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Results for “Sumoylation”

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PubMed2006

Sumoylation delimits KLF8 transcriptional activity associated with the cell cycle regulation.

KLF8 (Krüppel-like factor 8) is a member of the Krüppel transcription factor family that binds CACCC elements in DNA and activates or represses their target genes in a context-dependent manner. Here we present sumoylation as a novel mechanism that regulates KLF8 post-translationally. We found that KLF8 can be covalently modified by small ubiqitin-like modifier (SUMO)-1, SUMO-2, and SUMO-3 in vivo. We showed that KLF8 interacted with the PIAS family of SUMO E3 ligases PIAS1, PIASy, and PIASxalpha but not with E2 SUMO-conjugating enzyme Ubc9. Furthermore, we demonstrated that the E2 and E3 ligases enhanced the sumoylation of KLF8. In addition, site-directed mutagenesis identified lysine 67 as the major sumoylation site on KLF8. Lysine 67 to arginine mutation strongly enhanced activity of KLF8 as a repressor or activator to its physiological target promoters and as an inducer of the G(1) cell cycle progression. Taken together, our results demonstrated that sumoylation of KLF8 negatively regulates its transcriptional activity and cellular functions.

Amino Acid Sequence↗

PubMed2006

DJ-1 transcriptionally up-regulates the human tyrosine hydroxylase by inhibiting the sumoylation of pyrimidine tract-binding protein-associated splicing factor.

Loss-of-function mutations in DJ-1 cause a subset of familial Parkinson disease (PD). However, the mechanism underlying the selective vulnerability in dopaminergic pathway due to the inactivation of DJ-1 is unclear. Previously, we have reported that DJ-1 is a neuroprotective transcriptional co-activator interacting with the transcriptional co-repressor pyrimidine tract-binding protein-associated splicing factor (PSF). Here we show that DJ-1 and PSF bind and regulate the human tyrosine hydroxylase (TH) promoter. Inactivation of DJ-1 by small interference RNA (siRNA) results in decreased TH expression and l-DOPA production in human dopaminergic cell lines. Consistent with its role as a transcriptional regulator, DJ-1 specifically suppresses the global SUMO-1 modification. High molecular weight sumoylated protein species, including PSF, accumulate in the lymphoblast cells from the patients carrying pathogenic DJ-1 mutations. DJ-1 elevates the TH expression by inhibiting the sumoylation of PSF and preventing its sumoylation-dependent recruitment of histone deacetylase 1. Furthermore, siRNA silencing of DJ-1 decreases the acetylation of TH promoter-bound histones, and histone deacetylase inhibitors restore the DJ-1 siRNA-induced repression of TH. Therefore, our results suggest DJ-1 as a regulator of protein sumoylation and directly link the loss of DJ-1 expression and transcriptional dysfunction to impaired dopamine synthesis.

Humans↗

PubMed2005

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program.

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.

Amino Acid Sequence↗

PubMed2004

Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus Towne strain is not required for virus growth in cultured human fibroblasts.

Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus has been shown to increase transactivation activity in target reporter gene assays. This study examined the role of IE2 sumoylation in viral infection. A Towne strain-based bacterial artificial chromosome clone was generated encoding a mutated form of the IE2 protein with Lys-->Arg substitutions at positions 175 and 180, the two major sumoylation sites. When human fibroblast (HF) cells were infected with the reconstituted mutant virus, (i) viral growth kinetics, (ii) the accumulation of IE1 (UL123), IE2 (UL122), p52 (UL44) and pp65 (UL83) proteins and (iii) the relocalization of the cellular small ubiquitin-like modifier (SUMO)-1, p53 and proliferating cell nuclear antigen proteins into viral DNA replication compartments were comparable with those of the wild-type and the revertant virus. The data demonstrate that sumoylation of IE2 is not essential for virus growth in cultured HF cells.

Cells, Cultured↗

PubMed2005

Circadian clock control by SUMOylation of BMAL1.

The molecular machinery that governs circadian rhythmicity is based on clock proteins organized in regulatory feedback loops. Although posttranslational modification of clock proteins is likely to finely control their circadian functions, only limited information is available to date. Here, we show that BMAL1, an essential transcription factor component of the clock mechanism, is SUMOylated on a highly conserved lysine residue (Lys259) in vivo. BMAL1 shows a circadian pattern of SUMOylation that parallels its activation in the mouse liver. SUMOylation of BMAL1 requires and is induced by CLOCK, the heterodimerization partner of BMAL1. Ectopic expression of a SUMO-deficient BMAL1 demonstrates that SUMOylation plays an important role in BMAL1 circadian expression and clock rhythmicity. This reveals an additional level of regulation within the core mechanism of the circadian clock.

ARNTL Transcription Factors↗

PubMed2006

MafG sumoylation is required for active transcriptional repression.

A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity.

Amino Acid Sequence↗

PubMed2005

Ikaros SUMOylation: switching out of repression.

Ikaros plays a key role in lymphocyte development and homeostasis by both potentiating and repressing gene expression. Here we show that Ikaros interacts with components of the SUMO pathway and is SUMOylated in vivo. Two SUMOylation sites are identified on Ikaros whose simultaneous modification results in a loss of Ikaros' repression function. Ikaros SUMOylation disrupts its participation in both histone deacetylase (HDAC)-dependent and HDAC-independent repression but does not influence its nuclear localization into pericentromeric heterochromatin. These studies reveal a new dynamic way by which Ikaros-mediated gene repression is controlled by SUMOylation.

Active Transport, Cell Nucleus↗

PubMed2006

SUMOylation plays a role in gemcitabine- and bortezomib-induced cytotoxicity in human oropharyngeal carcinoma KB gemcitabine-resistant clone.

Bortezomib, a novel dipeptide boronic acid proteasome inhibitor, has been shown in previous studies to be synergistic with gemcitabine; however, the molecular mechanisms are not fully understood. Because post-translational modification of proteins, such as ubiquitination and SUMOylation, plays a critical role in governing cellular homeostasis, we explored this further by treating human oropharyngeal carcinoma KB wild-type (KBwt) and gemcitabine-resistant (KBGem) cells with gemcitabine and bortezomib in a time-dependent and sequence-dependent manner. Treatment with bortezomib at 4 to 8 hours post-gemcitabine significantly induced cell death in KBwt cell lines. However, in KBGem cells, bortezomib alone was just as cytotoxic. Using reporter assays, nuclear factor-kappaB (NF-kappaB) activity was found to be 5-fold higher in KBGem cells than that in KBwt cells, and the combination treatment decreased NF-kappaB activity by 44% in KBwt cells and 28% in KBGem cells, respectively. By Western blot analyses, treatment with gemcitabine and bortezomib resulted in a cleavage of NF-kappaB in KBwt but not in KBGem cells. SUMOylation capacity was modulated by transducing KBwt and KBGem cells with lenti-SUMO-1 or the unconjugatable lenti-SUMO-1aa followed by drug treatment. The expression of cyclins A, D1, and E was differentially regulated by SUMOylation capacity in KBGem but not in KBwt cells. We report herein that the activation of NF-kappaB signaling plays a critical role in eliciting KBwt cell survival against gemcitabine, whereas the role of SUMOylation in modulating the steady-state levels of key cell cycle regulator proteins seems more significant in KBGem cells.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

Sumoylation of LIN-1 promotes transcriptional repression and inhibition of vulval cell fates.

The LIN-1 ETS transcription factor inhibits vulval cell fates during Caenorhabditis elegans development. We demonstrate that LIN-1 interacts with UBC-9, a small ubiquitin-related modifier (SUMO) conjugating enzyme. This interaction is mediated by two consensus sumoylation motifs in LIN-1. Biochemical studies showed that LIN-1 is covalently modified by SUMO-1. ubc-9 and smo-1, the gene encoding SUMO-1, inhibit vulval cell fates and function at the level of lin-1, indicating that sumoylation promotes LIN-1 inhibition of vulval cell fates. Sumoylation of LIN-1 promoted transcriptional repression and mediated an interaction with MEP-1, a protein previously shown to associate with the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies showed that mep-1 inhibits vulval cell fates and functions at the level of lin-1. We propose that sumoylation of LIN-1 mediates an interaction with MEP-1 that contributes to transcriptional repression of genes that promote vulval cell fates. These studies identify a molecular mechanism for SUMO-mediated transcriptional repression.

Amino Acid Motifs↗

PubMed2005

Detection of sumoylated proteins.

Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is involved in regulation of protein function, nuclear targeting, and the formation of subcellular structures. Because SUMO is involved in such a plethora of functions and modifies numerous proteins it is important to identify proteins that are sumoylated in order to increase our understanding of how this modification affects protein function and localization. This overview describes techniques utilized for the detection of sumoylated proteins. The techniques covered include immunoprecipitation, an in vitro sumoylation assay, and gel shift mobility assays that have been used to identify SUMO-modified proteins.

Blotting, Western↗

PubMed2006

SUMOylation interferes with CCAAT/enhancer-binding protein beta-mediated c-myc repression, but not IL-4 activation in T cells.

The transcription factor C/EBPbeta transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPbeta also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPbeta represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPbeta-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPbeta. Whereas sumoylation does not affect the C/EBPbeta-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPbeta and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPbeta.

Animals↗

PubMed2026

Decoding SUMOylation as a metabolic stress sensor in aging and age-related disorders: Mechanisms, tissue specificity and therapeutic potential.

SUMOylation is a reversible post-translational modification increasingly recognized for its role in coordinating cellular responses to metabolic stress during aging. Emerging evidence indicates that it functions beyond a conventional modification, representing an adaptive stress‑responsive regulatory network that integrates metabolic, oxidative, inflammatory, and proteotoxic signals. Rather than acting on isolated pathways, this network finely tunes mitochondrial function, proteostasis, genome maintenance, immune balance, and epigenetic regulation. Accumulating evidence indicates that SUMO-dependent regulation exhibits remarkable tissue specificity, supporting mitochondrial adaptation and contractile integrity in skeletal muscle, shaping lipid and glucose metabolism in the liver, modulating proteotoxic stress and neuronal resilience in the brain, and contributing to immune cell differentiation and chronic low-grade inflammation during aging. In this review, we summarize current mechanistic insights into SUMO signaling across aging-relevant tissues, with particular emphasis on its functional interplay with other post-translational modifications, including ubiquitination and acetylation. We discuss how SUMOylation operates as a shared regulatory layer while enabling context-dependent outcomes that underlie diverse aging phenotypes and age-related disorders. Finally, we evaluate emerging translational approaches-ranging from pharmacological modulation of SUMO enzymes to lifestyle interventions such as caloric restriction and exercise-that highlight both the opportunities and challenges of targeting SUMO-regulated stress responses in aging. Together, this synthesis provides a framework for understanding how SUMOylation links metabolic stress to tissue-specific aging trajectories and therapeutic potential.

Aging↗

PubMed2006

SARS-CoV nucleocapsid protein binds to hUbc9, a ubiquitin conjugating enzyme of the sumoylation system.

SARS-CoV is a newly identified coronavirus (CoV) that causes severe acute respiratory syndrome (SARS). The SARS-CoV nucleocapsid (N) protein is an important structural and functional protein. To identify cellular proteins that interact with the SARS-CoV N protein and to elucidate the possible involvement of N protein in SARS-CoV pathogenesis, a human lymphocyte cDNA library was screened using a yeast two-hybrid system assay. hUbc9, a ubiquitin conjugating enzyme of sumoylation system, was found to interact specifically with the N protein, implying the post-translational sumoylation of the N protein. Mapping studies localized the critical N sequences for this interaction to amino acids 170-210, which includes the SR-rich motif. However, the consensus motif of sumoylation GK(62)EE in the N protein is not responsible for binding to hUbc9. Mutations of hUbc9 at the enzyme active site C93A or C93S severely impair the interaction with the N protein. The two proteins were also shown to colocalize in the cytoplasm of the transfected 293T cells. This is the first report demonstrating the interaction of hUbc9 with a structural protein of plus-strand RNA viruses, indicating a new drug target for SARS-CoV.

Binding Sites↗

PubMed2005

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo.

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1-6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif.

Amino Acid Sequence↗

PubMed2006

Aspects of the regulatory mechanisms of PPAR functions: analysis of a bidirectional response element and regulation by sumoylation.

Peroxisome proliferator-activated receptors (PPARs) constitute a subfamily of nuclear receptor superfamily. A wide variety of compounds including hypolipidemic agents, antidiabetic drugs, and long-chain fatty acids are the potential ligands of PPARs. To approach the regulatory mechanisms of PPARs, we studied on two subjects in this work. First, we identified a functional PPAR-binding site in the spacer region between the PEX11alpha and perilipin genes, which are arranged in tandem on the mouse genome. By gene reporter assays and in vivo as well as in vitro binding assays, we show that these genes are regulated tissue-selectively through this common binding site: The PEX11alpha gene is activated by PPARalpha in the liver, whereas the perilipin gene by PPARgamma in the adipose tissue. As the second subject, we found that PPARgamma2 is conjugated with small ubiquitin-related modifier (SUMO) at a specific lysine residue in the amino-terminal region. By site-directed mutagenesis combined with gene reporter assays and sumoylation analyses, we show that sumoylation represses the ligand-independent transactivating function carried by this region, and hence negatively regulates the whole transactivating competence of PPARgamma2. In addition, phosphorylation at a specific site in the amino-terminal region represses the transactivation by PPARgamma2 possibly through enhancing sumoylation.

Adipose Tissue↗

PubMed2005

Requirement of the coiled-coil domain of PML-RARalpha oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation.

Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-alpha (RARalpha) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(DeltaC-C)-RARalpha, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the DeltaC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the DeltaC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the DeltaC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARalpha. However, the RA-mediated induction of C/EBPbeta in cells expressing the DeltaC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation all contribute to the oncogenic activity of PML-RARalpha.

Active Transport, Cell Nucleus↗

PubMed2005

Modification of MDMX by sumoylation.

MDMX is a homolog of MDM2 and is critical for regulating p53 function during mouse development. MDMX level is regulated by MDM2-mediated poly-ubiquitination, which results in its accelerated degradation after DNA damage or expression of ARF. In this report, we demonstrate that MDMX can be modified by conjugation to SUMO-1 both in vivo and in vitro. We found that double mutation of two lysine residues, K254 and K379, abrogated MDMX sumoylation in vivo. Experiments using the sumoylation-deficient MDMX mutant showed that it undergoes normal ubiquitination and degradation by MDM2, normal nuclear translocation and degradation after DNA damage, and inhibits p53 with wild type efficiency. Therefore, sumoylation is not required for several activities of MDMX under our assay conditions.

Active Transport, Cell Nucleus↗

PubMed2005

SoxE factors function equivalently during neural crest and inner ear development and their activity is regulated by SUMOylation.

Sox9 and the closely related factor Sox10 are essential for the formation of neural crest precursor cells, and play divergent roles in the process by which these cells are subsequently directed to form specific derivatives. These group E Sox factors have also been implicated in the development of the vertebrate inner ear. Despite their importance, however, the mechanisms that allow SoxE proteins to regulate such a diverse range of cell types have remained poorly understood. Here we demonstrate that during vertebrate development, the activities of individual SoxE factors are well conserved and are regulated by SUMOylation. We show that SoxE mutants that cannot be SUMOylated, or that mimic constitutive SUMOylation, are each able to mediate a subset of the diverse activities characteristic of wild-type SoxE proteins. These findings provide important mechanistic insight into how the activity of widely deployed developmental regulatory proteins can be directed to specific developmental events.

Animals↗