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At least 19 recordsLinked to original sources
PubMed1997

Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p.

At least one essential function of Smt3p, a Saccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While we were unable to detect formation of a Ub approximately Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation.

Amino Acid Substitution↗
PubMed1997

The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.

SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1. We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus. The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain. Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.

Alleles↗
PubMed1996

Associations of UBE2I with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system.

The yeast RAD52-dependent pathway is involved in DNA recombination and double-strand break repair. Yeast ubiquitin-conjugating enzyme UBC9 participates in S- and M-phase cyclin degradation and mitotic control. Using the human RAD52 protein as the "bait" in a yeast two-hybrid system, we have identified a human homolog of yeast UBC9, designated UBE2I, that interacts with RAD52, RAD51, p53, and a ubiquitin-like protein UBL1. These interactions are UBE2I-specific, since another DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), does not interact with these proteins. The interaction of UBE2I with RAD52 is mediated by RAD52's self-association region. These results suggest that the RAD52-dependent processes, cell cycle control, p53-mediated pathway(s), and ubiquitination interact through human UBE2I.

Carrier Proteins↗
PubMed1997

Ubch9 conjugates SUMO but not ubiquitin.

Ubiquitin conjugating enzymes participate in the thioester cascade that leads to protein ubiquitination. Although Ubc9 is homologous to E2 ubiquitin conjugating enzymes we have shown that it is unable to form a thioester with ubiquitin, but can form a thioester with the small ubiquitin-like protein SUMO. Thus Ubc9 is a SUMO conjugating enzyme rather than a ubiquitin conjugating enzyme. Transacetylation of Ubc9 by SUMO is not mediated by the E1 ubiquitin activating enzyme, but by a distinct enzymatic activity. SUMO conjugation to target proteins is mediated by a different, but parallel pathway to ubiquitination.

Acetylation↗
PubMed2004

SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?

The small ubiquitin-related modifier SUMO posttranslationally modifies many proteins with roles in diverse processes including regulation of transcription, chromatin structure, and DNA repair. Similar to nonproteolytic roles of ubiquitin, SUMO modification regulates protein localization and activity. Some proteins can be modified by SUMO and ubiquitin, but with distinct functional consequences. It is possible that the effects of ubiquitination and SUMOylation are both largely due to binding of proteins bearing specific interaction domains. Both modifications are reversible, and in some cases dynamic cycles of modification may be required for activity. Studies of SUMO and ubiquitin in the nucleus are yielding new insights into regulation of gene expression, genome maintenance, and signal transduction.

Amino Acid Sequence↗
PubMed1996

PIC 1, a novel ubiquitin-like protein which interacts with the PML component of a multiprotein complex that is disrupted in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) arises following a reciprocal translocation t(15;17) that fuses PML with retinoic acid receptor alpha (RARA). The PML-RARA fusion protein targets and disrupts nuclear multiprotein complexes called PODs, ND10 or NBs, a process which is associated with a block in myeloid differentiation leading to APL. A human B-cell cDNA library was screened for PML-interacting clones and a single positive clone (PIC1) was isolated. The sequence of PIC1 shows 52% identity to a S. cerevisiae ubiquitin-like protein that was cloned as a suppressor of mutations in MIF2, a protein required for mitotic spindle integrity during anaphase. Transient transfection of NIH3T3 cells with PIC1 results in a nuclear staining pattern coincident with that of endogenous mouse PML. Further, cotransfection of PIC1 with human PML produces a completely overlapping staining pattern between the two proteins. An antibody raised against PIC1 detects a punctate staining pattern in HeLa cells that is coincident with endogenous human PML. There is no significant colocalisation observed between the staining of PML/ PML-RARA and PIC1 in an APL-derived cell line NB4, as compared to cells expressing only wild type PML. However, following all trans retinoic acid treatment of NB4 cells a significant relocalisation of PIC1 and PML is observed. PIC1 is the first identified NB-associated protein that interacts with PML, the function of which may lead to a fuller understanding of the molecular events leading to APL.

3T3 Cells↗
PubMed1996

Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin.

Fas/APO-1 and TNF receptor 1 share a common signaling motif in their cytoplasmic tail called the "death domain." Using the death domain as bait in the yeast two-hybrid system, several death domain-containing proteins that participate in cell death signaling have been identified. Here we report the isolation of a novel protein, sentrin, which interacts with Fas/APO-1 and TNF receptor 1 but not with FADD/MORT1 or CD40. Two-hybrid interaction assays reveal that sentrin associates only with the signal-competent forms of Fas/APO-1 or TNF receptor 1 death domains. Sentrin is a novel protein of 101 amino acids with homology to ubiquitin, Nedd8, and a Saccharomyces cerevisiae protein, Smt3. When overexpressed, sentrin provides protection against both anti-Fas/APO-1 and TNF-induced cell death.

Adaptor Proteins, Signal Transducing↗
PubMed2010

MAP Kinase: SUMO pathway interactions.

The convergence and coordinated cross talk of different signalling pathways forms a regulatory network which determines the biological outcome to environmental cues. The MAPK pathways are one of the important routes by which extracellular signals are transduced into intracellular responses. Through protein phosphorylation mechanisms, they can play a pivotal role in regulating other posttranslational modifications such as protein acetylation and ubiquitination. In addition, protein sumoylation has emerged as an important pathway which also functions through post-translational modification. The SUMO pathway modulates a diverse range of cellular processes including signal transduction, chromosome integrity, and transcription. Interestingly, recent studies have provided links between the SUMO and MAPK signalling pathways which converge to modulate transcription factor activity. This was first demonstrated by the observation that the activation of the ERK pathway caused de-sumoylation of the transcription factor, Elk-1. Furthermore, a growing number of links are now being made between the MAPK pathway and protein sumoylation. Given the nature of protein sumoylation in diverse biological functions, it is not surprising that the effect of MAPK pathways on sumoylation varies between different proteins. Here, we describe protocols that can be used in studying the cross talk between the MAPK and SUMO pathways, particularly at the level of gene regulation.

Amino Acid Sequence↗
PubMed1997

Preferential modification of nuclear proteins by a novel ubiquitin-like molecule.

Sentrin is a novel ubiquitin-like protein that protects cells against both anti-Fas and tumor necrosis factor-induced cell death. Antiserum recognizing the N terminus of sentrin revealed the presence of a 18-kDa sentrin monomer, a 90-kDa band (p90), and multiple high molecular mass bands. Because sentrin possesses the conserved Gly-Gly residues near the C terminus, it is likely that these additional bands represent conjugation of sentrin to other proteins in a manner that is similar to the ubiquitination pathway. Transient expression of hemagglutinin epitope-tagged sentrin mutants in COS cells demonstrated that the sentrin C terminus is cleaved, which allows it to be conjugated to other proteins via the conserved C-terminal Gly residue. Immunocytochemical staining and cell fractionation analysis demonstrated that sentrin monomer is localized predominantly to the cytosol. However, p90 and the majority of sentrinized proteins appeared to be localized to the nucleus. When the conserved Gly-Gly residues of sentrin were changed to Gly-Ala, only sentrin monomer and p90 but not the high molecular mass bands were observed. Thus, p90 generation appears to be required for the formation of high molecular mass bands in the nucleus. Taken together, sentrinization represents a novel pathway for nuclear protein modification, which is distinct from ubiquitination.

Amino Acid Sequence↗
PubMed1997

Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9.

Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.

Binding Sites↗
PubMed1996

A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

Ran is a nuclear Ras-like GTPase that is required for the bidirectional transport of proteins and ribnucleoproteins across the nuclear pore complex (NPC). A key regulator of the Ran GTP/GDP cycle is the 70-kD Ran-GTPase-activating protein RanGAP1. Here, we report the identification and localization of a novel form of RanGAP1. Using peptide sequence analysis and specific mAbs, RanGAP1 was found to be modified by conjugation to a ubiquitin-like protein. Immunoblot analysis and immunolocalization by light and EM demonstrated that the 70-kD unmodified from of RanGAP1 is exclusively cytoplasmic, whereas the 90-kD modified form of RanGAP1 is associated with the cytoplasmic fibers of the NPC. The modified form of RanGAP1 also appeared to associated with the mitotic spindle apparatus during mitosis. These findings have specific implications for Ran function and broad implications for protein regulation by ubiquitin-like modifications. Moreover, the variety and function of ubiquitin-like protein modifications in the cell may be more diverse than previously realized.

Amino Acid Sequence↗
PubMed1997

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.

Animals↗
PubMed2005

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Adenoviridae↗
PubMed1996

UBL1, a human ubiquitin-like protein associating with human RAD51/RAD52 proteins.

The RAD51/RAD52-dependent DNA repair pathway is involved in DNA recombination and DNA double-strand break repair in yeast. Although many proteins in the RAD51/RAD52-dependent DNA repair pathway have been identified in yeast, a novel protein(s) that functions with RAD51/RAD52 may also exist in humans. Using a yeast two-hybrid system, we have identified a 12-kDa protein that associates with the human RAD51 and RAD52 proteins. This protein shares significant amino acid homology with the yeast protein SMT3, which functionally associates with the yeast mitosis fidelity protein MIF2. It also shares moderate homology with ubiquitin and several other proteins, including the N-terminus of the RAD23 protein and a ubiquitin cross reacting protein. Therefore, the gene is tentatively designated UBL1 for ubiquitin-like 1. The UBL1 mRNA is expressed in many human tissues, most highly in testis. The UBL1 gene is mapped to chromosome 2q32.2-q33, and a related sequence may be located on chromosome 1q23-q25.

Amino Acid Sequence↗
PubMed2025

HDAC6-dependent deacetylation of SAE2 enhances SUMO1 conjugation for mitotic integrity.

Mammalian cells express three conjugatable SUMO variants: SUMO1 and the closely related SUMO2 and SUMO3 (together referred to as SUMO2/3). While some substrates are modified by both, others show a clear preference, though the basis for this selectivity remains unclear. Here, we examine a modification of the catalytic component of the human SUMO activation enzyme, SAE2. We find that lysine 164 of SAE2 undergoes HDAC6-dependent deacetylation during mitosis. A non-deacetylatable acetyl-mimetic mutant, SAE2-K164Q, selectively enhances SUMO2 over SUMO1 activation and conjugation, and distinguishes between SUMO1 and SUMO2/3 based on differences in their C-terminal tails. Complementation of SAE2-deficient or inhibited cells with SAE2-K164Q suppresses mitotic SUMO1 conjugation and promotes multipolar spindle formation. We identify NuMA as a SUMO E1-dependent substrate and demonstrate that mitotic defects caused by SAE2-K164Q or HDAC6 inhibition are rescued by SUMO1 overexpression or expression of a GFP-SUMO1-NuMA-K1766R fusion. These results support a model in which SAE1:SAE2 deacetylation during early mitosis promotes SUMO1 conjugation to ensure mitotic fidelity, highlighting a regulatory role for the SUMO-activating enzyme in the selection of SUMO proteins.

Humans↗
PubMed1997

Characterization of reaction intermediates of human excision repair nuclease.

Nucleotide excision repair in humans is a complex reaction involving 14 polypeptides in six repair factors for dual incisions on either sides of a DNA lesion. To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: purification of functional DNA.protein complexes, permanganate footprinting, and the employment as substrate of presumptive DNA reaction intermediates containing unwound sequences 5' to, 3' to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by substrate binding of XPA, RPA, XPC.HHR23B plus TFIIH (preincision complex 1, PIC1). In this complex the DNA was unwound on either side of the lesion by no more than 10 bases. Independent of the XPG nuclease function, the XPG protein stabilized this complex, forming a long lived preincision complex 2 (PIC2). The XPF.ERCC1 complex bound to PIC2, forming PIC3, which led to dual incisions and the release of the excised oligomer. With partially unwound DNAs, thymine cyclobutane dimer was excised at a fast rate independent of XPC.HHR23B, indicating that a major function of this protein is to stabilize the unwound DNA or to aid lesion unwinding in preincision complexes.

Base Sequence↗
PubMed1998

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.

Animals↗