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.     

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.     

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.     

Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.     

Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress

To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox‐sensitive proteins. We previously identified SUMO (small ubiquitin‐related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox‐regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM–Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS‐producing chemotherapeutic drugs etoposide/Vp16 and Ara‐C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress.     

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.     

Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358.

Ubc9, a conjugation enzyme for the ubiquitin-related modifier SUMO, is present predominantly in the nucleus and at the nuclear pore complex. The functional significance of its subcellular compartmentalization, however, remains to be elucidated. Here, we define a Pro-Glu-Asp-Ser-Thr-rich element containing 129 amino acid residues, designated IR1+2, on the human nucleoporin RanBP2/Nup358, which binds directly to Ubc9 with high affinity both in vitro and in vivo. When IR1+2 tagged with green fluorescence protein at its amino terminus (GFP-IR1+2) was transfected into COS-7 cells, we found that approximately 90% of the nuclear Ubc9 was sequestered in the cytoplasm. We also observed that both SUMO-1 and SUMO-2/3 were mislocalized, and promyelocytic leukemia protein PML formed an enlarged aggregate in the nucleus. Moreover, the homologous recombination protein Rad51 mislocalized to the cytoplasm, and Rad51 foci, a hallmark of functional association of Rad51 with damaged DNA, did not form efficiently even in the presence of a DNA strand breaker. These findings emphasize that the IR1+2 domain is a useful tool for manipulating the nuclear localization of Ubc9 and perturbing the subcellular localization of SUMOs and/or SUMOlated proteins, and they emphasize the important role of nuclear Ubc9 in the Rad51-mediated homologous recombination pathway, possibly by modulating intracellular trafficking of Rad51.     

Role of SUMO/Ubc9 in DNA Damage Repair and Tumorigenesis

DNA damage repair is an important cell function for genome integrity and its deregulation can lead to genomic instability and development of malignancies. Sumoylation is an increasingly important ubiquitin-like modification of proteins affecting protein stability, enzymatic activity, nucleocytoplasmic trafficking, and protein-protein interactions. In particular, several important DNA repair enzymes are subject to sumoylation, which appears to play a role in copping with DNA damage insults. Recent reports indicate that Ubc9, the single SUMO E2 enzyme catalyzing the conjugation of SUMO to target proteins, is overexpressed in certain tumors, such as lung adenocarcinoma, ovarian carcinoma and melanoma, suggestive of its clinic significance. This review summarizes the most important DNA damage repair pathways which are potentially affected by Ubc9/SUMO and their role in regulating the function of several proteins involved in the DNA damage repair machinery.     

Ubc9 is essential for viability of higher eukaryotic cells

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.     

Activity-dependent SUMOylation of the brain-specific scaffolding protein GISP

G-protein coupled receptor interacting scaffold protein (GISP) is a multi-domain, brain-specific protein derived from the A-kinase anchoring protein (AKAP)-9 gene. Using yeast two-hybrid screens to identify GISP interacting proteins we isolated the SUMO conjugating enzyme Ubc9. GISP interacts with Ubc9 in vitro, in heterologous cells and in neurons. SUMOylation is a post-translational modification in which the small protein SUMO is covalently conjugated to target proteins, modulating their function. Consistent with its interaction with Ubc9, we show that GISP is SUMOylated by both SUMO-1 and SUMO-2 in both in vitro SUMOylation assays and in mammalian cells. Intriguingly, SUMOylation of GISP in neurons occurs in an activity-dependent manner in response to chemical LTP. These data suggest that GISP is a novel neuronal SUMO substrate whose SUMOylation status is modulated by neuronal activity.     

Dynamin interacts with members of the sumoylation machinery

Dynamin is a GTP-binding protein whose oligomerization-dependent assembly around the necks of lipid vesicles mediates their scission from parent membranes. Dynamin is thus directly involved in the regulation of endocytosis. Sumoylation is a post-translational protein modification whereby the ubiquitin-like modifier Sumo is covalently attached to lysine residues on target proteins by a process requiring the concerted action of an activating enzyme (ubiquitin-activating enzyme), a conjugating enzyme (ubiquitin carrier protein), and a ligating enzyme (ubiquitin-protein isopeptide ligase). Here, we show that dynamin interacts with Sumo-1, Ubc9, and PIAS-1, all of which are members of the sumoylation machinery. Ubc9 and PIAS-1 are known ubiquitin carrier protein and ubiquitin-protein isopeptide ligase enzymes, respectively, for the process of sumoylation. We have identified the coiled-coil GTPase effector domain (GED) of dynamin as the site on dynamin that interacts with Sumo-1, Ubc9, and PIAS-1. Although we saw no evidence of covalent Sumo-1 attachment to dynamin, Sumo-1 and Ubc9 are shown here to inhibit the lipid-dependent oligomerization of dynamin. Expression of Sumo-1 and Ubc9 in mammalian cells down-regulated the dynamin-mediated endocytosis of transferrin, whereas dynamin-independent fluid-phase uptake was not affected. Furthermore, using high resolution NMR spectroscopy, we have identified amino acid residues on Sumo-1 that directly interact with the GED of dynamin. The results suggest that the GED of dynamin may serve as a scaffold that concentrates the sumoylation machinery in the vicinity of potential acceptor proteins.     

Two-hybrid interaction of a human UBC9 homolog with centromere proteins ofSaccharomyces cerevisiae

Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of theSaccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity toS. cerevisiae Ubc9p and 64% identity toSchizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses aS. cerevisiae ubc9 temperature-sensitive mutation, indicating that theUBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However,S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction betweenS. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G₂/M phase ofS. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.