.

Footnotes

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