UBE2S drives elongation of K11-linked ubiquitin chains by the Anaphase-Promoting Complex

Abstract

The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.uring protein ubiquitination, a lysine residue on a target protein is conjugated to ubiquitin through an enzymatic cascade involving E1 activating enzymes, E2 conjugating enzymes, and E3 ligases (1). Chain formation can proceed in the same manner by repetitively adding ubiquitins to one or more of seven lysines on conjugated ubiquitin, potentially generating ubiquitin chains of several different topologies (2, 3). The linkage, topology, and length of the ubiquitin chains are thought to determine the fate of the tagged protein: K48-linked ubiquitin for degradation and K63 for signaling DNA damage and inflammation (4).We have studied the linkage and topology of ubiquitin chains generated by the APC, a multisubunit E3 enzyme that controls cell-cycle progression (5). Recently, two interesting observations have been made about chain topology: First, different E2s might be required in concert to form long ubiquitin conjugates on APC substrates (6). Second, the density of ubiquitin, rather than the length of the chains, may qualify substrates for degradation (7).Like most E3 ligases, APC modifies multiple lysine residues on each substrate, generating a diverse mixture of products. Poor resolution obtained in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) hinders analysis. Even when distinct bands are observed, they may not correspond to single products. A band of a discrete molecular weight may reflect ubiquitination on multiple lysine residues or, alternatively, the formation of ubiquitin chains on single-lysine sites.Dissecting the topology of the ubiquitin chains requires a careful simplification of the ubiquitination reaction under meaningful conditions. To achieve that, following earlier work of Petroski and Deshaies (8), we have modified the well-characterized APC substrate, securin, and generated a model substrate that has only a single-lysine residue available for ubiquitination, thereby limiting chain formation to a single chain. By using this model substrate, we discovered that the ubiquitin conjugates formed on APC substrates are preferentially linked through ubiquitin lysine 11. Similar results have r...

Ube2s regulates Sox2 stability and mouse ES cell maintenance

“…This could be because of endogenous Ube2s proteins copurified with its E3 APC Cdc27 complex. 21 A previous study showed that Ube2s mediates K11-linked polyubiquitin chain assembly on substrates. 21 To verify this, we used a Ub-K11R mutant to perform the in vitro ubiquitination assay and found that this mutation significantly reduces the assembly of polyubiquitin chain on Sox2 (Figure 2d), suggesting that Ube2s modifies Sox2 through the formation of K11-linked polyubiquitin chains.…”

“…21 A previous study showed that Ube2s mediates K11-linked polyubiquitin chain assembly on substrates. 21 To verify this, we used a Ub-K11R mutant to perform the in vitro ubiquitination assay and found that this mutation significantly reduces the assembly of polyubiquitin chain on Sox2 (Figure 2d), suggesting that Ube2s modifies Sox2 through the formation of K11-linked polyubiquitin chains. Furthermore, Ube2s-overexpressed mES cells exhibit a robust elevation of polyubiquitinated Sox2 signal, confirming the capability of Ube2s in ubiquitinating Sox2 in vivo (Figure 2e).…”

“…Our results show that the Ubc domain is essential and sufficient for mediating the association between Ube2s and Sox2 ( Figure 1f, right panel). Interestingly, the enzymatically inactive C95S mutation 21 within the Ubc domain abrogates the interaction of Ube2s with Sox2 (Figure 1f). Consistently, full-length Ube2s with C95S mutation also fails to bind Sox2, thus confirming the critical role of C95 in this interaction (Figures 1g and h).…”

“…(d) Ube2s-mediated Sox2 ubiquitination in vitro. The reaction components for in vitro ubiquitination assay include purified His 6 -tagged Ub, the E3 APC Cdc27 complex co-immunoprecipitated by Cdc27 from mES cells 21 and purified E1 (Uba1), glutathione Stransferase (GST)-tagged Ube2c, GST-tagged Ube2s and His 6 -tagged Sox2 proteins. The reaction solution was analyzed by western blot with an anti-Sox2 antibody.…”

“…20,21 A recent study further clarifies that Ube2s governs the efficiency of substrate degradation through the formation of branched K11-linked polyubiquitin chains. 22 In this study, we identified Ube2s as a novel critical regulator of mES cells.…”