An increasing number of proteins that contain SUMO-binding motifs (SIMs) have been identified (10
). These proteins act as potential adapters that recognize sumoylated protein subpopulations and drive the downstream functional consequences of sumoylation. Here, we demonstrate that an E3 ligase, Pc2, is a bona fide SUMO-binding protein that contains two functional SIMs. We focused on the C-terminal SIM, SIM2, and demonstrated that this SIM makes an important contribution to the E3 ligase activity of Pc2. This finding was recently corroborated in another study (23
). We find that SIM2 in Pc2 is essential for efficient SUMO binding (Fig. and ) and is also important for its ability to promote both the sumoylation of its substrate CtBP1 and the sumoylation of Pc2 itself (Fig. ). SIM2 is also important in determining the subcellular localization of Ubc9 in the insoluble fraction and nuclear foci (Fig. and ). This relocalizing activity of Pc2 appears to be an important determinant of its E3 ligase activity, as colocalization with Ubc9 is required for both Pc2 sumoylation and the sumoylation of CtBP1. Thus, Pc2 acts as pivotal factor in the SUMO pathway and acts as a bridging factor that binds to SUMO, and this binding activity is then transmitted into further downstream sumoylation events.
An additional spatially distinct motif was identified in the C-terminal region of Pc2, the IIIT motif, which seems to function in part through reinforcing the activities of SIM2. For example, SUMO binding through SIM2 is potentiated by the presence of the IIIT motif (Fig. ). However, the IIIT motif does not act independently as a SIM (Fig. ) but instead might act as a functional module with SIM2 (our unpublished data).
SUMO binding through the SIM2 motif is important for the E3 ligase activity of Pc2 both in vitro
and in vivo
; therefore, either free SUMO or a sumoylated target must be important for this regulatory activity. One such target in vivo
appears to be Ubc9, as the level of recruitment of Ubc9 to subnuclear foci by Pc2 is much reduced when a nonconjugatable form of SUMO is introduced (Fig. ). Thus, it appears that SIM2 functions through conjugated SUMO moieties on targets. The E2 enzyme Ubc9 is covalently linked to SUMO via a thioester linkage in its active form (9
). In addition, Ubc9 can noncovalently bind to SUMO and was previously shown to be modified by SUMO through isopeptide linkages (4
). Indeed, Pc2 relocalizes Ubc9 to the insoluble compartment and to nuclear foci in a manner that is dependent on both SIM2 and the ability of Ubc9 to be able to be linked to SUMO via thioester linkages and to noncovalently bind to SUMO (Fig. ). This is highly reminiscent of a similar mechanism seen in the ubiquitin system, where ubiquitin-binding motifs have been shown to be able to recruit ubiquitin-loaded E2 enzymes (12
). Our data do, however, suggest a model whereby Pc2 acts via SIM2 to cause the redistribution of the active form of Ubc9 to nuclear foci, which can potentially function as sumoylation centers that ensure the specificity and efficiency of sumoylation (Fig. ). The simplest model invokes a direct recruitment of SUMO-linked Ubc9, as depicted, but we cannot rule out other potential indirect mechanisms though intermediary proteins that are themselves modified by SUMO. The proposed action of Pc2 in these centers was postulated previously due to its colocalization with SUMO machinery components, but its mechanism of action in this context was not clear (15
). The mechanism that we uncover is in keeping with the general model that E3 SUMO ligases act as adapters to bring the E2 enzyme (Ubc9) and substrates into close proximity for efficient SUMO conjugation (9
). Moreover, this proposed mode of action is also in agreement with the observation that Pc2 promotes substrate sumoylation subsequent to Ubc9 loading in vitro
), suggesting a two-step model of relocalization, followed by promoting SUMO transfer. A further prediction of this model is that the efficiency of Pc2 as an E3 ligase promoting substrate sumoylation should be affected by SIM2. This is indeed the case in both in vitro
and in vivo
experiments and is supported by the observation that the SUMO E3 ligase activity of Pc2 toward CtBP1 is also dependent on the presence of SIM2. Interestingly, the sumoylated form of CtBP1 is found in the insoluble fraction, further emphasizing the potential importance of the Ubc9-relocalizing activity of Pc2 (see Fig. S3 in the supplemental material). Previous studies demonstrated that the phosphorylation of Pc2 at T495 by HIPK2 promoted its E3 ligase activity, and this site is close to SIM2 (32
). It is therefore possible that this or other regulatory events might influence Pc2-SUMO interactions and hence control the E3 ligase activity of Pc2.
FIG. 7. Model illustrating the role of SIM2 (dark gray rectangle) in Pc2 that coordinates the SUMO machinery-substrate association. Pc2 utilizes its SIM2 motif to coordinate substrate (Sub)-Ubc9 interactions in the context of PcG bodies. The SIM2 motif in Pc2 (more ...)
Previous studies have identified a functionally important region of the E3 ligase RanBP2, which resembles the hydrophobic SIM (30
). A combination of structural and biochemical assays suggested that mechanistically, a SIM-SUMO interaction is important for the coordination of the thioester-linked Ubc9 with substrate lysine residues. However, no role in determining the subcellular localization of SUMO pathway components was examined. Other E3 ligases in the PIAS family contain SIMs that can bind to SUMO (38
). However, recent mutagenesis studies of PIAS1 indicated that these motifs have no role in their E3 ligase activity (39
). Thus, although different E3 ligases contain functional SIMs, their molecular roles might differ.
Other studies have shown that SUMO-binding activity and protein sumoylation are linked in several cases, such as with thymine-DNA glycosylase (TDG), Daxx, and SP100 (16
). However, the sumoylation status of PML was demonstrated to be independent from noncovalent SUMO interactions. Our results clearly demonstrate that SUMO-binding activity is indispensable for the efficient SUMO conjugation of Pc2 (Fig. ). In addition, many SUMO E3 ligases are found to be sumoylated, although the function of the sumoylation of these proteins remains unclear (18
). Interestingly, we have found that one of the functional consequences of Pc2 sumoylation is in promoting fgf5
expression during mESC differentiation (Fig. ), although it is not yet known whether the effect on this target gene is direct or indirect. However, we demonstrate that the sumoylation status of Pc2 plays no roles in (i) its ability to bind to SUMO (see Fig. S1E in the supplemental material), (ii) its subcellular localization (see Fig. S4), (iii) its ability to partition the SUMO machinery (Fig. ), and (iv) its E3 ligase activity toward substrates (data not shown).
Pc2 contains two SIMs, which both play a role in SUMO binding. While we have focused on SIM2, SIM1 also appears to be important for Pc2 function as an E3 ligase and also in determining its ability to relocalize other PcG proteins (23
), although it still remains unclear how the two SIMs cooperate to promote SUMO binding and subsequent Pc2 functions. While the binding of mono-SUMO is mediated by these two SIMs, it is not clear whether in some instances they might work together to recognize poly- or multisumoylated species. Such a scenario was demonstrated for RNF4, which contains four SIMs that bind weakly to mono- or di-SUMO and show high selectivity for poly-SUMO-2 chains (44
). Furthermore, a recent study suggested a difference in the binding of BLM to SUMO paralogues with resulting functional consequences for its own sumoylation (52
). However, while BLM discriminates between SUMO paralogues, the SIM2 in Pc2 does not show any obvious discriminatory activity (Fig. and see Fig. S2 in the supplemental material).
Functionally, we demonstrate the importance of SUMO binding via the SIM2 motif in the context of Pc2 function in ESC differentiation (Fig. ). Polycomb-repressive complexes (PRCs) play an important role in controlling embryonic stem cell properties, and Pc2 is part of these multiprotein complexes (19
). The identification of subnuclear foci as potential centers for protein sumoylation through the E3 ligase activity of Pc2 suggests that other components within PRCs may be subjected to SUMO modification and, thus, might lead to the regulation of the activities of PRCs. Indeed, in silico
analyses indicate that a number of components of PcG proteins contain SUMO consensus sites. Our data suggest that the sumoylation of Pc2 is important for its role in specifying cell fate, but in addition, it is tempting to speculate that the E3 ligase activity of Pc2 might contribute to the regulation of the activities of other components of the PRCs and, thus, contribute to ESC differentiation. Indeed, we show that lineage-specific markers are aberrantly expressed upon the depletion of Pc2, and this effect is specifically through a loss of the SUMO-binding activity of Pc2 (Fig. ). One functional consequence of the loss of SUMO-binding activity is the reduced sumoylation of Pc2 itself. This appears likely to be one of the important downstream functional consequences, as a Pc2 protein, which cannot be sumoylated (K492R mutant), is also defective in lineage-specific gene expression (Fig. ). Although the role of SIM2 in determining Pc2 function in ESCs is at least partially determined by modulating its E3 ligase activities, we cannot exclude additional roles through interactions with other SUMO-conjugated proteins. Further studies will be needed to identify the potential targets of Pc2 in the context of ESCs, which are subject to SUMO modification.
In summary, our findings demonstrate a novel important role for noncovalent SUMO binding via a SIM in promoting the activity of the E3 ligase Pc2. We provide novel mechanistic insights into how substrate selectivity can be achieved, at least in part, through the partitioning of a specific active E2-E3 complex into specialized subnuclear compartments. It remains to be determined whether the E1 SUMO-activating enzyme is also partitioned in subnuclear structures. The functional importance of SIM-SUMO interactions in promoting the E3 ligase activity of Pc2 is demonstrated in the context of the regulation of lineage-specific gene expression in mouse embryonic stem cells.