Evidence for Covalent Modification of the Nuclear Dot–associated Proteins PML and Sp100 by PIC1/SUMO-1

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1–specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.     

SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.     

SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1-conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis.     

SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.     

SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the PsiKXE motif, where Psi represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IkappaBalpha, containing the PsiKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation.     

A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast

Yeast SUMO (Smt3) and its mammalian ortholog SUMO-1 are ubiquitin-like proteins that can reversibly be conjugated to other proteins. Among the substrates for SUMO modification in vertebrates are RanGAP1 and RanBP2/Nup358, two proteins previously implicated in nucleocytoplasmic transport. Sumoylated RanGAP1 binds to the nuclear pore complex via RanBP2/Nup358, a giant nucleoporin, which was recently reported to act as a SUMO E3 ligase on some nuclear substrates. However, no direct evidence for a role of the SUMO system in nuclear transport has been obtained so far. By the use of conditional yeast mutants, we examined nuclear protein import in vivo. We show here that cNLS-dependent protein import is impaired in mutants with defective Ulp1 and Uba2, two enzymes involved in the SUMO conjugation reaction. In contrast, other transport pathways such as rgNLS-mediated protein import and mRNA export are not affected. Furthermore, we find that the yeast importin-alpha subunit Srp1 accumulates in the nucleus of ulp1 and uba2 strains but not the importin-beta subunit Kap95, indicating that a lack of Srp1 export might impair cNLS import. In summary, our results provide evidence that SUMO modification in yeast, as has been suspected for vertebrates, plays an important role in nucleocytoplasmic trafficking.     

The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo

RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.     

Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein

SUMO-1 is a member of a family of ubiquitin-like molecules that are post-translationally conjugated to various cellular proteins in a process that is mechanistically similar to ubiquitylation. To identify molecules that bind noncovalently to SUMO-1, we performed yeast two-hybrid screening with a SUMO-1 mutant that cannot be conjugated to target proteins as the bait. This screening resulted in the isolation of cDNAs encoding the b isoform of thymine DNA glycosylase (TDGb). A deletion mutant of TDGb (TDGb(Delta11)) that lacks a region shown to be required for noncovalent binding of SUMO-1 was also found not to be susceptible to SUMO-1 conjugation at an adjacent lysine residue, suggesting that such binding is required for covalent modification. In contrast, another mutant of TDGb (TDGb(KR)) in which the lysine residue targeted for SUMO-1 conjugation is replaced with arginine retained the ability to bind SUMO-1 non-covalently. TDGb was shown to interact with the promyelocytic leukemia protein (PML) in vitro as well as to colocalize with this protein to nuclear bodies in transfected cells. TDGb(KR) also colocalized with PML, whereas TDGb(Delta11) did not, indicating that the noncovalent SUMO-1 binding activity of TDGb is required for colocalization with PML. Furthermore, SUMO-1 modification of TDGb and PML enhanced the interaction between the two proteins. These results suggest that SUMO-1 functions to tether proteins to PML-containing nuclear bodies through post-translational modification and noncovalent protein-protein interaction.     

Structural and dynamic independence of isopeptide-linked RanGAP1 and SUMO-1

Although sumoylation regulates a diverse and growing number of recognized biological processes, the molecular mechanisms by which the covalent attachment of the ubiquitin-like protein SUMO can alter the properties of a target protein remain to be established. To address this question, we have used NMR spectroscopy to characterize the complex of mature SUMO-1 with the C-terminal domain of human RanGAP1. Based on amide chemical shift and 15N relaxation measurements, we show that the C terminus of SUMO-1 and the loop containing the consensus sumoylation site in RanGAP1 are both conformationally flexible. Furthermore, the overall structure and backbone dynamics of each protein remain unchanged upon the covalent linkage of Lys524 in RanGAP1 to the C-terminal Gly97 of SUMO-1. Therefore, SUMO-1 and RanGAP1 behave as "beads-on-a-string," connected by a flexible isopeptide tether. Accordingly, the sumoylation-dependent interaction of RanGAP1 with the nucleoporin RanBP2 may arise through the bipartite recognition of both RanGAP1 and SUMO-1 rather than through a new binding surface induced in either individual protein upon their covalent linkage. We hypothesize that this conformational flexibility may be a general feature contributing to the recognition of ubiquitin-like modified proteins by their downstream effector machineries.     

Enzymes of the SUMO modification pathway localize to filaments of the nuclear pore complex

SUMOs are small ubiquitin-related polypeptides that are reversibly conjugated to many nuclear proteins. Although the number of identified substrates has grown rapidly, relatively little is still understood about when, where, and why most proteins are modified by SUMO. Here, we demonstrate that enzymes involved in the SUMO modification and demodification of proteins are components of the nuclear pore complex (NPC). We show that SENP2, a SUMO protease that is able to demodify both SUMO-1 and SUMO-2 or SUMO-3 protein conjugates, localizes to the nucleoplasmic face of the NPC. The unique amino-terminal domain of SENP2 interacts with the FG repeat domain of Nup153, indicating that SENP2 associates with the nucleoplasmic basket of the NPC. We also investigated the localization of the SUMO conjugating enzyme, Ubc9. Using immunogold labeling of isolated nuclear envelopes, we found that Ubc9 localizes to both the cytoplasmic and the nucleoplasmic filaments of the NPC. In vitro binding studies revealed that Ubc9 and SUMO-1-modified RanGAP1 bind synergistically to form a trimeric complex with a component of the cytoplasmic filaments of the NPC, Nup358. Our results indicate that both SUMO modification and demodification of proteins may occur at the NPC and suggest a connection between the SUMO modification pathway and nucleocytoplasmic transport.     

Generation of SUMO-1 modified proteins in E. coli: towards understanding the biochemistry/structural biology of the SUMO-1 pathway

Here, we developed a binary vector system that introduces a synthetic SUMO-1 conjugation pathway into Escherichia coli and demonstrated that large amounts of sumoylated Ran GTPase activating protein 1 C-terminal region (RanGAP1-C2), Ran binding protein 2 internal repeat domain, p53 and promyelocytic leukemia were efficiently produced. The sumoylated recombinant RanGAP1-C2 appeared to retain the in vivo properties, since it was specifically sumoylated at lysine 517 as expected from in vivo studies. Our findings indicate the establishment of a biosynthetic route for producing large amounts of sumoylated recombinant proteins that will open up new avenues for studying the biochemical and structural aspects of the SUMO-1 modification pathway.     

Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1

The growth-suppressive properties of p53 are controlled by posttranslational modifications and by regulation of its turnover rate. Here we show that p53 can be modified in vitro and in vivo by conjugation to the small ubiquitin-like protein SUMO-1. A lysine residue at amino acid position 386 of p53 is required for this previously undescribed modification, strongly suggesting that this lysine residue serves as the major attachment site for SUMO-1. Unlike ubiquitin, attachment of SUMO-1 does not appear to target proteins for rapid degradation but rather, has been proposed to change the ability of the modified protein to interact with other cellular proteins. Accordingly, we provide evidence that conjugation of SUMO-1 to wild-type p53 results in an increased transactivation ability of p53. We suggest that posttranslational modification of p53 by SUMO-1 conjugation provides a novel mechanism to regulate p53 activity.     

The Nup358-RanGAP complex is required for efficient importin alpha/beta-dependent nuclear import

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin alpha/beta-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin beta, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin beta strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin beta recycling and reformation of novel import complexes.     

Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3

Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress.     

Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1

The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha. As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.     

Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation

Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.     

SUMO-1 transiently localizes to Cajal bodies in mammalian neurons

Cajal bodies (CBs) are nuclear organelles involved in the maturation of small nuclear ribonucleoproteins required for the processing of pre-mRNAs. They concentrate coilin, splicing factors and the survival of motor neuron protein (SMN). By using immunocytochemistry and transfection experiments with GFP–SUMO-1, DsRed1-Ubc9, GFP–coilin and GFP–SMN constructs we demonstrate the presence of SUMO-1 and the SUMO conjugating enzyme (Ubc9) in a subset of CBs in undifferentiated neuron-like UR61 cells. Furthermore, SUMO-1 is transiently localized into neuronal CBs from adult nervous tissue in response to osmotic stress or inhibition of methyltransferase activity. SUMO-1-positive CBs contain coilin, SMN and small nuclear ribonucleoproteins, suggesting that they are functional CBs involved in pre-mRNA processing. Since coilin and SMN have several putative motifs of SUMO-1 modification, we suggest that the sumoylation of coilin and/or SMN might play a role in the molecular reorganization of CBs during the neuronal differentiation or stress–response.     

Loss of IκBα-Mediated Control over Nuclear Import and DNA Binding Enables Oncogenic Activation of c-Rel

The IκBα protein is able both to inhibit nuclear import of Rel/NF-κB proteins and to mediate the export of Rel/NF-κB proteins from the nucleus. We now demonstrate that the c-Rel–IκBα complex is stably retained in the cytoplasm in the presence of leptomycin B, a specific inhibitor of Crm1-mediated nuclear export. In contrast, leptomycin B treatment results in the rapid and complete relocalization of the v-Rel–IκBα complex from the cytoplasm to the nucleus. IκBα also mediates the rapid nuclear shuttling of v-Rel in an interspecies heterokaryon assay. Thus, continuous nuclear export is required for cytoplasmic retention of the v-Rel–IκBα complex. Furthermore, although IκBα is able to mask the c-Rel-derived nuclear localization sequence (NLS), IκBα is unable to mask the v-Rel-derived NLS in the context of the v-Rel–IκBα complex. Taken together, our results demonstrate that IκBα is unable to inhibit nuclear import of v-Rel. We have identified two amino acid differences between c-Rel and v-Rel (Y286S and L302P) which link the failure of IκBα to inhibit nuclear import and DNA binding of a mutant c-Rel protein to oncogenesis. Our results support a model in which loss of IκBα-mediated control over c-Rel leads to oncogenic activation of c-Rel.