Modification SUMOylation Research Articles

Phosphorylation and SUMOylation of CRMP2 regulate the formation and maturation of dendritic spines

The posttranslational modifications of CRMP2 play an important role in axon outgrowth, cell polarization and dendritic morphogenesis. However, whether CRMP2 and its posttranslational modifications are involved in dendritic spine development specifically is not completely clear. Here, we show that CRMP2 can promote the formation and maturation of dendritic spines in cultured hippocampal neurons. Overexpression of CRMP2 results in an increase in the density of spines especially the mushroom-shape spines. The amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) are both enhanced and the intensity of PSD95 is strengthened in the neurons with CRMP2 overexpression. Furthermore, dephosphorylation of CRMP2 at Thr514 and deSUMOylation at Lys374 can further promote the formation and maturation of dendritic spines, whereas, no cross-talk is found between these two posttranslational modifications in the regulation of dendritic spine formation and maturation. Taken together, our data support a model in which phosphorylation and SUMOylation modification of CRMP2 independently promote the formation and maturation of dendritic spines and participate in the process of dendritic spine plasticity.

SUMOylation of IQGAP1 promotes the development of colorectal cancer

IQGAP1 is a conserved multifunctional protein implicated in tumorigenesis. An aberrant expression of IQGAP1 widely exists in many cancers, but the SUMOylation modification of IQGAP1 in carcinogenesis is unknown by now. Here we first time explore biological functions of IQGAP1 SUMOylation in promoting colorectal cancer progression in vitro and in vivo. The expression of IQGAP1 and its SUMOylation level are both increased in human colorectal carcinoma (CRC) cells and tissues. IQGAP1 is mainly SUMOylated by SUMO1 at the K1445 residue, which could stabilize IQGAP1 by reducing protein ubiquitination. IQGAP1 SUMOylation improves CRC cell growth, cell migration and tumorigenesis in vivo through activating the phosphorylation of ERK, MEK and AKT. While the SUMOylation site mutation at K1445 of IQGAP1 greatly reduces CRC cell proliferation, migration ability and tumor growth of CRC-xenograft mice by suppressing phosphorylation of ERK, MEK and AKT. Our findings discover the IQGAP1 SUMOylation is a novel regulatory mechanism to enhance tumorigenesis and development of CRC in vitro and in vivo.

Protein SUMOylation modification and its associations with disease

SUMOylation, as a post-translational modification, plays essential roles in various biological functions including cell growth, migration, cellular responses to stress and tumorigenesis. The imbalance of SUMOylation and deSUMOylation has been associated with the occurrence and progression of various diseases. Herein, we summarize and discuss the signal crosstalk between SUMOylation and ubiquitination of proteins, protein SUMOylation relations with several diseases, and the identification approaches for SUMOylation site. With the continuous development of bioinformatics and mass spectrometry, several accurate and high-throughput methods have been implemented to explore small ubiquitin-like modifier-modified substrates and sites, which is helpful for deciphering protein SUMOylation-mediated molecular mechanisms of disease.

SUMO co-expression modifies KV 11.1 channel activity.

The voltage-gated potassium channel KV 11.1 is the molecular basis for the IKr current, which plays an important role in cardiac physiology. Its malfunction is associated with both inherited and acquired cardiac arrhythmias. Native currents differ from those in experimental models, suggesting additional regulatory mechanisms. We hypothesized that the post-translational modification sumoylation fine-tunes channel activity. The functional effects of sumoylation on KV 11.1 were addressed by employing two-electrode voltage-clamp (TEVC) experiments in Xenopus laevis oocytes. Site-directed mutagenesis enabled a further analysis of the SUMO-target amino acids. We assessed protein expression levels and used confocal imaging for localization studies. Co-expression with Ubc9 and SUMO alters the electrophysiological properties of KV 11.1 leading to a decrease in steady-state current amplitude largely due to faster inactivation and alteration of deactivation kinetics. We identified three lysines (K21, K93 and K116) in the PAS domain as the putative SUMO-targets. This study indicates KV 11.1 as a sumoylation target and offers three main targets: K21, K93, and K116. Furthermore, it proposes an underlying mechanism for the observed kinetic impact of the PAS domain.

Roles of SUMOylation in Heart Development and Cardiovascular Diseases.

Heart is an extremely important organ, and cardiovascular disorders emerge as primary life-threatening disease in human life. Aberrant post-translational modifications (PTMs) on cardiac proteins are closely correlated with pathological abnormalities in heart. SUMOylation, one of the most prevalent PTMs with thousands of substrates throughout the cell including critical subcellular organelles, has been shown to precisely finetune the cell survival and proliferation during heart development, and delicately control the function of mitochondrion and sarcoplasmic reticulum in physiological heart functioning. The silver lining is pathologically cardiacspecific SUMOylation being considered as target for cardiovascular disease intervention and treatment. Here, we summarize the recent progress in heart-specific functions of the SUMOylation pathway. In particular, we discuss the biological significance of SUMO conjugation/deconjugation during heart development, and in physiological cardiovascular health involving cardiac mitochondrial function, cardiac contractility, stress adaption and protein homeostasis. We also discuss the crosstalks between sumoylation and other post-translational modifications such as acetylation and ubiquitination. These crosstalks not only shed light to our understanding of the regulatory mechanisms on cardiovascular disorders but also contributes to their future therapy.

Rifampicin pre-treatment inhibits the toxicity of rotenone-induced PC12 cells by enhancing sumoylation modification of α-synuclein

Our previous research revealed that rifampicin could protect PC12 (pheochromocytoma 12) cells from rotenone-induced cytotoxicity by reversing the aggregation of α-synuclein. Furthermore, increasing evidence indicated that the misfolded α-synuclein with SUMOylation, an important protein posttranslational modification, was easier to solubilize and was less toxic. Here, we investigated whether rifampicin could stabilize α-synuclein and prevent rotenone-induced PC12 cells from undergoing apoptosis by enhancing SUMOylation of α-synuclein. The expression of SUMO1 and SUMO2/3, the two main proteins responsible for the SUMOylation modification in PC12 cells, were detected by western blotting. Co-immunoprecipitation was performed to compare qualitatively the SUMOylation modification of α-synuclein. The cell viability and apoptosis rate were measured by a CCK-8 assay kit and flow cytometry, respectively. We targeted Ubc9 as a key enzyme in the SUMOylation modification pathway and knocked down the UBC9 gene using a short interfering RNA. Treatment with 150 μmol/L rifampicin, increased the expressions of SUMO1 and SUMO2/3 in cells by 1.5 times compared with the control group; meanwhile, the cell viability of rotenone-induced cells increased from 20 to 80% (P < 0.05). In addition, the increased SUMOylation activity in the cells stimulated by rifampicin was observed 18 h earlier compared with cells treated by rotenone alone. SUMOylation of α-synuclein was more significant in rifampicin-treated cells and Ubc9 upregulated cells. However, the same phenomenon and the protective effect of rifampicin were reversed after UBC9 knockout. In conclusion, rifampicin might reduce the cytotoxicity of rotenone-induced PC12 cells by promoting SUMOylation of α-synuclein.

Orthoreovirus outer-fiber proteins are substrates for SUMO-conjugating enzyme Ubc9

Reoviruses are potential anticancer agents due to their ability to induce cell death in tumor cells. Grass carp reovirus (GCRV) is one of the best characterized models on reovirus pathogenesis in vitro. However, there is little known about how SUMOylation affects reovirus pathogenesis. The SUMO conjugating enzyme 9 (Ubc9) determines the targets of SUMOylation. Here, the protein interactions between reovirus outer fiber proteins, specifically GCRV-104 VP55, and Ubc9 were probed using a yeast two-hybrid system. The N-terminal coiled-coil domain of VP55, containing a single lysine residue, was responsible for the interaction between VP55 and Ubc9 in yeast. In solid phase binding assays, a single amino acid mutation (K87R) prevented Ubc9 from binding to VP55. Overexpression of Ubc9 enhanced GCRV-104 infection efficiency, and knockdown of Ubc9 in CIK cells inhibited viral replication, which suggested that Ubc9 was a proviral factor. Furthermore, Ubc9 was shown to bind outer fiber proteins from type II GCRV, avian reovirus and mammalian reovirus in yeast. To our knowledge, this is the first study to show that Ubc9 binds to reovirus outer-fiber proteins and likely contributes to efficient orthoreovirus replication. These results suggest that SUMOylation modifications could be targeted to improve the therapeutic efficacy of oncolytic reovirus.

HDAC4 stabilizes SIRT1 via sumoylation SIRT1 to delay cellular senescence.

The nicotinamide adenine dinucleotide-dependent protein deacetylase silent information regulator 2 (Sir2) regulates cellular lifespan in several organisms. Histone deacetylase 4 (HDAC4) belongs to the class IIa group of HDACs; this class of HDACs is composed of proteins that are important regulators of gene expression that control pleiotropic cellular functions. However, the role of HDAC4 in cellular senescence is still unknown. This study shows that the expression patterns of HDAC4 and Sirtuin 1 (SIRT1; the mammalian homolog of Sir2) are positively correlated during cellular senescence. Moreover, the overexpression of HDAC4 delays senescence, whereas the knockdown of HDAC4 leads to premature senescence in human fibroblasts. Furthermore, it is demonstrated that HDAC4 increases endogenous SIRT1 expression by enhancing its sumoylation modification levels, thereby stabilizing its protein levels. This study, therefore, provides a new molecular mechanism for the regulation of cellular senescence.

HTLV Tax-1 and Tax-2 proteins enhance interferon regulatory factor 3 dependent promoter activation

The HTLV-1 infection is known to induce an alteration of type I interferon (IFN-I) signaling since it is capable of escaping IFN-mediated immune response in vitro and Tax-1 protein modulates the expression of factors involved in the interferon signaling. In the present study we have investigated the effect of Tax-1 and Tax-2 expression on the activation of an IFN-regulatory factor 3 (IRF3) regulated promoter through the recruitment of the IFN-I upstream IKKe and TANK-binding kinase 1 (TBK1) factors, two IkB-related kinase homologues, which are essential for the activation of IRF3 pathway. We have demonstrated that both Tax-1 and Tax-2 were detectable in immuno-complexes formed by IKKe and TBK1 in HEK 293T transfected cells, but did not interact with the IRF3 factor. The presence of Tax-1 and IKKe in transfected cells resulted in a significant activation of the IRF3 regulated promoter. A similar effect was measured in the presence of Tax-1 and TBK1. We have also observed that Tax-1 mutants defective in sumoylation and ubiquitination post-translational modification were impaired in their ability to form complexes with IKKe or TBK1 and in the transactivating activity on IRF3 dependent promoter.These data provide evidence for a role of Tax proteins in the activation of IFN-I pathway, mediated by interaction with IKKe and TBK1 kinases. The effects of the Tax interaction with factors that act upstream of interferon regulatory factor IRF3 should be taken into account to further explain the IFN-mediated immune response to HTLV-1 infection. This study is funded by AIRC-Cariverona.

Receptor trafficking and the regulation of synaptic plasticity by SUMO.

Timely and efficient information transfer at synapses is fundamental to brain function. Synapses are highly dynamic structures that exhibit long-lasting activity-dependent alterations to their structure and transmission efficiency, a phenomenon termed synaptic plasticity. These changes, which occur through alterations in presynaptic release or in the trafficking of postsynaptic receptor proteins, underpin the formation and stabilisation of neural circuits during brain development, and encode, process and store information essential for learning, memory and cognition. In recent years, it has emerged that the ubiquitin-like posttranslational modification SUMOylation is an important mediator of several aspects of neuronal and synaptic function. Through orchestrating synapse formation, presynaptic release and the trafficking of postsynaptic receptor proteins during forms of synaptic plasticity such as long-term potentiation, long-term depression and homeostatic scaling, SUMOylation is being increasingly appreciated to play a central role in neurotransmission. In this review, we outline key discoveries in this relatively new field, provide an update on recent progress regarding the targets and consequences of protein SUMOylation in synaptic function and plasticity, and highlight key outstanding questions regarding the roles of protein SUMOylation in the brain.

SUMOylation of the small GTPase ARL-13 promotes ciliary targeting of sensory receptors

Primary cilia serve as cellular antenna for various sensory signaling pathways. However, how the sensory receptors are properly targeted to the ciliary surface remains poorly understood. Here, we show that UBC-9, the sole E2 small ubiquitin-like modifier (SUMO)-conjugating enzyme, physically interacts with and SUMOylates the C terminus of small GTPase ARL-13, the worm orthologue of ARL13B that mutated in ciliopathy Joubert syndrome. Mutations that totally abolish the SUMOylation of ARL-13 do not affect its established role in ciliogenesis, but fail to regulate the proper ciliary targeting of various sensory receptors and consequently compromise the corresponding sensory functions. Conversely, constitutively SUMOylated ARL-13 fully rescues all ciliary defects of arl-13-null animals. Furthermore, SUMOylation modification of human ARL13B is required for the ciliary entry of polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease. Our data reveal a novel but conserved role for the SUMOylation modification of ciliary small GTPase ARL13B in specifically regulating the proper ciliary targeting of various sensory receptors.

Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

BackgroundRetroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway.ResultsThe comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1.ConclusionsBoth Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway.

SUMOylation of the small GTPase ARL-13 promotes ciliary targeting of sensory receptors

Primary cilia serve as cellular antenna for various sensory signaling pathways. However, how the sensory receptors are properly targeted to the ciliary surface remains poorly understood. Here, we show that UBC-9, the sole E2 small ubiquitin-like modifier (SUMO)-conjugating enzyme, physically interacts with and SUMOylates the C terminus of small GTPase ARL-13, the worm orthologue of ARL13B that mutated in ciliopathy Joubert syndrome. Mutations that totally abolish the SUMOylation of ARL-13 do not affect its established role in ciliogenesis, but fail to regulate the proper ciliary targeting of various sensory receptors and consequently compromise the corresponding sensory functions. Conversely, constitutively SUMOylated ARL-13 fully rescues all ciliary defects of arl-13-null animals. Furthermore, SUMOylation modification of human ARL13B is required for the ciliary entry of polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease. Our data reveal a novel but conserved role for the SUMOylation modification of ciliary small GTPase ARL13B in specifically regulating the proper ciliary targeting of various sensory receptors.

Developmental Regulation and Spatiotemporal Redistribution of the Sumoylation Machinery in the Rat Central Nervous System

BackgroundSmall Ubiquitin-like MOdifier protein (SUMO) is a key regulator of nuclear functions but little is known regarding the role of the post-translational modification sumoylation outside of the nucleus, particularly in the Central Nervous System (CNS).Methodology/Principal FindingsHere, we report that the expression levels of SUMO-modified substrates as well as the components of the sumoylation machinery are temporally and spatially regulated in the developing rat brain. Interestingly, while the overall sumoylation is decreasing during brain development, there are progressively more SUMO substrates localized at synapses. This increase is correlated with a differential redistribution of the sumoylation machinery into dendritic spines during neuronal maturation.Conclusions/SignificanceOverall, our data clearly demonstrate that the sumoylation process is developmentally regulated in the brain with high levels of nuclear sumoylation early in the development suggesting a role for this post-translational modification during the synaptogenesis period and a redistribution of the SUMO system towards dendritic spines at a later developmental stage to modulate synaptic protein function.

Posttranslational Modifications of Proteins in the Pathobiology of Medically Relevant Fungi

Posttranslational modifications of proteins drive a wide variety of cellular processes in eukaryotes, regulating cell growth and division as well as adaptive and developmental processes. With regard to the fungal kingdom, most information about posttranslational modifications has been generated through studies of the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, where, for example, the roles of protein phosphorylation, glycosylation, acetylation, ubiquitination, sumoylation, and neddylation have been dissected. More recently, information has begun to emerge for the medically important fungal pathogens Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, highlighting the relevance of posttranslational modifications for virulence. We review the available literature on protein modifications in fungal pathogens, focusing in particular upon the reversible peptide modifications sumoylation, ubiquitination, and neddylation.

Nuclear Factor of Activated T Cells (NFAT) Proteins Repress Canonical Wnt Signaling via Its Interaction with Dishevelled (Dvl) Protein and Participate in Regulating Neural Progenitor Cell Proliferation and Differentiation

The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.

Arresting times for PTEN

Arresting times for PTEN

Mechanisms, regulation and consequences of protein SUMOylation

The post-translational modification SUMOylation is a major regulator of protein function that plays an important role in a wide range of cellular processes. SUMOylation involves the covalent attachment of a member of the SUMO (small ubiquitin-like modifier) family of proteins to lysine residues in specific target proteins via an enzymatic cascade analogous to, but distinct from, the ubiquitination pathway. There are four SUMO paralogues and an increasing number of proteins are being identified as SUMO substrates. However, in many cases little is known about how SUMOylation of these targets is regulated. Compared with the ubiquitination pathway, relatively few components of the conjugation machinery have been described and the processes that specify individual SUMO paralogue conjugation to defined substrate proteins are an active area of research. In the present review, we briefly describe the SUMOylation pathway and present an overview of the recent findings that are beginning to identify some of the mechanisms that regulate protein SUMOylation.

Differences in BCL11A SUMOylation Are Associated with Differences in γ-Globin Expression in Primary Erythroid Cells.

Abstract 458Increased fetal hemoglobin (HbF) levels associated with acute erythropoietic stress in man and experimental baboons have been proposed to result from increased commitment of early progenitors that preferentially express γ-globin to the terminal erythroid differentiation pathway. The increased propensity of early progenitors to preferentially express γ-globin has been hypothesized to be due to the presence of trans-acting factors favoring γ-globin expression. Because increased HbF in response to acute erythropoietic stress does not occur in transgenic human β-globin gene locus mouse models, investigation of the mechanism responsible for this phenomenon requires the use of a primate model system. We investigated the role of DNA methylation and the trans-acting factor BCL11A in the mechanism responsible for increased HbF in a primary cell culture system designed to mimic conditions associated with acute erythropoietic stress. Erythroid progenitor cells (EPC) derived from CD34+ baboon bone marrow (BM) cells cultured in Iscove's medium containing 30% fetal bovine serum supplemented with 2 U/ml Epo, 200ng/ml SCF, and 1uM dexamethasone express high levels of γ-globin (0.47+ 0.09 γ/γ+β; n=6). Bisulfite sequence analysis performed to determine whether changes in DNA methylation of 5 CpG residues within the 5' γ-globin promoter regions were associated with increased γ-globin expression showed that DNA methylation levels were similar in BM erythroid cells from normal baboons expressing very low levels of HbF (<1%), bled baboons expressing moderately elevated levels of HbF (5-10%), and cultured erythroid progenitor cells expressing highly elevated levels of HbF (30-50%). Changes in γ-globin promoter DNA methylation were thus not associated with increased γ-globin expression in EPC cultures. Further experiments were therefore performed to investigate whether differences in BCL11A expression were associated with increased γ-globin in EPC cultures. Western blot assays performed using three different anti-BCL11A monoclonal antibodies recognizing epitopes present in the N terminus, core, and C terminus detected different BCL11A isoforms in cultured EPC and normal BM erythroid cells. The size of the predominant protein band detected in cultured EPC was 125kDa, corresponding to the reported size of the in vitro transcription/translation product encoded by the BCL11A-XL transcript (Liu et al, Mol Cancer 16:18, 2006). In contrast, the size of the predominant band observed in BM erythroid cells was 220kDa. The 220kDa isoform was not observed in cultured EPC. Higher molecular weight forms of BCL11A have been observed following co-transfection of vectors encoding BCL11A and SUMO-1 (Kuwata and Nakamura, Genes Cells 13:931, 2008). Therefore we investigated whether the post-translational modification SUMOylation was responsible for the difference in the size of the 125 and 220kDa isoforms. Immunoprecipitation experiments performed using either SUMO-1 or SUMO 2/3 antibodies followed by Western blot with anti-BCL11A antibody showed that the 220 kDa isoform, but not the 125kDa isoform, was immunoprecipitated by either anti-SUMO-1 or anti-SUMO-2/3 antibody, confirming that the 220 kDA isoform, but not the 125 kDa isoform, was SUMOylated. Western blot assays performed to investigate the relative levels of these isoforms in BM erythroid cells of normal baboons, phlebotomized baboons, and early gestational age (53d) baboon fetal liver showed that expression of the 125kDa isoform was increased in bled compared to normal unbled baboons, suggesting that the deSUMOylated BCL11A isoform was increased by erythropoietic stress. The relative levels of the 125 and 220 kDa isoforms were similar in bled BM and fetal liver, indicating that SUMOylation of BCL11A was not developmentally regulated. The absolute level of BCL11A was reduced in fetal liver erythroid cells compared to BM erythroid cells consistent with observations showing that the level of BCL11A expression is developmentally regulated in man (Sankaran et al, Nature epub 2009). We conclude that BCL11A is post-translationally modified by SUMOylation in primary BM erythroid cells, but not in cultured EPC expressing high levels of HbF and suggest that modulation of the level of BCL11A SUMOylation is important in the mechanism responsible for increased HbF levels during recovery from acute erythropoietic stress. Disclosures:No relevant conflicts of interest to declare.

Nuclear Tumor Necrosis Factor Receptor-associated Factor 6 in Lymphoid Cells Negatively Regulates c-Myb-mediated Transactivation through Small Ubiquitin-related Modifier-1 Modification

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-kappaB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5'-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.