Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex

The small ubiquitin-like modifier SUMO-1 is covalently attached to lysine residues on target proteins by a specific conjugation pathway involving the E1 enzyme SAE1/SAE2 and the E2 enzyme Ubc9. In an ATP-dependent manner, the C-terminus of SUMO-1 forms consecutive thiolester bonds with cysteine residues in the SAE2 subunit and Ubc9, before the Ubc9.SUMO-1 thiolester complex catalyzes the formation of an isopeptide bond between SUMO-1 and the epsilon-amino group of the target lysine residue on the protein substrate. The SUMO-1 conjugation pathway bears many similarities with that of ubiquitin and other ubiquitin-like protein modifiers (Ubls), and because of its production of a singly conjugated substrate and the lack of absolute requirement in vitro for E3 enzymes, the SUMO-1/Ubc9 system is a good model for the analysis of protein conjugation pathways that share this basic chemistry. Here we describe methods of both steady-state and half-reaction kinetic analysis of Ubc9, and use these techniques to determine the role of two residues, Asp(100) and Lys(101) of Ubc9 which are not found in E2 enzymes from other protein conjugation pathways. These residues are found close to the active site Cys in the tertiary structure of Ubc9, and although they are shown to inhibit the transesterification reaction from SAE1/SAE2, they are important for substrate recognition in the context of the thiolester complex with SUMO-1.     

The Mdm-2 amino terminus is required for Mdm2 binding and SUMO-1 conjugation by the E2 SUMO-1 conjugating enzyme Ubc9

Covalent attachment of SUMO-1 to Mdm2 requires the activation of a heterodimeric Aos1-Uba2 enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm2 by Ubc9, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Upon Sumo-1 conjugation, Mdm2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide ligase (E3) activity toward p53, thereby increasing its oncogenic potential. Because of the biological implication of Mdm2 sumoylation, we mapped Ubc9 binding on Mdm2. Here we demonstrate that Ubc9 can associate with Mdm2 only if amino acids 40-59 within the N terminus of Mdm2 are present. Mdm2 from which amino acids 40-59 have been deleted can no longer be sumoylated. Furthermore, addition of a peptide that corresponds to amino acids 40-59 on Mdm2 to a sumoylation reaction efficiently inhibits Mdm2 sumoylation in vitro and in vivo. In UV-treated cells Mdm2 exhibits reduced association with Ubc9, which coincides with decreased Mdm2 sumoylation. Our findings regarding the association of Ubc9 with Mdm2, and the effect of UV-irradiation on Ubc9 binding, point to an additional level in the regulation of Mdm2 sumoylation under normal growth conditions as well as in response to stress conditions.     

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.     

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.     

The type 2 dengue virus envelope protein interacts with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9)

Dengue viruses are mosquito-borne flaviviruses and may cause the life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its envelope protein is responsible mainly for the virus attachment and entry to host cells. To identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, we have performed a screening with the yeast-two-hybrid-based “Functional Yeast Array”. Interestingly, the small ubiquitin-like modifier-1 conjugating enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, we have demonstrated that it indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nuclear membrane and co-localized with Flag-Ubc9 proteins around the nuclear membrane in the cytoplasmic side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus propagation.     

The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification

SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence "PsiKXE," where Psi is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates.     

Caspase recruitment domain of procaspase-2 could be a target for SUMO-1 modification through Ubc9

To identify the binding proteins that regulate the function of procaspase-2, we screened for proteins using the yeast two-hybrid method and isolated human Ubc9 and SUMO-1 as the candidates. Ubc9 and SUMO-1 interacted with the caspase recruitment domain of procaspase-2 in its N-terminal. We elucidated the covalent modification of procaspase-2 by SUMO-1 in mammalian cells by immunoprecipitation followed by Western blot analysis. Procaspase-2 and SUMO-1 were co-localized by dot-like structures in the nucleus that are related to promyelocytic leukemia bodies. Interestingly, a conjugation-deficient mutant (K60R) procaspase-2 resulted in a delay of its enzyme maturation (appearance of p12 subunit) compared to that of wild-type. Thus, the modification with SUMO-1 may play a critical role in the nuclear localization and the activation (maturation) of procaspase-2.     

Solution structure of human SUMO-3 C47S and its binding surface for Ubc9

Small ubiquitin-related modifier SUMO-3 is a member of a growing family of ubiquitin-like proteins (Ubls). So far, four isoforms of SUMO have been identified in humans. It is generally known that SUMO modification regulates protein localization and activity. Previous structure and function studies have been mainly focused on SUMO-1. The sequence of SUMO-3 is 46% identical with that of SUMO-1; nevertheless, functional heterogeneity has been found between the two homologues. Here we report the solution structure of SUMO-3 C47S (residues 14-92) featuring the beta-beta-alpha-beta-beta-alpha-beta ubiquitin fold. Structural comparison shows that SUMO-3 C47S resembles ubiquitin more than SUMO-1. On the helix-sheet interface, a strong hydrophobic interaction contributes to formation of the globular and compact fold. A Gly-Gly motif at the C-terminal tail, extending away from the core structure, is accessible to enzymes and substrates. In vivo, SUMO modification proceeds via a multistep pathway, and Ubc9 plays an indispensable role as the SUMO conjugating enzyme (E2) in this process. To develop a better understanding of SUMO-3 conjugation, the Ubc9 binding surface on SUMO-3 C47S has been detected by chemical shift perturbation using NMR spectroscopy. The binding site mainly resides on the hydrophilic side of the beta-sheet. Negatively charged and hydrophobic residues of this region are highly or moderately conserved among SUMO family members. Notably, the negatively charged surface of SUMO-3 C47S is highly complementary in its electrostatic potentials and hydrophobicity to the positively charged surface of Ubc9. This work indicates dissimilarities between SUMO-3 and SUMO-1 in tertiary structure and provides insight into the specific interactions of SUMO-3 with its modifying enzyme.     

Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.     

Regulation of bcl-2 expression by Ubc9

Posttranslational modifications mediated by ubiquitin-like proteins have been implicated in regulating a variety of cellular pathways. Although small ubiquitin-like modifier (SUMO) is a new member of this family, it has caught a great deal of attention recently because of its novel and distinguished functions. Sumoylation is a multiple-step process, involving maturation, activation, conjugation and ligation. Ubc9 is an E2 conjugating enzyme essential for sumoylation. We have previously shown that suppression of sumoylation by a dominant negative Ubc9 mutant (Ubc9-DN) in the estrogen receptor (ER) positive MCF-7 cells is associated with alterations of tumor cell's response to anticancer drugs as well as tumor growth in a xenograft mouse carcinoma model. To dissect the underlying mechanism of Ubc9-associated alterations of drug responsiveness and tumor growth, we profiled gene expression for the cells expressing wild type Ubc9 (Ubc9-WT) and Ubc9-DN. We found that several tumorigenesis-related genes were downregulated in the Ubc9-DN cells. Within this group, we found that over 10 genes are known to be regulated by ER. Experiments using the estrogen response element fused to the luciferase reporter showed that the basal level of luciferase activity was significantly reduced in the Ubc9-DN cells when compared to the vector alone or the Ubc9-WT cells. Furthermore, we found that both the stability and the subcellular localization of steroid hormone receptor coactivator-1 (SRC-1) were altered in the Ubc9-DN cells. Together, these results suggest that Ubc9 might regulate bcl-2 expression through the ER signaling pathway, which ultimately contributes to the alterations of drug responsiveness and tumor growth.     

The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase

RanBP2/Nup358 is an essential protein with roles in nuclear transport and mitosis, and is one of the few known SUMO E3 ligases. However, why RanBP2 functions in vivo has been unclear: throughout the cell cycle it stably interacts with RanGAP1*SUMO1 and Ubc9, whose binding sites overlap with the E3 ligase region. Here we show that cellular RanBP2 is quantitatively associated with RanGAP1, indicating that complexed rather than free RanBP2 is the relevant E3 ligase. Biochemical reconstitution of the RanBP2/RanGAP1*SUMO1/Ubc9 complex enabled us to characterize its activity on the endogenous substrate Borealin. We find that the complex is a composite E3 ligase rather than an E2-E3 complex, and demonstrate that complex formation induces activation of a catalytic site that shows no activity in free RanBP2. Our findings provide insights into the mechanism of an important E3 ligase, and extend the concept of multisubunit E3 ligases from ubiquitin to the SUMO field.     

Smt3/SUMO and Ubc9 are required for efficient APC/C-mediated proteolysis in budding yeast

Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells. Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.     

Pellino-1, an adaptor protein of interleukin-1 receptor/toll-like receptor signaling,is sumoylated by Ubc9

Covalent modifications of the Pellino-1 protein are essential for transmitting innate immune response signals downstream, as the phosphorylation and polyubiquitination of Pellino-1 mediated by the IRAK proteins appear to have roles in regulating Pellino-1 function. In this study, we demonstrate that the Pellino-1 protein is post-translationally modified by small-ubiquitin-related modifier-1 (SUMO-1). Sumoylation assays with Pellino-1 and SUMO-1 expression plasmids reveal that the Pellino-1 protein is sumoylated in vitro and in vivo. Treatment of SUMO-1 specific protease 1 (SENP1) inhibited the sumoylation of the Pellino-1 protein and a GST pull-down assay as well as a yeast two hybrid assay showed that Pellino-1 binds to the SUMO-conjugating enzyme, Ubc9. Furthermore, we identified the five lysine residues of the Pellino-1 protein where SUMO-1 covalently attaches. Some of the sumoylated sites overlap with previously identified ubiquitination sites, suggesting competition between sumoylation and ubiquitination, as well as suggesting that the sumoylated Pellino-1 protein may have a cellular function distinct from previously identified functions.     

DNA repair and global sumoylation are regulated by distinct Ubc9 noncovalent complexes

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways.