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Originally published In Press as doi:10.1074/jbc.M411718200 on November 23, 2004

J. Biol. Chem., Vol. 280, Issue 6, 5004-5012, February 11, 2005
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Systematic Identification and Analysis of Mammalian Small Ubiquitin-like Modifier Substrates*{boxs}

Christian B. Gocke{ddagger}, Hongtao Yu§, and Jungseog Kang{ddagger}

From the Department of Pharmacology, the University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, October 14, 2004 , and in revised form, November 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Small ubiquitin-like modifier (SUMO) regulates diverse cellular processes through its reversible, covalent attachment to target proteins. Many SUMO substrates are involved in transcription and chromatin structure. Sumoylation appears to regulate the functions of target proteins by changing their subcellular localization, increasing their stability, and/or mediating their binding to other proteins. Using an in vitro expression cloning approach, we have identified 40 human SUMO1 substrates. The spectrum of human SUMO1 substrates identified in our screen suggests general roles of sumoylation in transcription, chromosome structure, and RNA processing. We have validated the sumoylation of 24 substrates in living cells. Analysis of this panel of SUMO substrates leads to the following observations. 1) Sumoylation is more efficient in vitro than in living cells. Polysumoylation occurs on several substrates in vitro. 2) SUMO isopeptidases have little substrate specificity. 3) The SUMO ligases, PIAS1 and PIASx{beta}, have broader substrate specificities than does PIASy. 4) Although SUMO1 and SUMO2 are equally efficiently conjugated to a given substrate in vitro, SUMO1 conjugation is more efficient in vivo. 5) Most SUMO substrates localize to the nucleus, and sumoylation does not generally affect their subcellular localization. Therefore, sumoylation appears to regulate the functions of its substrates through multiple, context-dependent mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Covalent conjugation of SUMO1 (sumoylation) is an important post-translational modification that regulates protein functions in eukaryotes (1-7). Three isoforms of SUMO, SUMO1, SUMO2, and SUMO3, exist in mammals (2). SUMO1 consists of 101 amino acids and shares about 50% sequence identity with SUMO2/3 and 18% sequence identity with ubiquitin (2).

Similar to the ubiquitin system (8, 9), conjugation of SUMO to substrate proteins is mediated by a cascade of enzymes, including SUMO isopeptidases (SENPs), SUMO-activating enzyme (a heterodimer of Aos1-Uba2), SUMO-conjugating enzyme (Ubc9), and SUMO ligases (1-7). SUMO precursor proteins are processed by a SUMO protease, exposing diglycine motifs at their C termini. In an ATP-dependent reaction, the active site cysteine of Aos1-Uba2 forms a thioester with the C terminus of SUMO. Aos1-Uba2 transfers SUMO to the Ubc9 SUMO-conjugating enzyme again as a thioester. Ubc9 then transfers SUMO to the {epsilon}-amino group of a lysine residue in the substrate, forming an isopeptide bond. Unlike ubiquitination, Ubc9 can catalyze efficient sumoylation of many substrates in the absence of SUMO ligases largely because of the ability of Ubc9 to directly recognize {Psi}KXE ({Psi}, a hydrophobic residue; X, any residue) sumoylation consensus motifs on substrates (10, 11). However, SUMO ligases can increase the rates of sumoylation, especially in vivo (1-7).

Several types of SUMO ligases have been identified, including the PIAS family of proteins (12), RanBP2 (13), and Pc2 (14). Interestingly, these SUMO ligases exhibit distinct patterns of subcellular localization (6). Furthermore, SUMO isopeptidases that function both in the maturation of SUMO precursors and in the removal of SUMO from modified substrates also exhibit distinct, defined patterns of subcellular localization. For instance, SENP1 resides in PML nuclear bodies (15). SENP2 localizes to the nucleoplasmic face of the nuclear envelope (16). SENP3 is enriched in the nucleolus (17). The distinct localization patterns of these SUMO ligases and isopeptidases suggest that sumoylation of a given substrate might be regulated by its localization within the cell.

Numerous SUMO substrates have been identified either individually or through proteomic efforts (1-7). The identities of these substrates implicate sumoylation in diverse cellular processes (1-7). Intriguingly, many transcription factors/cofactors and components of the chromatin remodeling complexes have been shown to be sumoylated (2-4). Sumoylation of transcription factors and cofactors inhibits transcription in some cases and activates transcription in others (2-4). The fact that sumoylation can affect the activity of transcription factors in seemingly opposite ways highlights the complex effects of sumoylation on protein functions. In contrast to ubiquitination, sumoylation of proteins does not generally target them for degradation. Instead, sumoylation appears to regulate the functions of the target proteins through several distinct yet not mutually exclusive mechanisms (1-7). First, sumoylation can affect the subcellular localization and trafficking of target proteins. For example, sumoylation of RanGAP1, a regulator of nucleocytoplasmic transport and the first SUMO-conjugated protein identified, is required for its recruitment to the nuclear pore complex (18, 19). Second, by competing with ubiquitin for the same lysine residues as attachment sites, sumoylation can antagonize ubiquitination and stabilize its target proteins, such as I{kappa}B (20). Third, attachment of the SUMO moiety to a target protein can create an additional surface for protein-protein interactions and enhance the binding between the SUMO target protein and its binding partner (19, 21). Therefore, sumoylation can regulate the function of its substrates in multiple ways.

To gain insights into the general cellular function of sumoylation in mammals, we have used an in vitro expression cloning (IVEC) strategy to identify mammalian substrates of the sumoylation pathway (22). Our approach complements the recent proteomic efforts in identifying SUMO target proteins, which is limited by the low steady-state levels of sumoylated forms of proteins and is further biased by the relative abundance of substrate proteins in cells (23-28). We have identified 40 human SUMO1 substrates in the IVEC screen. Many of these substrates are involved in transcription, RNA processing, maintenance of genome integrity, and chromatin remodeling. We have confirmed the sumoylation of 24 substrates in living cells. Using this panel of substrates, we have investigated the extent of polysumoylation of substrates, the conjugation selectivity of SUMO1 and SUMO2, and the specificities of SUMO isopeptidases and ligases. We have also systematically analyzed the effect of sumoylation on the subcellular localization of target proteins. Our study represents an important first step toward the understanding of the mechanism and function of sumoylation in mammalian cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The coding regions of SUMO1 (1-97), SUMO2, PIASy, SENP1, SENP2, and SENP3 were amplified from human fetal thymus cDNA library (BD Biosciences) by PCR. Full-length cDNA encoding the SUMO substrates identified in the IVEC screen (see below) were amplified either directly from the original clone (if the clone contained the entire open reading frame) or from human brain or fetal thymus cDNA libraries (BD Biosciences). The PCR products were digested and ligated into pCS2 mammalian expression vectors containing N-terminal Myc, HA, or GFP tags. Ubc9 was also cloned into a pCS2 vector containing a C-terminal FLAG tag. The SUMO1 {Delta}GG, SUMO1 KØ mutant, the dominant-negative Ubc9 mutant, SAP130 K785R, SAP130 K869R, and SAP130 K923R were constructed with the QuikChange site-directed mutagenesis kit (Qiagen). The pET11c-hAos1, pET28b-hUba2, and pET28b-hUbc9 vectors were gifts from C. Lima and K. Orth. The pGEX-ScUlp1 vector was obtained from M. Hochstrasser. The pGEX-PIASx{beta} and pGEX-PIAS1 vectors were provided by S. Muller. The Topo II{beta} vector was a gift from L. Liu.

Protein Expression and Purification—DNA fragments encoding the wild-type or KØ mutant of His6-SUMO1 and the wild-type His6-SUMO2 were subcloned into pET28a. These proteins were expressed in BL21(DE3) and purified using nickel-nitrilotriacetic acid beads per the manufacturer's protocols (Qiagen). Ubc9 was expressed and purified similarly. For the expression of Aos1-Uba2, pET11c-hAos1 and pET28b-hUba2 were co-transformed into BL21(DE3). The resulting Aos1-Uba2 complex was purified by nickel-nitrilotriacetic acid beads followed by gel filtration chromatography on a Superdex 200 column (Amersham Biosciences) to remove the excess amount of Aos1. The pGEX-ScUlp1 vector was transformed into BL21. The resulting GST-Ulp1 protein was purified using glutathione-agarose beads (Amersham Biosciences). All proteins were concentrated to between 1-5 mg/ml in a buffer containing 20 mM Tris (pH 7.7), 100 mM KCl, 1 mM dithiothreitol, and 10% glycerol and stored in aliquots at -80 °C.

IVEC and Sumoylation Assays—Five 96-well plates with 100 cDNAs per well of a human adult brain library (Promega) were used in the IVEC screen for SUMO1 substrates per the manufacturer's protocols. Briefly, 1 µl of DNA was in vitro transcribed and translated in reticulocyte lysate in the presence of [35S]methionine and subjected to in vitro sumoylation reactions, which contained 2 µl of in vitro transcribed and translated product, 2 µg of Aos1-Uba2, 0.5 µg of Ubc9, 1 µg of SUMO1, and 1 µl of energy mix (150 mM phosphocreatine, 20 mM ATP, 2 mM EGTA, 20 mM MgCl2, adjusted pH to 7.7). Reactions were adjusted to a final volume of 10 µl with the XB buffer (10 mM HEPES (pH 7.7), 1 mM MgCl2, 0.1 mM CaCl2, 100 mM KCl, and 50 mM sucrose). Control reactions contained water and XB buffer. After 2 h at 30 °C, reactions were stopped with 10 µl of 2x SDS sample buffer, boiled, and subjected to 10% SDS-PAGE followed by autoradiography. Positive pools were transformed into DH5{alpha}. A total of 96 individual clones per positive pool were picked and cultured overnight in LB/AMP medium on 96-well plates. Aliquots of cultures in each row and each column of these 96-well plates were combined separately. Plasmids were isolated from these cultures and tested in the sumoylation assay as described above. Positive clones were identified and sequenced. The SUMO1 conjugation efficiency of each substrate was quantified using the ImageQuant software (Amersham Biosciences). All other in vitro sumoylation reactions were performed similarly.

Cell Culture and Transfections—HeLa Tet-on cells (BD Biosciences) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml penicillin and streptomycin at 37 °C and 5% CO2. Plasmid transfections were performed at 30-60% confluency in 6-well plates using the Effectene transfection reagent according to the manufacturer's protocols (Qiagen). The small interfering RNA oligonucleotides against SENP1 and SENP2 were chemically synthesized at an in-house facility, and their sense strands contained the following sequences: 5'-CAGCUGUCCCACAGUGUAUTT-3' (SENP1) and 5'-GCCCAUGGUAACUUCUGCUTT-3' (SENP2). The annealing and transfection of the small interfering RNAs were performed using the Oligofectamine reagent (Invitrogen) as described (29). Cells were harvested 24-48 h post-transfection. The cell pellets were solubilized by the addition of 200 µl of 1x SDS sample buffer, briefly sonicated, and boiled. For each sample, 15 µl of lysate was separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blotting.

Immunofluorescence—HeLa Tet-on cells transfected with various plasmids were fixed with 4% paraformaldehyde, permeablized with 0.1% Triton-X100 in phosphate-buffered saline, and incubated with 1 µg/ml of anti-Myc (9E10, Roche Applied Science) or anti-FLAG (Sigma). After washing, fluorescent secondary antibodies (Molecular Probes) were added at 1:500 dilutions. The cells were again washed three times with phosphate-buffered saline, counter-stained with 4',6-diamidino-2-phenylindole, and viewed using a x63 objective on a Zeiss Axiovert 200M microscope. Images were acquired using the Intelligent Imaging software and pseudo-colored in Adobe Photoshop.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Human SUMO1 Substrates by IVEC—Since the discovery of SUMO in 1996, on a case by case basis, many proteins with diverse cellular functions have been shown to be modified by SUMO (1-7). In addition, several recent proteomic studies have utilized the affinity purification of tagged SUMO followed by mass spectrometry to identify new in vivo sumoylation substrates in budding yeast and mammalian cells (23-28). However, relatively few mammalian SUMO substrates have been identified in two such efforts, presumably because of the dynamic nature of sumoylation and desumoylation and the low steady-state levels of SUMO conjugates (24, 25, 28). As an alternative, we carried out an IVEC screen to identify SUMO1 substrates from a human brain cDNA plasmid library (22). We screened about 48,000 independent cDNA clones (five 96-well plates with ~100 cDNAs per well). However, because of potential redundancy and poor expression of certain clones in the library, the total number of genes screened is estimated to be ~10,000.

To illustrate the strategy of our screen, the identification of two substrates, STAF65{gamma} and ETV1, is shown in Fig. 1. Briefly, the pools of cDNA plasmids were in vitro translated in rabbit reticulocyte lysate in the presence of [35S]methionine. These 35S-labeled proteins were incubated in the absence or presence of purified recombinant Aos1-Uba2, Ubc9, SUMO1, and ATP. The samples were then resolved on SDS-PAGE followed by autoradiography. In the C5 well of plate B and the E5 well of plate E, we observed additional up-shifted bands in the presence of the sumoylation reaction mixture (Fig. 1, A and B). A secondary screen was carried out to identify the SUMO1 substrates within these two wells as STAF65{gamma} and ETV1, respectively (Fig. 1, C and D). The appearance of these up-shifted bands in both cases required the presence of all necessary sumoylation components, such as SUMO1, Aos1-Uba2, and Ubc9 (Fig. 2A). Addition of Ulp1 (30), a yeast SUMO isopeptidase, to the reaction mixtures greatly reduced the intensities of these up-shifted bands (Fig. 2A). These results confirm that STAF65{gamma} and ETV1 are efficiently sumoylated in vitro.



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  		FIG. 1.
Identification of STAF65{gamma} and ETV1 as SUMO1 substrates by IVEC. A and B, 12 pools of cDNAs from Row C of Plate B (A) or Row E of Plate E (B) from a human brain cDNA library were in vitro transcribed and translated in the presence of [35S]methionine and subjected to a control reaction (-) or SUMO reaction (+) that contained Aos1-Uba2, Ubc9, SUMO1, and ATP. The reaction mixtures were analyzed by SDS-PAGE followed by autoradiography. Putative substrates in pools B-C5 and E-E5 are boxed with dashed lines. The positions of the unsumoylated substrates are indicated by asterisks, whereas the putative SUMO conjugates are marked by brackets. C and D, secondary screen that identifies STAF65{gamma} (C) and ETV1 (D) as the SUMO1 substrates in pools B-C5 and E-E5, respectively. Reactions were performed as in A and B.

 



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  		FIG. 2.
Efficient multi- and polysumoylation of SUMO1 substrates in vitro. A, confirmation of STAF65{gamma} and ETV1 as SUMO1 substrates in vitro. Plasmids encoding STAF65{gamma} and ETV1 were transcribed and translated in rabbit reticulocyte lysate in the presence of [35S]methionine. The 35S-labeled proteins were incubated with SUMO reaction mixtures containing the indicated components and analyzed by SDS-PAGE followed by autoradiography. B, polysumoylation of substrates in vitro. The indicated in vitro translated 35S-labeled substrates were subjected to either a control reaction (-) or a SUMO reaction containing either His6-SUMO1 WT (WT) or the His6-SUMO1 KØ mutant (KØ). The reaction mixtures were analyzed by SDS-PAGE followed by autoradiography. PIAS1 was used as a positive control for polysumoylation.

 
From the screen, we identified 40 human proteins that were sumoylated efficiently in vitro (Table I). Six of the 40 substrates identified in the screen, including heterogeneous nuclear ribonucleoprotein M, topoisomerase II{beta}, PML, SART1, similar to MGC25497 and TFII-I, are known SUMO substrates (24, 25, 28, 31-33). A close homolog of SATB1, SATB2, has also been shown to be sumoylated in vivo (34). This confirmed the validity of our screen.


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  		TABLE I
Identification and characterization of human SUMO1 substrates

 
Multi- and Polysumoylation of Substrates in Vitro—We were amazed by the high efficiency of sumoylation of our substrates in vitro. Defined as the percentage of substrates converted to SUMO1 conjugates, the in vitro sumoylation efficiency ranged from 12-94% (Table I). In addition, many substrates formed SUMO-conjugates that contained multiple SUMO1 molecules and appeared as ladders on SDS-PAGE (Fig. 1 and supplemental Fig. S1). We tested whether these conjugates contained monosumoylation at multiple sites (multisumoylation), a SUMO1 chain at a single lysine (polysumoylation), or a combination of both. We first counted the number of {Psi}KXE motifs in our substrates. Many substrates contained multiple such motifs (Table I). However, there does not appear to be a strict correlation between the number of {Psi}KXE motifs and the efficiency of sumoylation. Furthermore, we randomly sequenced several cDNAs that were not sumoylated in vitro and found that some of them contained {Psi}KXE motifs (data not shown). Therefore, not surprisingly, the mere presence of {Psi}KXE motifs is not sufficient to target proteins for sumoylation.

Next we constructed a SUMO1 mutant (referred to as SUMO1 KØ) with all 11 lysine residues changed to arginines. SUMO1 KØ is not expected to form SUMO1 chains. Earlier studies have shown that SUMO1 can form chains on a fragment of the SUMO ligase RanBP2 (RanBP2{Delta}FG) in vitro (13). We first examined the autosumoylation of the SUMO ligases, PIAS1 (Fig. 2B) and RanBP2{Delta}FG (data not shown). The average molecular mass of SUMO1 KØ conjugates was much lower than that of SUMO1 WT conjugates, indicating that PIAS1 and RanBP2{Delta}FG underwent polysumoylation. Similarly, Ku80, Mi2, FLASH, Topo II{beta}, and heterogeneous nuclear ribonucleoprotein M also appeared to be polysumoylated (Fig. 2B and data not shown). In contrast, there was no significant difference between the gel banding patterns of SUMO1 WT and KØ conjugates of ETV1 (Fig. 2B). This suggested that ETV1 was only multisumoylated consistent with the fact that ETV1 contained 4 {Psi}KXE motifs (Table I). This also served as an important control for the conjugation efficiency of the SUMO1 KØ mutant. On a cautionary note, ubiquitination can occur at the N terminus of certain proteins (35). Though unlikely, we cannot rule out the possibility that SUMO1 KØ can still support SUMO chain formation at its N terminus. Regardless, our data clearly demonstrate that polysumoylation can occur on substrates other than SUMO ligases themselves. The remarkably efficient sumoylation of many substrates in the absence of SUMO ligases also indicates that Ubc9 can catalyze efficient SUMO conjugation in vitro.

Confirmation of Sumoylation of Substrates in Vivo—To determine whether the SUMO1 substrates identified in our in vitro screen were sumoylated in vivo, we cloned the Myc-tagged full-length cDNAs of 26 substrates into mammalian expression vectors and co-transfected HeLa cells with plasmids encoding GFP-SUMO1. Slower migrating species of the substrates were observed when the proteins were co-expressed with GFP-SUMO1 (Fig. 3A). These slower migrating bands were absent when these substrates were co-expressed with GFP-SUMO1 {Delta} GG that lacked the C-terminal diglycine motif and cannot be conjugated to substrates (Fig. 3A). Furthermore, overexpression of either the dominant-negative mutant of Ubc9 (DN Ubc9) or the SENP2 SUMO isopeptidase greatly reduced the intensity of these slower migrating bands (Fig. 3A). Therefore, these substrates are also sumoylated in living cells. Twenty-four of 26 substrates tested in this in vivo assay were shown to be sumoylated (Table I). In addition, there appeared to be a general positive correlation between the in vitro and in vivo sumoylation efficiencies. For example, substrates that were efficiently sumoylated in vitro, such as TFII-I, ETV1, STAF65{gamma}, and ZNF24, were also efficiently sumoylated in vivo (Fig. 3A). This further confirmed the validity of our in vitro screen.



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  		FIG. 3.
In vivo sumoylation of SUMO1 substrates identified by IVEC. A, Myc-tagged SUMO1 substrates identified by IVEC were co-transfected with the indicated plasmids into HeLa cells. Twenty-four to 48 h after transfection, the total cell lysates were blotted with anti-Myc. B, RNAi against either SENP1 or SENP2 enhances sumoylation of TFII-I and SATB1. HeLa cells were transfected with HA-SENP1 or HA-SENP2 plasmids together with small interfering RNAs against SENP1 or SENP2. The total cell lysates were blotted with anti-HA (two left panels). The Myc-TFII-I and Myc-SATB1 plasmids were co-transfected with small interfering RNA against SENP1 or SENP2 into HeLa cells. The total cell lysates were blotted with anti-Myc.

 
We emphasize that with a few exceptions most known SUMO substrates are not sumoylated efficiently in cells. For most substrates, sumoylation cannot be observed in the absence of SUMO overexpression, and less than 5% of a given protein is sumoylated even in the presence of SUMO overexpression. The underlying reason for the low steady-state levels of SUMO conjugates in cells is unclear at present. However, our data are entirely consistent with published reports for other bona fide SUMO1 substrates. In fact, PML is a well established SUMO1 substrate and is not efficiently sumoylated under the same conditions (Fig. 3A). Most of the other substrates identified in our IVEC screen are sumoylated more efficiently than PML and indeed more efficiently than many of the known SUMO substrates in the literature. In addition, several proteomic efforts aimed at identifying SUMO substrates were also performed in the presence of SUMO overexpression or under stress conditions that are known to artificially activate global cellular sumoylation.

Regulation of in Vivo Sumoylation by SUMO Isopeptidases and Ligases—Although the in vivo sumoylation of our substrates was generally more efficient than those reported for other known SUMO substrates, sumoylation of many of our substrates in vivo was nonetheless very inefficient as compared with their in vitro sumoylation. In particular, few substrates were visibly sumoylated in the absence of SUMO1 overexpression. We reasoned that the inefficient sumoylation of our substrates in human cells in the absence of SUMO overexpression might be partially caused by the actions of SUMO isopeptidases. To test this, we depleted HeLa cells of SENP1 or SENP2 with RNA interference (RNAi). RNAi against SENP1 and SENP2 greatly reduced the protein levels of ectopically expressed HA-SENP1 and HA-SENP2, confirming the efficiency of RNAi (Fig. 3B). Interestingly, knockdown of either SENP1 or SENP2 caused a significant increase in the sumoylation of TFII-I and a marginal increase in the sumoylation of SATB1 in the absence of SUMO1 overexpression (Fig. 3B). Therefore, sumoylation is negatively regulated by SUMO isopeptidases in vivo. However, RNAi against SENP1 or SENP2 did not increase the sumoylation of 13 other substrates tested (data not shown). This was not surprising given that there are multiple SENPs in mammals.

Next we tested the substrate specificity of these SUMO isopeptidases. Despite having lower levels of expression, HA-SENP2 was more efficient in reducing sumoylation of ZNF24 in HeLa cells and in removing conjugation of GFP-SUMO1 to other cellular proteins (supplemental Fig. S2A). Similar results were observed for MEF2C and ETV1 (data not shown). In fact, overexpression of HA-SENP2 efficiently reduced sumoylation of every single substrate tested in vivo (Fig. 3A and data not shown). This indicates that, as compared with SENP1 and SENP3, SENP2 is a more efficient SUMO isopeptidase and has little substrate specificity. At present, we do not know whether SENP2 is intrinsically a more efficient enzyme as compared with SENP1 and SENP3 or whether the distinct subcellular localization of these enzymes also contributes to the apparent difference in their efficiency to remove SUMO conjugates in vivo. Nevertheless, our findings indicate that overexpression of SENP2 is a reliable way to reduce the global levels of sumoylation in mammalian cells (supplemental Fig. S2B).

Because we did not include SUMO ligases in our IVEC screen and because the in vivo sumoylation of some of these substrates is relatively inefficient, we tested whether sumoylation of our SUMO1 substrates can be stimulated by PIAS1, PIASx{beta}, and PIASy in vivo. We co-expressed PIASx{beta} with 22 of our substrates in HeLa cells. Sumoylation of 11 substrates were clearly stimulated by PIASx{beta} (Fig. 4A and Table I). PIASx{beta} did not stimulate the sumoylation of all substrates (Table I). In fact, sumoylation of PML was reduced in the presence of ectopically expressed PIASx{beta} (Fig. 4A). We do not know why overexpression of PIASx{beta} inhibited sumoylation of certain substrates. It is possible that autosumoylation of PIASx{beta} or sumoylation of its cellular targets might consume/preoccupy components of sumoylation pathway such as Ubc9 and SUMO. We also tested several substrates with PIAS1 (data not shown). PIAS1 appeared to have similar substrate specificity to PIASx{beta}. Next we tested whether PIASy stimulated sumoylation of our substrates. Among 6 of the 11 PIASx{beta} substrates, only sumoylation of LOC339287 was enhanced by PIASy despite the fact that PIASy was expressed to much higher levels than PIASx{beta} (Fig. 4B). This indicates that PIASx{beta} can stimulate sumoylation of many (but not all) substrates in vivo and that PIAS1 and PIASx{beta} might have broader substrate specificities than PIASy.



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  		FIG. 4.
Stimulation of sumoylation by PIASx{beta} and PIASy in vivo. A and B, Myc-tagged SUMO1 substrates were co-expressed in HeLa cells with the indicated proteins. Twenty-four h after transfection, the total cell lysates were blotted with anti-Myc or anti-HA. PIASx{beta} failed to stimulate the sumoylation of PML, which served as a negative control.

 
Conjugation Selectivity of SUMO1 and SUMO2—SUMO2 and SUMO3 are 96% identical, whereas SUMO1 is about 50% identical to SUMO2/3. Although all three SUMO isoforms are conjugated to substrates by the same enzymes, it has been suggested that SUMO1 and SUMO2/3 might display different substrate specificity (2). Because we performed our IVEC screen with SUMO1, we used our panel of SUMO1 substrates to compare the substrate specificity of SUMO1 and SUMO2 in vitro and in vivo (Fig. 5). All substrates tested were modified equally efficiently by both SUMO1 and SUMO2 in vitro (Fig. 5A). For ZNF24, modification of SUMO2 even appeared to be more efficient (Fig. 5A). Thus, there does not seem to be an inherent difference in substrate specificity between SUMO1 and SUMO2 in the absence of SUMO ligases. Next we compared conjugation of SUMO1 and SUMO2 to two substrates, ZNF24 and Ku80, in HeLa cells. Surprisingly, SUMO1 modification was much stronger for both substrates (Fig. 5B). The protein levels of GFP-SUMO1 and GFP-SUMO2 were similar (Fig. 5B). Global modification of SUMO2 to other cellular proteins was also weaker than SUMO1 conjugation (Fig. 5B). These data suggest that conjugation of SUMO2 to target proteins might be more tightly regulated in vivo.



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  		FIG. 5.
Conjugation selectivity of SUMO1 and SUMO2. A, SUMO1 and SUMO2 are conjugated equally efficiently to a set of SUMO1 substrates identified in our IVEC screen. The indicated substrates were in vitro-translated in the presence of [35S]methionine and incubated with buffer alone (-) or SUMO reaction mixtures containing His6-SUMO1 (1) or His6-SUMO2 (2). The reaction mixtures were analyzed by SDS-PAGE followed by autoradiography. B, Myc-tagged ZNF24 or Ku80 were co-expressed in HeLa cells with vector alone (-), GFP-SUMO1 (1), or GFP-SUMO2 (2). Twenty-four h after transfection, the total cell lysates were blotted with anti-GFP and anti-Myc antibodies.

 
Regulation of Subcellular Localization by Sumoylation— Next we determined the subcellular localization of our SUMO1 substrates. Consistent with their functions, most of our substrates are enriched in the nucleus (Fig. 6A, Table I). Overexpression of GFP-SUMO1 alone did not significantly alter the localization of any of our substrates (Fig. 6A and data not shown). A significant fraction of ETV1, STAF65{gamma}, or SAP130 was sumoylated when co-transfected with GFP-SUMO1. However, there was no noticeable difference in their localization patterns in the presence or absence of GFP-SUMO1 overexpression (Fig. 6A). Therefore, sumoylation does not generally lead to changes in subcellular localization of target proteins.



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  		FIG. 6.
Subcellular localization of SUMO1 substrates. A, HeLa Tet-on cells were transfected with the indicated plasmids, fixed, and stained with anti-Myc and 4',6-diamidino-2-phenylindole (DAPI). The scale bar indicates 10 µm. B, HeLa Tet-on cells were transfected with the indicated plasmids, fixed, and stained with anti-FLAG. GFP is shown in green, whereas anti-FLAG staining is shown in red. C, lysates from HeLa cells transfected with the indicated plasmids were blotted with anti-Myc. The position of the SAP130-SUMO conjugate is indicated by an arrow. D, HeLa Tet-on cells were transfected with the indicated plasmids, fixed, and stained with anti-Myc. GFP is shown in green, whereas anti-Myc staining is shown in red.

 
We noticed that co-expression of Ubc9 and GFP-SUMO1 greatly enhanced the global level of sumoylation (supplemental Fig. S2) and caused accumulation of GFP-SUMO1 in nuclear foci (Fig. 6B). The formation of these nuclear foci was dependent on SUMO-conjugation as these foci were not observed in cells expressing GFP-SUMO1 {Delta}GG or the dominant-negative mutant of Ubc9 (Fig. 6B). The nature of these nuclear foci was currently unknown. However, they did not perfectly co-localize with PML nuclear bodies (data not shown). Therefore, sumoylation of unknown cellular target proteins is likely responsible for the formation of these nuclear foci. We then tested whether any of our substrates were recruited to these nuclear structures. Interestingly, when both Ubc9 and GFP-SUMO1 were overexpressed, SAP130 (Fig. 6D) and to lesser extents TFII-I and Topo II{beta} (data not shown) were enriched in these nuclear foci. To determine whether the recruitment of SAP130 to these foci was also dependent on its own sumoylation, we mutated the lysine residues of three {Psi}KXE motifs of SAP130 to arginines. The K785R mutation abolished sumoylation of SAP130 in vivo, indicating that K785 was the major acceptor site for SUMO1 (Fig. 6C). SAP130 K785R was still recruited to the nuclear foci when co-expressed with Ubc9 and GFP-SUMO1 (Fig. 6D), indicating that sumoylation of SAP130 itself was not required for its recruitment to these nuclear foci.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Consistent with several recent studies (23-28), the majority of SUMO1 substrates identified in our screen are involved in transcription, RNA processing, DNA repair, and chromatin remodeling. Some novel, well studied representatives from these categories are: MEF2C, an important transcription factor involved in muscle differentiation (36); Symplekin, a factor for polyadenylation of pre-mRNA (37); Ku80, a DNA damage repair factor (38); and Mi2, an ATPase of the NuRD chromatin remodeling complex (39).

Intriguingly, Wohlschlegel et al. showed that in budding yeast there is a significant clustering of SUMO substrates in multisubunit macromolecular complexes (26). Though the number of human substrates identified in our screen is too small for rigorous statistical analysis, we also noticed a tendency of our substrates to be subunits of large protein complexes. For example, TFII-I and BHC110 (BRAF-HDAC complex p110) both associate with a novel HDAC1/2-containing complex called the BRAF-HDAC complex (40). STAF65{gamma} and GCN5 are both subunits of a histone acetyltransferase complex called STAGA (41). Therefore, sumoylation might generally regulate the activity, stability, and/or biogenesis of large macromolecular complexes with functions in the nucleus.

It remains an open question how sumoylation affects the functions of its target proteins. Unlike ubiquitination that generally targets proteins for degradation (8), sumoylation does not appear to have one defined, general role. Instead, sumoylation has been shown to increase protein stability through antagonizing ubiquitination, to change the localization and/or the kinetics of trafficking of substrates within the cell, and to mediate protein-protein interactions (1-7). Consistent with these findings, we have not yet identified a prevailing mechanism by which sumoylation regulates the functions of the SUMO substrates identified in our screen. For example, sumoylation does not generally affect the steady-state localizations of target proteins. However, it remains possible that sumoylation affects the kinetics of trafficking of these proteins within the cell. Consistent with this notion, sumoylation regulates the kinetics of nucleocytoplasmic shuttling of Elk1 (42). In addition, we have shown that up-regulation of sumoylation may lead to the formation of certain nuclear structures, which then recruits other proteins into these structures. The recruitment of SAP130 into these unknown nuclear foci is very reminiscent of the recruitment of p53, Daxx, and other proteins to the PML nuclear bodies (43-45). Similar to SAP130, mutations of the sumoylation sites within p53 or Daxx did not affect their recruitment to the PML bodies (43, 44), although sumoylation of PML is essential for their recruitment (45). Future studies are needed to address the nature of the nuclear foci formed by the overexpression of both Ubc9 and GFP-SUMO1. Our results presented herein are consistent with the notion that sumoylation might regulate the functions of its substrates with multiple, context-dependent mechanisms.

In conclusion, we have identified 34 novel human SUMO1 target proteins. The identities of these SUMO1 substrates point to important functions of sumoylation in regulating transcription and chromatin structure. Analysis of this panel of SUMO1 substrates has also yielded valuable information about several properties of sumoylation, including the extent of polysumoylation in vitro, the specificities of SUMO isopeptidases and ligases, the conjugation selectivity of SUMO1 and SUMO2, and the effect of sumoylation on the subcellular localization of its substrates.


   FOOTNOTES
 
* This work is partially supported by National Institutes of Health Grant GM61542, the Packard Foundation, the W. M. Keck Foundation, the March of Dimes Foundation, and the Leukemia and Lymphoma Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

{ddagger} These authors contributed equally to this work. Back

§ The Michael L. Rosenberg Scholar in Biomedical Research. To whom correspondence should be addressed. Tel: 214-648-9697; Fax: 214-648-2971; E-mail: hongtao.yu{at}utsouthwestern.edu.

1 The abbreviations used are: SUMO, small ubiquitin-like modifier; SENP, sentrin-specific protease; Uba2, ubiquitin-activating enzyme 2; Ubc9, ubiquitin-conjugating enzyme 9; Aos1, activation of Smt3p; PIAS, protein inhibitor of activated signal transducers and activators of transcription; PML, promyelocytic leukemia protein; IVEC, in vitro expression cloning; HA, hemagglutinin; GFP, green fluorescent protein; RNAi, RNA interference. Back


   ACKNOWLEDGMENTS
 
We thank Frauke Melchior, Stefan Muller, Mark Hochstrasser, Christopher Lima, Kimberly Orth, David Wotton, Leroy Liu, and Eric Olson for reagents. We also thank Josh Bembenek for preparation of recombinant Aos1/Uba2, SUMO1, GST-Ulp1, and Ubc9 proteins, Ryan Potts for reading the manuscript critically, and Zhanyun Tang for helpful discussions. We also thank an anonymous reviewer for insightful comments that improved the manuscript.



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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