Small Ubiquitin-like Modifier (SUMO) Modification of Natively Unfolded Proteins Tau and α-Synuclein*

Sumoylation is an important post-translational modification that provides a rapid and reversible means for controlling the activity, subcellular localization, and stability of target proteins. We have examined the covalent attachment of the small ubiquitin-like modifier (SUMO) proteins to tau and α-synuclein, two natively unfolded proteins that define several neurodegenerative diseases. Both brain proteins were preferentially modified by SUMO1, as compared with SUMO2 or SUMO3. Tau contains two SUMO consensus sequences, and mutational analyses identified Lys340 as the major sumoylation site. Although both tau and α-synuclein are targets for proteasomal degradation, only tau sumoylation was affected by inhibitors of the proteasome pathway. Tau is a microtubule-associated protein, whose ability to bind and stabilize microtubules is negatively regulated by phosphorylation. Treatment with the phosphatase inhibitor, okadaic acid, or the microtubule depolymerizing drug, colchicine, up-regulated tau sumoylation. This suggests that SUMO modification may preferentially target a free soluble pool of the substrate. These findings revealed a new, possibly regulatory, modification of tau and α-synuclein that may also have implications for their pathogenic roles in neurodegenerative diseases.

Small ubiquitin-like modifier proteins (SUMO) 2 display similarities to ubiquitin in both the structure and the biochemistry of their conjugation (for review, see Ref. 1). SUMO isoforms are expressed in humans and display cell type-specific expression levels and distinct, although not exclusive, subcellular localizations (2). Each SUMO paralog is expressed as a precursor protein that undergoes processing by a C-terminal hydrolase (3). Once cleaved, the mature protein has a diglycine motif exposed at the C terminus and is ready to enter a multistep enzymatic pathway, which is similar but quite distinct from ubiquitination. Mature SUMO proteins are primed in an ATP-dependent manner by the SUMO-activating (E1) enzyme Sua1/hUba2 (4,5). Activated SUMO is then transferred, through a trans-esterification reaction, to a unique conjugating (E2) enzyme, Ubch9 (6,7). The final step is the formation of an isopeptide bond between the C-terminal glycine of SUMO and the lysine ⑀amino group of the target substrate. A majority of the acceptor lysine residues are found within a SUMO consensus motif ⌿KX(E/D), in which ⌿ corresponds to a hydrophobic residue.
Although E1 and E2 are sufficient for SUMO conjugation to various substrates (8,9), it is assumed that SUMO E3 ligases catalyze sumoylation at non-consensus sites, increase the rate of modification, or ensure substrate specificity (10 -13). Sumoylation is a highly dynamic and reversible process as specific proteases can rapidly remove SUMO from their substrates (for review, see Ref. 14). In contrast to ubiquitin, which mainly tags proteins for proteasome-mediated degradation, covalent modification by SUMO can have a number of functional consequences for the target proteins. For example, sumoylation modulates proteinprotein interactions, affects subcellular localization and, in some cases, antagonizes the proteasome pathway by competing with ubiquitin (for review, see Ref. 1). Despite the rapidly growing number of SUMO substrates identified, in most cases, the physiological function and regulation of sumoylation remain elusive and may vary according to the nature of the target. Tau and ␣-synuclein belong to the family of natively unfolded proteins as they display an extended conformation in vitro with little ordered secondary structure (15,16). Both proteins are highly soluble and heat-resistant. They are highly expressed in the brain and are associated with several neurodegenerative disorders including Alzheimer and Parkinson disease (reviewed in Ref. 17). As with other amyloidogenic proteins, tau and ␣-synuclein undergo a pathological transition from random coil to a ␤-pleated sheet conformation that is accompanied by extensive aggregation and fibril formation (18 -20). Post-translational modifications affect both protein structure and function and may also contribute to protein dysfunction.
Tau is a phosphoprotein with up to 30 tightly regulated phosphorylation sites, and hyperphosphorylation is a common feature of paired helical filaments in Alzheimer disease (21). Similarly, ␣-synuclein inclusions in the form of Lewy bodies are a pathological hallmark of Parkinson disease and other ␣-synucleinopathies (22). In addition to phosphorylation (23), ␣-synuclein is also subject to nitration (24). Besides chemical modifications, proteins can also be modified by the conjugation of other polypeptides such as ubiquitin. It has been shown that both tau and ␣-synuclein are degraded by the proteasome in a ubiquitinindependent (25,26) and -dependent manner (27)(28)(29)(30). Alzheimer neurofibrillary tangles are strongly immunoreactive for ubiquitin (31,32), and tau is also a substrate for the CHIP-Hsc70 complex (27,28) as well as the ubiquitin E3 ligase TRAF6 (29). Recently, ubiquitination of ␣-synuclein as well as the sites of the modification within both the soluble and the filamentous forms of the protein have been reported (33).
In the present study, we examined the sumoylation of these two native unfolded proteins, tau and ␣-synuclein, and showed that tau undergoes SUMO modification at a defined consensus motif. Additional evidence suggests that there is a dynamic interplay between tau sumoylation and proteasome inhibition. Functional studies involving phosphorylation and microtubule stability indicated that free soluble tau is targeted for covalent SUMO modification. These findings indicated a novel pathway for tau and ␣-synuclein regulation that may have unique consequences for the cellular regulation of these proteins and possibly their disease-related processes.

MATERIALS AND METHODS
Plasmids-The plasmids encoding N-terminally HA-tagged (peptide YPYDVPDYA) full-length SUMO1, SUMO2, and SUMO3 were generously provided by Dr R. T. Hay (University of St. Andrews, UK). These correspond to the human protein sequences as described previously (34). PCR was used to generate His epitope tag (peptide AHHHHHHV), using the corresponding HA-tagged plasmids as templates. All SUMO constructs were cloned into pcDNA3 vectors and confirmed by DNA sequencing. Human wild-type tau 4R2N cDNA in bacterial expression vector pBluescript II was subcloned into pcDNA3, and ␣-synuclein was cloned into pcDNA6. Single mutants Tau (K340R, K385R), ␣-synuclein (K96R, K102R), and the conjugation-deficient double mutant SUMO1 (G96A,G97A) were generated by site-directed mutagenesis according to the manufacturer's instructions (Stratagene) and confirmed by DNA sequencing.
Cell Culture and Transfection-Human embryonic kidney 293 cells (HEK293) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected in reduced serum medium at ϳ50% confluence using Lipofectamine (Invitrogen), according to the manufacturer's instructions. For each transfection, cells were incubated in the presence of 5 g of plasmid DNA encoding tau or ␣-synuclein, in the presence or absence of 5 g of His-SUMO isoforms as indicated. Where necessary, pcDNA3 empty vector was used to bring the total amount of DNA to 10 g. After 20 h of transfection, medium was replaced, and cells were incubated for an additional 24 h. Cells were treated for the final 16 h with 5 M MG132 (Me 2 SO), 20 nM okadaic acid (ethanol), or 1 M colchicine (H 2 O). Treated cells were harvested and washed in phosphate-buffered saline.
Purification of His-tagged SUMO Conjugates-Transfected and treated cells were lysed in buffer (8 M urea, 100 mM NaH 2 PO 4 , pH 8.0, 10 mM ␤-mercaptoethanol, 1% Triton X-100, 10 mM iodoacetamide, 5 mM imidazole) containing complete protease inhibitor mixture (Roche Applied Science). Lysates were briefly sonicated to reduce viscosity and cleared by centrifugation. The protein content was determined using the Bradford assay. Clarified lysates were mixed with 35 l of Ni 2ϩnitrilotriacetic acid-agarose prewashed with lysis buffer containing 20 mM imidazole and incubated for 2 h at 4°C. The beads were washed by centrifugation once with lysis buffer (pH 8.0) containing 10 mM imidazole and twice with lysis buffer (pH 6.4) containing 10 mM imidazole. His-tagged SUMO conjugates were eluted (lysis buffer, pH 6.4, containing 300 mM imidazole), diluted in Laemmli sample buffer, and analyzed by Western blotting.
Western Blotting-Proteins were separated by electrophoresis on precast 4 -20% polyacrylamide gels (Invitrogen) and transferred onto nitrocellulose (Amersham Biosciences). SUMO1 and SUMO2/3 antibodies were purchased from Zymed Laboratories Inc.. Anti-tau antibody CP27 was generously provided by Dr Peter Davies (Albert Einstein College of Medicine, New York, NY). Anti-␣-synuclein antibody (Syn1, clone 42) was purchased from Pharmingen. Horseradish peroxidaseconjugated anti-mouse and anti-rabbit IgG were used as secondary antibodies (Jackson ImmunoResearch). Immunoreactive bands were visualized by enhanced chemiluminescence using ECL detection kit (Amersham Biosciences), according to the manufacturer's instructions. Western blots presented were representative of 3-5 experiments, which displayed comparable results.

RESULTS
Sumoylation of Tau and ␣-Synuclein-Tau and ␣-synuclein are two natively unfolded proteins frequently found in intracellular filamentous inclusions that define several neurodegenerative diseases. Both proteins are regulated through various post-translational modifications such as phosphorylation (21,23) and ubiquitination (27)(28)(29)33). Sumoylation plays an important role in many cellular processes. Recent reports have also implicated sumoylation in neurodegeneration (35)(36)(37)(38)(39), and many proteins involved in these pathologies were found to be SUMO targets (40 -42). We therefore evaluated whether the two brain proteins, tau and ␣-synuclein, were sumoylated. HEK293 cells were transfected with plasmids expressing tau or ␣-synuclein along with the different Histagged SUMO isoforms. Cells were lysed under denaturing conditions, and total His-tagged SUMO substrates were isolated by nickel affinity chromatography.
In the presence of SUMO1 and, to a lesser extent, SUMO2 and SUMO3, higher molecular weight tau species were observed (Fig. 1A, arrow). Unmodified tau appeared as a single band at ϳ64 kDa. The most intense tau immunoreactive band at ϳ98 kDa is compatible with the conjugation of a single SUMO protein (ϳ20 kDa). An additional higher molecular weight SUMO1-and tau-positive band was also observed ( Fig. 1A, arrowhead). This could correspond to conjugation of more than one SUMO molecule to different tau target lysines (multisumoylation) or the extension of a SUMO chain on a single lysine (polysumoylation). Sumoylation of tau was not detected in cells lacking SUMO expression. In addition, co-transfection of tau and the conjugation-deficient SUMO1 GG-AA mutant abolished the higher molecular weight bands, consistent with the loss of a covalent modification (Fig. 1A).
For ␣-synuclein and, in contrast to tau, a single primary sumoylated species at ϳ36 kDa was observed when it was co-expressed with the His-tagged SUMO isoforms. Human ␣-synuclein was modified primarily by SUMO1, and to a lesser extent, by SUMO2 and SUMO3 (Fig. 1B, arrow). The SUMO/␣-synuclein derivatives were also specific to the presence of SUMO expression as they were absent from cells transfected with empty vector. No polysumoylation or multisumoylation of ␣-synuclein was detected, and no bands were observed with the conjugation-deficient SUMO1 GG-AA mutant, suggesting a specific modification.
Total SUMO conjugates were visualized using SUMO1-and SUMO2/3-specific antibodies (Fig. 1, C and D). The overall sumoylation by these proteins was comparable, which demonstrates that the observed bands were not the result of differences in expression levels or activity of the transfected SUMO proteins. This indicates a specific SUMO1 conjugation to tau and ␣-synuclein. Probing total cell extracts for the conjugation-deficient SUMO1 GG-AA mutant revealed that it existed exclusively as a monomeric species, consistent with the loss of tau and ␣-synuclein modification (Fig. 1C, asterisk). Cumulatively, these results demonstrate that both natively unfolded proteins tau and ␣-synuclein can be preferentially sumoylated by SUMO1.
Mapping SUMO Modification Sites of Tau and ␣-Synuclein-A majority of SUMO-accepting lysines are defined by the consensus motif ⌿KX(E/D), where ⌿ corresponds to a hydrophobic residue, K is the target lysine for covalent conjugation, X is any amino acid, and the final amino acid is a glutamate or aspartate (E/D) residue. Tau (VK340SE, AK385TD) and ␣-synuclein (VK96KD, GK102NE) have two putative SUMO consensus motifs ( Figs. 2A and 3A). Mutagenesis was used to examine sumoylation at these sites, and individual lysine-to-arginine mutants were generated. The tau mutants (K340R and K385R) were expressed at similar levels as the wild-type protein (data not shown). The single mutation K385R had no effect on SUMO1 modification of tau (Fig. 2B). This tau mutant was sumoylated to the same extent as the wild-type substrate, suggesting that the consensus motif containing Lys 385 is not a target for SUMO1 conjugation. In contrast, sumoylation of the tau K340R mutant was significantly reduced, and virtually no SUMO1 conjugates were observed (Fig. 2B). These findings indicate that Lys 340 represents one of the major SUMO1 acceptor sites for both mono-and poly/multisumoylation.
To determine whether sumoylation of ␣-synuclein was also the result of a specific consensus motif, similar mutagenesis of the two potential target lysine residues (Lys 96 and Lys 102 ) was performed. Co-expression of the K102R mutant ␣-synuclein resulted in a slight decrease in the level of sumoylation as compared with wild type (Fig. 3B). This suggested that Lys 102 may be one site of SUMO1 conjugation, but it is unlikely to be a primary target residue. Despite repeated attempts, the other putative SUMO site, Lys 96 , could not be directly investigated due to complications with antibody recognition. Lys 96 lies within the sequence recognized by the Syn1 antibody, and the K96R substitution   disrupted the epitope, which rendered the mutant protein undetectable (data not shown). To resolve this problem, other anti-␣-synuclein antibodies such as the monoclonal Ab211 (Zymed Laboratories Inc.) and a polyclonal antibody raised against human ␣-synuclein (peptide epitope 107 APQEGILEDMPVDPDNEAY 125 ) 3 were examined. However, these were not suitable for our particular investigation since both antisera exhibited nonspecific bands in the molecular weight range expected for sumoylated ␣-synuclein (data not shown). However, the fact that the Syn1 antibody recognizes sumoylated ␣-synuclein strongly suggests that Lys 96 is not a major SUMO target site (Fig. 1). It would be predicted that steric hindrance caused by SUMO1 conjugation at Lys 96 would also result in epitope disruption and failure of antigen-antibody recognition caused by the K96R substitution as described above.
In addition to Lys 96 and Lys 102 , ␣-synuclein contains 13 other lysine residues that are localized mainly within the core repeats. It has also been shown that sumoylation is not exclusively restricted to consensus motifs (13,43,44), and therefore, SUMO1 modification may occur at one or more of these other sites. This is the case for ␣-synuclein ubiquitination, which is confined to lysine residues within the N-terminal half of the protein and does not involve either Lys 96 or Lys 102 (33). These findings suggest that sumoylation of ␣-synuclein may be dispersed within the protein sequence, and further investigations are required to map the precise modification sites. However, our findings indicated that Lys 102 may be one minor sumoylation target that contributes to this process.
Dynamic Interplay between Sumoylation and Proteasome Inhibition-Lysine residues are common targets for several post-translational modifications, including acetylation, methylation, ubiquitination, and sumoylation. Therefore, it is possible that SUMO conjugation to a target lysine may prevent and/or regulate other modifications at the same site. This paradigm has been reported for the substrates NF-B essential modulator (NEMO) (45), proliferating cell nuclear antigen (PCNA) (46), Smad4 (43,47), and Huntingtin (41), which are either sumoylated or ubiquitinated at the same lysine in response to different signaling events. One of the most extensively characterized proteins is IB␣ (48), in which conjugation of SUMO directly antagonizes the ubiquitin-proteasome pathway by competing with ubiquitin for a single target lysine. It has been proposed that this provides a new mechanism to regulate protein stability.
Tau and ␣-synuclein can be degraded by the proteasome through ubiquitin-dependent (27)(28)(29)(30) as well as independent processes (25,26). Given the specific conjugation of SUMO1 to both proteins, it is possible that there is similar competition between the two pathways that could be perturbed by proteasome inhibition. Cells co-expressing tau or ␣-synuclein and His-tagged SUMO1 were treated with MG132 prior to purification of SUMO1 conjugates. Proteasome inhibition significantly increased the levels of monomeric tau (2.7-fold Ϯ 1.2; n ϭ 3), consistent with a reduced catabolism of the free pool of protein (Fig. 4A). Under these conditions in which the conjugation of ubiquitin was up-regulated in response to MG132 treatment (Fig. 4B), a marked decrease in tau sumoylation was observed (Fig. 4C).
Proteasome inhibition also significantly increased ␣-synuclein levels (1.7-fold Ϯ 0.3; n ϭ 3) Fig. 4D). However, SUMO modification of ␣-synuclein remained largely unaffected in response to MG132-mediated proteasome inhibition (Fig. 4E). The exact function of ␣-synuclein is unclear, but it has been suggested that it may be involved in the regulation of cell sensitivity to proteasome inhibition and could have a potential role in cell death pathways (49). In a preliminary investigation, we examined the effect of SUMO1 on ␣-synuclein-mediated sensitivity to the proteasome inhibitor, MG132, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell viability assays. We observed ϳ20% cell death following proteasome inhibition as compared with untreated control cells, similar to that seen in previous investigations (49). However, no significant effect on MG132-mediated cell death was observed for cells co-expressing ␣-synuclein and SUMO1 as compared with ␣-synuclein alone (data not shown). Therefore, the functional significance for SUMO1 conjugation of ␣-synuclein remains to be determined.
Although tau and ␣-synuclein belong to the same family of natively unfolded proteins and are also SUMO substrates, the effect of proteasome inhibition reveals that both proteins are most likely regulated through distinct mechanisms. This is reflected in their different responses in terms of sumoylation despite similar effects induced by changes in the ubiquitin-proteasome system.

Stimulation of Tau Sumoylation by Phosphorylation and Microtubule
Depolymerization-Phosphorylation is known to have significant structural and functional effects on proteins. Several natively unfolded and partially disordered proteins are regulated by this post-translational modification (reviewed in Ref. 50). The resulting conformational changes can alter protein-protein interactions and are a common pathological feature of paired helical filament-tau aggregation and fibrillogenesis. By changing the conformation of tau, phosphorylation might also modulate its ability to interact with SUMO enzymes. To examine the relationship between tau phosphorylation and sumoylation, cells were co-transfected with human tau and His-tagged SUMO1 and treated with 20 nM okadaic acid. Treatment with this phosphatase inhibitor has been shown to increase phosphorylation of tau at Ser 202 as detected by the phospho-specific anti-tau antibody AT8 (29) (data not shown).  showing increased levels of monomeric tau in total cell extracts following proteasome inhibition. B, total extracts in A probed for ubiquitin showed the up-regulation of ubiquitin conjugates in response to proteasome inhibition. C, nickel affinity-purified SUMO1 substrates for tau reveal a decreased level of sumoylation by proteasome inhibitor MG132 as compared with untreated controls. D, total extracts from treated and untreated cells probed for ␣-synuclein using Syn1 antibody indicated an increase in ␣-synuclein levels by the proteasome inhibitor. E, nickel affinity-purified SUMO1 substrates probed for ␣-synuclein (␣-syn) showed no effect of MG132 on level of ␣-synuclein sumoylation.
Total SUMO substrates were isolated using nickel affinity chromatography, and tau sumoylation was analyzed by Western blotting. This indicated that the conjugation of SUMO1 to tau was stimulated in response to okadaic acid treatment (Fig. 5A). It has been well documented that the binding of tau to microtubules is negatively regulated by phosphorylation (51). The okadaic acid-induced sumoylation of phosphorylated tau suggests the possibility that soluble tau may be a preferred target for SUMO1. To examine this further, cells co-expressing tau and His-SUMO1 were treated with 1 M colchicine prior to purification of sumoylated substrates. As illustrated in Fig. 5B, colchicine-induced microtubule depolymerization markedly increased tau sumoylation. This supports the conclusion that once released from the microtubules, the soluble pool of tau is free to interact with SUMO enzymes and undergo covalent modification.

DISCUSSION
Tau and ␣-synuclein belong to a family of natively unfolded proteins that lack typical secondary structure in the absence of a binding partner. Under pathological conditions, these disordered proteins can also undergo conformational changes that lead to protein aggregation and deposition. In the case of tau and ␣-synuclein, their pathological fibrillization results in the formation of intracellular inclusions and neurodegeneration.
Post-translational modifications are important regulatory mechanisms of protein structure and function. Both tau and ␣-synuclein are subject to modifications such as phosphorylation, glycosylation, and ubiquitination. Sumoylation is the covalent attachment of SUMO proteins to target lysines through an enzymatic pathway similar but distinct from ubiquitin. SUMO modification is becoming increasingly recognized as an important post-translational modification affecting proteinprotein interactions, subcellular localization, and protein stability. Although sumoylation has been traditionally considered as a nuclear process, recent proteomic studies revealed that a large fraction of cytosolic proteins is targeted for SUMO modification (52,53). In the current study, we have shown that the two natively unfolded proteins, tau and ␣-synuclein, are sumoylated in vitro. Both cytoplasmic proteins are specifically conjugated to SUMO1 with very little modification seen for the related SUMO2 or SUMO3. In contrast to ␣-synuclein, which is exclusively monosumoylated, the sumoylation profile of tau suggested the conjugation of single and multiple SUMO molecules.
Mutagenesis analyses identified Lys 340 , which lies within a SUMO consensus sequence, as being a major site for SUMO1 conjugation to tau. The presence of a glycine/proline stretch upstream of this target lysine is likely to facilitate conjugation of the ubiquitin-like molecule at this site (54). The lysine-to-arginine K340R mutation prevented the conjugation of both single and multiple SUMO molecules. Although we cannot rule out the possibility that an initial conjugation of SUMO at Lys 340 is required for subsequent sumoylation of other target lysines, the observation indicated that the SUMO-tau derivatives displaying the highest molecular weight shift were modified by a polymeric SUMOs rather than the conjugation of single SUMO proteins to different lysines. Whether this polymeric chain contains other isoforms, in addition to SUMO1, remains to be determined. A previous report suggested the ability of SUMO1 to self-polymerize (11) despite the lack of consensus motifs such as those found within SUMO2 and SUMO3 (34). Alternatively, mixed polymeric SUMO chains can be formed (34,55,56), and the possibility of SUMO1 acting as a capping protein of SUMO2/3 chains has been previously hypothesized (34,54,55) but has yet to be demonstrated experimentally.
Several proteins are substrates for ubiquitin and SUMO conjugation, and in some instances, the modification targets identical lysine residues. As recently reviewed, the cross-talk between both the ubiquitination and the sumoylation pathways appears to be more complex than the simple competition for target lysines (57). For tau, treatment with the proteasome inhibitor MG132 significantly elevated the levels of tau, which is accompanied by increased ubiquitination (27)(28)(29), but decreased sumoylation was observed. It is possible that there is a direct competition between SUMO and ubiquitin for conjugation to the similar target lysines given that tau is also ubiquitinated within the C-terminal microtubule-binding domains, which also contain the Lys 340 sumoylation site (58). Alternatively, the observed decrease in SUMO1 conjugation could be the result of mistrafficking of ubiquitinated tau to other subcellular compartments.
Proteasome-mediated degradation of tau also involves both ubiquitindependent (27)(28)(29) and ubiquitin-independent processes (25). Therefore, one can speculate that sumoylation may prevent tau turnover via both mechanisms, through the inhibition of ubiquitination or prevention of its translocation into the proteasome catalytic chamber. Interestingly, sumoylation has recently been implicated in the pathogenesis of several neurodegenerative diseases that may, for example, be linked to proteasome dysfunction (35)(36)(37)(38)(39). Recent reports have revealed that a growing number of proteins involved in neurodegeneration, such as ataxin-1 (40), Huntingtin (41), DJ-1 (42), and tau and ␣-synuclein (current work), are sumoylated, which represents an as yet unexplored pathway in terms of their function, cellular regulation, and potential involvement in the disease process.
Proteasome impairment is a common feature in aging (59) and several neurodegenerative disorders (60), and neuronal inclusions are frequently immunopositive for ubiquitin. Down-regulation of tau sumoylation in response to proteasome failure may explain why neurofibrillary tangles in Alzheimer disease do not cross-react with anti-SUMO antibodies (38). However, we cannot rule out the possibility that the experimental conditions need to be optimized for immunohistochemistry. This has been illustrated, for example, by the fact that initial studies reported that inclusions in ␣-synucleinopathies were SUMO-negative (38). However, more recent investigations have demonstrated co-localization of ␣-synuclein deposits with SUMO1 (39). Our observation of specific ␣-synuclein sumoylation further supports the observation of SUMO immunoreactivity in the characteristic inclusions found in Parkinson disease and multiple system atrophy (39).
Functional studies suggested that SUMO was preferentially conjugated to a pool of free soluble tau. Treatment with the phosphatase inhibitor, okadaic acid, increases tau phosphorylation and was found to stimulate tau sumoylation. A similar phenomenon has been reported for the heat-shock factor HSF1 (61). Hyperphosphorylation is a hallmark of neurofibrillary tangles in Alzheimer disease, is thought to alter tau conformation and to prevent tau association with microtubules. FIGURE 5. Sumoylation of tau is induced by phosphorylation and microtubule depolymerization. HEK293 cells were co-transfected with plasmids expressing wild-type tau and His-SUMO1 and treated with 20 nM okadaic acid, a phosphatase inhibitor (A) or 1 M colchicine, a microtubule-destabilizing drug (B), for 16 h. His-tagged SUMO1 substrates were purified, and sumoylated tau was detected by Western blotting. Significant increases in the degree of SUMO1 conjugation were observed upon treatment with both compounds.
Similarly, colchicine promotes microtubule breakdown, releases tau and other associated proteins, and was found to up-regulate tau sumoylation. Cumulatively, these results suggest that microtubule-unbound tau is a preferred target for SUMO conjugation. This is also consistent with the fact that the major target, Lys 340 , is located within the fourth microtubule-binding repeat.
This domain is thought to adopt a ␤-strand structure upon binding to microtubules and would be inaccessible for SUMO modification when bound to tubulin (reviewed in Ref. 62). The release of tau from the surface of the microtubule would expose this domain and permit access to sumoylation enzymes. It has also been suggested for soluble tau that the microtubule-binding repeats are subject to intramolecular interactions with either the N-terminal projection domain or the C-terminal region (reviewed in Ref. 63), potentially preventing SUMO conjugation. Such an inhibitory fold can be relieved, or at least partially neutralized by phosphorylation (reviewed in Ref. 64), potentially explaining the okadaic acid-mediated increase in tau sumoylation.
In the present study, we report the preferential conjugation of SUMO1 to two natively unfolded proteins, tau and ␣-synuclein. This novel modification pathway was dependent upon changes in proteasome-mediated protein turnover as well as other functional modifications such as phosphorylation. Given that both tau and ␣-synuclein define a number of neurodegenerative diseases and the recent implication of SUMO conjugation in these processes, the contribution of sumoylation in the pathogenesis of tauopathies and ␣-synucleinopathies warrants further investigations.