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The Fast-Growing Business of SUMO Chains
Molecular Cell
Short Review The Fast-Growing Business of SUMO Chains Helle D. Ulrich1,* 1Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, EN6 3LD, UK *Correspondence: [email protected] DOI 10.1016/j.molcel.2008.10.010
Like ubiquitin itself, the small ubiquitin-related modifier SUMO can form polymeric chains on many of its targets. Recent analyses have provided evidence for a number of distinct biological functions of the poly-SUMO signal. Posttranslational modifications modulate the properties of proteins in a reversible manner that does not require de novo protein synthesis, thus allowing the cell to react quickly and flexibly to changes in its environment. Beyond the spectrum of smallmolecule modifications, such as phosphorylation, acetylation or methylation, covalent attachment of entire protein moieties is used by eukaryotic cells as a means to influence activities or interactions of intracellular targets. Most, if not all, of these modifier proteins belong to a group of structurally related molecules termed the ubiquitin family (Kerscher et al., 2006). Conjugation to the target protein—usually through an isopeptide linkage between the modifier’s carboxy (C) terminus and the 3-amino group of an internal lysine (K) in the target—is mediated by a cascade of dedicated enzymes involved in the activation of the modifier (E1), its transfer (E2), and finally, substrate selection (E3). In most instances, the modification is reversible through the action of specific isopeptidases. Among the members of the ubiquitin family, ubiquitin itself is famous for its ability to form polymeric chains in which one ubiquitin moiety is linked to another via one of ubiquitin’s seven lysines. The most prominent biological function of the ubiquitin system, the initiation of regulated protein degradation by the 26S proteasome, is mediated by polyubiquitin chains linked uniformly via K48. Alternative, often nonproteolytic functions have been assigned to chains linked via other lysines (Pickart and Fushman, 2004). Central to the physiological significance of the polyubiquitin signal is an appropriate read-out by downstream effector proteins bearing ubiquitin-binding domains that differentiate between monoubiquitin and different types of polymeric chains (Hurley et al., 2006). How this selectivity is achieved is a subject of ongoing research. It has become clear, however, that the ability to form chains is not unique to ubiquitin. SUMO, the small ubiquitin-related modifier, is functionally at least as versatile as ubiquitin. It is involved in the regulation of transcription, in various aspects of genome stability including DNA repair pathways and chromosome segregation, and in the control of nucleocytoplasmic transport (Hay, 2005). Although in most of these roles it is believed to act as a monomeric conjugate, there is now growing evidence for a contribution of poly-SUMO chains to at least some aspects of SUMO function. Evidence for the Formation of Poly-SUMO Chains In order to understand how poly-SUMO chains are formed, it is instructive to consider the mechanism of substrate recognition by the SUMO conjugation system (Hay, 2005; Kerscher et al.,
2006). In contrast to the ubiquitin system, where the ligases (E3) as opposed to the conjugating enzymes (E2) largely determine substrate selectivity, the SUMO-specific E2, Ubc9, plays an important role in target selection. Ubc9 binds directly to a short consensus motif of the sequence JKXE/D (where J is a bulky aliphatic residue and X stands for any amino acid), provided that its conformation is not constrained by elements of secondary structure. Consequently, most SUMO targets are modified at lysine residues situated within this type of consensus sequence. Two of the three mammalian SUMO isoforms known to engage in conjugation, SUMO-2 and SUMO-3, bear such a motif in their unstructured amino (N)-terminal regions, centered on K11 (Figure 1A). Similarly, the budding yeast SUMO homolog, Smt3, harbors three sumoylation consensus sites around K11, K15, and K19. Hence, these lysines lend themselves as internal acceptor sites for polymerization, and Ubc9 readily catalyzes the in vitro assembly of free SUMO-2/3 or Smt3 chains linked via the expected lysines. SUMO-1 forms chains much less efficiently in vitro, although polymers of both SUMO-1 and SUMO-2/3 have been observed to form via nonconsensus lysines (Cooper et al., 2005; Matic et al., 2008; Pedrioli et al., 2006). A possible mechanism for SUMO polymerization has been suggested based on a noncovalent interaction between SUMO and a surface on Ubc9 opposite its catalytic center (Capili and Lima, 2007; Knipscheer and Sixma, 2007). By means of this interaction, dimerization of two Ubc9 molecules would position the N terminus of a SUMO-2/3 molecule bound noncovalently to one Ubc9 in a way that would allow conjugation of another SUMO moiety bound as a thioester to the other Ubc9 molecule (Figure 1B). SUMO ligases generally enhance free chain formation and often stimulate the assembly of long polymeric conjugates on suitable substrates in vitro, but at least under some experimental conditions their action appears to result in enhanced chain formation via nonconsensus lysines (Bylebyl et al., 2003; Cooper et al., 2005; Pedrioli et al., 2006). In vivo, the detection of poly-SUMO chains is less straightforward, as they are difficult to distinguish from large monosumoylated conjugates and substrates modified by mono-SUMO at multiple sites. The formation of a SUMO-SUMO linkage on a physiological substrate was first demonstrated for the histone deacetylase HDAC4 (Tatham et al., 2001). The protein harbors a single sumoylation site at K559; yet, cotransfection of HDAC4 with tagged SUMO-3 yielded two conjugates, the larger one depending on the presence of K11 in the modifier. In contrast, cotransfection with SUMO-1 gave rise to a single conjugate,
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Molecular Cell
Short Review a massive redistribution of SUMO-2 and -3 to nuclear PML bodies (Mukhopadhyay et al., 2006). In vitro, the enzyme preferentially cleaves SUMO-2/3 conjugates of three or more moieties, particularly on substrates with a single acceptor lysine, suggesting that it, like Ulp2, primarily acts on canonical poly-SUMO chains. These findings indicate the necessity for an active control over the levels of poly-SUMO chains in yeast and mammals in order to prevent adverse effects for the cell.
Figure 1. SUMO Can Form Polymeric Chains (A) Sequence alignment of the N termini of human SUMO isoforms with ubiquitin and with budding yeast Smt3. Sumoylation consensus motifs are highlighted in yellow with the acceptor lysine depicted in red. (B) Proposed mechanism of poly-SUMO chain formation via dimerization of the conjugating enzyme Ubc9. SUMO is shown in gray, with the C-terminal glycines highlighted in red, and Ubc9 is shown in brown. The active-site cysteine involved in thioester formation is indicated.
indicating that the consensus sumoylation site in SUMO-3 is used in vivo for chain assembly. More recently, the existence of SUMO-SUMO linkages via K11 of SUMO-2 and -3 in cell extracts was verified by mass spectrometry via detection of the relevant branched peptides (Matic et al., 2008). The transcriptional regulator HIF-1a was identified as another conjugation target for poly-SUMO chains. Interestingly, this study also revealed the presence of SUMO-1 conjugated to K11 of SUMO-2 or -3, suggesting that SUMO-1 may act as a chain terminator in vivo. In budding yeast, Smt3 chains are formed via all three sumoylation consensus sites, K11, K15, and K19 (Bylebyl et al., 2003). Short oligomeric Smt3 chains have recently been detected on the replication factor PCNA (Windecker and Ulrich, 2008). It is unknown, however, whether the bulk of the high-molecular weight SUMO conjugates observed in yeast and in higher eukaryotes corresponds to SUMO polymers attached to substrate proteins or to free, unanchored chains. Disassembly of Poly-SUMO Chains Yeast cells lacking the SUMO-specific isopeptidase Ulp2 display enhanced levels of high-molecular-weight poly-Smt3 conjugates, suggesting that this deconjugating enzyme normally limits the accumulation of poly-Smt3 chains (Bylebyl et al., 2003). The ulp2 mutant exhibits a highly pleiotropic phenotype, including defects in chromosome segregation and recovery from checkpoint-induced cell-cycle arrest, poor growth, and enhanced sensitivity to various stress conditions (Bylebyl et al., 2003; Schwartz et al., 2007). Although some of these defects may in part be caused by an inappropriate accumulation of monomeric conjugates to specific target proteins, evidence for a contribution of poly-Smt3 chains comes from the observation that many aspects of the ulp2 phenotype are suppressed to a large extent by mutating K11, 15, and 19 of Smt3, i.e., by preventing chain formation (Bylebyl et al., 2003). Similarly, depletion of the Ulp2-related mammalian isopeptidase SENP6/SUSP1 causes an accumulation of poly-SUMO-2/3 conjugates in human cells, resulting in
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Physiological Relevance of the Poly-SUMO Signal Detection of polysumoylation in vivo raises the question of whether this type of conjugation fulfills a specific signaling function distinct from that of mono-SUMO. In general, SUMO is believed to act mainly by modulating the protein-protein interactions of its targets. On one hand, attachment of SUMO can potentially mask a binding site for another factor, thereby preventing either a noncovalent interaction or possibly the attachment of another posttranslational modifier. In this manner, SUMO has been proposed to antagonize the effects of ubiquitin on a number of common substrate proteins (Ulrich, 2005). It is difficult to imagine, however, how polysumoylation in this context could have any effect not achieved with equal efficiency by the attachment of a single SUMO molecule. On the other hand, sumoylation can create an additional binding surface allowing the preferential interaction of a downstream effector with the modified target. This mode of function requires the specific recognition of the SUMO moiety by an interaction domain within the effector protein. In yeast as well as higher eukaryotes, a single type of SUMO-interaction motif (SIM) has been identified so far (Kerscher, 2007). It consists of a short hydrophobic core that is often either preceded or followed by a stretch of acidic and/or phosphorylated residues and aligns as a b strand with a matching region on the surface of SUMO. SIM-SUMO interactions are known to mediate the downstream effects of SUMO on many of its targets, such as the recruitment of corepressors by sumoylated transcription factors in higher eukaryotes or of the antirecombinogenic helicase Srs2 by sumoylated PCNA in budding yeast (Kerscher, 2007; Ulrich, 2005). Even intramolecular interactions can be induced by sumoylation, such as the conformational change upon modification of the thymidine DNA glycosylase TDG that is believed to result from the binding of an internal SIM to the covalently attached SUMO moiety (Kerscher, 2007; Ulrich, 2005). In principle, SIM-SUMO interactions could be enhanced by SUMO polymerization, either simply by an increase in the local SUMO concentration that would facilitate rebinding after dissociation, or, given the 1:1 stoichiometry of the interaction, by the presence of multiple SIMs in the effector protein, possibly by means of its oligomerization. It should be noted, however, that the same effect could potentially be achieved by the attachment of multiple units of mono-SUMO to closely spaced acceptor sites on the target. As discussed below, a distinction between the effects of polysumoylation and multiple monosumoylation is not always possible, but in some instances, there is good evidence for a contribution of the SUMO-SUMO linkage to in vivo function. Structural Roles of Poly-SUMO Chains In budding yeast, poly-SUMO chains are not essential for viability, as smt3(3R) mutants, bearing lysine-to-arginine substitutions
Molecular Cell
Short Review Figure 2. SUMO-Dependent Ubiquitin Ligases Promote Degradation of Sumoylated Proteins (A) Domain arrangements of SUMO-dependent ubiquitin ligases from human (H. sapiens), budding yeast (S. cerevisiae), and fission yeast (S. pombe). The symbols within the RING domains indicate whether the corresponding domains have ubiquitin ligase activity. (B) A polysumoylated substrate (blue) is recognized in a SIM-dependent manner by a SUMOdependent ubiquitin ligase (showing the S. pombe Slx8-Rfp1 complex as an example). Ubiquitin (black) is conjugated to the outermost SUMO moiety as well as to the substrate protein itself. Following ubiquitylation, the modified substrate is degraded by the 26S proteasome. It is unknown whether the SUMO moieties are degraded along with the substrate or whether deconjugation occurs before proteolysis.
at positions 11, 15 and 19, exhibit no obvious defect during vegetative growth (Bylebyl et al., 2003). The only detectable phenotype is a significantly reduced sporulation efficiency, suggesting a potential role of SUMO polymerization in meiosis (Cheng et al., 2006). This notion is further supported by the observation that Smt3 colocalizes with the synaptonemal complex (SC), a structure that bridges homologous chromosomes along their lengths when they align in preparation for meiotic division. One component of the SC, Zip1, was found to interact with Smt3 and several sumoylated proteins, and a second component, Zip3, contains an SP-RING domain related to the Siz- and PIAS-type SUMO ligases and was shown to promote Smt3 chain formation in vitro. In the smt3(3R) mutant, Zip1 is not properly localized, resulting in an abnormal SC structure that is likely the cause of the sporulation defect. It is unclear whether Zip1 is recruited to the SC primarily by polysumoylated proteins or whether unanchored Smt3 chains are a constituent of the SC. Overall, however, these observations indicate that Smt3 chains play a role in maintaining the structural integrity of the SC, possibly by recruiting the Zip1 protein to its correct location. A structural role for SUMO-2/3 chains in mammalian cells was recently proposed by Zhang et al., who identified the microtubule motor protein CENP-E as a SUMO-interacting protein (Zhang et al., 2008). CENP-E, whose localization to kinetochores is important for the alignment of chromosomes at the metaphase plate during mitosis, was found to bind specifically to SUMO-2/3 chains, but not to mono-SUMO or SUMO-1 polymers in vitro. Its correct localization was found to depend on an internal SIM. Overexpression of the SUMO-specific isopeptidase SENP2 or depletion of Ubc9 caused a delocalization of CENP-E and resulted in a cell-cycle arrest in prometaphase. CENP-E itself is a target for SUMO-2/3 modification, as are several other kinetochore-associated proteins, such as BubR1 and Nuf2; however, it remains to be determined whether these SUMO substrates are relevant for proper CENP-E localization. Although the in vitro binding preferences of CENP-E strongly suggest that SUMO-
SUMO linkages are important for its function, an interaction with closely spaced multiple mono-SUMO conjugates has not rigorously been excluded. Nevertheless, these findings point to a contribution of poly-SUMO-2/3 chains to kinetochore function. Poly-SUMO Chains as a Ubiquitylation Signal A series of publications has recently revealed that poly-SUMO chains can exert a signaling function that stands in stark contrast to SUMO’s perceived role as a ubiquitin antagonist. By means of a newly discovered class of ubiquitin ligases, attachment of polySUMO chains to a substrate was shown to promote its subsequent ubiquitylation and degradation (Lallemand-Breitenbach et al., 2008; Mullen and Brill, 2008; Prudden et al., 2007; Sun et al., 2007; Tatham et al., 2008; Uzunova et al., 2007; Xie et al., 2007). The hallmark of the relevant family of ubiquitin E3s is the presence of multiple SIMs by which they recognize polysumoylated proteins as ubiquitylation targets (Figures 2A and 2B). In mammalian cells, the RING finger E3 RNF4 bears four closely spaced SIMs in its N-terminal region (Figure 2A). All of them contribute to SUMO binding, and although their affinity for SUMO is not isotype-specific, collectively they prefer polymeric chains formed via K11 of SUMO-2 or -3 over monomeric SUMO (Tatham et al., 2008). Polysumoylated PML, a structural component of nuclear bodies, was identified as an in vivo substrate of RNF4-dependent ubiquitylation. Attachment of ubiquitin to both PML and the SUMO moieties was found to target the sumoylated conjugate for degradation by the 26S proteasome. The same was found to apply for an oncogenic fusion protein of PML with retinoic acid receptor, PML-RARa, a causative agent of acute promyelocytic leukemia (APL). Treatment with arsenic trioxide, known to induce PML-RARa degradation and to cure the disease, was shown to promote sumoylation of the fusion protein, which in turn triggered its proteasomal degradation in an RNF4-dependent manner (Lallemand-Breitenbach et al., 2008). A significance of poly-SUMO chains was suggested by the notion that silencing of SUMO-2/3 had a stronger effect on
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Short Review protein levels than silencing of SUMO-1, but the residual effect of SUMO-1 left open the possibilites of either noncanonical SUMO-SUMO linkages or a contribution of multiple monoSUMO adducts. SUMO-dependent ubiquitin ligases in lower eukaryotes appear to function as heterodimers where one subunit, Slx8, harbors RING-dependent ubiquitin ligase activity, while the other one, dimerizing with Slx8 via RING-RING interactions, is primarily responsible for SUMO recognition (Figure 2A). The complexes from budding and fission yeast display very similar biochemical activities, analyzed in vitro with model substrates such as linear SUMO fusions to GST or to S. cerevisiae Rad52, a known SUMO substrate, or the S. pombe protein Rad60, which harbors two SUMO-like domains in its C terminus. However, neither of these proteins appears to be a substrate of SUMO-dependent ubiquitin ligases in vivo, and physiological targets remain to be identified. Inactivation of the SIMs causes an accumulation of unidentified high-molecular-weight SUMO conjugates and results in a general DNA damage sensitivity phenotype that is consistent with a role of Slx8 and its interaction partners in genome maintenance. Like in mammalian cells, the fate of ubiquitylated SUMO conjugates appears to be proteasome-mediated degradation, as treatment with proteasome inhibitors caused an accumulation of mixed ubiquitin-SUMO conjugates in S. cerevisiae (Uzunova et al., 2007). The dependence of these conjugates on the presence of K11, 15, and 19 of Smt3 indicates that the relevant ubiquitylation signal is indeed a poly-SUMO chain. This notion was further confirmed by a detailed biochemical analysis of the Slx8-Slx5 complex from budding yeast with a model substrate (Mullen and Brill, 2008). The use of various Smt3 mutants clearly demonstrated that polysumoylation, as opposed to multiple monosumoylation, effectively triggered ubiquitylation. Interestingly, however, the linkage within the poly-Smt3 chain was not critical, as lysines other than K11, K15, and K19 were able to form chains in vitro that served as productive ubiquitylation signals. In addition to the target protein itself, the N-terminal lysines of the outermost Smt3 moiety served as predominant acceptor sites for ubiquitylation. Intriguingly, the Slx8-Slx5 complex has also been reported to catalyze the formation of poly-Smt3 chains (Ii et al., 2007), but this in vitro activity may simply be a consequence of the SIM domains bringing several Smt3 molecules into close proximity, thus facilitating their ligation by Ubc9. This new family of SUMO-directed ubiquitin ligases illustrates how polysumoylation can function as a ubiquitylation signal and thereby indirectly influence protein levels (Figure 2B). The notion that polysumoylated proteins are degraded in vivo raises the question of whether this process serves as a regulatory device for the selective removal of specific proteins in a controlled manner or whether polysumoylated proteins are toxic by-products of the SUMO conjugation system that the cell needs to eliminate in order to prevent the malfunction of certain biological pathways. While the sporulation defect of the smt3(3R) mutant indicates a positive contribution to cellular metabolism, the phenotypes of cells deficient in the disassembly of poly-SUMO chains argues for a need to avoid an uncontrolled accumulation of poly-SUMO conjugates (Bylebyl et al., 2003; Mukhopadhyay et al., 2006).
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Poorly Defined Roles of Poly-SUMO Chains There are cases of polysumoylation that appear to fit neither of the models presented above, although they have important implications for the cell. For instance, Li et al. were able to show that cleavage of the amyloid precursor protein (APP) by b-secretase, which releases the amyloidogenic Ab peptide in the course of Alzheimer’s disease, is indirectly influenced by sumoylation (Li et al., 2003). APP itself does not appear to be a sumoylation target. However, whereas overexpression of wild-type SUMO-3 promoted a nonamyloidogenic mode of APP processing, thus lowering the levels of secreted Ab peptide, overexpression of the K11R mutant of SUMO-3 had the opposite effect. Hence, K11-linked poly-SUMO chains appear to play a role in preventing the accumulation of the pathological Ab peptide. The target protein(s) relevant for this phenomenon have yet to be identified. A possible role in targeting to the nucleolus was recently proposed for the polysumoylation of budding yeast topoisomerase II (Takahashi and Strunnikov, 2008). Insertions of linear tandem arrays of Smt3 close to the natural sumoylation sites within the enzyme were designed to mimic its modified forms. Whereas insertion of a single Smt3 moiety afforded pericentromeric localization, constructs with multiple inserts were found in the nucleolus. Likewise, highly sumoylated topoisomerase II accumulated in the nucleolus of ulp2 deletion mutants. This SUMO-dependent targeting mechanism appears to be physiologically relevant, as nonsumoylatable mutants of the enzyme or deletion of the SUMO ligase genes SIZ1 and SIZ2 cause signifcant rDNA instability. However, as topoisomerase II is naturally sumoylated at multiple sites, it is quite possible that several closely spaced monomeric SUMO adducts could function equally well as a polymeric chain in targeting the enzyme to its correct location. Finally, it should be noted that poly-SUMO chains may not necessarily have to convey a special signal. In the case of budding yeast PCNA, sumoylation is known to recruit the antirecombinogenic helicase Srs2 (Ulrich, 2005). Although short chains are formed in vivo on two acceptor lysines, prevention of polysumoylation in an smt3(3R) mutant was found to have no effect on the function of sumoylated PCNA at the replication fork (Windecker and Ulrich, 2008). Conclusion The examples discussed above illustrate that polysumoylation can fulfill signaling functions but also promote the assembly of large macromolecular complexes. In either situation, however, the effects of poly-SUMO chains appear to be mediated by the same type of conserved SIM-SUMO interaction. Considering the nature of this interaction and the restricted choice of acceptor lysines within the SUMO sequence in vivo, it is therefore unlikely that the SUMO system is able to functionally differentiate between alternative linkages as observed in the case of polyubiquitin chains. It should be noted that a functional equivalence of canonical and alternative SUMO-SUMO linkages has yet to be proven. Generally, however, the preference of downstream effectors for poly-, as opposed to multiple mono-SUMO adducts, will likely be dictated by the geometry and affinities of the respective SIMs involved in chain recognition, and the diversity of the poly-SUMO signal may turn out to be much more limited than that conveyed by polyubiquitin. In order to gain a better
Molecular Cell
Short Review understanding of the mechanisms by which SUMO chains carry out their respective functions, it will be essential to identify the cellular proteins bearing this modification in the relevant physiological context. Despite many open questions, the emerging roles of polymeric SUMO chains once more emphasize the versatility of ubiquitin-like protein modifiers. REFERENCES
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Short Review The Fast-Growing Business of SUMO Chains Helle D. Ulrich1,* 1Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, EN6 3LD, UK *Correspondence: [email protected] DOI 10.1016/j.molcel.2008.10.010
Like ubiquitin itself, the small ubiquitin-related modifier SUMO can form polymeric chains on many of its targets. Recent analyses have provided evidence for a number of distinct biological functions of the poly-SUMO signal. Posttranslational modifications modulate the properties of proteins in a reversible manner that does not require de novo protein synthesis, thus allowing the cell to react quickly and flexibly to changes in its environment. Beyond the spectrum of smallmolecule modifications, such as phosphorylation, acetylation or methylation, covalent attachment of entire protein moieties is used by eukaryotic cells as a means to influence activities or interactions of intracellular targets. Most, if not all, of these modifier proteins belong to a group of structurally related molecules termed the ubiquitin family (Kerscher et al., 2006). Conjugation to the target protein—usually through an isopeptide linkage between the modifier’s carboxy (C) terminus and the 3-amino group of an internal lysine (K) in the target—is mediated by a cascade of dedicated enzymes involved in the activation of the modifier (E1), its transfer (E2), and finally, substrate selection (E3). In most instances, the modification is reversible through the action of specific isopeptidases. Among the members of the ubiquitin family, ubiquitin itself is famous for its ability to form polymeric chains in which one ubiquitin moiety is linked to another via one of ubiquitin’s seven lysines. The most prominent biological function of the ubiquitin system, the initiation of regulated protein degradation by the 26S proteasome, is mediated by polyubiquitin chains linked uniformly via K48. Alternative, often nonproteolytic functions have been assigned to chains linked via other lysines (Pickart and Fushman, 2004). Central to the physiological significance of the polyubiquitin signal is an appropriate read-out by downstream effector proteins bearing ubiquitin-binding domains that differentiate between monoubiquitin and different types of polymeric chains (Hurley et al., 2006). How this selectivity is achieved is a subject of ongoing research. It has become clear, however, that the ability to form chains is not unique to ubiquitin. SUMO, the small ubiquitin-related modifier, is functionally at least as versatile as ubiquitin. It is involved in the regulation of transcription, in various aspects of genome stability including DNA repair pathways and chromosome segregation, and in the control of nucleocytoplasmic transport (Hay, 2005). Although in most of these roles it is believed to act as a monomeric conjugate, there is now growing evidence for a contribution of poly-SUMO chains to at least some aspects of SUMO function. Evidence for the Formation of Poly-SUMO Chains In order to understand how poly-SUMO chains are formed, it is instructive to consider the mechanism of substrate recognition by the SUMO conjugation system (Hay, 2005; Kerscher et al.,
2006). In contrast to the ubiquitin system, where the ligases (E3) as opposed to the conjugating enzymes (E2) largely determine substrate selectivity, the SUMO-specific E2, Ubc9, plays an important role in target selection. Ubc9 binds directly to a short consensus motif of the sequence JKXE/D (where J is a bulky aliphatic residue and X stands for any amino acid), provided that its conformation is not constrained by elements of secondary structure. Consequently, most SUMO targets are modified at lysine residues situated within this type of consensus sequence. Two of the three mammalian SUMO isoforms known to engage in conjugation, SUMO-2 and SUMO-3, bear such a motif in their unstructured amino (N)-terminal regions, centered on K11 (Figure 1A). Similarly, the budding yeast SUMO homolog, Smt3, harbors three sumoylation consensus sites around K11, K15, and K19. Hence, these lysines lend themselves as internal acceptor sites for polymerization, and Ubc9 readily catalyzes the in vitro assembly of free SUMO-2/3 or Smt3 chains linked via the expected lysines. SUMO-1 forms chains much less efficiently in vitro, although polymers of both SUMO-1 and SUMO-2/3 have been observed to form via nonconsensus lysines (Cooper et al., 2005; Matic et al., 2008; Pedrioli et al., 2006). A possible mechanism for SUMO polymerization has been suggested based on a noncovalent interaction between SUMO and a surface on Ubc9 opposite its catalytic center (Capili and Lima, 2007; Knipscheer and Sixma, 2007). By means of this interaction, dimerization of two Ubc9 molecules would position the N terminus of a SUMO-2/3 molecule bound noncovalently to one Ubc9 in a way that would allow conjugation of another SUMO moiety bound as a thioester to the other Ubc9 molecule (Figure 1B). SUMO ligases generally enhance free chain formation and often stimulate the assembly of long polymeric conjugates on suitable substrates in vitro, but at least under some experimental conditions their action appears to result in enhanced chain formation via nonconsensus lysines (Bylebyl et al., 2003; Cooper et al., 2005; Pedrioli et al., 2006). In vivo, the detection of poly-SUMO chains is less straightforward, as they are difficult to distinguish from large monosumoylated conjugates and substrates modified by mono-SUMO at multiple sites. The formation of a SUMO-SUMO linkage on a physiological substrate was first demonstrated for the histone deacetylase HDAC4 (Tatham et al., 2001). The protein harbors a single sumoylation site at K559; yet, cotransfection of HDAC4 with tagged SUMO-3 yielded two conjugates, the larger one depending on the presence of K11 in the modifier. In contrast, cotransfection with SUMO-1 gave rise to a single conjugate,
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Short Review a massive redistribution of SUMO-2 and -3 to nuclear PML bodies (Mukhopadhyay et al., 2006). In vitro, the enzyme preferentially cleaves SUMO-2/3 conjugates of three or more moieties, particularly on substrates with a single acceptor lysine, suggesting that it, like Ulp2, primarily acts on canonical poly-SUMO chains. These findings indicate the necessity for an active control over the levels of poly-SUMO chains in yeast and mammals in order to prevent adverse effects for the cell.
Figure 1. SUMO Can Form Polymeric Chains (A) Sequence alignment of the N termini of human SUMO isoforms with ubiquitin and with budding yeast Smt3. Sumoylation consensus motifs are highlighted in yellow with the acceptor lysine depicted in red. (B) Proposed mechanism of poly-SUMO chain formation via dimerization of the conjugating enzyme Ubc9. SUMO is shown in gray, with the C-terminal glycines highlighted in red, and Ubc9 is shown in brown. The active-site cysteine involved in thioester formation is indicated.
indicating that the consensus sumoylation site in SUMO-3 is used in vivo for chain assembly. More recently, the existence of SUMO-SUMO linkages via K11 of SUMO-2 and -3 in cell extracts was verified by mass spectrometry via detection of the relevant branched peptides (Matic et al., 2008). The transcriptional regulator HIF-1a was identified as another conjugation target for poly-SUMO chains. Interestingly, this study also revealed the presence of SUMO-1 conjugated to K11 of SUMO-2 or -3, suggesting that SUMO-1 may act as a chain terminator in vivo. In budding yeast, Smt3 chains are formed via all three sumoylation consensus sites, K11, K15, and K19 (Bylebyl et al., 2003). Short oligomeric Smt3 chains have recently been detected on the replication factor PCNA (Windecker and Ulrich, 2008). It is unknown, however, whether the bulk of the high-molecular weight SUMO conjugates observed in yeast and in higher eukaryotes corresponds to SUMO polymers attached to substrate proteins or to free, unanchored chains. Disassembly of Poly-SUMO Chains Yeast cells lacking the SUMO-specific isopeptidase Ulp2 display enhanced levels of high-molecular-weight poly-Smt3 conjugates, suggesting that this deconjugating enzyme normally limits the accumulation of poly-Smt3 chains (Bylebyl et al., 2003). The ulp2 mutant exhibits a highly pleiotropic phenotype, including defects in chromosome segregation and recovery from checkpoint-induced cell-cycle arrest, poor growth, and enhanced sensitivity to various stress conditions (Bylebyl et al., 2003; Schwartz et al., 2007). Although some of these defects may in part be caused by an inappropriate accumulation of monomeric conjugates to specific target proteins, evidence for a contribution of poly-Smt3 chains comes from the observation that many aspects of the ulp2 phenotype are suppressed to a large extent by mutating K11, 15, and 19 of Smt3, i.e., by preventing chain formation (Bylebyl et al., 2003). Similarly, depletion of the Ulp2-related mammalian isopeptidase SENP6/SUSP1 causes an accumulation of poly-SUMO-2/3 conjugates in human cells, resulting in
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Physiological Relevance of the Poly-SUMO Signal Detection of polysumoylation in vivo raises the question of whether this type of conjugation fulfills a specific signaling function distinct from that of mono-SUMO. In general, SUMO is believed to act mainly by modulating the protein-protein interactions of its targets. On one hand, attachment of SUMO can potentially mask a binding site for another factor, thereby preventing either a noncovalent interaction or possibly the attachment of another posttranslational modifier. In this manner, SUMO has been proposed to antagonize the effects of ubiquitin on a number of common substrate proteins (Ulrich, 2005). It is difficult to imagine, however, how polysumoylation in this context could have any effect not achieved with equal efficiency by the attachment of a single SUMO molecule. On the other hand, sumoylation can create an additional binding surface allowing the preferential interaction of a downstream effector with the modified target. This mode of function requires the specific recognition of the SUMO moiety by an interaction domain within the effector protein. In yeast as well as higher eukaryotes, a single type of SUMO-interaction motif (SIM) has been identified so far (Kerscher, 2007). It consists of a short hydrophobic core that is often either preceded or followed by a stretch of acidic and/or phosphorylated residues and aligns as a b strand with a matching region on the surface of SUMO. SIM-SUMO interactions are known to mediate the downstream effects of SUMO on many of its targets, such as the recruitment of corepressors by sumoylated transcription factors in higher eukaryotes or of the antirecombinogenic helicase Srs2 by sumoylated PCNA in budding yeast (Kerscher, 2007; Ulrich, 2005). Even intramolecular interactions can be induced by sumoylation, such as the conformational change upon modification of the thymidine DNA glycosylase TDG that is believed to result from the binding of an internal SIM to the covalently attached SUMO moiety (Kerscher, 2007; Ulrich, 2005). In principle, SIM-SUMO interactions could be enhanced by SUMO polymerization, either simply by an increase in the local SUMO concentration that would facilitate rebinding after dissociation, or, given the 1:1 stoichiometry of the interaction, by the presence of multiple SIMs in the effector protein, possibly by means of its oligomerization. It should be noted, however, that the same effect could potentially be achieved by the attachment of multiple units of mono-SUMO to closely spaced acceptor sites on the target. As discussed below, a distinction between the effects of polysumoylation and multiple monosumoylation is not always possible, but in some instances, there is good evidence for a contribution of the SUMO-SUMO linkage to in vivo function. Structural Roles of Poly-SUMO Chains In budding yeast, poly-SUMO chains are not essential for viability, as smt3(3R) mutants, bearing lysine-to-arginine substitutions
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Short Review Figure 2. SUMO-Dependent Ubiquitin Ligases Promote Degradation of Sumoylated Proteins (A) Domain arrangements of SUMO-dependent ubiquitin ligases from human (H. sapiens), budding yeast (S. cerevisiae), and fission yeast (S. pombe). The symbols within the RING domains indicate whether the corresponding domains have ubiquitin ligase activity. (B) A polysumoylated substrate (blue) is recognized in a SIM-dependent manner by a SUMOdependent ubiquitin ligase (showing the S. pombe Slx8-Rfp1 complex as an example). Ubiquitin (black) is conjugated to the outermost SUMO moiety as well as to the substrate protein itself. Following ubiquitylation, the modified substrate is degraded by the 26S proteasome. It is unknown whether the SUMO moieties are degraded along with the substrate or whether deconjugation occurs before proteolysis.
at positions 11, 15 and 19, exhibit no obvious defect during vegetative growth (Bylebyl et al., 2003). The only detectable phenotype is a significantly reduced sporulation efficiency, suggesting a potential role of SUMO polymerization in meiosis (Cheng et al., 2006). This notion is further supported by the observation that Smt3 colocalizes with the synaptonemal complex (SC), a structure that bridges homologous chromosomes along their lengths when they align in preparation for meiotic division. One component of the SC, Zip1, was found to interact with Smt3 and several sumoylated proteins, and a second component, Zip3, contains an SP-RING domain related to the Siz- and PIAS-type SUMO ligases and was shown to promote Smt3 chain formation in vitro. In the smt3(3R) mutant, Zip1 is not properly localized, resulting in an abnormal SC structure that is likely the cause of the sporulation defect. It is unclear whether Zip1 is recruited to the SC primarily by polysumoylated proteins or whether unanchored Smt3 chains are a constituent of the SC. Overall, however, these observations indicate that Smt3 chains play a role in maintaining the structural integrity of the SC, possibly by recruiting the Zip1 protein to its correct location. A structural role for SUMO-2/3 chains in mammalian cells was recently proposed by Zhang et al., who identified the microtubule motor protein CENP-E as a SUMO-interacting protein (Zhang et al., 2008). CENP-E, whose localization to kinetochores is important for the alignment of chromosomes at the metaphase plate during mitosis, was found to bind specifically to SUMO-2/3 chains, but not to mono-SUMO or SUMO-1 polymers in vitro. Its correct localization was found to depend on an internal SIM. Overexpression of the SUMO-specific isopeptidase SENP2 or depletion of Ubc9 caused a delocalization of CENP-E and resulted in a cell-cycle arrest in prometaphase. CENP-E itself is a target for SUMO-2/3 modification, as are several other kinetochore-associated proteins, such as BubR1 and Nuf2; however, it remains to be determined whether these SUMO substrates are relevant for proper CENP-E localization. Although the in vitro binding preferences of CENP-E strongly suggest that SUMO-
SUMO linkages are important for its function, an interaction with closely spaced multiple mono-SUMO conjugates has not rigorously been excluded. Nevertheless, these findings point to a contribution of poly-SUMO-2/3 chains to kinetochore function. Poly-SUMO Chains as a Ubiquitylation Signal A series of publications has recently revealed that poly-SUMO chains can exert a signaling function that stands in stark contrast to SUMO’s perceived role as a ubiquitin antagonist. By means of a newly discovered class of ubiquitin ligases, attachment of polySUMO chains to a substrate was shown to promote its subsequent ubiquitylation and degradation (Lallemand-Breitenbach et al., 2008; Mullen and Brill, 2008; Prudden et al., 2007; Sun et al., 2007; Tatham et al., 2008; Uzunova et al., 2007; Xie et al., 2007). The hallmark of the relevant family of ubiquitin E3s is the presence of multiple SIMs by which they recognize polysumoylated proteins as ubiquitylation targets (Figures 2A and 2B). In mammalian cells, the RING finger E3 RNF4 bears four closely spaced SIMs in its N-terminal region (Figure 2A). All of them contribute to SUMO binding, and although their affinity for SUMO is not isotype-specific, collectively they prefer polymeric chains formed via K11 of SUMO-2 or -3 over monomeric SUMO (Tatham et al., 2008). Polysumoylated PML, a structural component of nuclear bodies, was identified as an in vivo substrate of RNF4-dependent ubiquitylation. Attachment of ubiquitin to both PML and the SUMO moieties was found to target the sumoylated conjugate for degradation by the 26S proteasome. The same was found to apply for an oncogenic fusion protein of PML with retinoic acid receptor, PML-RARa, a causative agent of acute promyelocytic leukemia (APL). Treatment with arsenic trioxide, known to induce PML-RARa degradation and to cure the disease, was shown to promote sumoylation of the fusion protein, which in turn triggered its proteasomal degradation in an RNF4-dependent manner (Lallemand-Breitenbach et al., 2008). A significance of poly-SUMO chains was suggested by the notion that silencing of SUMO-2/3 had a stronger effect on
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Short Review protein levels than silencing of SUMO-1, but the residual effect of SUMO-1 left open the possibilites of either noncanonical SUMO-SUMO linkages or a contribution of multiple monoSUMO adducts. SUMO-dependent ubiquitin ligases in lower eukaryotes appear to function as heterodimers where one subunit, Slx8, harbors RING-dependent ubiquitin ligase activity, while the other one, dimerizing with Slx8 via RING-RING interactions, is primarily responsible for SUMO recognition (Figure 2A). The complexes from budding and fission yeast display very similar biochemical activities, analyzed in vitro with model substrates such as linear SUMO fusions to GST or to S. cerevisiae Rad52, a known SUMO substrate, or the S. pombe protein Rad60, which harbors two SUMO-like domains in its C terminus. However, neither of these proteins appears to be a substrate of SUMO-dependent ubiquitin ligases in vivo, and physiological targets remain to be identified. Inactivation of the SIMs causes an accumulation of unidentified high-molecular-weight SUMO conjugates and results in a general DNA damage sensitivity phenotype that is consistent with a role of Slx8 and its interaction partners in genome maintenance. Like in mammalian cells, the fate of ubiquitylated SUMO conjugates appears to be proteasome-mediated degradation, as treatment with proteasome inhibitors caused an accumulation of mixed ubiquitin-SUMO conjugates in S. cerevisiae (Uzunova et al., 2007). The dependence of these conjugates on the presence of K11, 15, and 19 of Smt3 indicates that the relevant ubiquitylation signal is indeed a poly-SUMO chain. This notion was further confirmed by a detailed biochemical analysis of the Slx8-Slx5 complex from budding yeast with a model substrate (Mullen and Brill, 2008). The use of various Smt3 mutants clearly demonstrated that polysumoylation, as opposed to multiple monosumoylation, effectively triggered ubiquitylation. Interestingly, however, the linkage within the poly-Smt3 chain was not critical, as lysines other than K11, K15, and K19 were able to form chains in vitro that served as productive ubiquitylation signals. In addition to the target protein itself, the N-terminal lysines of the outermost Smt3 moiety served as predominant acceptor sites for ubiquitylation. Intriguingly, the Slx8-Slx5 complex has also been reported to catalyze the formation of poly-Smt3 chains (Ii et al., 2007), but this in vitro activity may simply be a consequence of the SIM domains bringing several Smt3 molecules into close proximity, thus facilitating their ligation by Ubc9. This new family of SUMO-directed ubiquitin ligases illustrates how polysumoylation can function as a ubiquitylation signal and thereby indirectly influence protein levels (Figure 2B). The notion that polysumoylated proteins are degraded in vivo raises the question of whether this process serves as a regulatory device for the selective removal of specific proteins in a controlled manner or whether polysumoylated proteins are toxic by-products of the SUMO conjugation system that the cell needs to eliminate in order to prevent the malfunction of certain biological pathways. While the sporulation defect of the smt3(3R) mutant indicates a positive contribution to cellular metabolism, the phenotypes of cells deficient in the disassembly of poly-SUMO chains argues for a need to avoid an uncontrolled accumulation of poly-SUMO conjugates (Bylebyl et al., 2003; Mukhopadhyay et al., 2006).
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Poorly Defined Roles of Poly-SUMO Chains There are cases of polysumoylation that appear to fit neither of the models presented above, although they have important implications for the cell. For instance, Li et al. were able to show that cleavage of the amyloid precursor protein (APP) by b-secretase, which releases the amyloidogenic Ab peptide in the course of Alzheimer’s disease, is indirectly influenced by sumoylation (Li et al., 2003). APP itself does not appear to be a sumoylation target. However, whereas overexpression of wild-type SUMO-3 promoted a nonamyloidogenic mode of APP processing, thus lowering the levels of secreted Ab peptide, overexpression of the K11R mutant of SUMO-3 had the opposite effect. Hence, K11-linked poly-SUMO chains appear to play a role in preventing the accumulation of the pathological Ab peptide. The target protein(s) relevant for this phenomenon have yet to be identified. A possible role in targeting to the nucleolus was recently proposed for the polysumoylation of budding yeast topoisomerase II (Takahashi and Strunnikov, 2008). Insertions of linear tandem arrays of Smt3 close to the natural sumoylation sites within the enzyme were designed to mimic its modified forms. Whereas insertion of a single Smt3 moiety afforded pericentromeric localization, constructs with multiple inserts were found in the nucleolus. Likewise, highly sumoylated topoisomerase II accumulated in the nucleolus of ulp2 deletion mutants. This SUMO-dependent targeting mechanism appears to be physiologically relevant, as nonsumoylatable mutants of the enzyme or deletion of the SUMO ligase genes SIZ1 and SIZ2 cause signifcant rDNA instability. However, as topoisomerase II is naturally sumoylated at multiple sites, it is quite possible that several closely spaced monomeric SUMO adducts could function equally well as a polymeric chain in targeting the enzyme to its correct location. Finally, it should be noted that poly-SUMO chains may not necessarily have to convey a special signal. In the case of budding yeast PCNA, sumoylation is known to recruit the antirecombinogenic helicase Srs2 (Ulrich, 2005). Although short chains are formed in vivo on two acceptor lysines, prevention of polysumoylation in an smt3(3R) mutant was found to have no effect on the function of sumoylated PCNA at the replication fork (Windecker and Ulrich, 2008). Conclusion The examples discussed above illustrate that polysumoylation can fulfill signaling functions but also promote the assembly of large macromolecular complexes. In either situation, however, the effects of poly-SUMO chains appear to be mediated by the same type of conserved SIM-SUMO interaction. Considering the nature of this interaction and the restricted choice of acceptor lysines within the SUMO sequence in vivo, it is therefore unlikely that the SUMO system is able to functionally differentiate between alternative linkages as observed in the case of polyubiquitin chains. It should be noted that a functional equivalence of canonical and alternative SUMO-SUMO linkages has yet to be proven. Generally, however, the preference of downstream effectors for poly-, as opposed to multiple mono-SUMO adducts, will likely be dictated by the geometry and affinities of the respective SIMs involved in chain recognition, and the diversity of the poly-SUMO signal may turn out to be much more limited than that conveyed by polyubiquitin. In order to gain a better
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Short Review understanding of the mechanisms by which SUMO chains carry out their respective functions, it will be essential to identify the cellular proteins bearing this modification in the relevant physiological context. Despite many open questions, the emerging roles of polymeric SUMO chains once more emphasize the versatility of ubiquitin-like protein modifiers. REFERENCES
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