Mechanisms, regulation and consequences of protein SUMOylation

THE SUMO (SMALL UBIQUITIN-LIKE MODIFIER) FAMILY OF PROTEINS

SUMO proteins are essential for the normal function of all eukaryotic cells. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe each contain one SUMO protein, Smt3 and Pmt3 respectively. Deletion of the Pmt3 gene results in severe growth impairment [1] and deletion of Smt3 causes a loss of cell viability [2,3].

In mammals there are four SUMO paralogues, designated SUMO-1 to SUMO-4. SUMO-1 (also known as UBL1 [4], PIC1 [5], sentrin [6], GMP1 [7] and Smt3c [8]) is an 11 kDa protein that was independently isolated by several groups as a binding partner of the RAD51/52 nucleoprotein filament proteins, which mediate DNA strand exchange, and PML (promyelocytic leukaemia), a component of multiprotein nuclear complexes [4–6]. The first reports that SUMO-1 functions as a covalent protein modifier described SUMOylation of the nuclear pore protein RanGAP1 (Ran-GTPase-activating protein 1) [7,9].

SUMO-2 (also known as sentrin 2 [10], Smt3b [8] and GMP-related protein [7]) and SUMO-3 (also known as sentrin 3 [10] and Smt3a [8]) were initially predicted from database searches [8] and the proteins were subsequently isolated and shown to conjugate substrate proteins [10,11]. SUMO-2 and -3 differ from each other by only three N-terminal residues and are often referred to collectively as SUMO-2/3. SUMO-2/3, however, share only approximately 50 % similarity with SUMO-1 (Figure 1). Interestingly, SUMO-2/3 are capable of forming chains on substrate proteins through internal lysine residues [12]. Although SUMO-1 chains have been reported in vitro [13], in vivo it appears that SUMO-1 may act as a chain terminator on SUMO-2/3 polymers [14].

A SUMO-4 isoform has also been proposed from DNA sequence analysis, which predicts a 95-residue protein with 87 % amino acid similarity to SUMO-2. Conjugation of an exogenously expressed mature form of SUMO-4 has been reported in conditions of extreme cellular stress [15]. However, in contrast with the other SUMO genes the SUMO-4 gene lacks introns, raising the possibility that it may be a pseudogene [16]. In addition, despite the presence of SUMO-4 mRNA in kidney, lymph node and spleen [16,17], endogenous SUMO-4 protein has not been detected. Finally, SUMO-4 has a proline residue in a critical position, which makes it unclear, even if it is endogenously expressed, whether the precursor SUMO-4 protein could undergo maturation to conjugate to target proteins [18].

THE SUMO CYCLE

KNOCKOUT STUDIES ON SUMOylation

The critical importance SUMOylation in mammals has been confirmed through knockout and knockdown studies of Ubc9. As Ubc9 is required for the conjugation of every SUMO paralogue, deletion of Ubc9 prevents all SUMO conjugation. Removal of Ubc9 in the chicken DT40 lymphocyte cell line results in abnormalities in chromosome segregation, nuclear organization and ultimately cell death by apoptosis [72]. Similarly, Ubc9-knockout mice die at an early embryonic stage due to defects in chromosomal segregation at mitosis and aberrant nuclear organization [73].

Intriguingly, Alkuraya et al. [74] identified a genomic insertion disrupting the SUMO-1 gene in a Caucasian female with cleft lip and palate. This gene insertion decreased SUMO-1 expression at both the mRNA and protein levels. In an attempt to confirm that SUMO-1 haplo-insufficiency leads to cleft lip and palate, they generated mice hetero- or homo-zygous for a β-galactosidase insertion in the SUMO-1 gene. Four of 46 heterozygote pups exhibited cleft palate and other animals died in late embryonic and early postnatal periods, indicating that SUMO-1 is also required for other developmental functions in addition to palatogenesis [74]. However, two more recent studies have reported that SUMO-1-knockout mice are viable and display an apparently normal phenotype [75,76]. These results suggest that SUMO-2/3 may act to compensate for the loss of SUMO-1. Furthermore, neither of these later studies detected a link between SUMO-1 deletion and cleft lip or palate. In support of a model in which SUMO-2/3 can compensate for a lack of SUMO-1, RanGAP1, which is preferentially modified by SUMO-1, displayed enhanced modification by SUMO-2/3 in SUMO-1-knockout embryonic extracts [76]. Thus, taken together, these results seem to indicate that, at least under extreme circumstances, SUMO-2/3 can effectively compensate for the lack of SUMO-1.

FUNCTIONAL HETEROGENEITY WITHIN THE SUMO FAMILY

Although some substrates are preferentially modified by one SUMO isoform (for example RanGAP1 and the PML-nuclear body component Sp100 are modified by SUMO-1 or SUMO-2/3 respectively [77,78]), many substrates can be modified by both SUMO-1 and SUMO-2/3 [78]. It is unclear how the SUMOylation machinery distinguishes between SUMO-1 and SUMO-2/3 for specific conjugation to protein targets. Furthermore, for most substrates that can be modified by either isoform the functional differences of SUMO-1 compared with SUMO-2/3 conjugation have yet to be defined. Nonetheless, observations that SUMO-1 and SUMO-2/3 differ in their conjugation dynamics and show discrete patterns of localization throughout the cell cycle indicate specific regulation of the SUMO paralogues [77,79]. For example, resting levels of unconjugated SUMO-1 and SUMO-2/3 differ. COS-7 cells contain a large free pool of SUMO-2/3, but there is very little free SUMO-1, suggesting SUMO-2/3 may act as a cellular reserve of SUMO [77]. Indeed, various cellular stresses invoke a massive increase in SUMO-2/3 conjugation in many cell types (for reviews see [80–85]).

The discovery of SUMO-targeted ubiquitin ligases also illustrates differences in SUMO-1 and SUMO-2/3 function and highlights the interplay between the SUMO and ubiquitin systems (for recent reviews see [86,87] and the SUMO and ubiquitin section). Ubiquitin, like SUMO, post-translationally modifies proteins at lysine residues and has been most well-characterized for its role in targeting substrate proteins for proteasomal degradation (for reviews see [88–90]). Recently, a number of ubiquitin ligases, responsible for ubiquitin conjugation to substrate proteins, have been reported that bind SUMO chains on substrate proteins, leading to the ubiquitination of the substrate protein and, ultimately, its degradation. For example, the protein PML can be modified by both SUMO-1 and SUMO-2/3. SUMO-1 modification of PML is required for of its localization to nuclear domains known as PML nuclear bodies [91]. Formation of SUMO-2/3 chains, however, leads to the degradation of PML via the recruitment of the SUMO-binding ubiquitin ligase RNF4 (ring finger protein 4), which mediates the ubiquitination of the SUMO chains, targeting the whole complex for degradation [92–94]. Thus attachment of SUMO chains to a substrate protein can act as a distinct signal from mono-SUMOylation through recruitment of proteins that specifically recognize polymeric SUMO chains.

NON-COVALENT SUMO BINDING

As mentioned briefly above, in addition to covalent SUMOylation of specific lysine residues on substrate proteins, an increasing number of proteins have been shown to bind SUMO or SUMO chains non-covalently via SIMs (SUMO-interacting motifs) (for a review see [95]). This is analogous to the ubiquitin system where the covalently coupled ubiquitin on substrate proteins can recruit ubiquitin-binding proteins that bind non-covalently. At least 20 different non-covalent ubiquitin interaction motifs have been identified [96]. Similarly, protein SUMOylation provides an interaction platform for the recruitment of SIM-containing effector proteins. The SIMs identified so far generally comprise a hydrophobic core surrounded by acidic flanking residues or phosphorylatable serine residues [97–100]. In addition to SIMs mediating the effects of SUMOylation, a growing number of proteins have been identified for which SUMOylation is dependent on the presence of the SIM in the substrate. This suggests that recruitment of SUMO-loaded Ubc9 may represent a general mechanism of substrate recognition and, possibly, paralogue specificity, through paralogue-specific binding to the substrate protein.

As discussed above, SUMOylation by SUMO-1 or SUMO-2/3 can lead to different functional consequences. SUMO paralogue-specific interacting proteins are likely to account for at least some of these differences. For example, CoREST1 (co-repressor for RE1-silencing transcription factor) is a nuclear protein that forms part of a protein complex that creates a transcriptionally repressive environment required for the silencing of neuronal-specific genes in non-neuronal cells [101–104]. CoREST contains a SIM that binds to promoter proteins which have been SUMOylated by SUMO-2, but not SUMO-1, and this interaction mediates the recruitment of the CoREST1 complex to target genes [105]. The SUMO-2-specific SIM in CoREST is similar to but distinct from other reported SIMs, containing a hydrophobic core (Ile-Asp-Ile-Glu-Val), with an N-terminal acidic stretch. Building on this discovery of a SUMO-2-specific SIM, database searches for proteins harbouring similar motifs have identified several other nuclear proteins that bind SUMO-2, but not SUMO-1 in vitro [105].

MOLECULAR CONSEQUENCES OF SUMOylation

Although the functional consequences of SUMO conjugation to a particular substrate protein are difficult to predict overall, protein modification by SUMO may lead to one of three non-mutually exclusive effects ([106]; Figure 3). First, SUMOylation may mask the binding site of a protein that interacts with the substrate protein, essentially acting to occlude the interaction in a SUMOylation-dependent manner. An example of this is SUMOylation of the ubiquitin-conjugating enzyme E2-25k, which inhibits its interaction with the ubiquitin E1 enzyme [107] leading to a decrease in its capability to conjugate ubiquitin to substrate proteins (see the SUMO and ubiquitin section). Secondly, and conversely, the covalently attached SUMO may act as an interaction ‘hub’ that recruits new interacting proteins to the substrate either by direct non-covalent interaction with the SUMO moiety, or via a novel interaction domain created at the SUMO-substrate interface. For example, SUMOylation of RanGAP1 promotes its interaction with RanBP2 and its relocation from the cytosol to the nuclear pore [7,9], poly-SUMOylation of PML can recruit the ubiquitin ligase RNF4 [92–94] and, as discussed below, SUMOylation of the transcription factor Elk1 [ETS (E twenty-six)-like kinase 1] can recruit the histone deacetylase HDAC2 [108] (see the SUMO and acetylation section). Thirdly, SUMOylation can lead to a conformational change in the SUMOylated substrate, directly regulating its function. An example of this is TDG (thymine–DNA glycosylase), which is discussed in more detail below.

THE SUMO ENIGMA

An initially counter-intuitive observation regarding SUMOylation is that only small proportion of the available substrate protein need be SUMOylated to achieve maximal effect. This phenomenon has been referred to as the ‘SUMO enigma’ [109]. Although the underlying mechanisms for this effect on many SUMO-modified proteins have not been determined, elegant explanations based on the highly dynamic nature of the SUMOylation cycle have been proposed to account for this effect in some specific cases. For example, in neurons, the SUMO modification of GluR6 (glutamate receptor subunit 6) leads to its endocytosis at the plasma membrane [110]. Although only a small proportion of SUMOylated GluR6 can be detected at a given time point, a large proportion of GluR6 undergoes SUMO-mediated endocytosis. The low level of detectable SUMOylated GluR6 is probably due to the action of SENPs rapidly removing SUMO. Nonetheless, the functional effects of GluR6 SUMOylation persist after deSUMOylation, namely GluR6 is endocytosed from the neuronal surface. Thus once SUMOylation has mediated endocytosis, SUMO can be removed and any GluR6 SUMOylated previously will have a different cellular localization to GluR6 that has never been modified.

Similar explanations have been used to account for the potent repression of transcription by protein SUMOylation. Multiple transcription factors can undergo SUMOylation and in many cases this leads to repression of transcription (for reviews see [111,112]). For some transcription factors, such as Sp3 (stimulating protein 3) or LEF-1 (lymphoid enhancer-binding factor 1), SUMOylation causes their relocation to nuclear subdomains not associated with transcriptional activity [35,113]. Thus, as with GluR6, the SUMOylation-mediated effects persist after the removal of SUMO.

SUMO modification of promoter-occupied transcription factors has also been reported to recruit chromatin-modifying proteins, such as the HDACs, or repressor proteins, such as DAXX (death-domain-associated protein) through non-covalent interactions [108,114,115]. SUMO-mediated HDAC recruitment leads to local histone deacetylation, resulting in a more condensed chromatin structure that favours transcriptional repression. Thus recruitment of HDACs to a SUMOylated transcription factor will induce a repressive transcriptional environment that persists after removal of SUMO from the substrate. Similarly, recruitment of repressor proteins, such as DAXX, may lead to the formation of ‘repressor complexes’. Again, SUMO-mediated sequestration of transcription factors into repressive complexes would persist after removal of the SUMO.

The DNA-modifying enzyme TDG provides another example of how low-level substrate SUMOylation can result in robust effects. TDG recognizes and excises mismatched base pairs and SUMOylation is required for every enzymatic cycle [116]. When TDG removes the mismatched base, it binds strongly to the DNA as a result of its high affinity for the abasic site created by the base excision [117]. This high affinity interaction causes a break in the reaction cycle that is released by TDG SUMOylation, which decreases its affinity for DNA and releases it into the nucleoplasm [116]. Subsequent removal of SUMO by SENPs allows TDG to undergo a further round of activity. Thus, although only a very small proportion of TDG is modified at any one time due to the rapid deconjugation of SUMO in the nucleoplasm, the effects of SUMOylation are critical to every reaction cycle.

Taken together these observations suggest that although only a small proportion of a substrate may be modified at any given time point, the majority of a substrate will be SUMOylated over time and undergo a SUMO-induced functional change that remains after the protein has been deSUMOylated.

REGULATION OF SUMOYLATION

The list of SUMO substrates is increasing rapidly but how SUMOylation of the vast majority of these proteins is regulated remains unclear. However, a number of common themes are beginning to emerge. Although numerous stimuli have been reported that lead to global changes in cellular SUMOylation, it is likely that SUMOylation is controlled in a substrate-specific manner. It seems that SUMOylation of many substrates is regulated through a complex interplay between SUMOylation and other post-translational modifications of the substrate protein. Furthermore, in addition to co-regulating substrate proteins, there is also direct reciprocal interplay between SUMOylation and other post-translational modifications through modification of the proteins involved in their enzymatic pathways (Figure 4). A number of illustrative examples of this complex cross-regulation are discussed below.

RELATIONSHIP BETWEEN SUMOylation AND OTHER POST-TRANSLATIONAL MODIFICATIONS

SUMO-REGULATION OF Ubc9

Component proteins of the SUMOylation pathway themselves are also subject to regulation by SUMOylation. The sole SUMOylation E2 enzyme Ubc9 was identified as a SUMO substrate. SUMOylation of yeast Ubc9 was initially observed in in vitro SUMOylation assays at high Ubc9 concentrations, however it was unclear if this in vitro modification was physiologically relevant [156]. Subsequent proteomic studies identified SUMO modification of Ubc9 in both yeast and mammalian cells [157–159]. In vitro SUMOylation assays followed by MS were used to identify Lys¹⁴ of Ubc9 as the SUMOylation site [160]. SUMOylation occurs at a different lysine residue (Lys¹⁵³) on yeast Ubc9 indicating that SUMOylation of Ubc9 may perform distinct functions in yeast and mammals [160]. SUMOylation of Ubc9 does not appear to influence Ubc9–SUMO-1 thioester formation but rather it alters the capability of Ubc9 to modify particular SUMO substrates. For example, SUMOylation of RanGAP1 by SUMOylated Ubc9 was dramatically reduced, whereas SUMO modification of the nuclear antigen Sp100 is enhanced when Ubc9 is SUMOylated. This is consistent with the idea that SUMOylation of Ubc9 influences its target specificity towards a particular subset of substrates. Interestingly, Sp100 contains a SIM, which is required for the enhanced SUMOylation of Sp100 by SUMOylated Ubc9, suggesting that SIM-mediated recruitment of SUMOylated Ubc9 to substrates may represent a novel mechanism of substrate selection [160].

In addition to direct SUMO modification of Ubc9, other proteins in the SUMO pathway, including the deSUMOylating enzyme SENP1 and, analogous to the well-documented automodification of ubiquitin E3s, the SUMOylation E3 enzymes RanBP2, Pc2, TLS, TOPORS and the PIAS proteins are also themselves substrates of SUMOylation [32,42,49,51,69,161]. SUMOylation of RanBP2 appears to be involved in SUMO paralogue specificity (see below) but the functions of SUMOylation of SENP1 and the other E3 enzymes are currently unclear.

MECHANISMS OF SUMO PARALOGUE SPECIFICITY

Although many substrate proteins can be modified by either SUMO-1 or SUMO-2/3 it is clear that many others are SUMOylated in a paralogue-specific manner [78]. As both SUMO-1 and SUMO-2/3 use identical core conjugation machinery, a major challenge is to determine how this paralogue specificity is achieved and there has been recent progress towards understanding aspects of this for some substrates (Figure 5).

CONCLUDING REMARKS AND FUTURE DIRECTIONS

It is clear that protein SUMOylation is a fundamentally important signalling pathway in all eukaryotic cells. There have been rapid advances in our understanding of the mechanisms and consequences of SUMOylation and recent work has begun to shed light on the complex regulation and the mechanisms of paralogue specificity. Future work will continue to delineate and refine the inter-relationships between SUMO and other post-translational modifications that act in combination to control all aspects of cell differentiation, development and function.